Study of the genetic basis of denitrification in pure culture denitrifiers ...

Study of the genetic basis ofdenitrification in pure culture

denitrifiers isolated fromactivated sludge and soil

Kim Heylen

Dissertation submitted in fulfillment of the requirements for the degree of Doctor ( Ph.D.)

in Sciences, Biotechnology

December 2007

Promotor: Prof. Dr. Paul De Vos

Co-promotor: Prof. Dr. Ir. Willy Verstraete

EXAMINATION COMMITTEE

Prof. Dr. Savvas Savvides (chairman)

Faculty of Sciences, UGent, Ghent

Prof. Dr. Paul De Vos (promotor)


Prof. Dr. Ir. W. Verstraete (co-promotor)

Faculty of Bioscience Engineering, UGent, Ghent

Prof. Dr. Ir. N. Boon

Faculty of Bioscience Engineering, UGent, Ghent

Prof. Dr. Ir. Mike S.M. Jetten

Faculty of Science, Radboud Universiteit Nijmegen, The Netherlands

Dr. C. Etchebehere

Faculty of Science, Universidad de la República, Uruguay

Prof. Dr. Anne Willems


"Eindelijk ist gedaan. Oef!"

Dat is zowat het eerste gevoel dat in me opkomt. Niet dat de jaren hier steeds kommer

en kwel waren - ik heb zo genoten van de vele reizen, de leuke (en minder leuke) collega's, de

verhuis van het achtste naart vierde, de (zatte) recepties, de nieuwjaarsetentjes, de

vrijdagavonden bij Christiane, de feestweken... Achterafgezien is alles zeer vlot gegaan, maar

als je er middenin zit, ervaar je dat wel anders. Iemand heeft me ooit gezegd dat naarmate je

meer twijfelt, je beter bezig bent. Voor mij is dat een beetje de samenvatting van vier jaar

doctoreren. Eigenlijk ben ik voor het oog van mijn collega's volwassen geworden... En is het

nu tijd voor het serieuzere werk. Natuurlijk had ik dit doctoraat nooit alleen kunnen volbrengen.

Vele mensen hebben hun steentje bijgedragen, via wetenschappelijk input, of gewoon door er

te zijn in moeilijke en/of zotte momenten. Ik ben niet echt prozaïsch aangelegd, maar ik doe

een poging om iedereen te bedanken.

Eigenlijk is het allemaal begonnen met mijn master thesis bij Geert en Margo. Margo

heeft me al de kneepjes van het microbiologische vak geleerd, en is eigenlijk 'verantwoordelijk'

voor al mijn latere werk. Ik heb me tijdens die thesis super geamuseerd, niet in het minste door

Liesbeth Masco, Robin en Evie. En ik heb er een goei vriendin aan overgehouden, en mijn

ventje...

Na die thesis begon een doctoraat op het labo voor Microbiologie me wel aan te

spreken. Allé, na een beetje rondvragen, kon ik toch bij Paul een doctoraat starten rond

denitrificatie. Dus, een verhuis naar het achtste. Iedereen is dit natuurlijk al vergeten, maar ik

heb me daar toen een half jaar zeer rustig en stil gehouden (ja, dat kan ik ook). Mijn partner

in crime werd Bram. Gelukkig bezat hij alle kwaliteiten die ik nodig had in een buurman:

dezelfde voorliefde voor harde, ietwat cynische humor, bestand tegen mijn gezaag en geklaag

en in staat om me telkens weer vertrouwen te geven in mijn werk. Natuurlijk was er ook

Liesbeth Lebbe, onze ancien. Altijd goed gezind, zo zot als een achterdeur, een echte spring-

in-'t-veld, maar ook steeds bezorgd en een luisterend oor ter beschikking. En dan nog een

verdomd goei laborante. Zo maken ze er geen twee.

Als eerste wapenfeit moest ik van Paul helemaal alleen naar een congres in Marburg.

Tof! Maar eigenlijk was dit een fantastisch begin voor mijn doctoraat. Bij mijn terugkeer ben

ik met veel goesting en volle overgave begonnen aan mijn werk. Dit is het perfecte voorbeeld

van Pauls input voor mijn doctoraat: veel impulsen, veel vertrouwen en veel vrijheid. Waarvoor

veel dank. Natuurlijk waren Prof Verstraete en Nico er ook om mijn wetenschappelijke

wandel te begeleiden. Het was een unieke kans om te mogen samenwerken met een 'instituut'

als Prof. Verstraete. Uw eigenzinnige perceptie en uw immense creativiteit waren een zeer

goede leerschool. Bedankt voor uw vertrouwen in mij. En wat zou ik de laatste vier jaar

gedaan hebben zonder Nico. Steeds maar een telefoontje of emailtje weg, steeds bereid om te

helpen of om een zoveelste draft van een paper na te lezen. Nen hele dikke merci.

Alsof soort soort zoekt, werd ons labootje uitgebreid met nog enkele prettig gestoorde

figuren, zoals Caroline en Joachim, en later ook nog An en Emly. Zij zorgden samen met

Liesbeth voor een enorm plezante werksfeer. Soms was effectief 'werken' een beetje moeilijk

(tijdens het zingen van Strangers in the Night bijvoorbeeld), maar dat werd ruimschoots

gecompenseerd door leuke dynamiek en het sympathieke groepsgevoel. Jeroen Adam bracht

dan weer wat zen opt labo. An heeft gaandeweg een beetje de rol van Bram overgenomen en

fungeerde de laatste maanden als mijn klankbord. Blijkbaar kon ik toch niet zonder. Natuurlijk

zijn er nog andere collega's die een verschil hebben gemaakt. Bij de start van mijn doctoraat

had ik twee voorbeelden, en eigenlijk is dat niet meer veranderd. Door het niveau van hun

werk en hun aanpak heb ik altijd opgekeken naar Peetse en Tom Coenye. En door onze

babbels, was het nu op een vrijdagavond in een bruin café, na een nieuwjaarsetentje of in een

trendy bar in Toronto, hebben ze me een figuurlijke schop onder mijn kont gegeven en

aangezet tot nadenken. De andere doctoraatstudenten waren ook een steun en toeverlaat.

Vooral dan Ilse. Het was enorm leuk samen de toerist uit te hangen. En dan nog Jeanine en

Paul Segers, twee gemotiveerde mensen zonder wie ons labo nooit zo vlot zou draaien.

Uiteraard zijn er naast het werk nog enkele mensen die een groot verschil hebben

gemaakt en mijn leven de laatste vier jaar. Ira en Ariane zijn er altijd geweest. We hebben

elkaar misschien niet veel gezien, vooral het laatste jaar niet, maar gewoon weten dat jullie

steeds voor me klaarstonden als ik het weer eens niet zag zitten of om die eerste publicatie te

vieren, dat was voor mij enorm belangrijk. Sven, wat had ik zonder jou het laatste jaar gedaan.

Een huis verbouwen en doctoreren in hetzelfde jaar, weeral typisch voor mij. Godzijdank dat

je positieve ingesteldheid ons door alles doorsleurt. En last but not least, mijn ouders. Ik weet

eigenlijk niet hoe ik moet omschrijven wat zij voor mij betekenen en betekend hebben tijdens

mijn 'academische loopbaan'. Al de kansen die jullie mij gegeven hebben en jullie voortdurende

onvoorwaardelijke steun heeft mij gemaakt tot wie ik ben. Ik zie jullie graag…

Enorm bedankt,

Kim.

CONTENTS

Background and aim..........................................................................................................9

1. Introduction.........................................................................................................................11

1.1. Denitrification......................................................................................................14

1.1.1. Definition..............................................................................................14

1.1.2. Importance...........................................................................................15

1.2. Distribution of the denitrifying ability...............................................................18

1.3. Key enzymes of the denitrification process....................................................19

1.3.1. Nitrite reduction..................................................................................19

1.3.2. Nitric oxide reduction..........................................................................21

1.4. Current denitrification research........................................................................24

1.5. Conceptual framework of the thesis.................................................................26

1.5.1. Cultivation-dependent research and alternatives..........................26

1.5.2. Strategy followed and overview of chapters...................................27

1.6. References............................................................................................................28

2. Isolation, characterization and identification of denitrifying bacteria from the en-

vironment.................................................................................................................................35

2.1. Cultivation of heterotrophic denitrifying bacteria: optimization of

isolation conditions and diversity study................................................................37

2.2. Diversity of heterotrophic denitrifiers isolated from soil using a

multiple-media set......................................................................................................53

2.3. Back & forth.........................................................................................................65

3. Functional phylogenetic analysis of pure culture denitrifiers......................................69

3.1. The incidence of nirS and nirK and their genetic heterogeneity in

cultivated denitrifiers...............................................................................................71

3.2. Nitric oxide reductase (norB) gene sequence analysis reveals

discrepancies with nitrite reductase (nir)gene phylogeny in cultivated

denitrifiers.............................................................................................................89

3.3.Functional gene study on heterotrophic denitrifiers isolated from soil

..............................................................................................................................103

3.4. Back & forth.......................................................................................................119

4. Screening for denitrification genes undectable with PCR..........................................123

4.1. Simple screening method for norB genes suggests extra enzymatic

redundancy for the denitrification process..........................................................125

4.2. Back & forth.......................................................................................................139

5. Description of novel bacterial species involved in the nitrogen cycle.......................141

5.1. Stenotrophomonas terrae sp. nov. and Stenotrophomonas humi sp.

nov., to novel nitrate-reducing Stenotrophomonas species isolated from soil

..............................................................................................................................143

5.2. Acidovorax caeni sp. nov., a novel denitrifying species with genetically

diverse isolates from activated sludge.................................................................155

5.3. Back & forth.......................................................................................................167

6. Concluding remarks..........................................................................................................171

Addendum.............................................................................................................................177

Summary.............................................................................................................................183

Samenvatting......................................................................................................................187

Curriculum vitae................................................................................................................191

BACKGROUND AND AIM

Since the initial description and introduction of the term denitrification in 1882

[29] and the first isolation of denitrifying bacteria four years later [30], research has

been focused on isolation and identification of denitrifiers and unraveling the

biochemistry and ecology of these pure cultures. With the advent of molecular

techniques, the focus shifted to the environmental monitoring of the whole

denitrification process in situ. However, the major challenge of this ecological

research is the correlation between the structural and functional biodiversity, as

denitrification is such a phylogenetically dispersed trait. Denitrification genes can

be used as functional markers, but their phylogenetic information content was not

clear, after several reports of differences between functional gene phylogeny and

organism phylogeny. The aim of this thesis was to investigate the genetic basis of

denitrification in a broad range of pure culture denitrifiers. The taxonomic value of

the genes encoding the key enzymes was assessed, and their detection, incidence

and phylogeny were investigated. Because most denitrification research had been

done with reference strains, new isolation procedures from activated sludge and

soil were performed through the development of new defined elective growth media.

9

1

INTRODUCTION

Extensive reviews, all focusing on different aspects of denitrification, are available, for

example on the history of the denitrification research [66], methods for measuring

denitrification [33], and cell and molecular biology [115]. In this literature overview, only

aspects of denitrification necessary for general situation or specifically linked to the

presented work are discussed. The focus lay on denitrifying bacteria. Therefore, denitrifying

archaea (see review [9]), fungi [97] and recently discovered foraminifers [75] will not be

discussed.

INTRODUCTION

13

1.1. DENITRIFICATION

1.1.1. Definition

Denitrification is a step-wise dissimilatory reduction of nitrate (NO3

-) or nitrite (NO2

-)

over nitric oxide (NO) and dinitric oxide (N2O), also named nitrous oxide, to nitrogen gas

(N2), coupled to electron transport phosphorylation. Denitrification is a modular process

and is accomplished in four enzymatic steps, catalyzed by four metalloproteins (Table

1.1).

Table 1.1. Overview of the denitrification process

1 nar, nitrate reductase gene; nir, nitrite reductase gene; nor, nitric oxide reductase gene;nos, nitrous oxide reductase gene, ² Taken from [114]

Each reduction step of the denitrification process has a positive redox couple, higher than

0.35V, which is comparable to that of oxygen reduction (O2/H

2O couple). Therefore, not

every step of the process is necessary to have a net conservation of energy. In fact, bacteria

that express only part of the denitrification electron transport chain are common.

Denitrification sensu stricto contains nitrite and nitric oxide respiration, causing loss of

fixed nitrogen [115]. This may optionally be preceded or followed by other reactions. N2O

respiration can proceed independently from denitrification, though not every denitrifying

bacterium will grow on N2O. Nitrate respiration, terminating at the level of nitrite, is the

most widely distributed nitrogenous oxide respiration variant among prokaryotes. In fact,

the majority of nitrate-respiring bacteria are not able to denitrify. On the other hand, some

denitrifying species are not able to respire nitrate, but start denitrification from nitrite. The

existence of these truncated variants of denitrification hampers the identification of bacteria

as true denitrifiers sensu stricto. The situation is even more complicated with other processes

also generating N2O or N

2 from nitrate or nitrite, such as dissimilatory nitrate or nitrite

reduction to ammonium (DNRA), nitrate assimilation [96], anaerobic ammonium oxidation

(anammox) [46], or methanotrophic nitrate assimilation coupled to chemodenitrification of

nitrite [74]. The distinctive feature of denitrification is the coupling to the electron transport

Overal reaction1:

NO3- → NO2

- → NO → N2O → N2

Separate reactions²:

NO3- + 2e- + 2H+ → NO2

- + H2O ∆G0’ = -161.1 kJ/mol E0’ = +420 mV

NO2- + e- + 2H+ → NO + H2O ∆G0’ = -76.2 kJ/mol E0’ = +374 mV

2NO + 2e- + 2H+ → N2O + H2O ∆G0’ = -306.3 kJ/mol E0’ = +1177 mV

N2O + 2e- + 2H+ → N2 + H2O ∆G0’ = -339.5 kJ/mol E0’ = +1352 mV

nar nir nor nos

CHAPTER 1

14

phosphorylation. Therefore, the two major criteria for respiratory denitrification are (i)

production of N2O or N

2 from nitrate or nitrite, and (ii) the coupling of this reduction to

energy conservation, and thus growth yield, increasing proportional to nitrate or nitrite

concentrations [58].

DNRA and anammox are two other processes that convert a nitrogen oxide in an anaerobic

environment, and therefore can go in substrate competition with denitrification in the

environment. DNRA (Figure 1.1) reduces nitrate to nitrite, similar to denitrification. Nitrite

is than further reduced to ammonium, in excess of the reduced nitrogen needed for growth.

The production of ammonium and the only sporadic production of nitrous oxide facilitate

differentiation from denitrification. In contrast, the anammox process can disguise itself as

denitrification. Anaerobic bacteria oxidize ammonium with nitrite and produce dinitrogen

gas (Figure 1.1). In addition, anammox bacteria themselves can produced ammonium through

nitrate reduction via dissimilatory nitrate reduction to ammonium, which is than oxidize

with nitrite [51]. The net result, the conversion of nitrate over nitrite to nitrogen gas, is

identical to denitrification. Because of its recent discovery, more research is necessary to

unravel the whole anammox process in nature and its competition with denitrification.

Nevertheless, the anammox bacteria known to date can be easily differentiated from common

denitrifying bacteria through distant phylogeny within the planctomycetes and their very

slow growth.

1.1.2. Importance

Denitrification is a very important biogeochemical process because it completes the global

nitrogen cycle (Figure 1.1) and returns fixed nitrogen to the atmosphere, where it is again

available for fixation by diazotrophic bacteria. Because of its global character, denitrification

has a large impact in a whole range of ecosystems and processes.

Figure 1.1. Nitrogen cycle

INTRODUCTION

15

NO3-NO2

-

N2

NH4+

nitrification

denitrification

DNRAnitrate assimilation

dinitr

ogen

fixa

tion

N-containing biomolecules

anam

mox

NO3-NO2

-

N2

NH4+

nitrification

denitrification

DNRAnitrate assimilation

dinitr

ogen

fixa

tion

N-containing biomolecules

anam

mox

When denitrification was first discovered, the primary focus ecosystem of the researchers

was agricultural soil, because the process is responsible for the loss of nitrogen, the nutrient

most limiting to crop production. Because of soil’s three-dimensional matrix, nutrient

conditions, and denitrifying activity, are variable in space and time. Especially in the

rhizosphere, large populations of denitrifiers can be active, removing nitrogen and making

soils sources of N2O [53]. Fertilizer losses to denitrification range from virtually none to

over 70 percent, but losses are more commonly in the range of 20 to 30% [94]. As a result,

more fertilizer is used in agriculture, causing nitrate leaching and runoff from fertilized

fields into surface waters and groundwater.

This excessive applications of fertilizers, together with intensive exploitation of farms and

a significant contribution from industry, have increased the nitrogen load discharged to

receiving waterways [101], leading to a decrease of water quality, contamination and

eutrophication of receiving waters and health problems related to oxidized forms of nitrogen.

As a result, more stringent regulations have been approved in an effort to deal with this

problem. In Europe, directive 91/271/EEC (1991) prescribes the nitrogen standards for

treated wastewater discharges. The most economic options for removal of nitrogen from

wastewater are biological processes; the most developed system is the nitrification-

denitrification process. This two-stage process consists of an initial nitrification stage,

accomplished by autotrophic bacteria, in which ammonia is oxidized to nitrite by ammonia-

oxidizing bacteria and nitrite is subsequently oxidized to nitrate by nitrite-oxidizing bacteria.

Numerous facultative heterotrophic bacteria then carry out a second denitrification stage,

where nitrate is reduced to molecular nitrogen, using substrates from the wastewater as

electron donor. Conditions favoring these two processes differ significantly, a problem

which can be overcome by spatial (in different regions of the same reactor or in separate

reactors) or temporal (by intermittent aeration) separation in the waste treatment process.

Denitrification can also be useful for the destruction of other pollutants such as hydrocarbons.

Aerobic bioremediation of hydrocarbon-contaminated sites, although studied for over a

century, is not optimal as the oxygen availability is usually the rate-limiting parameter, due

to its low solubility in water and its low rate of transport through saturated porous

matrices such as soil and sediments. Therefore, the use of nitrate as electron acceptor is

advantageous, also compared with other anaerobic electron acceptors such as sulphate or

ferric iron, because it is water soluble, not costly, not seriously toxic and does not interact

with other inorganic species present. The use of denitrifiers in pollutant removal is also

attractive because denitrification is a facultative trait and denitrifiers have the highest

growth yield and are the easiest to grow of any bacteria capable of anoxic growth [96].

CHAPTER 1

16

Denitrifying bacteria are able to degrade hydrocarbons such as BTEX compounds (benzene,

toluene, ethylbenzene, and xylenes), which are primarily contaminants of concern in aquifer

water and sediments, due to leakage of underground petroleum storage tanks and spills at

petroleum production wells, refineries, pipelines and distribution terminals.

Denitrification releases N2 but also N

2O to the atmosphere. The latter is the third largest

greenhouse gas contributor to global warming, next to CO2 and CH

4. While its radiative

warming effect is substantially less than that of CO2, nitrous oxide is 300 times more

persistent in the atmosphere [43]. Thus, denitrification in all different environments, intrinsic

to nature or stimulated by human activity, contributes to the depletion of ozone and global

warming. Soil and oceans are considerable sources of N2O. Recently, it has been shown that

earthworms also emit nitrous oxide, next to nitrogen gas [50]. These emissions appear to be

primarily due to soil-derived denitrifying bacteria subjected to the in situ conditions of the

earthworm gut (anoxia, high quality organic carbon, and nitrate or nitrite), which are highly

favorable for denitrification. Up to 56% of the in situ emission of N2O from certain soils

might be derived from earthworms [22].

In addition, denitrification possibly has a role in bacterial pathogenicity [68]. The ability to

respire nitrogen oxides confers an advantage to a pathogen in adapting to intracellular life.

For example Fritz et al. [25] showed that anaerobic nitrate reduction is essential for the

metabolism of Mycobacterium bovis in the lungs, liver and kidneys of immuno-competent

mice. Also, the ability to reduce NO could be beneficial for pathogenic bacteria, because

macrophages generate NO, which is cyto- and genotoxic, to kill invasive bacteria.

INTRODUCTION

17

1.2. DISTRIBUTION OF THE DENITRIFYING ABILITY

Among the biogeochemical cycles on earth, there are no inorganic biotransformations that

are carried out by wider distributed and diverse organisms than is the case for denitrification

[96].

An annotated survey by Zumft [114] more than a decade ago, listed almost 130 bacterial

species within more than 50 genera. True denitrifying bacteria are found in Alpha-, Beta-,

Gamma- and Epsilonproteobacteria, Firmicutes, and Bacteroidetes. Three groups of bacteria

are classically seen as not containing true denitrifiers [96]: a) Gram-positives other than

Bacillus, b) the Enterobacteriaceae, and c) obligate anaerobes. Gram-positive bacteria are

somewhat neglected in denitrification research, although the genus Bacillus has long been

recognized for harboring denitrifying strains in, e.g. the species B. subtilis [77] and B.

azotoformans [58]. Nevertheless, other Gram-positive bacteria have been confirmed for

respiratory denitrification, for example Corynebacterium nephridii [58] and several

Actinomycetes [14, 85]. Probably even more Gram-positive bacteria will be recognized as

denitrifiers when within this group, specific tests and further characterization continues.

Within the enterobacteria, the facultative anaerobe metabolism is coupled to respiratory

nitrate or nitrite reduction to ammonium, and never to denitrification, as both processes are

mutually exclusive in one bacterium. Previous reports on denitrification in enterobacteria

were shown to be unsupported [114]. No denitrifying obligatory anaerobic bacteria are

known to date.

Denitrifiers can have diverse modes of energy conservation, namely organotrophs -

fermentors, extremophiles, sporeformers, magnetotactic bacteria, pathogens -,

chemolithotrophs or phototrophs. And although the denitrification process is generally

anoxic, denitrifiers have different oxygen thresholds and some even need oxygen to perform

the process. For example Paracoccus pantotrophus (previously named Thiosphaera

pantotropha) can denitrify under complete air saturation [54], while denitrifying nitrifiers

are obligate aerobes and thus need oxygen to complete denitrification. These denitrifying

nitrifiers are a good example of chemolithotrophic denitrification, which was discovered

when production of N2O from nitrite by Nitrosomonas europaea was observed [71, 82].

Other ammonia-oxidizing bacteria can also perform both processes, suggesting that nitrifier

denitrification can be a universal trait among betaproteobacterial ammonium-oxidizing bacteria

[84]. All other major branches of the nitrogen cycle can also be associated with denitrification,

except ammonification, as mentioned above. Especially denitrification in diazotrophic

bacteria has been reported frequently, for example in Rhizobium [63], Bradyrhizobium

[99], Sinorhizobium [13], Azoarcus [113].

CHAPTER 1

18

1.3. KEY ENZYMES OF THE DENITRIFICATION PROCESS

The conversion of nitrite to nitric oxide by nitrite reductase is the crucial step in denitrification

because it converts fixed nitrogen to gaseous NO. This nitric oxide is further reduced to

N2O through the action of nitric oxide reductase. The control of both reductases in

denitrifying bacteria is regulated coordinately to assure removal of NO by the latter reductase

or, if this is not possible, by down-regulation of nitrite reduction [116]. The steady-state

concentration of free NO during denitrification is in the nanomolar range, because, although

NO metabolism is innate to denitrifiers, NO is also toxic for this group of bacteria.

1.3.1. Nitrite reduction

In denitrifying bacteria, two structurally different types of nitrite reductases occur, which

are distinguishable by their prosthetic groups, either cytochrome cd1 or copper. Both enzymes

are mutually exclusive within one cell. They can be present within the same genus, and

even the same species, as was reported for Alcaligenes faecalis [4]. Their interchangeability

is limited: CuNiR can replace cytochrome cd1 NiR, which was demonstrated using the

CuNiR encoding gene nirK from Pseudomonas aureofaciens in a mutationally cytochrome

cd1-free background of P. stutzeri [32], but requirements for heme d

1 biosynthesis makes

the reverse replacement impossible. It is generally assumed that cytochrome cd1 NiR is

numerically dominant in the environment, while CuNiR can be found in a greater variety of

physiological groups and bacteria from different habitats. These conclusions were based on

pure culture results and therefore could reflect biases of culture methods. Within the

proteobacteria, neither NiR enzyme has been found in exclusive association with a particular

taxon. However, to date, only CuNiR is observed in less conventional denitrifiers, such as

nitrifying bacteria [10], bacilli [20] or archaea [41, 42].

CuNiR are trimetric enzymes located in the periplasm. Each subunit is close to 40 kDa,

contains one type I and one type II Cu and comprises of two domains (Figure 1.2). The

type I Cu site is located on domain I, ligated to His95, Cys136, His145 and Met150

(numbering from “Achromobacter cycloclastes”), where its functional as the electron entry

site. The principal electron donors to CuNiR are azurin [21, 116] and pseudoazurin [48,

56], while cytochromes appear less frequently involved [81]. Type II Cu is the substrate-

binding site of nitrite reductase [55], which is coordinated by three histidines, His100,

His135, and His306, with the latter histidine provided by the adjacent subunit (Figure

1.2.). The electrons donated to the type I Cu center are transferred to the catalytic type II

Cu center through a chemical path involving Asp and His. In the reaction of CuNiR with

INTRODUCTION

19

nitrite to form nitric oxide, a cuprous nitrosyl complex functions as the key intermediate

[40, 92].

In contrast, the respiratory nitrite reductase of Bacillus halodenitrificans [20], B.

azotoformans [89] and Geobacillus stearothermophilus [39] is a dimeric, membrane-bound

Cu-containing enzyme with the catalytic side oriented towards the inside of the cell [98].

Its primary structure is homologeous to that of Gram-negative bacteria, also with type 1

and type 2 Cu. Their electron donors are not known.

A single copy nirK gene encodes CuNiR.

Figure 1.2. Copper-containing nitrite reductase from “Achromobacter cycloclastes”. Schematic representation ofthe trimer and position of Cu atoms. Full circles represent the six Cu atoms coordinated by cysteine (C), hystidine(H), and methionine (M). Domains I and II are related to domains I’, II’ and I”, II” by a crystallographic threefoldaxis. The type II Cu is bound by three histidines between the subunit interfaces. Taken from [114].

The cytochrome cd1 NiR is a homodimeric enzyme with a subunit mass of around 60 kDa,

located in the periplasm. The prosthetic groups are non-covalently bound heme c and

heme d1, which are both present in each subunit to render cytochrome cd

1 a tetraheme

protein. The smallest domain contains the heme c and acts as the electron transfer center.

This c-heme-binding peptide is located nearby the amino-terminus of the protein. A

hydrophobic patch on top of the heme c domain of cytochrome cd1, can form the docking

site for a complementary hydrophobic patch in electron donors (e.g. azurin, cytochrome

c551

, pseudoazurin), bringing the metal centers very closely together [108]. Not being

highly discriminatory, this type of recognition provides a rationale for the interchangeability

of electron donors observed among denitrification components. The heme d1 is localized in

the largest domain and is the catalytic center [107].

CHAPTER 1

20

The ligation of both hemes in oxidized state differs in different denitrifiers [94]. For example,

in Paracoccus panthotrophus, heme c binds to two histidines and heme d1 has two ligands,

a proximal histidine and a distal tyrosine. However, through reduction, the distal ligand of

heme d1 is lost, causing an exchange of one of the axial ligands of the heme c, replacing a

histidine by a methionine. In Pseudomonas aeruginosa, heme c is coordinated by one

histidine and one methionine, and does not modify after reduction, and the distal ligand of

heme d1 is a hydroxyl ion. The nitrogen atom of nitrite is bound to the iron of heme d

1,

while hydrogen bonds bind on of the oxygen atoms to two conserved histidines in the

distal pocket of heme d1. The physiological reaction of cytochrome cd

1 is the protonation

of nitrite and removal of water to yield NO.

The cytochrome cd1 nitrite reductase is encoded by the single copy nirS gene.

1.3.2. Nitric oxide reduction

The N-N bond formation takes place during the reduction of nitric oxide to nitrous oxide.

This step is catalyzed by the membrane-bound nitric oxide reductase (NOR). Three classes

of NOR’s have been identified in bacteria: cNOR, qNOR and qCuNOR (Figure 1.3.).

They mainly differ in electron donors and in the number and type of electron transfer

centers present. The active site is, however, thought to be highly homologous in these

three classes. Compared to the other denitrification enzymes, less is known about NOR

since no tridimensional structure is yet obtained and active site structural data has been

mainly inferred from spectroscopic studies [94]. So, the mechanism of NO reduction to

N2O is not clear, and reports in the literature are contradictory.

The cNOR is a membrane-bound cytochrome bc complex, composed of two subunits of

about 17 and 53 kDa [117]. NORC, the small subunit, is anchored to the cytoplasmic

membrane. It retains one heme c, which is ligated to one conserved histidine and one

methionine, and is responsible for mediating electron transfer from the periplasmic electron

donors, such as cytochrome c and cupredoxin, to the catalytic subunit (Figure 1.3A). NORB,

the largest subunit, contains two b hemes and one non-heme iron, forming one low-spin

heme b and a binuclear active site. The low-spin heme b will act as a provider of electrons

to the active site (Figure 1.3A), and is ligated to two conserved histidines. Four conserved

histidines are possible ligands of this binuclear site. Proximity, provided by the binuclear

site, appears to be a factor in N-N bond formation. The catalytic site is located closer to the

periplasmic than the cytoplasmic face of the membrane, releasing N2O into the periplasm.

This enzyme has only been found in denitrifying bacteria. Other names used are NORB or

INTRODUCTION

21

short-chain NOR (scNOR). The catalytic subunit is encoded by the single copy cnorB

gene.

While possessing a similar primary structure to cNOR, qNOR is a single subunit enzyme

(Figure 1.3B) that accepts electrons from quinols [17]. Primary sequence analysis shows

that qNOR is constituted by an N-terminus extension similar to the NORC subunit, and a

C-terminus region homologous to NORB subunit. This enzyme has been found in both

denitrifying and non-denitrifying bacteria [7], the latter being mostly pathogenic [36].

Other names used are NORZ or long-chain NOR (lcNOR). The qnorB gene encodes this

monomer NOR.

A third type of nitric oxide reductase, qCuANOR, was purified from Bacillus azotoformans

[90] and is formed by two subunits. The largest catalytic subunit is similar to the NORB

subunit, the smaller subunit does not posses a heme c but uses a copper A site to achieve

electron transport to the catalytic subunit (Figure 1.3C). This enzyme can use menaquinol

as well as cytochrome c551

as electron donor, the former is suggested to be active in NO

detoxification, while the latter would be functional in denitrification [90]. This qCuANOR

has only been found in B. azotoformans and the encoding genes are not yet identified.

Bacterial NO reductases belong to the superfamily of the heme-copper oxidases including

cytochrome oxidases, [36], which are the terminal enzymes of the aerobic electron transport

chain that catalyze the reduction of O2 to H

2O. The common phylogeny between heme-

copper terminal oxidases and bacterial NO reductases was proposed because of structural

similarities [35, 80, 100]: (i) the large catalytic subunit displays significant sequence

homology, (ii) crucial residues (including six metal-binding histidines) are conserved, (iii)

topology of the catalytic subunit predicts 12 transmembrane helices, and (iv) both enzyme

types contain a bimetallic center, consisting of a heme-iron and a second metal, which is Cu

in oxidases and qCuANOR, and Fe in the cNOR and qNOR. The common phylogeny is

supported by examples of both reductases using both O2 and NO as alternative electron

acceptors [26, 31].

CHAPTER 1

22

Figure 1.3. Schematic representation of NOR enzymes: (A) cNOR, (B) qNOR, and (C) qCuANOR. Dashedarrows represent the proposed electron transfer pathway from a periplasmic electron donor to the active site.

Taken from [94].

INTRODUCTION

23

1.4. CURRENT DENITRIFICATION RESEARCH

Knowledge on the physiology, biochemistry and molecular regulatory mechanisms of several

pure culture denitrifiers provided information to develop molecular tools for environmental

studies, allowing investigation of the unknown not-yet-cultured denitrifying diversity. As

a result, the last decade, denitrification research focused on environmental (culture-

independent) analysis.

Because of their taxonomic diversity, denitrifying bacteria could not be studied through the

conventional 16S rRNA gene sequence approach, but rather the functional genes were used

for cultivation-independent study. Primers were first developed for nirS and nirK genes [4,

34], so most denitrification research to date focuses on these nir genes. Environmental

studies assessed the nir sequence diversity through clone library sequencing [76, 79, 112],

T-RFLP studies [2, 3, 5, 72, 109, 111], and DGGE analysis [95], mainly in soil and marine

environments. Most studies revealed major environmental gene clusters, showing little

overlap with the clusters harboring genes from isolated strains, suggesting the presence of

yet uncharacterized denitrifiers in the environment [5, 12, 62, 72, 109]. Generally, nirK

sequences were retrieved from more diverse habitats but were more closely related, while

nirS amplicons could not always be obtained from environmental samples but seemed to be

very diverse [5, 72, 111], and represented spatially distinct sequence populations [5, 62].

Yet the reverse was also observed [64, 79].

Next to functional diversity, both quantification and activity of denitrifying bacteria in the

environment are essential to determine the influence of these microbial populations on the

overall denitrification process [69, 70]. Quantification of denitrifiers in the environment

was assessed based on nir genes through southern hybridization [59], PCR-based techniques,

such as competitive PCR [61, 73], MPN-PCR [61], and real-time PCR [37, 38, 49, 103], or

microarrays [93, 110]. This revealed denitrifier abundance between 104 to 109 nir gene

copies per gram soil, which was greatly underestimated by cultivation, but indicated a low

proportion of potential denitrifiers to total bacteria in soils [38]. Also recently, a group-

specific cnorB real-time PCR was developed, for quantification of Pseudomonas and rhizobia

in soil [19]. In spite of all these efforts, the presence of functional genes determined by

DNA probing only indicates the denitrifying potential, regardless of whether they are

retrieved from active denitrifying bacteria in this environment. So, also reverse transcription

PCR amplification was necessary to study only active denitrifiers in the environment on

mRNA level [62, 83].

CHAPTER 1

24

All these molecular approaches tried to correlate diversity, quantity and activity of the

denitrifying community to environmental controls (reviewed by Wallenstein [102]).

Unfortunately, other traits of the organism, not connected to denitrification, but rather to

its phylogeny, can also play a crucial factor in environmental selection. The correlation

between structural and functional biodiversity is one of the major challenges in microbial

ecology. But already in the first studies using functional probes, discrepancies were observed

between nirS gene diversity and phylogenetic diversity of denitrifying pseudomonads

[104, 105]. Functional gene sequences from a dozen or so bacterial genomes [67] and

halobenzoate degrading denitrifying isolates [86] confirmed that functional gene phylogeny

does not always relate to 16S rRNA gene phylogeny. Also, failure to detect nir genes in

denitrifying strains closely related to those in which nir genes were detected [86] suggested

a great nir gene diversity in highly related bacteria. In addition, denitrification genes nirK

and cnorB can be detected in pure culture nitrifiers [10, 11, 28, 45]. Functional genes in

nitrifier denitrifiers can be closely related to those of denitriers, making differentiation

between both metabolic guilds, i.e. denitrification and nitrification, based on functional

sequences very difficult.

Thus, sequence analysis of denitrification genes in cultivation-independent studies is very

useful to assess the functional diversity present in the environment. Yet, the functional

significance of biotic diversity is currently unknown due to the unclear phylogenetic

information content of the denitrification genes. However, to evaluate the importance of

organism diversity versus functional diversity for the denitrification process in the

environment, knowledge of the denitrifiers’ identity is indispensable. For this, researchers

need to return to the basis of the denitrification research, i.e. cultivation, because to date,

the collection of the bacterium is the most straightforward method to obtain information

that can correlate its denitrification capacity, functional genes and phylogenetic position.

INTRODUCTION

25

1.5. CONCEPTUAL FRAMEWORK OF THE THESIS

1.5.1. Cultivation-dependent research and alternatives

As mentioned above, cultivation is the easiest method to obtain information on both the

denitrification capacity, the functional genes and the overall phylogenetic position of an

organism. Unfortunately, it is generally known that cultivation does not retrieve all diversity

present in a given biotope due to ‘the great plate count anomaly’ [87], which is dependent

of the environment of choice [1]. However, DNA-based techniques are also not free of

biases: DNA extraction protocols are not suitable for all bacteria, PCR primers can be

biased towards specific bacterial groups, amplified genes might not be functional, and

extra-cellular DNA can persist in the environment. A renewed interest in cultivation has

yielded new approaches and insights to isolate not-yet-cultivated bacteria, inspired by the

necessity of the microorganism itself to gain in-depth understanding of its physiology or to

access its metabolic pathway [52].

Conventional agar plating on complex media selects for microorganisms that are fast-

growing, grow in high cell densities, are resistant to high nutrient concentrations and are

able to grow on solid media. Dilution culturing [8] or extinction culturing [15, 16] overcome

the interference of these fast-growing ‘bacterial weeds’ and allow isolation of previously

uncultured bacteria. Other techniques, such as diffusion growth chambers, incubate samples

in their natural environment by restricting cell movement but allowing chemical exchange

with the environment [47]. However, also simple adjustments, such as prolonged incubation

time, other solidifying agents, or lower nutrient concentrations can significantly increase

the cultivability of a sample [44, 78, 88].

Most described denitrifier cultivation studies were carried out using complex media. Tiedje

[95] recommended TSA supplemented with 0.1% KNO3 as the most favorable general

medium for isolation of most heterotrophic denitrifiers [23, 65], resulting in a growth rate

two times faster than nutrient broth plus nitrate [18, 57]. Nitrate broth and agar were also

used for soil studies [14, 27, 106]. Several denitrifier cultivation studies were performed on

soil [14, 27, 106], activated sludge [14, 23, 24, 57, 60] or marine environments [6, 64].

CHAPTER 1

26

1.5.2. Strategy followed and overview of chapters

The aim of this thesis was to investigate the genetic basis of denitrification in a broad range

of pure culture denitrifiers. Therefore, new isolation procedures for activated sludge and

soil were performed through the development of new defined elective growth media. This

medium optimization focused on the heterotrophic, mesophilic, anaerobic denitrifiers,

knowingly ignoring other possible denitrifier metabolisms. This choice was motivated by

the probable numerically unimportance of very specialized physiologies in the studied

environments [95]. The pure culture denitrifiers were used to assess the taxonomic value

of the genes encoding the key enzymes, and to investigate their detection, incidence and

phylogeny.

The description of the performed work is organizated into the following chapters:

CHAPTER 2 - New defined growth media were developed to specifically isolate unknown

and less-conventional denitrifiers from the environment. An evolutionary algorithm was

used to determine the optimal components of the growth medium and their relative amounts

to isolate the highest possible denitrifier diversity. Denitrifying bacteria retrieved from

activated sludge and soil were polyphasically identified.

CHAPTER 3 - The genes encoding the key enzymes of the denitrification process – nirK,

nirS, cnorB and qnorB – were studied in pure culture denitrifiers, isolated from activated

sludge and soil. Their taxonomic value, detection, incidence and phylogeny were investigated.

CHAPTER 4 - In many denitrifying isolates, functional genes were not detected with

available PCR protocols. A dot-blot screening method for norB genes was developed to

determine the cause: unsuitable primers or unknown enzymatic redundancy of the

denitrification process.

CHAPTER 5 - The isolation campaigns of soil and activated sludge generated bacteria

involved in the nitrogen cycle that were only distantly related to currently described bacterial

taxa. Some of these isolates were subjected to a polyphasic characterization and described

as novel species.

Each chapter ends with a ‘BACK & FORTH’ section, containing a global discussion of the

subject with hindsight reflections, updated information and/or extra experimental work.

INTRODUCTION

27

1.6. REFERENCES1. Amann RI, Ludwig W, Schleifer K-H (1995) Phylogenetic identification and in situ detection of

individual microbial cells without cultivation. Microbiol Rev 59:143-169

2. Avrahami S, Conrad R, Braker G (2002) Effect of soil ammonium concentration on N2O release and on the

community structure of ammonia oxidizers and denitrifiers. Appl Environ Microbiol 68:5685-5692

3. Braker G, Ayala del Rio HL, Devol AH, Fesefeldt A, Tiedje JM (2001) Community structure of denitrifiers,

Bacteria and Archaea along redox gradients in Pacific Northwest marine sediments by terminal restriction

fragment length polymorphism analysis of amplified nitrite reductase (nirS) and 16S rRNA genes. Appl

Environ Microbiol 67:1893-1901

4. Braker G, Fesefeldt A, Witzel K-P (1998) Development of PCR primer systems for amplification of nitrite

reductase genes (nirK and nirS) to detect denitrifying bacteria in environmental samples. Appl Environ Microbiol

64:3769-3775

5. Braker G, Zhou J, Wu L, Devol AH, Tiedje JM (2000) Nitrite reductase genes (nirK and nirS) as functional

markers to investigate diversity of denitrifying bacteria in pacific northwest marine sediment communities.

Appl Environ Microbiol 66:2096-2104

6. Brettar I, Moore ERB, Höfle MG (2001) Phylogeny and abundance of novel denitrifying bacteria isolated

from the water column of the Central Baltic Sea. Microbiol Ecol 42:295-305

7. Büsch A, Friedrich B, Cramm R (2002) Characterization of the norB gene, encoding nitric oxide reductase,

in the nondenitrifying Cyanobacterium Synechocystis sp. strain PCC6803. Appl Environ Microbiol 68:668-

672

8. Button DK, Schut F, Quang P, Martin R, Roberston BR (1993) Viability and isolation of marine bacteria by

dilution culture: theory, procedures, and initial results. Appl Environ Microbiol 59:881-891

9. Cabello P, Roldàn MD, Moreno-Viviàn C (2004) Nitrate reduction and the nitrogen cycle in archaea.

Microbiology 150:3527-3546

10. Casciotti KL, Ward BB (2001) Dissimilatory nitrite reductase genes from autotrophic ammonia-oxidizing

bacteria. Appl Environ Microbial 67:2213-2221

11. Casciotti KL, Ward BB (2005) Phylogenetic analysis of nitric oxide reductase gene homologues from aerobic

ammonia-oxidizing bacteria. FEMS Microbiol Ecol 52:197-205

12. Casto-Gonzàlez M, Braker G, Farias L, Ulloa O (2005) Communities of nirS denitrifiers in the water column

of the oxygen minimum zone in the eastern South Pacific. Environ Microbiol 7:1298-1306

13. Chan Y-K, Barran LR, Broomfield ESP (1989) Denitrification activity of phage types representative of two

populations of indigenous Rhizobium meliloti. Can J Microbiol 35:737-740

14. Chèneby D, Philippot L, Hartmann A, Hénault C, Germon J-C (2000) 16S rDNA analysis for characterisation

of denitrifying bacteria isolated from three agricultural soils. FEMS Microbiol Ecol 34:121-128

15. Cho J-C, Giovannoni SJ (2004) Cultivation and growth chracateristics of a diverse group of oligotrophic

marine Gammaproteobacteria. Appl Environ Microbiol 70:432-440

16. Connon SA, Giovannoni SJ (2002) High-throughput methods for culturing microorganisms in very-low-nutrient

media yield diverse new marine isolates. Appl Environ Microbiol 68:3878-3885

17. Cramm R, Pohlmann A, Friedrich B (1999) Purification and characterization of the single-component nitric

oxide reductase from Ralstonia eutropha H16. FEBS Lett 460:6-10

18. Dandie CE, Burton DL, Zebarth BJ, Trevors JT, Goyer C (2007) Analysis of denitrification genes and

comparison of nosZ, cnorB and 16S rDNA from culturable denitrifying bacteria in potato cropping systems.

Syst Appl Microbiol 30:128-138

19. Dandie CE, Miller MN, Burton DL, Zebarth BJ, Trevors JT, Goyer C (2007) Nitric oxide reductase-targeted

real-time PCR quantification of denitrifier populations in soil. Appl Environ Microbiol 73:4250-4258

20. Denariaz G, Payne WJ, LaGall J (1991) The denitrifying nitrite reductase of Bacillus halodenitrificans.

Biochim Biophys Acta 1056:225-232

21. Dodd FE, Hasnain SS, Hunter WN, Abraham ZHL, Debenham M, Kanzler H, Eldridge M, Eady RR, Ambler

RP, Smith BE (1995) Evidence for two distinct azurins in Alcaligenes xylosoxidans (NCIMB 11015): potential

electron donors to nitrite reductase. Biochemistry 34:10180-10186

22. Drake HL, Horn MA (2006) Earthworms as a transient heaven for terrestrial denitrifying microbes: a review.

Eng Life Sci 6:261-265

23. Etchebehere C, Errazquin I, Barrandeguy E, Dabert P, Moletta R, Mixi L (2001) Evaluation of the denitrifying

microbiota of anoxic reactors. FEMS Microbiol Ecol 35:259-265

24. Etchebehere C, Errazquin MI, Dabert P, Muxi L (2002) Community analysis of a denitrifying reactor treating

landfill leachate. FEMS Microbiol Ecol 40:97-106

CHAPTER 1

28

25. Fritz C, Maass S, Kreft A, Bange FC (2002) Dependence of Mycobacterium bovis BCG on anaerobic nitrate

reductase for persistence is tissue specific. Infect Immun 70:286-291

26. Fujiwara T, Fukumori Y (1996) Cytochrome cb-type nitric oxide reductase with cytochrome c oxidase activity

from Paracoccus denitrificans ATCC 35512. J Bacteriol 178:1866-1871

27. Gamble TN, Betlach MR, Tiedje JM (1977) Numerically dominant denitrifying bacteria from world soils.


28. Garbeva P, Baggs EM, Prosser JI (2007) Phylogeny of nitrite reductase (nirK) and nitric oxide reductase

(norB) genes from Nitrosospira species isolated from soil. FEMS Microbiol Lett 266:83-89

29. Gayon U, Dupetit G (1882) Sur la fermentation des nitrates. C R Acad Sci 95:644-646

30. Gayon U, Dupetit G (1886) Recherches sur la reduction de nitrate par les infinement petits. Mem Soc Sci

Phys Nat Bordeaux Ser.3 2:201-307

31. Giuffrè A, Stubauer G, Sarti P, Brunori M, Zumft WG, Buse G, Soulimane T (1999) The heme-copper oxidases

of Thermus thermophilus catalyze the reduction of nitric oxide: evolutionary implications. PNAS 96:14718-

14723

32. Glockner AB, Jüngst A, Zumft WG (1993) Copper-containing nitrite reductase from Pseudomonas aureofaciens

is functional in a mutationally cytochrome cd1-free backround (NirS-) of Pseudomonas stutzeri. Arch Microbiol

160:18-26

33. Groffman PM, Altabet MA, Böhlke JK, Butterbach-Bahl K, David MB, Firestone MK, Giblin AE, Kana TM,

Nielsen LP, Voytek MA (2006) Methods for measuring denitrification: diverse approaches to a difficult problem.

Ecol Appl 16:2091-2122

34. Hallin S, Lindgren P-E (1999) PCR detection of genes encoding nitrite reductase in denitrifying bacteria. Appl


35. Hendriks J, Gohlke Y, Saraste M (1998) From NO to O2: Nitric oxide and dioxygen in bacterial respiration. J

Bioenerg Biomembr 30:15-24

36. Hendriks J, Oubrie A, Castresana J, Urbani A, Gemeinhardt S, Saraste M (2000) Nitric oxide reductases in

bacteria. Biochim Biophys Acta 1459:266-273

37. Henry S, Baudoin E, Lopez-Gutiérrez JC, Martin-Laurent F, Brauman A, Philippot L (2004) Quantification of

denitrifying bacteria in soils by nirK gene targeted real-time PCR. J Microbiol Meth 59:327-335

38. Henry S, Bru D, Stres B, Hallet S, Philippot L (2006) Quantitative detection of the nosZ genes, encoding

nitrous oxide reductase, and comparison of the abundance of 16S rRNA, narG, nirK and nosZ genes in soils.


39. Ho TP, Jones AM, Hollocher TC (1993) Denitrification enzymes of Bacillus stearothermophilus. FEMS

Microbiol Lett 114:135-138

40. Hulse CI, Averill BA, Tiedje JM (1989) Evidence for a copper-nitrosyl intermediate in denitrification by the

copper-containing nitrite reductase of Achromobater cycloclastes. J Am Chem Soc 111:2322-2323

41. Ichiki H, Tanaka Y, Mochizuki K, Yoshimatsu K, Sakurai T, Fujiwara T (2001) Purification, characterization

and genetic analysis of Cu-containing dissimilatory nitrite reductase from a denitrifying halophilic archaeon,

Haloarcula marismortui. J Bacteriol 183:4149-4156

42. Inatomi K, Hochstein LI (1996) The purification and properties of a copper nitrite reductase from Haloferax

denitrificans. Curr Microbiol 32:72-76

43. Intergovernmental Panel on Climate Change (2001) Climate Change 2001, the scientific basis. Cambridge

University Press, Cambridge

44. Janssen PH, Yates PS, Grinton BE, Taylor PM, Sait M (2002) Improved culturability of soil bacteria and

isolation in pure culture of novel members of the divisions Acidobacteria, Actinobacteria, Proteobacteria,

and Verrucomicrobia. Appl Environ Microbiol 68:2391-2396

45. Jason J, Cantera L, Stein L (2007) Molecular diversity of nitrite reductase genes (nirK) in nitrifying bacteria.


46. Jetten MSM, Logemann S, Muyzer G, Roberston LA, de Vries S, Van Loosdrecht MCM, Kuenen JG (1997)

Novel principles in the microbial conversion of nitrogen compounds. Antonie van Leeuwenhoek 71:75-93

47. Kaeberlein T, Lewis K, Epstein S (2002) Isolating ‘uncultivable’ microorganisms in pure culture in a

simulated environment. Science 296:1127-1129

48. Kakutani T, Watanabe H, Arima K, Beppu T (1981) A blue protein as an inactivating factor for nitrite reductase

from Alcaligenes faecalis strain S-6. J Biochem 89:463-472

49. Kandeler E, Deiglmayr K, Tscherko D, Bru D, Philippot L (2006) Abundance of narG, nirS, nirK, and nosZ

genes of denitrifying bacteria during primary succession of a glacier foreland. Appl Environ Microbiol 72:5957-

5962

INTRODUCTION

29

50. Karsten GR, Drake HL (1997) Denitrifying bacteria in the earthworm gastrointestinal tract and in vivo

emission of nitrous oxide (N2O) by earthworms. Appl Environ Microbiol 63:1878-1882

51. Kartal B, Kuypers MMM, Lavik G, Schalk J, Op den Camp HJM, Jetten, MSM, Strous M (2007) Anammox

bacteria disguised as denitrifiers: nitrate reduction to dinitrogen gas via nitrite and ammonium. Environ

Microbiol 9:635-642

52. Keller M, Zengler K (2004) Tapping into microbial diversity. Nature Rev Microbiol 2:141-150

53. Knowles R (1982) Denitrification. Microbiol Rev 46:43-70

54. Kuenen JG, Robertson LA (1988) Ecology of nitrification and denitrification. In: The nitrogen and sulphur

cycle, Cole JA, Ferguson SJ (eds.) Cambridge University Press, Cambridge, pp.161-218

55. Libby E, Averill BA (1992) Evidence that the type 2 copper centers are the site of nitrite reduction by

Achromobacter cycloclastes nitrite reductase. Biochem Biophys Res Commun 187:1529-1535

56. Liu M-Y, Liu M-C, Payne WJ, LeGall J (1986) Properties and electron transfer specificity of copper proteins

from the denitrifier “Achromobacter cycloclastes”. J Bacteriol, 166:604-608

57. Magnusson G, Edin H, Dalhammar G (1998) Characterization of efficient denitrifying bacteria strains isolated

from activated sludge by 16S rRNA analysis. Wat Sci Tech 38:63-68

58. Mahne I, Tiedje J.M (1995) Criteria and methodology for identifying respiratory denitrifiers. Appl Environ

Microbiol 6: 1110-1115

59. Mergel A, Schmitz O, Mallmann T, Bothe H (2001) Relative abundance of denitrifying and dinitrogen-fixing

bacteria in layers of a forest soil. FEMS Microbiol Ecol 36:33-42

60. Merzouki M, Delgenès J-P, Bernet N, Moletta R, Venlemlih M (1999) Polyphosphate-accumulating and

denitrifying bacteria isolated from anaerobic-anoxic and anaerobic-aerobic sequencing batch reactors. Curr

Microbiol 38:9-17

61. Michotey V, Méjean V, Bonin P (2000) Comparison of methods for quantification of cytochrome cd1-

denitrifying bacteria in environmental marine samples. Appl Environ Microbiol 66:1564-1571

62. Nogales B, Timmis KN, Nedwell DB, Osborn AM (2002) Detection and diversity of expressed denitrification

genes in estuarine sediments after reverse transcription-PCR amplification from mRNA. Appl Environ Microbiol

68:5017-5025

63. O’Hara GW, Daniel RM (1985) Rhizobial denitrification: a review. Soil Biol Biochem 17:1-9

64. Oakley BB, Francis CA, Roberts KJ, Fuchsman CA, Srinivasan S, Staley JT (2007) Analysis of nitrite reductase

(nirK and nirS) genes and cultivation reveal depauperate community of denitrifying bacteria in the Black Sea

suboxic zone. Environ Microbiol. 9:118-130

65. Park SJ, Yoon JC, Shin K-S, Kim EH, Yim S, Cho Y-J, Sung GM, Lee D-G, Kim SB, Lee D-U, Woo S-H,

Koopman B (2007) Dominance of endospore-forming bacteria on a rotating activated Bacillus contactor

biofilm for advanced wastewater treatment. J Microbiol 45:113-121

66. Payne WJ (1981) Denitrification. John Wiley & Sons, New York

67. Philippot L (2002) Denitrifying genes in bacterial and Archaeal genomes. Biochim Biophys 1577:355-376

68. Philippot L (2005) Denitrification in pathogenic bacteria: for better or worst? Trends Microbiol 13:191-

192

69. Philippot (2005) Use of functional genes to quantify denitrifiers in the environment. Biochem Soc Trans

34:101-103

70. Philippot L, Hallin S (2005) Finding the missing link between diversity and activity using denitrifying

bacteria as a model functional community. Curr Opinion Microbiol 8:234-239

71. Poth M, Focht DD (1985) 15N kinetic analysis of N2O production by Nitrosomonas europaea: an examination

of nitrier denitrification. Appl Environ Microbiol 49:1134-1141

72. Priemé A, Braker G, Tiedje JM (2002) Diversity of nitrite reductase (nirK and nirS) gene fragments in forested

upland and wetland soils. Appl Environ Microbiol 68:1893-1900

73. Qiu X-Y, Hurt RA, Wu L-Y, Chen C-H, Tiedje JM, Zhou J-Z (2004) Detection and quantification of copper-

denitrifying bacteria by quantitative competitive PCR. J Microbiol Meth 59:199-210

74. Ren T, Roy R, Knowles R (2000) Production and consumption of nitric oxide by three methanotrophic

bacteria. Appl Environ Microbiol 66:3891-3897

75. Risgaard-Petersen N, Langezaal AM, Ingvardsen S, Schmid MC, Jetten MSM, Op den Camp HJM, Derksen

JWM, Piña-Ochoa, Eriksson SP, Nielsen LP, Revsbeech NP, Cedhagen T, van der Zwaan GJ (2006) Evidence

of complete denitrification in a benthic foraminifer. Nature 443:93-96

76. Rösch C, Mergel A, Bothe H (2002) Biodiversity of denitrifying and dinitrogen-fixing bacteria in an acid

forest soil. Appl Environ Microbiol 68:3818-3829

CHAPTER 1

30

77. Sakai K, Ikehata Y, Ikenaga Y, Wakayama M, Moriguchi M (1996) Nitrite oxidation by heterotrophic bacteria

under various nutritional and aerobic conditions. J Ferm Bioeng 82: 613-617

78. Sait M, Hugenholtz P, Janssen PH (2002) Cultivation of globally distributed soil bacteria from phylogenetic

lineages previously only detected in cultivation-independent surveys. Environ Microbiol 4:654-666

79. Santoro AE, Boehm AB, Francis CA (2006) Denitrifier community composition along a nitrate and salinity

gradient in a coastal aquifer. Appl Environ Microbiol 72:2102-2109

80. Saraste M, Castresana J (1994) Cytochrome oxidase evolved by tinkering with denitrification enzymes.

FEBS Lett 341:1-4

81. Sawada E, Satoh ET, Kitamura H (1978) Purification and properties of a dissimilatory nitrite reductase of a

denitrifying phototrophic bacterium. Plant Cell Physiol 19:1339-1351

82. Schmidt I, van Spanning RJM, Jetten MSM (2004) Denitrification and ammonia oxidation by Nitrosomonas

europaea wild-type and NirK- and NorB-deficient mutants. Microbiology 150:4107-4114

83. Sharma S, Aneja MK, Mayer J, Munck JC, Schloter M (2005) Diversity of transcripts of nitrite reductase genes

(nirK and nirS) in rhizospheres of grain legumes. Appl Environ Microbiol 71:2001-2007

84. Shaw LJ, Nicol GW, Smith Z, Fear J, Prosser JI, Baggs EM (2006) Nitrosospira spp. can produce nitrous oxide

via a nitrifier denitrification pathway. Environ Microbiol 8:214-222

85. Shoun H, Kano M, Baba I, Takaya N., Matsuo M (1998) Denitrification by actinomycetes and purification of

dissimilatory nitrite reductase and azurin from Streptomyces thioluteus. J Bacteriol 180: 4413-4415

86. Song B, Ward BB (2003) Nitrite reductase genes in halobenzoate degrading denitrifying bacteria. FEMS

Microbiol Ecol 43:349-357

87. Staley JT, Konopka A (1985) Measurements of in situ activities of nonphotosynthetic microorganisms in

aquatic and terrestrial habitats. Ann Rev Microbiol 39:321-346

88. Stevenson BS, Eichorst SA, Wertz JT, Schmidt TM, Breznak JA (2004) New strategies for cultivation and

detection of previously uncultured microbes. Appl Environ Microbiol 70:4748–4755

89. Suharti, de Vries S (2005) Membrane-bound denitrification in the Gram-positive bacterium Bacillus

azotoformans. Biochem Soc Trans 33:130-133

90. Suharti, Heering HA, de Vries S (2004) NO reductase from Bacillus azotoformans is a bifunctional enzyme

accepting electrons from menaquinol and a specific endogenous membrane-bound cytochrome c551

. Biochemistry

43:13487-13495

91. Suharti S, Strampraad MJ, Schröder T, De Vries S (2001) A novel copper A containing menaquinol NO

reductase from Bacillus azotoformans. Biochemistry 40:2632-2639

92. Suzuki S, Yoshimura T, Kohzuma T, Shidara S, Masuko M, Sakurai T, Iwasaki H (1989) Spectroscopic evidence

for a copper-nitrosyl intermediate in nitrite reduction by blue copper-containing nitrite reductase. Biochem

Biophys Res Commun 164:1366-1372

93. Taroncher-Oldenburg G, Griner EM, Francis CA, Ward BB (2003) Oligonucleotide microarray for the study

of functional gene diversity in the nitrogen cycle in the environment. Appl Environ Microbiol 69:1159-

1171

94. Tavares P, Pereira AS, Moura JJG, Moura I (2006) Metalloenzymes of the denitrification pathway. J Inorg

Biochem 100:2087-2100

95. Throbäck IN, Enwall K, Jarvis Ã, Hallin S (2004) Reassessing PCR primers targeting nirS, nirK and nosZ

genes for community surveys of denitrifying bacteria with DGGE. FEMS Microbiol Ecol 49:401-417

96. Tiedje JM (1988) Ecology of denitrification and dissimilatory nitrate reduction to ammonium. In:

Environmental microbiology of anaerobes, A.J.B. Zehnder (ed.), John Wiley & Sons, New York, 179-244

97. Tielens AGM, Rotte C, van Hellemond JJ, Martin W (2002) Mitochondria as we don’t know them. Trends

Biochem Sci 27:564-572

98. Urata K, Satoh T (1991) Enzyme localization and orientation of the active site of dissimilatory nitrite reductase

from Bacillus firmus. Arch Microbiol 156:24-27

99. Vairinhos F, Wallace W, Nicholas DJD (1989) Simultaneous assimilation and denitrification of nitrate by

Bradyrhizobium japonicum. J Gen Microbiol 135:189-193

100. Van der Oost J, de Boer APN, de Gier JL, Zumft WG, Stouthamer AH, Spanning RJM (1994) The heme-copper

oxidase family consists of three distinct types of terminal oxidases and is related to nitric oxide reductase.

FEMS Microbiol Lett 121:1-10

101. Vitousek PM, Mooney HA, Lubchenco J, Melillo JM (1997) Human dominantion of Earth’s ecosystem.

Science 277:494-499

102. Wallenstein MD, Myrold DD, Firestone M, Voytek M (2006) Environmental controls on denitrifying

communities and denitrification rates: insights from molecular methods. Ecol Appl 16:2143-2152

INTRODUCTION

31

103. Wallenstein MD, Vilgalys RJ (2005) Quantitative analyses of nitrogen cycling genes in soils. Pedbiologica

49:665-672

104. Ward BB (1995) Diversity of culturable denitrifying bactera: limits of rDNA RFLP analysis and probes for

the functional gene, nitrite reductase. Arch Microbiol 163:167-175

105. Ward BB (1996) Nitrification and denitrification: probing the nitrogen cycle in aquatic environments.

Microb Ecol 32:247-261

106. Weier KL, MacRae IC (1992) Denitrifying bacteria in the profile of a brigalow clay soil beneath a permamnent

pasture and a cultivated crop. Soil Biol Biochem 24:919-923

107. Williams PA, Fülöp V, Garman EF, Saunders NFW, Ferguson SJ, Hajdu J (1997) Haem-ligand switching

during catalysis in crystals of a nitrogen-cycle enzyme. Nature 389:406-412

108. Williams PA, Fülöp V, Leung Y-C, Chan C, Moir JWB, Howlett G, Ferguson SJ, Radford SE, Hajdu J (1995)

Pseudospecific docking surfaces on electron transfer proteins as illustrated by pseudoazurin, cytochrome

c550

and cytochrome cd1 nitrite reductase. Nat Struct Biol 2:975-982

109. Wolsing M, Priemé A (2004) Observation of high seasonal variation in community structure of denitrifying

bacteria in arable soil receiving artificial fertilizer and cattle manure by determining T-RFLP of nir gene

fragments. FEMS Microbiol Ecol 48:261-271

110. Wu L, Thompson DK, Li G, Hurt RA, Tiedje JM, Zhou Z (2001) Development and evaluation of functional

gene arrays for detection of selected genes in the environment. Appl Environ Microbiol 67:5780-5790

111. Yan T, Fields MW, Wu L, Zu Y, Tiedje JM, Zhou J (2003) Molecular diversity and characterization of nitrite

reductase gene fragments (nirK and nirS) from nitrate- and uranium-contaminated groundwater. Environ

Micrbiol 5:13-24

112. Yoshie S, Noda N, Tsuneda S, Hirata A, Inamori Y (2004) Salinity decreases nitrite reductase gene diversity

in denitrifying bacteria of wastewater treatment systems. Appl. Environ. Microbiol. 70:3152-3157

113. Zhou J, Fries MR, Chee-Sanford JC, Tiedje JM (1995) Phylogenetic analyses of a new group of denitrifiers

capable of anaerobic growth on toluene and description of Azoarcus tolulyticus sp. nov. Int J Syst Bacteriol

45:500-506

114. Zumft WG (1992) The denitrifying Prokaryotes, In: Balows A, Trüper HG, Dworkin M, Harder W, Schleifer

K-H (Eds.), The Prokaryotes. A handbook on the biology of bacteria: ecophysiology, isolation, identification,

applications, 5nd ed., vol. 1. Springer-Verlag, New York, pp. 554-582

115. Zumft, W.G. (1997) Cell biology and molecular basis of denitrification. Microbiol Mol Biol Rev 61:533-616

116. Zumft W (2005) Nitric oxide reductase of prokaryotes with emphasis on the respiratory, heme-copper oxidase

type. J Inorg Biochem 99:194-215

117. Zumft WG, Gotzmann DJ, Frunzke K, Viebrock A (1987) Novel terminal oxidoreductases of anaerobic

respiration (denitrification) from Pseudomonas. In: W.R.Ullrich, P.J. Aparicio, P.J.Syrett, and F. Castillo

(ed.) Inorganic nitrogen metabolism. Springer-Verlag KG, Berlin, Germany, pp. 61-67

CHAPTER 1

32

ISOLATION, CHARACTERIZATION AND

IDENTIFICATION OF DENITRIFYING BACTERIA FROM

THE ENVIRONMENT

2

2.1 CULTIVATION OF HETEROTROPHIC DENITRIFYINGBACTERIA: OPTIMIZATION OF ISOLATIONCONDITIONS AND DIVERSITY STUDY

Redrafted from: Heylen K, Vanparys B, Wittebolle L, Verstraete W, Boon N, De Vos P

(2006) Cultivation of denitrifying bacteria: optimization of isolation conditions and

diversity study. Appl Environ Microbiol 72:2637-2643

SUMMARY

An evolutionary algorithm was applied to study the complex interactions between medium

parameters and their effect on the isolation of heterotrophic denitrifying bacteria, both in

number and diversity. Growth media with a pH of 7, a nitrogen concentration of 3 mM,

supplemented with 1 ml of vitamin solution but without sodium chloride or riboflavin

turned out most successful for isolation of denitrifiers from activated sludge. The use of

ethanol or succinate as carbon source and a molar C/N ratio of 2.5, 20 or 25 were also

favourable. After testing 60 different medium parameter combinations and comparison

with each other as well as with the standard medium TSA supplemented with nitrate, three

growth media were highly suitable for cultivation of denitrifying bacteria. All evaluated

isolation conditions were used to study the cultivable heterotrophic denitrifier diversity

of activated sludge from a municipal wastewater treatment plant. 199 denitrifiers were

isolated, the majority of which belonged to the Betaproteobacteria (50.4%) and the

Alphaproteobacteria (36.8%). Representatives of Gammaproteobacteria (5.6%),

Epsilonproteobacteria (2%), Firmicutes (4%) and one isolate of the Bacteroidetes were

also found. This study revealed a much more diverse denitrifying community than

previously described in cultivation-dependent research on activated sludge.

38

INTRODUCTION

For nearly two decades, molecular biology provided the tool to successfully overcome the

‘great plate count anomaly’ and allow study of the uncultured microbial diversity [3]. The

growing awareness that molecular methods cannot, or in very few cases can only indirectly,

investigate the function of specific microorganisms in the environment has raised the interest

in new cultivation efforts and approaches once again [14, 15, 34]. Simple adjustments to

the classical cultivation approach such as prolonging the incubation time and avoiding

complex or nutrient rich growth media have successfully resulted in cultivation of previously

uncultured bacteria [12, 30].

A physiological trait like denitrification, the respiratory reduction of nitrate and nitrite to

N2O and nitrogen gas, is not limited to specific microbial taxa, and is therefore studied

culture-independently through the relevant functional genes [6, 25, 32]. To date, it is however

not clear to what extent, if at all, these functional genes contain phylogenetic information.

Phillipot [23] showed that the phylogeny of nir and nor genes, coding for the key enzymes

nitrite reductase and NO reductase in the denitrification pathway, does not always agree

with the phylogeny of the 16S rRNA gene. New isolation and cultivation approaches are

therefore imperative to provide the basis for further research on phylogenetic and functional

gene diversity.

Isolation of specific physiologic groups of bacteria such as denitrifiers requires knowledge

of the interaction of a large number of medium components and growth conditions. Genetic

or evolutionary algorithms (EA’s) are heuristical optimization programs based on the

Darwinistic principles of evolution by natural selection [10]. An EA supports a rational

selection of possible combinations of medium parameters to be tested in practice, with the

advantage that it does not assume a model [10]. Highly complex optimization problems in

various domains as diverse as improvement of silage additives [8] and electricity estimations

[21] have been resolved with EAs. In microbiology, their use was so far limited to

optimization of fermentation media [36, 37] and conditions for transconjugant formation

[5].

This paper discusses the optimization of isolation conditions for heterotrophic denitrifying

bacteria. The interaction between different medium parameters was investigated with an

evolutionary algorithm. Using a minimal mineral medium as basis, different combinations of

medium parameters were applied as isolation medium for denitrifiers, with activated sludge

of a municipal wastewater treatment plant as inoculum, and the diversity of cultured

denitrifiers was assessed.

CHAPTER 2

39

MATERIALS & METHODS

InoculumActivated sludge samples were taken at a municipal wastewater treatment plant with subsequent anoxic and aerated

tanks (Bourgoyen-Ossemeersen, Gent, Belgium). Samples (20 ml) were collected from an anoxic tank at the start of

each new batch of growth media and immediately processed. Homogenisation of the flocs was performed using a

needle (diameter 0.8 mm) and a 50 ml syringe. After homogenisation, a dilution series of the sample (100 to 10-8) was

made and spread plated on the growth media.

EA experimental designEach medium parameter can have different values, which can be different levels in concentration, in temperature, but

also different sources of carbon or nitrogen. A combination of these values determines the composition of a growth

medium. [The use of the term ‘growth medium’ refers to the composition of the medium and the culture conditions.]

Different growth media are grouped into batches. Based on the success or fitness of the growth media of previous

batches, a new batch is calculated by the EA. Therefore, the values of the medium parameters of the best scoring

growth media are recombined in a new batch of growth media. As a result the average fitness of this new batch

should increase.

Eleven medium parameters with different values were selected as variables for the EA. The number of possible

combinations of all parameters with their different values was 1,197,504. Each growth medium made up of a

combination of medium parameter values was tested for the suitability to isolate denitrifiers and was assigned a

fitness value. This fitness contained two selection parameters: (i) the number of denitrifying isolates, and (ii) the

diversity of the denitrifying isolates. The first selection parameter was represented by the ratio between the number

of isolated denitrifiers and the total number of isolates (RATIOden

) per growth medium. The second selection parameter

required knowledge of the identity of the isolated denitrifiers. For this purpose, FAME analysis was chosen as a fast

identification method. The observed diversity onto genus level was represented for each growth medium by a Simpson’s

reciprocal diversity index 1/D,

1/D = N × (N-1)/E[ni × (n

i-1)] (eq.1)

with N = number of denitrifying isolates per medium, ni = number of denitrifying isolates per medium belonging to

genus i. When only one denitrifier was isolated, the diversity index was zero; when all denitrifiers were assigned to

the same genus by FAME analysis, the diversity index was set at one. A fitness was calculated for each medium, i.e.

based on the results of both selection parameters, both equally weighted:

Fitness = RATIOden

× 1/D (eq.2)

The fitness of a given growth medium would increase if both the number of denitrifying isolates grown on this

medium and the diversity of these denitrifying isolates would increase. The combination of medium parameters with

the highest fitness will therefore be most suited to use as growth medium for denitrifiers.

Evolutionary algorithm

The Simple Evolutionary Algorithm for Optimization (seao) software [31] is available in an easy-to-use graphical

user interface and can be freely downloaded (http://www.cran.r-project.org). The configuration and parameterisation

of the seao for the experimental optimization of the medium composition used the following settings: number of

medium parameters at 11, number of growth media at 15; all previous batches to be used for calculation of the next

batch of growth media; the selection type was fitness based (rescaling = 0); recombination rate at 90%; mutation

followed a uniform distribution (i.e. all possible values have the same chance of being chosen), with a spread of 1.0

and a rate of 15. For the initial batch of growth media, the EA randomly combined medium parameter values into 15

different growth media.

Growth mediaAll growth media were based on the mineral medium described by Stanier et al. [29]. Eleven medium parameters

with different values were selected for optimization with the EA: pH at 6.5, 7, 7.5 or 8; temperature at 20°C or 37°C;

sodium acetate-trihydrate, glycerol, sodium pyruvate, methanol, ethanol, glucose or sodium succinate as carbon

source; molar C/N ratio at 1, 2.5, 5, 7.5, 10, 12.5, 15, 17.5, 20, 22.5 or 25; potassium nitrate or potassium nitrite

as nitrogen source; nitrogen concentration at 3 mM, 6 mM, 9 mM, 12 mM, 15 mM or 18 mM; no addition of sodium

chloride or a sodium chloride concentration of 0.34 M; 0, 1 or 2 ml addition of vitamin solution (17) containing

4 mg 4-aminobenzoic acid, 2 mg D-(+)-biotin, 10 mg nicotinic acid, 5 mg calcium D-(+)-panthothenate, 15 mg

pyridoxin hydrochloride, 4 mg folic acid, 1 mg lipoic acid in 100 ml 10 mM NaH2PO

4, at pH 7.1; 0, 1 or 2 ml addition

ISOLATION, CHARACTERIZATION AND IDENTIFICATION OF DENITRIFYING BACTERIA FROM THE ENVIRONMENT

40

of riboflavin solution (17) containing 2.5 mg riboflavin in 100 ml 25 mM NaH2PO

4, at pH 3.2; 0, 1 or 2 ml addition

of thiamine solution (17) containing 10 mg thiamine hydrochlorid in 100 ml 25 mM NaH2PO4, at pH 3.4 (0 ml; 1

ml; 2 ml); cobalamin solution (17) containing 50 mg cyanocobalamin per liter distilled water. A pH indicator was

added (10 µM): bromothymolblue for growth media with a pH of 6.5, phenol red for growth media with a pH of 7

or higher. Trypticase soy agar (Oxoid) was supplemented with 10 mM KNO3 and 10 µM phenol red.

IsolationA dilution series (100 to 10-8) of activated sludge was spread plated (100 µl) on 15 different growth media per batch,

as determined by the EA. The inoculated growth media were incubated for two weeks in an anaerobic chamber

(composition gas mixture 8%CO2/8%H

2/84%N

2). From each growth medium and supplemented TSA, 20 isolates

were picked from the highest dilution still showing growth and further purified and sub-cultured on the same medium

(G4M3 was tested in triplicate).

Denitrification testsAll purified isolates were incubated in liquid isolation medium for one week at isolation conditions. Tests for nitrate

and nitrite reduction were performed using the Griess reagents [27]. Selection for denitrifiers was based on the

results of the reduction tests and the pH indicator [19]. This selection approach was validated by the confirmation of

the denitrifying activity of all isolates of the first batch with N2O measurements. All isolates of the first batch

presumed to denitrify were grown in 50-ml culture flasks with 10 ml liquid isolation medium. The headspace of the

vials was replaced with filter-sterilised argon by evacuating five times and refilling. Acetylene (10%) was added to

stop the reduction of N2O to N

2. After one-week incubation, a gas sample (1 ml) was taken with a gas-tight syringe

and N2O was measured with a gas chromatograph (Shimadzu GC-14B) equipped with an electron capture detector,

a precolumn (1m) and a Porapak column (2m, 80-100 mesh).

Fatty acids methyl ester analysis (FAME)A qualitative and quantitative analysis of cellular fatty acid compositions was performed with the gas-liquid

chromatographic procedure as described by Sasser [26]. The resulting profiles were identified with the Microbial

Identification software (MIDI) using the TSBA database version 5.0 (Microbial ID, Newark, DE, USA). In batch 4,

some denitrifiers could not be grown in the standard conditions (medium and incubation time) for FAME analysis.

The genus identification was then obtained by 16S rRNA gene sequence analysis and used in the same way for the

determination of the diversity.

DNA extraction & 16S rRNA gene sequence analysisDNA was extracted from each denitrifying isolate using the guanidium-thiocyanate-EDTA-sarkosyl method described

by Pitcher et al. [22] for fast-growing strains and using alkaline lysis for slow-growing isolates. For alkaline lysis,

one colony was suspended in an eppendorf tube with 20 µl of lysis buffer (2.5 ml 10% SDS; 5 ml 1M NaOH; 92,5

ml MilliQ water). After 15 min at 95°C, 180 ml MilliQ water was added, the tube was centrifuged for 5 min at

13,000 g and the supernatant was transferred to a new tube. DNA extracts were stored at –20°C until use. PCR

amplification was performed as described by Heyrman et al. [9]. The PCR-amplified 16S rRNA gene products were

purified using the Nucleofast® 96 PCR system (Millipore). For each sequence reaction a mixture was made using 3

µl purified and concentrated PCR product, 1 µl of BigDye™ Termination RR mix version 3.1 (Applied Biosystems),

1.5 µl of BigDye™ buffer (5x), 1.5 µl sterile milliQ water and 3 µl (20 ng/µl) of one of the 6 sequencing primers

used [9]. The temperature-time profile was as follows: 30 cycles of denaturation for 15 s at 96°C, primer annealing

for 1 s at 35°C and extension for 4 min at 60°C. The sequencing products were cleaned up, as described by Naser et

al. [20]. Sequence analysis of the partial 16S rRNA gene (first 300-500 bp) was performed using an Applied Biosystems

3100 DNA Sequencer according to protocols provided by the manufacturer. Sequences were assembled using the

BioNumerics 4.0 software (Applied Maths). A reliable identification was obtained in two steps: (i) a BLAST search

[2] of the 16S rRNA gene sequence of an isolate retrieved 50 sequences with the highest sequence similarities to the

query sequence, (ii) all type strains of all species of all genera mentioned in the BLAST report were compared in an

exhaustive pair wise manner with the query sequence of each strain in BioNumerics 4.0. The strains were assigned

to a genus based on the obtained 16S rRNA gene sequence similarity.

Nucleotide sequence accession numbersThe nucleotide sequence data generated in this study have been deposited in Genbank/EMBL/DDBJ with accession

numbers AM083989 to AM084186.

CHAPTER 2

41

RESULTS

EA experiment

An evolutionary algorithm was used to optimize the isolation conditions for denitrifiers.

The influence of eleven medium parameters with different values and their combinations

on the number and diversity of isolated denitrifying bacteria was examined. Sixty different

growth media, i.e. combinations of medium parameter values, were investigated in four

subsequent batches, with 15 growth media per batch. Activated sludge from a municipal

wastewater treatment plant was used as inoculum. Table 2.1 gives an overview of the

composition and the fitness results of each growth medium per batch.

2,48

3,864,09

4,50

0,48 0,540,86 0,87

0

1

2

3

4

5

1 2 3 4

Batch

Fitn

ess

va

lue

Maxim al Fi tness Average Fi tness

Figure 2.1. The average and maximal fitness value for each batch of growth media. The fitness value of a growthmedium represents the success of a combination of medium parameters to render a high (relative) number of denitrifyingisolates, highly diverse in genus assignment.

The success of a growth medium was determined as a fitness value (Figure 2.1). This

fitness selected for (i) a high number of denitrifying bacteria, and (ii) a high diversity of

denitrifying bacteria (see Materials and Methods). For the first batch, the EA randomly

combined medium parameter values into 15 growth media. Batch 1 gave an average fitness

of 0.48. In total 269 isolates were examined and 34 were detected as denitrifiers. The

maximal fitness of batch 1 (i.e. 2.48) was assigned to growth medium G1M1, with a

nitrite concentration of 3mM, a molar C/N ratio of 20, with succinate as carbon source, no

sodium chloride or riboflavin added, addition of 1ml vitamin solution, 2 ml thiamine

solution and 2 ml cobalamin solution, a pH of 6.5 and incubated at 37°C. The EA calculated

a second batch, hereby selecting for those medium parameter values that contributed to a

high fitness in the previous batch. In batch 2, 217 isolates were examined, 33 isolates were

detected as denitrifiers and an average fitness of 0.54 was measured. Results of batch 1 and


42

2 appeared very similar, except for the maximal fitness, which increased to 3.86 in batch 2

(Figure 2.1). Growth medium G2M11, giving the maximal fitness, differed from the best

scoring medium of batch 1 only in the pH, 7 instead of 6.5. Some growth media in batch 1

and 2 showed no growth, not even from the undiluted activated sludge sample, while

others showed growth but less than 20 colonies. This greatly limited the total number of

isolates and subsequently the number of denitrifiers in these batches. Batch 3 was calculated

based on the fitness results of batch 1 and 2. For this third batch, the average fitness

increased to 0.86 (Figure 2.1), 315 isolates were examined and 56 denitrifiers were detected,

a clear increase for all three features compared to batch 1 and 2. The maximal fitness (i.e.

4.09) was found for growth medium G3M12, differing from the two former best scoring

media in values of most medium parameters: a pH of 7.5, ethanol as carbon source, a low

molar C/N ratio of 2.5, a nitrate concentration of 18 mM, 1ml of thiamin solution, no

cobalamin solution added and incubation temperature of 20°C. The EA calculated batch 4

based on the three preceding batches. Again, an increased number of denitrifying bacteria

was isolated, 69 denitrifiers from a total of 300 examined isolates. The maximal fitness of

4.50 was assigned to G4M3, which differed from G2M11 only in the use of nitrate instead

of nitrite as a nitrogen concentration. Arbitrarily chosen, this growth medium was also

tested in triplicate to investigate the reproducibility of the evolutionary algorithm. The

fitness value differed between the three repeats, due to a difference in diversity of the

isolated denitrifiers (Table 2.1). The average fitness value (i.e. 0.87) reached a plateau in

batch 4, which led to the decision to stop the EA. Supplemented TSA was tested parallel

with each batch. The average fitness for supplemented TSA was 0.625.

CHAPTER 2

43

Table 2.1. Overview of all combinations of medium parameters tested in this study. Values for all medium parameterswere determined by the EA, based on the composition of the most successful combination in previous batches. Theresults of the experimental parameters of the EA are given for each growth medium: RATIO

den, Simpson’s reciprocal

diversity index 1/D and fitness. The average values per batch for the three parameters are also given.

Growth Medium1 pH T (°C) C source2 C/N ratio3 N source N (mM) NaCl (M) Vitamin (ml) Riboflavin (ml) Thiamine (ml) Cobalamin (ml) RATIOden 1/D Fitness G1M1 6.5 37 succinate (4) 20.0 nitrite 3 0 1 0 2 2 0.60 4.13 2.48 G1M2 7.5 37 methanol (1) 10.0 nitrite 18 0.34 0 0 2 2 0.00 0.00 0.00 G1M3 8.0 20 acetate (2) 25.0 nitrate 12 0 0 2 0 1 0.20 6.00 1.20 G1M4 8.0 37 glucose (6) 15.0 nitrate 12 0.34 2 2 1 1 0.05 0.00 0.00 G1M5 7.0 20 pyruvate (4) 7.5 nitrite 3 0.34 0 0 2 0 0.33 3.00 1.00 G1M6 6.5 37 methanol (1) 10.0 nitrate 9 0 1 0 1 2 0.00 0.00 0.00 G1M7 7.0 20 methanol (1) 12.5 nitrite 3 0 0 2 2 0 0.05 0.00 0.00 G1M8 8.0 37 glucose (6) 7.5 nitrate 6 0 2 1 2 0 0.10 0.50 0.20 G1M9 7.0 20 pyruvate (4) 7.5 nitrite 3 0.34 2 2 0 2 0.00 0.00 0.00 G1M10 7.0 37 glucose (6) 5.0 nitrate 15 0 0 1 1 0 0.05 0.00 0.00 G1M11 8.0 37 glucose (6) 7.5 nitrate 12 0 2 1 0 0 0.00 0.00 0.00 G1M12 7.0 37 glucose (6) 2.5 nitrate 15 0 2 1 0 2 0.00 0.00 0.00 G1M13 6.5 37 methanol (1) 15.0 nitrate 15 0 1 1 0 0 0.00 0.00 0.00 G1M14 7.0 20 succinate (4) 2.5 nitrate 3 0.34 0 2 1 1 0.30 3.75 1.13 G1M15 7.0 20 ethanol (2) 2.5 nitrate 18 0 1 0 1 0 0.20 6.00 1.20

Average G1 0.13 1.56 0.48 G2M1 6.5 37 succinate (4) 20.0 nitrite 3 0 1 2 2 1 0.00 0.00 0.00 G2M2 6.5 37 ethanol (2) 2.5 nitrite 3 0 1 0 2 2 0.00 0.00 0.00 G2M3 6.5 37 succinate (4) 20.0 nitrate 3 0 1 2 0 1 0.00 0.00 0.00 G2M4 8.0 37 acetate (2) 25.0 nitrite 12 0 1 0 2 2 0.20 1.00 0.20 G2M5 7.0 20 pyruvate (4) 7.5 nitrite 3 0.34 0 1 1 0 0.00 0.00 0.00 G2M6 7.0 20 ethanol (2) 25.0 nitrate 18 0 1 0 2 0 0.24 10.00 2.38 G2M7 6.5 20 pyruvate (4) 20.0 nitrite 3 0 1 0 2 2 0.35 1.90 0.66 G2M8 6.5 37 succinate (4) 10.0 nitrite 3 0.34 1 0 2 2 0.00 0.00 0.00 G2M9 7.0 20 ethanol (2) 2.5 nitrate 18 0 0 0 2 0 0.10 2.00 0.20 G2M10 7.0 37 pyruvate (4) 7.5 nitrite 3 0.34 1 0 1 0 0.00 0.00 0.00 G2M11 7.0 37 succinate (4) 20.0 nitrite 3 0 1 0 2 2 0.43 9.00 3.86 G2M12 8.0 37 succinate (4) 12.5 nitrite 3 0 1 0 2 2 0.10 2.00 0.20 G2M13 8.0 37 succinate (4) 20.0 nitrite 15 0 1 0 2 2 0.10 1.00 0.10 G2M14 6.5 37 succinate (4) 20.0 nitrite 12 0 1 0 2 2 0.10 0.00 0.00 G2M15 7.0 20 ethanol (2) 2.5 nitrate 12 0 1 0 0 2 0.15 3.00 0.45

Average G2 0.12 1.99 0.54 G3M1 8.0 20 succinate (4) 2.5 nitrite 3 0 1 2 0 2 0.29 1.00 0.29 G3M2 7.0 20 succinate (4) 20.0 nitrate 3 0.34 0 2 1 1 0.30 3.75 1.13 G3M3 7.5 37 ethanol (2) 17.5 nitrite 18 0 0 2 1 1 0.09 1.00 0.09 G3M4 7.0 20 acetate (2) 2.5 nitrate 3 0.34 1 1 2 0 0.43 5.14 2.20 G3M5 7.0 37 ethanol (2) 20.0 nitrite 3 0 1 0 2 2 0.43 3.27 1.40 G3M6 6.5 20 succinate (4) 15.0 nitrite 3 0 1 0 2 2 0.00 0.00 0.00 G3M7 7.0 37 succinate (4) 1.0 nitrate 12 0.34 1 0 1 1 0.25 5.00 1.25 G3M8 7.0 37 succinate (4) 20.0 nitrite 3 0 1 0 2 2 0.00 0.00 0.00 G3M9 7.5 20 pyruvate (3) 2.5 nitrite 3 0 0 0 1 0 0.15 1.60 0.90 G3M10 7.0 20 ethanol (2) 2.5 nitrate 18 0 1 0 0 0 0.95 1.00 0.95 G3M11 7.0 20 ethanol (2) 12.5 nitrate 6 0 1 0 2 1 0.05 0.00 0.00 G3M12 7.5 20 ethanol (2) 2.5 nitrate 18 0 1 0 1 0 0.27 15.00 4.09 G3M13 7.0 20 ethanol (2) 25.0 nitrate 9 0 2 2 2 2 0.14 3.00 0.41 G3M14 7.0 37 glucose (6) 20.0 nitrite 3 0 1 0 2 0 0.10 1.00 0.10 G3M15 7.5 37 succinate (4) 20.0 nitrate 3 0 1 0 2 2 0.09 1.00 0.09

Average G3 0.24 2.78 0.86 G4M1 7.0 20 glucose (6) 20.0 nitrite 9 0 1 0 2 2 0.00 0.00 0.00 G4M2 6.5 20 succinate (4) 7.5 nitrate 3 0.34 0 0 2 0 0.00 0.00 0.00 G4M34 7.0 37 succinate (4) 20.0 nitrate 3 0 1 0 2 2 0.20 6.00 1.20

0.30 2.50 0.75 0.30 15.0 4.50

G4M4 6.5 20 pyruvate (3) 25.0 nitrite 3 0 1 0 0 2 0.05 0.00 0.00 G4M5 7.0 20 ethanol (2) 2.5 nitrate 18 0 0 2 1 1 0.15 3.00 0.45 G4M6 7.0 20 pyruvate (3) 20.0 nitrate 3 0 1 1 1 0 0.45 6.00 2.70 G4M7 7.0 37 succinate (4) 1.0 nitrate 12 0.34 1 0 2 2 0.20 3.00 0.60 G4M8 7.0 37 succinate (4) 20.0 nitrate 12 0 1 0 1 2 0.25 3.33 0.83 G4M9 6.5 20 pyruvate (3) 12.5 nitrate 3 0 2 1 2 0 0.05 0.00 0.00 G4M10 7.0 20 acetate (2) 2.5 nitrite 3 0 1 0 0 2 0.85 2.77 2.36 G4M11 6.5 37 ethanol (2) 25 nitrate 18 0 1 0 2 0 0.00 0.00 0.00 G4M12 7.0 20 succinate (4) 10.0 nitrate 12 0.34 1 0 1 2 0.25 1.67 0.42 G4M13 7.0 20 ethanol (2) 20.0 nitrate 18 0 0 0 1 0 0.05 0.00 0.00 G4M14 7.0 20 ethanol (2) 2.5 nitrate 18 0 1 0 0 0 0.10 1.00 0.10 G4M15 7.0 20 succinate (4) 25.0 nitrate 3 0.34 1 2 0 1 0.25 3.33 0.83

Average G4 0.20 2.80 0.87


44

Experimental course of medium parameters

A detailed look at the experimental course of each medium parameter defined by the EA

revealed convergence to one ‘optimal’ value for five medium parameters (Figure 2.2).

The percentage of growth media with the same medium parameter value is directly correlated

with its contribution to a higher fitness in the preceding batches. Thus, a pH value of 7, a

nitrogen concentration of 3 mM, the addition of 1 ml of vitamin solution, and the exclusion

of sodium chloride and riboflavin solution contributed to the success of an elective growth

medium for denitrifiers (Figure 2.2). The other medium parameters diverged to different

values. Both temperature values were equally selected over four batches, with an increasing

preference for 20°C in batch 3 and 4. Cobalamin converged to either exclusion or the

addition of 2 ml. For the nitrogen source, both nitrite and nitrate were equally selected,

with an increasing preference for the latter in batch 4. For thiamine, all three possible

values were equally selected. Although no ‘optimal’ value could be determined, carbon

source and molar C/N ratio diverged to respectively two (i.e. ethanol and succinate) and

three values (i.e. 2.5, 20, 25), which were therefore more favourable for isolation of

denitrifiers than the other possible values. The best scoring growth medium of batch 1, 2

and 4 incorporated most or all of the ‘optimal’ values determined for the medium parameters;

only the composition of the best scoring medium of batch 3 deviated from this.

Figure 2.2. The percentage of growth media with a certain value for a medium parameter is represented for eachbatch. The experimental course of five medium parameters (A to E) converge to one value: pH (A), nitrogenconcentration (B), sodium chloride concentration (C), vitamin solution (D), and riboflavin solution (E). The percentageof growth media with the same value for a medium parameter is directly correlated with its contribution to a higherfitness in the preceding batches.

A.

0

10

20

30

40

50

60

70

80

1 2 3 4Batch

% o

f gr

owth

med

ia

6,5 7 7,5 8

B.

0

10

20

30

40

50

60

70

1 2 3 4Batch

% o

f gr

owth

med

ia

3mM 6mM 9mM

12mM 15mM 18mM

C.

0

10

20

30

40

50

60

70

80

90

1 2 3 4Batch

% o

f gr

owth

med

ia

0mM 0,34mM

D.

0

10

20

30

40

50

60

70

80

90

100

1 2 3 4Batch

% o

f gr

owth

med

ia

0ml 1ml 2ml

E.

0

10

20

30

40

50

60

70

80

90

1 2 3 4Batch

% o

f gr

owth

med

ia

0ml 1ml 2ml

CHAPTER 2

45

Diversity of the denitrifying populations in activated sludge

192 denitrifying isolates were distinguished in a total of 1101 isolates obtained on the 60

evaluated growth media, while seven out of 80 isolates obtained on supplemented TSA

were able to denitrify. After FAME analysis, 198 denitrifying isolates were reliably identified

onto genus level (Table 2.2) via partial 16S rRNA gene sequence analysis (no 16S rRNA

gene amplicon could be obtained for one isolate). The majority of the denitrifiers belonged

to the Betaproteobacteria (50.5% or 100 isolates). Sixty-eight strains were assigned to

Acidovorax, Alicycliphilus, Comamonas and Diaphorobacter of the Comamonadaceae,

and were isolated predominantly from growth media with ethanol or succinate as carbon

source, coupled with nitrate or nitrite as nitrogen source respectively. Thirty-one isolates

were assigned to Azospira, Azovibrio, Dechloromonas, Thauera and Zoogloea of the

Rhodocyclaceae, the majority of which were isolated on growth medium with succinate as

carbon source and pH value of 7. One isolate belonged to the genus Aquaspirillum of the

Neisseriaceae. The second largest group of denitrifiers belonged to the Alphaproteobacteria

(37.3% or 74 isolates): 22 isolates belonged to Brucella and Ochrobactrum of the

Brucellaceae, 8 isolates to Rhizobium and Sinorhizobium of the Rhizobiaceae, 43 isolates to

Paracoccus and Pannonibacter of the Rhodobacteraceae, and 1 isolate assigned to

Methylobacterium represented the Methylobacteraceae. The Gammaproteobacteria were

represented by 11 isolates belonging to Pseudomonas (5.6%). Four isolates (2%) belonging

to Arcobacter were Epsilonproteobacteria. Eight isolates (4%) belonging to Bacillus,

Trichococcus, Enterococcus, Paenibacillus and Staphylococcus were Firmicutes. One isolate

of the genus Chryseobacterium belonging to the Flavobacteriaceae represented the

Bacteroidetes. No clear trends were observed in the composition of the growth media used

for isolation of members of the Alpha-, Gamma-, Epsilonproteobacteria and Firmicutes.

Table 2.2. (next two pages) An overview of all cultured genera found in activated sludge from a municipal WWTP.All growth media used for isolation of each genus are given, together with the number of isolates.


46

CHAPTER 2

Tax

on

om

ical

po

siti

on

T

ype

stra

in w

ith

hig

hes

t 16

S r

RN

A g

ene

seq

uen

ce s

imila

rity

to

qu

ery

seq

uen

ce

Gro

wth

med

ium

(C

lass

, Fam

ily, G

enu

s)

(str

ain

nu

mb

er,

% s

equ

ence

sim

ilari

ty,

acce

ssio

n n

um

ber

) [#

iso

late

s]

Alp

hap

rote

ob

acte

ria

Bru

cella

ceae

Bru

cella

B

ruce

lla o

vis

AT

CC

258

40T

99.5

%

L261

68

G3M

5 [1

]

Och

rob

actr

um

O

chro

bact

rum

ant

hrop

i D

SM

688

2T 97

.8%

-10

0%

D12

794

G1M

3 [1

], G

1M14

[3],

G2M

6 [1

], G

2M7

[1],

G3M

4 [5

], G

3M5

[3],

G3M

G

4M3[

1]

O

chro

bact

rum

inte

rmed

ium

LM

G 3

301T

99.2

%

U70

978

G2M

11 [1

], G

2M12

[1]

O

chro

bact

rum

triti

ci

DS

M 1

3340

T 10

0%

AJ2

4258

4 G

3M5

[2]

M

eth

ylo

bac

tera

ceae

M

eth

ylo

bac

teri

um

M

ethy

loba

cter

ium

suo

mie

nse

DS

M 1

4458

T 95

.1%

A

Y00

9404

G

2M4

[1]

R

hiz

ob

iace

ae

R

hiz

ob

ium

R

hizo

bium

gia

rdin

ii C

IP 1

0550

3T 97

.2%

U

8634

4 G

3M12

[1]

R

hizo

bium

gal

licum

M

SD

J110

9T 97

.6%

U

8634

3 G

1M15

[1]

R

hizo

bium

rad

ioba

cter

A

TC

C 1

9358

T 97

.2%

- 1

00%

A

J389

904

G3M

2 [1

], G

1M15

[1]

R

hizo

bium

sul

lae

DS

M 1

4623

T 97

.6%

Y

1017

0 G

1M15

[1]

Sin

orh

izo

biu

m

Sin

orhi

zobi

um m

orel

ense

LC

04T

97.0

% -

97.

2%

AY

0243

35

G1M

1 [1

], G

2M11

[1],

G2M

12 [1

]

Rh

od

ob

acte

race

ae

P

arac

occ

us

P

arac

occu

s am

inop

hilu

s

AT

CC

496

73T

97.6

%

AY

0141

76

G1M

3 [2

]

Par

acoc

cus

alca

liphi

lus

AT

CC

511

99T

97.6

% -

97.

8%

AY

0141

77

G1M

3 [1

], G

2M7

[1],

G3M

4 [1

], G

3M7

[1],

G3M

12 [1

], G

4M15

[2],

G1

P

arac

occu

s am

inov

oran

s A

TC

C 4

9632

T 97

.4%

- 9

9.7%

D

3224

0 G

1M14

[3],

G2M

3 [3

], G

3M4

[1],

G3M

5 [1

], G

4M3

[1],

G4M

12 [5

], G

4M

Par

acoc

cus

caro

tinifa

cien

s E

-396

T 98

.4%

- 9

8.7%

A

B00

6899

G

3M4

[1],

G3M

7 [2

]

Par

acoc

cus

pant

otro

phus

A

TC

C 3

5512

T 10

0%

Y16

933

G3M

5 [1

], G

3M14

[2]

P

arac

occu

s ye

ei

CC

UG

468

22T

97.8

%

AY

0141

73

G3M

4 [1

], G

3M12

[1],

G3M

13 [1

]

Par

acoc

cus

vers

utus

A

TC

C 2

5364

T 99

.9%

- 1

00%

A

Y01

4174

G

1M7

[1],

G2M

4 [1

], G

2M7

[1],

G3M

5 [1

]

P

ann

on

ibac

ter

Pan

noni

bact

er p

hrag

mite

tus

DS

M 1

4782

T 10

0%

AJ4

0070

4 G

3M2

[1]

Bet

apro

teo

bac

teri

a

C

om

amo

nad

acea

e

Aci

do

vora

x

Aci

dovo

rax

aven

ae s

ubsp

. citr

ulli

AT

CC

296

25T

98.1

% -

98.

3%

AF

0787

61

G2M

6 [1

], G

2M7

[1]

A

cido

vora

x de

fluvi

i D

SM

126

44T

99.4

% -

100

%

Y18

616

G2M

15 [1

], G

3M2

[1],

G3M

10 [2

], G

3M11

[1],

G3M

12 [1

], G

3M13

G4M

13 [1

], G

4M14

[1]

A

cido

vora

x te

mpe

rans

A

TC

C 4

9665

T 97

.8%

- 9

9.5%

A

F07

8766

G

1M1

[4],

G1M

8 [1

], G

2M15

[2]

Alic

yclip

hilu

s

Alic

yclip

hilu

s de

nitr

ifica

ns

DS

M 1

4773

T 97

.7%

- 9

9.8%

A

J418

042

G1M

1 [3

], G

3M1

[1]

Co

mam

on

as

Com

amon

as a

quat

ica

AT

CC

113

30T

99.0

% -

99.

8%

AJ4

3034

4 G

3M1

[2]

C

omam

onas

den

itrifi

cans

A

TC

C 7

0093

6T 98

.3%

- 9

9.9%

A

F23

3877

G

1M15

[1],

G2M

4 [1

], G

2M7

[2],

G2M

9 [1

], G

2M11

[1],

G3M

1 [2

G

3M3

[2],

G3M

9 [1

], G

3M15

[1],

G4M

4 [1

], G

4M5

[3],

G4M

8 [3

], G

4M

D

iap

ho

rob

acte

r

Dia

phor

obac

ter

nitr

ored

ucen

s D

SM

159

85T

99.2

% -

99.

8%

AB

0643

17

G1M

1 [3

], G

2M11

[2],

G3M

1 [1

], G

3M12

[1],

G4M

14 [1

]

47


N

eiss

erac

eae

A

qu

asp

irill

um

A

quas

piril

lum

met

amor

phum

D

SM

183

7T 98

.9%

Y

1861

8 G

4M3

[1]

R

ho

do

cycl

acea

e

Azo

spir

a A

zosp

ira o

ryza

e LM

G 9

096T

99.9

% -

100%

A

F01

1347

G

2M11

[1],

G4M

3 [1

]

A

zovi

bri

o

Azo

vibr

io r

estr

ictu

s LM

G 9

099T

100%

A

F01

1346

G

2M9

[1]

Dec

hlo

rom

on

as

Dec

hlor

omon

as a

gita

ta

AT

CC

700

666T

100%

A

F04

7462

G

4M3

[4],

G4M

7 [2

]

Dec

hlor

omon

as d

enitr

ifica

ns

DS

M 1

5892

T 97

.2%

- 9

9.8%

A

J318

917

G4M

3 [1

], G

4M6

[1]

Th

auer

a

Tha

uera

aro

mat

ica

DS

M 6

984T

99.2

% -

99.

3%

X77

118

TS

A [2

]

Tha

uera

am

inoa

rom

atic

a D

SM

147

42T

99.5

% -

100

%

AJ3

1567

7 G

4M3

[5],

G4M

7 [2

], G

4M8

[2]

T

haue

ra c

hlor

oben

zoic

a A

TC

C 7

0072

3T 99

.4%

A

F12

3264

T

SA

[1]

T

haue

ra m

eche

rnic

hens

is

DS

M 1

2266

T 98

.0%

Y

1759

0 G

2M13

[1]

T

haue

ra p

heny

lace

tica

DS

M 1

4743

T 95

.0%

- 9

9,5%

A

J315

678

G2M

13 [1

], G

3M15

[1],

G4M

6 [2

], T

SA

[1]

T

haue

ra s

elen

atis

A

TC

C 5

5363

T 99

.1%

Y

1759

1 T

SA

[1]

Zo

og

loea

Z

oogl

oea

ram

iger

a A

TC

C 1

9544

T 96

.4%

X

7491

3 G

4M6

[1]

Gam

map

rote

ob

acte

ria

Pse

ud

om

on

adac

eae

P

seu

do

mo

nas

P

seud

omon

as a

erug

inos

a A

TC

C 1

0145

T 99

.9%

- 1

00%

A

F09

4713

G

1M1

[1],

G2M

11 [1

]

Pse

udom

onas

alc

alig

enes

A

TC

C 1

4909

T 10

0%

Z76

653

G2M

6 [2

], G

2M7

[1]

P

seud

omon

as m

endo

cina

A

TC

C 2

5411

T 95

.3%

- 9

5.6%

A

J308

310

G3M

9 [1

], T

SA

[1]

P

seud

omon

as n

itror

educ

ens

AT

CC

336

34T

99.1

% -

99.

3%

D84

021

G1M

10 [1

], G

3M9

[1]

P

seud

omon

as p

utid

a A

TC

C 1

2633

T 98

.9%

A

J308

313

G4M

9 [1

]

Pse

udom

onas

stu

tzer

i A

TC

C 1

7588

T 10

0%

AF

0947

48

G2M

11 [1

]

Ep

silo

np

rote

ob

acte

ria

Cam

pyl

ob

acte

race

ae

Arc

ob

acte

r A

rcob

acte

r cr

yaer

ophi

lus

CC

UG

178

01T

99.3

% -

99.

8%

L146

24

G4M

6 [2

]

Arc

obac

ter

skirr

owii

AT

CC

511

32T

94.6

%

L146

25

G4M

6 [1

]

Arc

obac

ter

nitr

ofig

ilis

AT

CC

333

09T

95.4

%

L146

27

G4M

6 [1

] F

irm

icu

tes

Bac

illac

eae

B

acill

us

B

acill

us c

laus

ii

AT

CC

700

160T

99.7

%

X76

440

G4M

3 [1

]

Bac

illus

moj

aven

sis

AT

CC

515

16T

98.6

% -

98.

7%

X68

416

G1M

4 [1

]

G1M

8 [1

]

Car

no

bac

tera

ceae

Tri

cho

cocc

us

Tric

hoco

ccus

floc

culif

orm

is

DS

M 2

094T

100%

A

J306

611

G4M

6 [1

]

En

tero

cocc

acea

e

En

tero

cocc

us

E

nter

ococ

cus

cass

elifl

avus

A

TC

C 2

5788

T 99

.2%

- 1

00%

A

F03

9903

G

1M5

[1]

G

2M11

[1]

P

aen

ibac

illac

eae

P

aen

ibac

illu

s P

aeni

baci

llus

agar

idev

oran

s D

SM

135

5T 98

.6%

A

J345

023

G3M

12 [1

]

Sta

ph

ylo

cocc

acea

e

Sta

ph

ylo

cocc

us

S

taph

yloc

occu

s ho

min

is

AT

CC

278

44T

99.9

%

L37

601

TS

A [1

] B

acte

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48

DISCUSSION

Little is known about the denitrifying diversity present in activated sludge, as straightforward

cultivation-independent approaches are not suitable and cultivation-dependent research is

limited. Magnusson et al. [18] performed an isolation campaign on nutrient agar with

activated sludge from five different municipal WWTPs and found only denitrifying

Proteobacteria, belonging to the Rhodobacteraceae, Comamonadaceae and

Pseudomonadaeae. After applying 60 different defined isolation conditions, a much more

important heterotrophic denitrifier diversity was found, although proteobacteria were still

predominant. Denitrifying representatives of Alpha-, Beta-, Gamma- and

Epsilonproteobacteria, Firmicutes and Bacteroidetes were found, and apart from genera

conventionally known to harbour denitrifiers such as Pseudomonas, Ochrobactrum,

Comamonas and Acidovorax, genera less frequently observed in cultivation studies of

denitrifiers were also encountered. The Rhodocyclaceae were well represented, encompassing

besides the genus Thauera also the recently described genera Azospira and Azovibrio [24],

and Dechloromonas [1, 11]. Furthermore, possibly new species belonging to Thauera and

Zoogloea were retrieved. Recent efforts to identify denitrifiers in activated sludge in a

cultivation-independent manner by combining fluorescent in situ hybridisation (FISH)

with microautoradiography (MAR) [35] recognised the Azoarcus-Thauera group of the

Rhodocyclaceae as probably the most abundant denitrifiers in industrial WWTPs. The

genus Arcobacter was previously found in significant numbers in activated sludge [28], but

its function was undetermined. In this study, four denitrifying Arcobacter strains were

isolated, demonstrating that the genus can contribute to the denitrification process in

activated sludge systems. The denitrifying potential of Bacteroidetes and Firmicutes strains

- Bacillus, Paenibacillus, Staphylococcus, Trichococcus and enterococci - known from

cultivation-independent studies to be numerically less important in WWTPs than the

Proteobacteria [13], was also established.

This study shows the applicability of an EA for the optimization of growth media. The

progressive improvement of the average and maximal fitness value in each successive

batch confirms the iterative nature of an EA. The maximal fitness value of each batch of

the newly designed media was significantly higher than the average fitness of supplemented

TSA, which is a widely applied growth medium for denitrifier isolation [33]. Highly suitable

elective growth media were developed, rendering between 40 and 80% heterotrophic

denitrifiers. Comparable data are unavailable for cultivation-dependent studies on activated

sludge; for soil, 10% of all isolates on supplemented nitrate broth were denitrifiers [7].

After evaluation of 60 different combinations of medium parameters, the three best scoring

CHAPTER 2

49

growth media G2M11, G3M12 and G4M3, can be recommended for isolation of denitrifiers

in the future.

The isolation conditions for denitrifiers were optimised heuristically. Convergence of a

medium parameter to one value indicates no interaction with other medium parameters.

The EA determined that five medium parameters converged to one ‘optimal’ value. Because

of their independence of the overall medium composition, these parameters can be fixed at

these values in further optimization studies, while other medium parameters are varied.

Although halotolerant and halophilic denitrifiers are known [16], exclusion of sodium

chloride appeared to increase the isolation of denitrifiers. This observation may be correlated

with the use of activated sludge as inoculum. Riboflavin did not result in an enhanced

retrieval of denitrifiers, which contradicts an earlier report on the reduction of the doubling

time for Paracoccus denitrificans when adding riboflavin under denitrifying conditions

[4]. The same study showed an increase in the nitrite reductase activity, thus decreasing

the accumulation of nitrite, with ethanol as carbon source. The suitability of ethanol as

carbon source for denitrifiers was also confirmed here. In contrast to previous optimization

studies in microbiology with an EA [5, 8, 36], the reproducibility of the fitness was assessed.

The observed non-reproducibility of the genus diversity determination was probably

attributed to (i) the limited number of investigated isolates per growth medium, due to

logistics and time, (ii) the use of FAME analysis for genus identification, and/or (iii) other

possible parameters, not included in the EA.

Weuster-Botz [37] stated that ‘a combination of highly directed random searches to explore

the n-dimensional variable space with a genetic algorithm, and subsequent application of

classical statistical experimental design is recommended for media development’. The here

reported work can be seen as the initial step for elective medium design and development

for denitrifying bacteria and provides the basis for further cultivation-dependent research

on denitrifiers. Furthermore, through this study, new growth media are available that favour

growth of heterotrophic denitrifiers exhibiting a high natural diversity. Also, a large set of

denitrifying isolates has been obtained that can be further subjected to research concerning

denitrification, e.g. functional gene sequence analysis. Similar large-scale cultivation studies

can have future value for physiological interesting bacterial groups that are difficult to

study, for instance filamentous or nitrifying bacteria.


50

REFERENCES1. Achenbach LA, Michaelidou U, Bruce RA, Fryman J, Coates JD (2001) Dechloromonas agitata gen. nov.,

sp. nov. and Dechloromonas suillum gen. nov., two novel environmentally dominant (per)chlorate-reducing

bacteria and their phylogenetic postion. Int J Syst Evol Microbiol 51:527-533

2. Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ (1990) Basic local alignment search tool. J Mol Biol

215:403-410

3. Amann, RI, Ludwig W, Schleifer K-H (1995) Phylogenetic identification and in situ detection of individual

microbial cells without cultivation. Microbiol Rev 59:143-169

4. Blaszczyk M (1993) Effect of medium composition on the denitrification of nitrate by Paracoccus denitrificans.


5. Boon N, Depuydt S, Verstraete W (2006) Evolutionary algorithms and flow cytometry to examine the

parameters influencing transconjugant formation. FEMS Microbiol Ecol 55:17-27

6. Braker G, Zhou J, Wu L, Devol AH, Tiedje JM (2000) Nitrite Reductase genes (nirK and nirS) as functional

markers to investigate diversity of denitrifying bacteria in Pacific Northwest marine sediment communities.


7. Chèneby D, Philippot L, Hartmann A, Hénault C, Germon J-C (2000) 16S rDNA analysis for characterisation

of denitrifying bacteria isolated from three agricultural soils. FEMS Microbiol Ecol 34:121-128

8. Davies ZS, Gilbert RJ, Merry RJ, Kell DB, Theodorou MK, Griffith GW (2002) Efficient improvement of

silage additives by using genetic algorithms. Appl Environ Microbiol 66:1435-1443

9. Heyrman J, Swings J (2001) 16S rDNA sequence analysis of bacterial isolates from Biodeteriorated mural

paintings in the Servilia tomb (necropolis of Carmona, Seville, Spain). Syst Appl Microbiol 24:417-422

10. Holland J (1992) Adaptation in natural and artificial systems: an introductory analysis with applications

to biology, control and artificial intelligence. MIT Press, Cambridge, MA

11. Horn, MA, Ihssen J, Matthies C, Schramm A, Acker G, Drake HL (2005) Dechloromonas denitrificans sp.

nov., Flavobacterium denitrificans sp. nov., Paenibacillus anaericanus sp. nov. and Paenibacillus terrae

strain MH72, N2O-producing bacteria isolated from the gut of the earthworm Aporrectodea caliginosa. Int

J Syst Evol Microbial 55:1255-1565

12. Joseph SJ, Hugenholtz P, Sangwan P, Osborne CA, Janssen PH (2003) Laboratory cultivation of widespread

and previously uncultured soil bacteria. Appl Environ Microbiol 69:7210-7215

13. Juretschko S, Loy A, Lehner A, Wagner M (2002) The microbial community of a nitrifying-denitrifying activated

sludge from an industrial sewage treatment plant analysed by the full-cycle rRNA approach. Syst Appl Microbiol

25:84-99

14. Kaeberlein T, Lewis K, Epstein SS (2002) Isolating ‘uncultivable’ microorganisms in pure culture in a simulated

environment. Science 296:1127-1129

15. Keller M, Zengler K (2004) Tapping into microbial diversity. Nature Rev Microbiol 2:141-150

16. Kim S-G, Bae H-S, Oh H-M, Lee S-T (2003) Isolation and characterisation of novel halotolerant and/or

halophilic denitrifying bacteria with versatile metabolic pathways for the degradation of trimethylamine. FEMS

Microbiol Lett 225:263-269

17. Kniemeyer O, Probian C, Roselló-Mora R, Harder J (1999) Anaerobic mineralization of quanternary carbon

atoms: isolation of denitrifying bacteria on dimethylmalonate. Appl Environ Microbiol 65:3319-3324

18. Magnusson G, Edin H, Dalhammar G (1998) Characterisation of efficient denitrifying bacteria strains isolated

from activated sludge by 16S rDNA analysis. Wat Sci Tech 38:63-68

19. Mazoch J, Kucera I (2002) Detection, with a pH indicator, of bacterial mutants unable to denitrify. J Microbiol

Meth 51:105-109

20. Naser S, Thompson FL, Hoste B, Gevers D, Vandemeulebroecke K, Cleenwerk I, Thompson CC, Vancanneyt

M, Swings J (2005) Phylogeny and identification of Enterococci using atpA gene sequence analysis. J Clin

Microbiol 43:2224-2230

21. Ozturk H K, Ceylan H, Canyurt OE, Hepbaslin A (2005) Electricity estimation using genetic algorithm approach:

a case study of Turkey. Energy 30:1003-1012

22. Pitcher DG, Saunders LA, Owen NA (1989) Rapid extraction of bacterial genomic DNA with guanidium thio-

cyanate. Lett Appl Microbiol 8:151-156

23. Phillipot L (2002) Denitrifying genes in bacterial and Archaeal genomes. Biochim Biophys Acta 1577:355-

376

24. Reinhold-Hurel B, Hurek T (2000) Reassessment of the taxonomic structure of the diazotrophic genus Azoarcus

sensu lato and description of three new genera and new species, Azovibrio restrictus gen. nov., sp. nov.,

CHAPTER 2

51

Azospira oryzae gen. nov., sp. nov. and Azonexus fungiphilus gen. nov., sp. nov. Int J Syst Evol Microbial

50:649-659

25. Rösch C, Mergel A, Bothe H (2002) Biodiversity of denitrifying and dinitrogen-fixing bacteria in an acid


26. Sasser M (1990) Identification of bacteria by gas chromatography of cellular fatty acids. MIDI Technical

Note #101. www.midi-inc.com

27. Smibert RM, Krieg NR (1994) Phenotypic characterization. In: Methods for General and Molecular

Bacteriology, p. 649. Edited by Gerhardt P, Murray RGE, Wood WA, Krieg NR, Washington: American

Society for Microbiology

28. Snaidr J, Amann R, Huber I, Ludwig W, Schleifer K-H (1997) Phylogenetic analysis and in situ identification

of bacteria in activated sludge. Appl Environ Microbiol 63:2884-2896

29. Stanier RY, Palleroni NJ, Doudoroff M (1966) The aerobic Pseudomonads: a taxonomic study. J Gen Microbiol

43:159-271

30. Stevenson BS, Eichorst SA, Wertz JT, Schmidt TM, Breznak JA (2004) New strategies for cultivation and

detection of previously uncultured microbes. Appl Environ Microbiol 70:4748-4755

31. Sys K, Boon N, Verstraete W (2004) Development and validation of evolutionary algorithm software as an

optimization tool for biological and environmental applications. J Microbial Meth 57:309-322

32. Throbäck IN, Enwall K, Jarvis Å, Hallin S (2004) Reassessing PCR primers targeting nirS, nirK and nosZ



Environmental microbiology of anaerobes, Zehnder AJB (ed.), John Wiley & Sons, New York, pp.179-244

34. Tyson GW, Banfield JF (2005) Cultivating the uncultivated: a community genomics perspective. Trends


35. Wagner M, Loy A (2002) Bacterial community composition and function in sewage treatment systems. Curr

Opin Biotechnol 13:218-227

36. Weuster-Botz D, Wandrey C (1995) Medium optimization by genetic algorithm for continuous production of

formate dehydrogenase. Process Biochem 30:63-571

37. Weuster-Botz D (2002) Experimental design for fermentation media development: statistical design or global

random search? J Biosc Bioeng 90:73-483

38. Zumft WG (1997) Cell biology and molecular basis of denitrification. Microbiol Mol Biol Rev 61:533-616


2.2 DIVERSITY OF HETEROTROPHIC DENITRIFIERSISOLATED FROM SOIL USING A MULTIPLE-MEDIASET

Redrafted from: Heylen K, Boon N, Verstraete W, De Vos P (2007) Functional gene study

on heterotrophic denitrifiers isolated from soil. Microbiol Ecol, Submitted

SUMMARY

Culturing bacteria from environmental samples is still the most straightforward way to

identify the denitrifying diversity present. The aim of this study was to generate more

knowledge on the denitrifying diversity present in soil. Therefore, a multiple-media set,

formed by three defined growth media, developed in a previous study to electivly isolate of

denitrifiers, and the commonly used complex medium TSA, was applied. A total of 112

denitrifiers were isolated and identified through FAME analysis and partial 16S rRNA gene

sequence analysis. Among the retrieved denitrifiers, bacilli, mostly isolated with nitrite as

nitrogen source, were numerically dominant (71%), next to rhizobia (16%). The four media

used in the multiple-media set were compared and evaluated on (i) a high ratio denitrifiers-

to-anaerobes, (ii) a high denitrifier diversity, both retrieved from (iii) the highest dilution

containing denitrifiers. Growth medium G3M12, containing nitrate and ethanol and incubated

at 20°C, scored best. However, the use of a multiple-media set is recommended to cover a

wide denitrifying diversity.

54

INTRODUCTION

Denitrification, i.e. the respiratory reduction of nitrate and nitrite to N2O and nitrogen gas,

is an important bacterial process in soil. It has since long been a concern in agriculture

because nitrogen is often most limiting in crop yields, and fertilizer losses due to

denitrification commonly range between 20 and 30 % (Tiedje 1988). Recently, its production

of N2O has renewed interest in the process, since N

2O contributes to global warming and

the greenhouse effect. Knowledge on the composition of the denitrifying community would

enable in situ monitoring of key players and their response to different environmental

controls, which could lead to management models of soil resulting in optimal fertilizer use

and decreasing N2O emissions.

However, this physiological trait is difficult to study culture-independently. The identity

of a soil bacterium does not yield information on its denitrification capacities, due to the

phylogenetically widespread occurrence of this trait [23]. In addition, the functional gene

sequences contain limited taxonomic information [17]. Other approaches that combine

identifying the taxonomic position and metabolism of microorganisms, such as fluorescent

in situ hybridisation in combination with microautoradiography [12] or stable-isotope

probing [19] are not (yet) suitable for large-scale diversity studies and/or for the scoped

physiological characteristics. As a result, the contributions of different microbial taxa to

the denitrification process in soil are scarcely known [13].

Conventional culturing approach makes a valid alternative for cultivation-independent

analysis, as the denitrifier itself is available for both phylogenetic and functional study. The

aim of this study was to confirm that the newly developed media can recover a higher

diversity of denitrifiers, compared to conventional TSA. Therefore, a multiple-media set,

formed by three defined growth media, developed in a previous study to electivly isolate of

denitrifiers [9], and the commercial complex medium TSA, was applied. The usefullness of

these media to generate new insights in denitrifying diversity in soil was assessed.

CHAPTER 2

55

MATERIALS & METHODS

Inoculum and isolation conditionsA soil sample was collected from the upperlayer (1-20 cm) of a luvisol test field in Melle, Belgium. The texture class

of the test field is sand-loam (composition 8.6% clay/11.6% loam/75.8% fine sand/4% rough sand). The soil matrix

was suspended in a sterile solution of 1% NaPO3 (3 g soil in 300ml) by stirring at room temperature [4]. A dilution

series (10-2 to 10-10) of the matrix was spread plated (100 µl) on four growth media: (i) TSA (Trypticase Soy Agar,

Oxoid), supplemented with 10 mM potassium nitrate and 10 µM phenol red, incubated at 37°C; (ii) G2M11,

containing 3mM potassium nitrite, 15 mM sodium succinate and 10 µM phenol red, at pH 7 and supplemented with

vitamins, incubated at 37°C; (iii) G3M12, containing 18 mM potassium nitrate, 22.5 mM ethanol, 10 µM phenol

red, at pH 7.5 and supplemented with vitamins, incubated at 20°C; and (iv) G4M3, containing 3 mM potassium

nitrate, 15 mM sodium succinate, 10 µM phenol red, at pH 7, and supplemented with vitamins, incubated at 37°C.

Media were solidfied with 1.7% agar. Defined growth media G2M11, G3M12 and G4M3 were developed and

described previously [9]. The inoculated growth media were incubated for two weeks in an anaerobic chamber

(composition gas mixture 8%CO2/8%H

2/84%N

2). From each medium, visibly different colony types were randomly

picked and further purified by sub-culturing under isolation conditions.

Nitrogen reduction testsNitrate and nitrite reduction tests were performed in liquid isolation medium as described by Smibert & Krieg [22].

N2O measurements were performed to confirm denitrification. All isolates were grown in 50-ml culture flasks with

10 ml liquid isolation medium. The headspace of the vials was replaced with filter-sterilized argon by evacuating five

times and refilling. Acetylene (10%) was added to stop the reduction of N2O to N2. After one-week incubation, a gas

sample (1 ml) was taken with a gas-tight syringe and N2O was measured with a gas chromatograph (Shimadzu GC-

14B) equipped with an electron capture detector, a precolumn (1 m) and a Porapak column (2 m, 80-100 mesh).

Fatty Acids Methyl Ester (FAME) analysisQualitative and quantitative analyses of cellular fatty acid compositions for all denitrifiers were performed by the

gas-liquid chromatographic procedure described by Sasser [20]. The whole-cell FAME profiles were identified and

quantified with the Microbial Identification System software package (MIS, TSBA database version 5.0).

DNA extraction & 16S rRNA gene sequence analysisDNA was extracted from each isolate using the alkaline lysis method. For alkaline lysis, one colony was suspended

in an eppendorf tube with 20 µl of lysis buffer (2.5 ml 10% SDS; 5 ml 1M NaOH; 92.5 ml MilliQ water). After 15

min at 95°C, 180 ml MilliQ water was added, the tube was centrifuged for 5 min at 13,000 g and the supernatant was

transferred to a new tube. DNA extracts were stored at –20°C until use. The 16S rRNA gene PCR amplification was

performed as described by Heyrman et al. [10]. The PCR-amplified 16S rRNA gene products were purified using the

Nucleofast® 96 PCR system (Millipore). For each sequence reaction a mixture was made using 3 µl purified and

concentrated PCR product, 0.5 µl of BigDye™ Termination RR mix version 3.1 (Perkin Elmer), 1.75 µl of BigDye™

buffer (5x), 1.75 µl sterile milliQ water and 3 µl (20 ng/µl) of one of the 6 sequencing primers. The sequencing

products were cleaned up, as described by Naser et al. [15]. The temperature-time profile was as follows: 30 cycles

of denaturation for 15 s at 96°C, primer annealing for 1 s at 35°C and extension for 4 min at 60°C. Sequence analysis

of the partial 16S rRNA gene (first 300-500 bp) was performed using the Applied Biosystems 3100 DNA Sequencer

according to protocols provided by the manufacturer. Sequences were assembled using the BioNumerics 4.6 software

(Applied Maths). A reliable genus identification was obtained in two steps: (i) a BLAST search [1] of the 16S rRNA

gene sequence of an isolate retrieved 50 sequences with the highest sequence similarities to the query sequence, (ii)

the 16S rRNA gene sequence of all type strains of all species mentioned in the BLAST report were compared in an

exhaustive pair wise manner with the query sequence in BioNumerics 4.6. Isolates were assigned to a genus based

on the FAME analysis and the obtained 16S rRNA gene sequence similarity.

Nucleotide sequence accession numbersThe sequence data generated in this study have been deposited in Genbank/EMBL/DDBJ. The accession numbers

of the 16S rRNA genes can be found in the Table 2.3.


56

RESULTS & DISCUSSION

A previous screening of sixty defined growth media showed the high suitability of three

media, G2M11, G3M12 and G4M3, for elective isolation of denitrifiers [9]. Thus far, their

applicability has only been assessed with activated sludge as inoculum. Here, the three

growth media are used for the cultivation of denitrifying isolates from soil. TSA

supplemented with nitrate was also included, as complex commercial growth media are

still frequently used to isolate denitrifiers. This study focused on heterotrophic denitrifiers

– defined growth media contained between 45 and 60 mM carbon – able to start

denitrification from either nitrate, the nitrogen source in G3M12, G4M3, and TSA, or

nitrite, the nitrogen source in G2M11.

Isolation and identification of heterotrophic soil denitrifiers

A total of 249 isolates were obtained from plating the soil suspension on the four media.

112 isolates were capable of performing denitrification in their isolation medium. Half of

the denitrifiers (50,9% or 57 isolates) were retrieved from G2M11, 20,5% (23 isolates)

from G3M12, 22,3% (25 isolates) from G4M3, and 6,3% (7 isolates) from TSA. All

denitrifiers were identified at the genus level through a biphasic identification approach,

using both FAME analysis (data not shown) and partial 16S rRNA gene sequence analysis.

An overview of the isolates, their identification, and other isolation information is included

in Table 2.3.

The majority of the denitrifiers belonged to the Firmicutes (77% or 83 isolates). Three

isolates were assigned to two different Staphylococcus species, while eighty isolates belonged

to Bacillus, showing high sequence similarities with seven different species. The denitrifying

capacity of Bacillus has long been recognized, but is not well investigated [21]. Other

cultivation studies also identified members of Bacillus as as soil denitrifiers [4,24]. Here,

bacilli were mostly retrieved from medium G2M11, the only medium with nitrite as nitrogen

source, indicating a possible preference for starting denitrification from nitrite. Although

this hypothesis is hard to substantiate because literature almost exclusively reports on

(commercial) denitrification tests including nitrate, nitrite tolerance or nitrite dependence

of many Bacillus strains has been described previously [24]. Most of the isolated denitrifying

bacilli were highly related with the dominant active bacteria found in Drentse A grassland

soils through direct ribosome isolation [5], namely B. drentensis, B. bataviensis and B. soli

[11].

CHAPTER 2

57

The other 29 denitrifiers belonged to the Proteobacteria. All 18 isolates of the

Alphaproteobacteria belonged to the family of the Rhizobiaceae, with representatives of

Ensifer, Rhizobium and Sinorhizobium. Rhizobial denitrification is speculated to yield

selective advantage for nodulation in situ through (i) neutralization of the nitrate inhibition

of nodulation, and (ii) generation of ATP for N2 fixation, but also enables survival and

growth of free-living rhizobia when other energy sources are limited [16]. Rhizobia have

not been frequently isolated in cultivation-dependent denitrification studies, despite their

widespread occurrence and a high persistence in agricultural and other soil [16]. Their

underrepresentation in denitrifier isolation campaigns could be attributed to the used growth

media: rhizobia do not grow very well on general complex media [23]. In this study,

rhizobia were mostly isolated from G3M12, containing nitrate and ethanol. Only three

representatives of Cupriavidus and eight of Pseudomonas were isolated. The numerical

dominance of Beta- and Gammaproteobacteria among soil isolates as described previously

[4,6,24] was not observed in our study. Also, this can probably be attributed to the

isolation media used.


58

Table 2.3. Identification of all isolated denitrifiers. Isolation medium and dilution is given. Shading indicates thedenitrifying taxa found in the highest dilution on each isolation medium.

S tra in n r G e n us M e d ium D ilu tio n A c c es s io n n r T yp e s tra in with h ig h e s t 16 S rR NA g e n e s equ en c e id e n tific a tio n s im ila r ity to q u e ry s e q ue n ce (% s e q . s im ilar ity, s p ec ie s n am e, s tra in n u m b e r, a cc e ss io n n um be r)

R -3 1 55 3 B a c illu s s p. G 2 M 1 1 -3 AM 6 9 1 5 7 6 9 9 % B ac illus s oli L M G 218 3 8 T (AJ5 4 25 13) R -3 3 77 3 B a c illu s s p. G 2 M 1 1 -3 AM 6 9 1 5 7 9 9 9 ,9% Ba cillus d ren te ns is L M G 21 8 31 T (AJ 5 4 25 06 ) R -3 2 85 1 B a c illu s s p. G 2 M 1 1 -3 AM 6 9 1 5 8 0 1 0 0% B ac illus d ren te n sis L M G 2 18 31 T (AJ 5 4 250 6) R -3 3 77 4 B a c illu s s p. G 2 M 1 1 -3 AM 6 9 1 5 9 2 9 8 ,6% Ba cillus d ren te ns is L M G 21 8 31 T (AJ 5 4 25 06 ) R -3 1 55 4 B a c illu s s p. G 2 M 1 1 -3 AM 6 9 1 6 0 5 9 8 ,6% Ba cillus d ren te ns is L M G 21 8 31 T (AJ 5 4 25 06 ) R -3 1 54 6 B a c illu s s p. G 2 M 1 1 -3 AM 6 9 1 6 0 4 9 9 % B ac illus ba ta v ie ns is L M G 21 83 3 T (AJ 5 42 50 8 ) R -3 2 70 5 B a c illu s s p. G 2 M 1 1 -2 AM 4 0 3 6 3 4 9 8 ,8% Ba cillus d ren te ns is L M G 21 8 31 T (AJ 5 4 25 06 ) R -3 2 84 9 B a c illu s s p. G 2 M 1 1 -2 AM 4 0 3 6 3 5 9 8 ,8% Bacillu s so li L M G 21 8 38 T (AJ 5 42 51 3) R -3 2 78 1 B a c illu s s p. G 2 M 1 1 -2 AM 4 0 3 6 5 1 9 9 % B ac illus ba ta v ie ns is L M G 21 83 3 T (AJ 5 42 50 8 ) R -3 2 77 9 B a c illu s s p. G 2 M 1 1 -2 AM 4 0 3 6 5 0 9 9 % B ac illus ba ta v ie ns is L M G 21 83 3 T (AJ 5 42 50 8 ) R -3 2 70 9 B a c illu s s p. G 2 M 1 1 -2 AM 4 0 3 6 3 1 9 9 ,1% Ba cillus b a tav ien sis L M G 2 18 33 T (AJ 5 4 25 08 ) R -3 2 70 6 B a c illu s s p. G 2 M 1 1 -2 AM 4 0 3 5 8 5 1 0 0% B ac illu s lic he n ifo rm is LM G 12 36 3 T (X 68 416 ) R -3 2 71 5 B a c illu s s p. G 2 M 1 1 -2 AM 4 0 3 6 1 1 9 9 ,1% Bacillu s so li L M G 21 8 38 T (AJ 5 42 51 3) R -3 1 76 9 B a c illu s s p. G 2 M 1 1 -2 AM 4 0 3 6 4 0 1 0 0% B ac illu s lic he n ifo rm is LM G 12 36 3 T (X 68 416 ) R -3 2 70 0 B a c illu s s p. G 2 M 1 1 -2 AM 4 0 3 6 1 0 9 8 ,6% Ba cillus b a tav ien sis L M G 2 18 33 T (AJ 5 4 25 08 ) R -3 1 77 0 B a c illu s s p. G 2 M 1 1 -2 AM 4 0 3 6 1 8 9 9 % B ac illus ba ta v ie ns is L M G 21 83 3 T (AJ 5 42 50 8 ) R -3 2 78 7 B a c illu s s p. G 2 M 1 1 -2 AM 4 0 3 6 2 3 9 9 % B ac illus ba ta v ie ns is L M G 21 83 3 T (AJ 5 42 50 8 ) R -3 2 78 9 B a c illu s s p. G 2 M 1 1 -2 AM 4 0 3 6 3 7 9 8 ,7% Ba cillus d ren te ns is L M G 21 8 31 T (AJ 5 4 25 06 ) R -3 2 51 6 B a c illu s s p. G 2 M 1 1 -2 AM 6 9 1 5 5 7 9 8 ,9% Bacillu s so li L M G 21 8 38 T (AJ 5 42 51 3) R -3 2 52 1 B a c illu s s p. G 2 M 1 1 -2 AM 6 9 1 5 5 8 9 8 ,9% Bacillu s so li L M G 21 8 38 T (AJ 5 4 25 13 ) R -3 2 52 3 B a c illu s s p. G 2 M 1 1 -2 AM 6 9 1 5 5 9 9 8 ,9% Ba cillus b a tav ien sis L M G 2 18 33 T (AJ 5 4 25 08 ) R -3 2 70 3 B a c illu s s p. G 2 M 1 1 -2 AM 6 9 1 5 6 0 9 8 ,9% Ba cillus b a tav ien sis L M G 2 18 33 T (AJ 5 4 25 08 ) R -3 1 85 6 B a c illu s s p. G 2 M 1 1 -2 AM 6 9 1 5 6 1 9 8 ,8% Bacillu s so li L M G 21 8 38 T (AJ 5 4 25 13 ) R -3 2 69 4 B a c illu s s p. G 2 M 1 1 -2 AM 6 9 1 5 6 3 9 9 ,1% Bacillu s so li L M G 21 8 38 T (AJ 5 42 51 3) R -3 3 81 9 B a c illu s s p. G 2 M 1 1 -2 AM 6 9 1 5 6 4 9 9 % B ac illus d re n te ns is L M G 2 1 83 1 T (AJ 5 42 50 6 ) R -3 2 71 7 B a c illu s s p. G 2 M 1 1 -2 AM 6 9 1 5 6 9 9 9 % B ac illus d re n te ns is L M G 2 1 83 1 T (AJ 5 42 50 6 ) R -3 2 70 4 B a c illu s s p. G 2 M 1 1 -2 AM 6 9 1 5 7 0 9 8 ,9% Bacillu s so li L M G 21 8 38 T (AJ 5 4 25 13 ) R -3 3 77 6 B a c illu s s p. G 2 M 1 1 -2 AM 6 9 1 5 7 1 9 8 ,9% Ba cillu s n ov alis L M G 21 83 7 T (AJ 5 42 512 ) R -3 2 70 7 B a c illu s s p. G 2 M 1 1 -2 AM 6 9 1 5 7 4 9 8 ,7% Ba cillus b a tav ien sis L M G 2 18 33 T (AJ 5 4 25 08 ) R -3 2 52 6 B a c illu s s p. G 2 M 1 1 -2 AM 6 9 1 5 7 8 9 9 % B ac illus s o li L M G 2 183 8 T (AJ 54 25 1 3 ) R -3 2 69 5 B a c illu s s p. G 2 M 1 1 -2 AM 6 9 1 5 8 1 9 9 ,1% Ba cillus d ren te ns is L M G 21 8 31 T (AJ 5 4 25 06 ) R -3 2 71 3 B a c illu s s p. G 2 M 1 1 -2 AM 6 9 1 5 8 2 9 8 ,7% Bacillu s so li L M G 21 8 38 T (AJ 5 42 51 3) R -3 2 70 8 B a c illu s s p. G 2 M 1 1 -2 AM 6 9 1 5 8 3 9 9 ,1% Ba cillus b a tav ien sis L M G 2 18 33 T (AJ 5 4 25 08 ) R -3 2 52 8 B a c illu s s p. G 2 M 1 1 -2 AM 6 9 1 5 8 5 9 8 ,6% Ba cillus d ren te ns is L M G 21 8 31 T (AJ 5 4 25 06 ) R -3 2 53 3 B a c illu s s p. G 2 M 1 1 -2 AM 6 9 1 5 8 6 9 8 ,7% Ba cillus b a tav ien sis L M G 2 18 33 T (AJ 5 4 25 08 ) R -3 2 52 2 B a c illu s s p. G 2 M 1 1 -2 AM 6 9 1 5 8 7 9 8 ,9% Ba cillus b a tav ien sis L M G 2 18 33 T (AJ 5 4 25 08 ) R -3 2 69 6 B a c illu s s p. G 2 M 1 1 -2 AM 6 9 1 5 8 8 9 8 ,6% Ba cillus d ren te ns is L M G 21 8 31 T (AJ 5 4 25 06 ) R -3 2 51 7 B a c illu s s p. G 2 M 1 1 -2 AM 6 9 1 5 9 0 9 8 ,7% Ba cillus b a tav ien sis L M G 2 18 33 T (AJ 5 4 25 08 ) R -3 2 53 4 B a c illu s s p. G 2 M 1 1 -2 AM 6 9 1 5 9 1 9 8 ,9% Ba cillus b a tav ien sis L M G 2 18 33 T (AJ 5 4 25 08 ) R -3 2 51 8 B a c illu s s p. G 2 M 1 1 -2 AM 6 9 1 5 9 6 9 8 ,9% Ba cillus b a tav ien sis L M G 2 18 33 T (AJ 5 4 25 08 ) R -3 2 52 9 B a c illu s s p. G 2 M 1 1 -2 AM 6 9 1 5 9 8 9 8 ,9% Ba cillus b a tav ien sis L M G 2 18 33 T (AJ 5 4 25 08 ) R -3 2 52 4 B a c illu s s p. G 2 M 1 1 -2 AM 6 9 1 5 9 9 9 8 ,7% Bacillus so li L M G 21 83 8 T (AJ 54 2 51 3) R -3 1 84 1 B a c illu s s p. G 2 M 1 1 -2 AM 6 9 1 6 0 0 9 9 % B ac illus d re n te ns is L M G 2 1 83 1 T (AJ 5 42 50 6 ) R -3 3 82 0 B a c illu s s p. G 2 M 1 1 -2 AM 6 9 1 6 0 2 9 9 % B ac illus s o li L M G 2 183 8 T (AJ 54 25 1 3 ) R -3 2 70 2 B a c illu s s p. G 2 M 1 1 -2 AM 6 9 1 6 0 3 9 8 ,9% Ba cillus d ren te ns is L M G 21 8 31 T (AJ 5 4 25 06 ) R -3 2 53 1 B a c illu s s p. G 2 M 1 1 -2 AM 6 9 1 6 0 8 9 8 ,7% Bacillu s so li L M G 21 8 38 T (AJ 5 4 25 13 ) R -3 2 52 5 B a c illu s s p. G 2 M 1 1 -2 AM 6 9 1 6 0 9 9 8 ,8% Bacillu s so li L M G 21 8 38 T (AJ 5 4 25 13 ) R -3 2 69 9 B a c illu s s p. G 2 M 1 1 -2 AM 6 9 1 6 1 0 9 8 ,9% Ba cillus b a tav ien sis L M G 2 18 33 T (AJ 5 4 25 08 ) R -3 2 53 5 B a c illu s s p. G 2 M 1 1 -2 AM 6 9 1 6 1 1 9 9 % B ac illus ba ta v ie ns is L M G 21 83 3 T (AJ 5 42 50 8 ) R -3 2 53 0 B a c illu s s p. G 2 M 1 1 -2 AM 6 9 1 6 1 2 9 8 ,9% Bacillu s so li L M G 21 8 38 T (AJ 5 4 25 13 ) R -3 2 78 4 B a c illu s s p. G 2 M 1 1 -2 AM 6 9 1 6 1 4 9 8 ,9% Ba cillus b a tav ien sis L M G 2 18 33 T (AJ 5 4 25 08 ) R -3 2 84 8 B a c illu s s p. G 2 M 1 1 -2 AM 6 9 1 6 2 0 9 8 ,9% Bacillu s so li L M G 21 8 38 T (AJ 5 4 25 13 ) R -3 1 85 5 B a c illu s s p. G 2 M 1 1 -2 AM 6 9 1 6 2 1 9 8 ,9% Bacillu s so li L M G 21 8 38 T (AJ 5 4 25 13 ) R -3 1 85 2 B a c illu s s p. G 2 M 1 1 -2 AM 6 9 1 6 2 5 9 8 ,9% Ba cillus b a tav ien sis L M G 2 18 33 T (AJ 5 4 25 08 ) R -3 2 51 9 B a c illu s s p. G 2 M 1 1 -2 AM 6 9 1 6 2 7 9 8 ,2% Ba cillus m a n na nily tic us AM -0 0 1 T (AB 0 438 6 4 ) R -3 2 77 8 B a c illu s s p. G 2 M 1 1 -2 AM 6 9 1 6 2 9 9 9 % B ac illus ba ta v ie ns is L M G 21 83 3 T (AJ 5 42 50 8 ) R -3 4 18 1 S ta p h yloc occu s s p. G 2 M 1 1 -2 AM 4 0 3 6 3 8 1 0 0% S ta ph y lo co cc us wa rne ri L M G 1 3 35 4 T (L376 0 3) R -3 3 77 1 S ta p h yloc occu s s p. G 2 M 1 1 -2 AM 4 0 3 6 4 3 1 0 0% S ta ph y lo co cc us wa rne ri L M G 1 3 35 4 T (L376 0 3) R -3 2 76 9 S in o rh izob iu m s p. G 3 M 1 2 -5 AM 6 9 1 6 1 5 1 0 0% S in o rh izob iu m m o re len se LM G 2 1 33 1 T (AY 02 43 35 ) R -3 2 54 9 S in o rh izob iu m s p. G 3 M 1 2 -4 AM 6 9 1 6 2 8 1 0 0% S in o rh izob iu m m o re len se LM G 2 1 33 1 T (AY 02 43 35 ) R -3 1 81 7 P s eu d o m onas s p. G 3 M 1 2 -4 AM 6 9 1 6 1 8 9 9 ,8% Ps eu do m o n as k ilo ne ns is D S M 13 54 7 T (AJ2 92 4 26 ) R -3 1 81 5 P s eu d o m onas s p. G 3 M 1 2 -4 AM 6 9 1 6 3 0 9 9 ,8% Ps eu do m o n as k ilo ne ns is D S M 13 54 7 T (AJ2 92 4 26 ) R -3 1 75 9 S in o rh izob iu m s p. G 3 M 1 2 -3 AM 4 0 3 6 4 7 1 0 0% S in o rh izob iu m m o re len se LM G 2 1 33 1 T (AY 02 43 35 ) R -3 2 73 7 S in o rh izob iu m s p. G 3 M 1 2 -3 AM 4 0 3 6 3 2 1 0 0% S in o rh izob iu m m o re len se LM G 2 1 33 1 T (AY 02 43 35 ) R -3 2 54 6 S in o rh izob iu m s p. G 3 M 1 2 -3 AM 6 9 1 6 2 6 1 0 0% S in o rh izob iu m m o re len se LM G 2 1 33 1 T (AY 02 43 35 ) R -3 1 76 3 S in o rh izob iu m s p. G 3 M 1 2 -3 AM 6 9 1 6 3 1 1 0 0% S in o rh izob iu m m o re len se LM G 2 1 33 1 T (AY 02 43 35 ) R -3 1 76 4 S in o rh izob iu m s p. G 3 M 1 2 -3 AM 4 0 3 6 4 8 1 0 0% S in o rh izob iu m m o re len se LM G 2 1 33 1 T (AY 02 43 35 ) R -3 2 54 4 E n s ife r s p. G 3 M 1 2 -3 AM 4 0 3 5 9 3 1 0 0% E ns ife r m ed ic ae A3 2 1 T (Z 78 20 4 ) R -3 1 76 2 R h izo biu m s p. G 3 M 1 2 -3 AM 4 0 3 5 8 4 9 8 ,3% Rh izo biu m rad io b ac te r L M G 14 0 T (AJ 3 8990 4 ) R -3 1 75 8 P s eu d o m onas s p. G 3 M 1 2 -3 AM 4 0 3 6 0 0 1 0 0% Ps e ud o m on as k ilo ne ns is D S M 1 35 47 T (AJ 29 24 26 ) R -3 1 76 5 P s eu d o m onas s p. G 3 M 1 2 -3 AM 4 0 3 6 0 4 1 0 0% Ps e ud o m on as k ilo ne ns is D S M 1 35 47 T (AJ 29 24 26 ) R -3 2 54 1 P s eu d o m onas s p. G 3 M 1 2 -3 AM 6 9 1 6 1 7 1 0 0% Ps e ud o m on as k ilo ne ns is D S M 1 35 47 T (AJ 29 24 26 )

R -3 1 76 1 P s eu d o m onas s p. G 3 M 1 2 -3 AM 6 9 1 6 2 2 9 9 ,8% Ps eu do m o n as b ras sicace a ru m C IP 10 70 5 9 T

(AF 1 0 0 32 1) R -3 3 77 7 S ta p h yloc occu s s p. G 3 M 1 2 -3 AM 6 9 1 5 6 2 1 0 0% S ta ph y lo co cc us e p id e rm id is L M G 10 47 4 T (D83 3 63 ) R -3 2 54 2 S in o rh izob iu m s p. G 3 M 1 2 -3 AM 4 0 3 6 0 6 1 0 0% S in o rh izob iu m m o re len se LM G 2 1 33 1 T (AY 02 43 35 ) R -3 1 75 7 E n s ife r s p. G 3 M 1 2 -3 AM 6 9 1 5 7 2 1 0 0% E ns ife r m ed ic ae A3 2 1 T (Z 78 20 4 ) R -3 2 72 5 R h izo biu m s p. G 3 M 1 2 -3 AM 4 0 3 6 0 5 1 0 0% R hizo b iu m rad iob ac te r L M G 14 0 T (AJ 3 899 04 ) R -3 2 53 9 R h izo biu m s p. G 3 M 1 2 -3 AM 6 9 1 5 8 4 9 8 ,5% Rh izo biu m rad io b ac te r L M G 14 0 T (AJ 3 8990 4 ) R -3 2 55 2 R h izo biu m s p. G 3 M 1 2 -3 AM 6 9 1 5 6 5 9 7 ,7% Rh izo biu m rad io b ac te r L M G 14 0 T (AJ 3 8990 4 ) R -3 2 55 3 P s eu d o m onas s p. G 3 M 1 2 -3 AM 6 9 1 6 1 9 9 9 ,8% Ps eu do m o n as k ilo ne ns is D S M 13 54 7 T (AJ2 92 4 26 ) R -3 2 72 6 P s eu d o m onas s p. G 3 M 1 2 -3 AM 6 9 1 6 2 3 9 9 ,8% Ps eu do m o n as k ilo ne ns is D S M 13 54 7 T (AJ2 92 4 26 ) R -3 1 81 6 S in o rh izob iu m s p. G 4 M 3 -4 AM 4 0 3 6 5 3 1 0 0% S in o rh izob iu m m o re len se LM G 2 1 33 1 T (AY 02 43 35 ) R -3 1 85 7 R h izo biu m s p. G 4 M 3 -4 AM 6 9 1 6 1 6 9 7 ,2% Rh izo biu m rad io b ac te r L M G 14 0 T (AJ 3 8990 4 ) R -3 2 84 5 B a c illu s s p. G 4 M 3 -4 AM 4 0 3 6 4 2 9 9 % B ac illus ba ta v ie ns is L M G 21 83 3 T (AJ 5 42 50 8 ) R -3 2 66 1 B a c illu s s p. G 4 M 3 -4 AM 6 9 1 5 9 3 9 8 ,8% Ba cillus b a tav ien sis L M G 2 18 33 T (AJ 5 4 25 08 ) R -3 1 84 8 B a c illu s s p. G 4 M 3 -4 AM 6 9 1 5 9 4 9 8 ,9% Ba cillus b a tav ien sis L M G 2 18 33 T (AJ 5 4 25 08 ) R -3 2 66 5 B a c illu s s p. G 4 M 3 -4 AM 6 9 1 6 0 7 9 8 ,9% Ba cillus b a tav ien sis L M G 2 18 33 T (AJ 5 4 25 08 ) R -3 1 54 9 R h izo biu m s p. G 4 M 3 -3 AM 4 0 3 6 2 1 9 8 ,5% Rh izo biu m rad io b ac te r L M G 14 0 T (AJ 3 8990 4 ) R -3 1 83 7 R h izo biu m s p. G 4 M 3 -3 AM 4 0 3 6 1 4 9 8 ,5% Rh izo biu m rad io b ac te r L M G 14 0 T (AJ 3 8990 4 ) R -3 2 66 3 B a c illu s s p. G 4 M 3 -3 AM 4 0 3 6 4 6 1 0 0% B ac illus d ren te n sis L M G 2 18 31 T (AJ 5 4 250 6) R -3 1 84 6 B a c illu s s p. G 4 M 3 -3 AM 4 0 3 6 4 5 9 9 % B ac illus d re n te ns is L M G 2 1 83 1 T (AJ 5 42 50 6 ) R -3 1 55 0 B a c illu s s p. G 4 M 3 -3 AM 4 0 3 6 0 2 1 0 0% B ac illus d ren te n sis L M G 2 18 31 T (AJ 5 4 250 6) R -3 1 83 5 B a c illu s s p. G 4 M 3 -3 AM 4 0 3 6 0 1 1 0 0% B ac illus d ren te n sis L M G 2 18 31 T (AJ 5 4 250 6) R -3 1 54 7 B a c illu s s p. G 4 M 3 -3 AM 4 0 3 5 8 3 1 0 0% B ac illus d ren te n sis L M G 2 18 31 T (AJ 5 4 250 6) R -3 1 83 4 B a c illu s s p. G 4 M 3 -3 AM 4 0 3 6 2 4 9 8 ,8% Ba cillus b a tav ien sis L M G 2 18 33 T (AJ 5 4 25 08 ) R -3 1 83 6 B a c illu s s p. G 4 M 3 -3 AM 6 9 1 5 6 6 1 0 0% B ac illus d ren te n sis L M G 2 18 31 T (AJ 5 4 250 6) R -3 1 83 8 B a c illu s s p. G 4 M 3 -3 AM 6 9 1 5 6 8 1 0 0% B ac illus d ren te n sis L M G 2 18 31 T (AJ 5 4 250 6) R -3 2 84 4 B a c illu s s p. G 4 M 3 -3 AM 6 9 1 5 7 3 9 9 ,3% Bacillu s so li L M G 21 8 38 T (AJ 5 42 51 3) R -3 2 65 6 B a c illu s s p. G 4 M 3 -3 AM 6 9 1 5 7 5 1 0 0% B ac illus d ren te n sis L M G 2 18 31 T (AJ 5 4 250 6) R -3 1 55 3 B a c illu s s p. G 4 M 3 -3 AM 6 9 1 5 7 6 9 9 % B ac illus s oli L M G 218 3 8 T (AJ 54 2 51 3) R -3 1 55 2 B a c illu s s p. G 4 M 3 -3 AM 6 9 1 5 7 7 9 8 ,9% Ba cillus b a tav ien sis L M G 2 18 33 T (AJ 5 4 25 08 ) R -3 2 66 2 B a c illu s s p. G 4 M 3 -3 AM 6 9 1 5 8 9 9 8 ,7% Ba cillus b a tav ien sis L M G 2 18 33 T (AJ 5 4 25 08 ) R -3 1 83 2 B a c illu s s p. G 4 M 3 -3 AM 6 9 1 5 9 5 9 8 ,8% Ba cillus d ren te ns is L M G 21 8 31 T (AJ 5 4 25 06 ) R -3 1 85 0 B a c illu s s p. G 4 M 3 -3 AM 6 9 1 5 9 7 1 0 0% B ac illus d ren te n sis L M G 2 18 31 T (AJ 5 4 250 6) R -3 1 84 5 B a c illu s s p. G 4 M 3 -3 AM 6 9 1 6 0 1 9 8 ,9% Bacillu s so li L M G 21 8 38 T (AJ 5 4 25 13 ) R -3 1 84 9 B a c illu s s p. G 4 M 3 -3 AM 6 9 1 6 0 6 1 0 0% B ac illus d ren te n sis L M G 2 18 31 T (AJ 5 4 250 6) R -3 1 54 1 B a c illu s s p. TS A -5 AM 4 0 3 6 3 0 1 0 0% B ac illus d ren te n sis L M G 2 18 31 T (AJ 5 4 250 6) R -3 2 57 5 B a c illu s s p. TS A -5 AM 6 9 1 6 1 3 1 0 0% B ac illus d ren te n sis L M G 2 18 31 T (AJ 5 4 250 6) R -3 1 54 3 C u p ria vid us s p. TS A -5 AM 4 0 3 5 9 9 9 9 ,3% Cu p riavidu s n ec a to r L M G 8 4 53 T (AF 1 9 17 3 7 ) R -3 1 54 2 C u p ria vid us s p. TS A -5 AM 4 0 3 5 9 7 9 9 ,3% Cu p riavidu s n ec a to r L M G 8 4 53 T (AF 1 9 17 3 7 ) R -3 1 54 4 C u p ria vid us s p. TS A -5 AM 6 9 1 5 6 7 9 9 ,5% Cu p riavidu s n ec a to r L M G 8 4 53 T (AF 1 9 17 3 7 ) R -3 1 83 0 B a c illu s s p. TS A -4 AM 4 0 3 6 0 9 9 9 ,5% Ba cillu s ps e ud o m yc oide s L M G 1 89 93 T (AF0 1 3 12 1 ) R -3 2 57 4 B a c illu s s p. TS A -4 AM 6 9 1 6 2 4 9 9 ,2% Ba cillu s ps e ud o m yc oide s L M G 1 89 93 T (AF0 1 3 12 1 )

CHAPTER 2

59

Assessment of applied growth media

Important for universal applicability of elective growth media for denitrifier isolation is (i)

the growth of a high number of denitrifiers, (ii) a high diversity of denitrifiers, both retrieved

from (iii) the highest dilution containing denitrifiers. These three factors can be used for

evaluation of the four growth media, applied simultaneously to the same soil sample.

These results are compared to a cultivation study of activated sludge using the same growth

media [9], and a cultivation study on three agricultural soils using nitrate agar as isolation

medium [4].

(i) An overview of the total number of isolates and their nitrogen reducing capacities per

growth medium is given in Table 2.4. G4M3 and G2M11 yielded a high ratio denitrifiers-

to-total-isolates, appr. 55% and 75% respectively. These ratios were significantly higher

than for TSA and G3M12, due to the high number of nitrate reducers on the latter growth

media, casting doubt on the suitability of G3M12 for denitrifier isolation. Compared to

results from literature achieved with nitrate agar, yielding between 2.2 and 22.8% [4],

media G4M3 and G2M11 numerically scored much better for elective isolation of

heterotrophic denitrifiers from soil. Both media also gave significant better results with

soil than with activated sludge, 20-30% and 43% respectively, while G3M12 and TSA

yielded a similar ratio for both inocula.

Table 2.4. An overview of the number of isolates with the nitrogen reduction capacities specified and grouped perisolation medium. Percentages per isolation medium are given. Exact numbers are mentioned between brackets.

(ii) The retrieved denitrifying diversity per growth medium was scored. The observed

diversity onto genus level - genus identification was based on both FAME analysis and

16S rRNA gene sequence analysis - was represented for each growth medium by the

Simpson’s reciprocal diversity index 1/D,

1/D = N × (N-1)/E[ni × (n

i-1)] (eq. 3)

with N = number of denitrifying isolates per medium, ni = number of denitrifying isolates

per medium belonging to genus i. Growth medium G3M12 retrieved the highest denitrifier

diversity (Figure 2.3). The results for G2M11 and G4M3 were comparable and significantly

Metabolism of nitrogen compounds Growth media for isolation and tests

(under isolation conditions) TSA (31) G2M11 (75) G3M12 (98) G4M3 (45)

No reduction of nitrate and/or nitrite (27) 0% (0) 24% (18) 1% (1) 17,7% (8)

Nitrate reduction (110) 77,4% (24) ND 75,5% (74) 26,7% (12)

Denitrification (112) 22,6% (7) 76% (57) 23,5% (23) 55,6% (25)


60

lower than TSA, due to the numerical dominance of bacilli retrieved on both media. G2M11,

G3M12 and G4M3 clearly retrieved less diversity from soil than from activated sludge

(Figure 2.3). The difference in retrieved cultured diversity between both habitats may be

correlated with the continuous enrichment of denitrifiers in activated sludge, contrasting

with the controlling factor of denitrification in soil, which is not the process itself but the

competition for carbon with aerobic heterotrophs [23]. Also, the different culturability of

the microbiota of both habitats can be involved: activated sludge contains 1-15% culturable

bacteria, while this is only 0.3% in soil [2]. However, the growth media could also have a

biased towards activated sludge, the sole inoculum during medium development [9]. In

contrast, little difference in retrieved denitrifier diversity was observed between activated

sludge and soil with supplemented TSA, suggesting an intrinsic average diversity index of

2. The cultured denitrifying diversity in the comparable study was nicely spread over five

different taxa, with 15-25% of Pseudomonas, Ralstonia, Frateuria, Bacillus, and

Streptomyces, while we predominantly found Bacillus. Thus, nitrate agar could be more

suitable for denitrifier isolation. Yet, it is difficult to compare results from different soil

samples. First, these cited results are collected from three different agricultural soils, and

the results per soil were not given. Second, because of soil’s three-dimensional matrix,

nutrient conditions, and thus denitrifying activity, are variable in space and time.

4,0

7,8

2,1 2,3

15,0

9,0

1,1

1,4

0

2

4

6

8

10

12

14

16

act ivatedsludge

soil act ivatedsludge



soil

G2M 11 G3M 12 G4M 3 TSA

1/D

Figure 2.3. Retrieved denitrifying diversity from soil, expressed as Simpson’s Reciprocal Diversity Index 1/D, pergrowth medium. The results of both activated sludge (taken from [9]) and soil (this study) are given.

CHAPTER 2

61

(iii) When taking the isolation dilution as a rough measurement of their in situ abundance

(Table 2.3), the relevance of the retrieved denitrifier diversity can be evaluated. TSA and

G3M12 scored best, with retrieval of at least 106 CFU g-1 denitrifiers, while G4M3 cultured

an order of magnitude less, and G2M11 scored worst with 103 CFU g-1 denitrifiers. Based

on these abundance estimates, Bacillus, Sinorhizobium and Cupriavidus harbor the culturable

denitrifiers dominant in this soil sample, followed by Pseudomonas. Unfortunately,

comparison with other cultivation studies or results from activated sludge is not possible

due to lack of data. However, our results comply with quantitative PCR data of soil using

the nirK gene, which is a single copy gene coding for copper-containing nitrite reductase

[25], reporting nirK densities of 104 to 109 nir gene copies per gram soil, depending on the

technique used and the type of soil [7, 8, 14, 18].

Conclusions

The assessment of a growth medium to cultivate a specific group of bacteria depends on

the criteria used. Here we defined three criteria: the number of denitrifiers retrieved, their

diversity and estimated abundance. Although G2M11, and to a lesser extent, G4M3, retrieved

a high number of denitrifying isolates, G3M12 yieled a much higher denitrifying diversity

from both soil and activated sludge and, based on the used criteria, this is the optimal

medium to study denitrifiers. Also, the results of supplemented TSA seem to be independent

of the studied environment. However, application of a multiple-media set generates more

information than the use of individual growth media. The estimated abundance of

Sinorhizobium, and Cupriavidus and Bacillus would not have been revealed without use

of G3M12 or supplemented TSA respectively. The use of a set of different growth media is

thus recommended for estimating general [3] or specific bacterial diversity. Now, an

investigation of the role and abundance of members of these three genera in situ, for example

with fluorescent in situ hybridization in combination with microautoradiography [12], is

warranted.


62

REFERENCES1. Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ (1990) Basic local alignment search tool. J

Mol Biol 215:403-410

2. Amann RI, Ludwig W, Schleifer K-H (1995) Phylogenetic identification and in situ detection of individual

microbial cells without cultivation. Microbiol Rev 59:143-169

3. Balestra GM, Misaghi IJ (1997) Increasing the efficiency of the plate counting method for estimating

bacterial diversity. J Microbiol Meth 30:111-117

4. Chèneby D, Philippot L, Hartmann A, Hénault C, Germon J-C (2000) 16S rDNA analysis for

characterisation of denitrifying bacteria isolated from three agricultural soils. FEMS Microbiol Ecol

34:121-128

5. Felske A, Wolterink A, Van Lis R, Akkermans ADL (1998) Phylogeny of the main bacterial 16S rRNA

sequences in Drentse A grassland soils (The Netherlands). Appl Environ Microbiol 64: 871-879

6. Gamble TN, Betlach MR, Tiedje TM (1977) Numerically dominant denitrifying bacteria from world

soils. Appl Environ Microbiol 33:926-939

7. Henry S, Baudoin E, Lopez-Gutiérrez JC, Martin-Laurent F, Brauman A, Philippot L (2004) Quantification

of denitrifying bacteria in soils by nirK gene targeted real-time PCR. J Microbiol Meth 59:327-335

8. Henry S, Bru D, Stres B, Hallet S, Philippot L (2006) Quantitative detection of the nosZ genes, encoding

nitrous oxide reductase, and comparison of the abundance of 16S rRNA, narG, nirK and nosZ genes in


9. Heylen K, Vanparys B, Wittebolle L, Verstraete W, Boon N, De Vos P (2006) Cultivation of denitrifying

bacteria: optimization of isolation conditions and diversity study. Appl Environ Microbiol 72:2637-

2643

10. Heyrman J, Swings J (2001) 16S rDNA sequence analysis of bacterial isolates from biodeteriorated

mural paintings in the Servilia tomb (necropolis of Carmona, Seville, Spain). Syst Appl Microbiol

24:417-422

11. Heyrman J, Vanparys B, Logan NA, Balcaen A, Rodríguez-Díaz M, Felske A, De Vos P (2004) Bacillus

novalis sp. nov., Bacillus vireti sp. nov., Bacillus soli sp. nov., Bacillus bataviensis sp. nov. and Bacillus

drentensis sp. nov., from the Drentse A grasslands. Int J Syst Evolution Microbiol 54:47-57

12. Lee N, Nielsen PH, Andreasen KH, Juretschko S, Nielsen JL, Schleifer K-H, Wagner M (1999)

Combination of fluorescent in situ hybridization and microautoradiography – a new tool for structure-

function analysis in microbial ecology. Appl Environ Microbiol 65:1289-1297

13. Mergel A, Schmitz O, Mallmann T Bothe H (2001) Relative abundance of denitrifying and dinitrogen-

fixing bateria in layers of a forest soil. FEMS Microbiol Ecol 36:33-42

14. Michotey V, Méjean V, Bonin P (2000) Comparison of methods for quantification of cytochrome cd1-

denitrifying bacteria in environmental marine samples. Appl Environ Microbiol 66:1564-1571

15. Naser S, Thompson FL, Hoste B, Gevers D, Vandemeulebroecke K, Cleenwerk I, Thompson CC,

Vancanneyt M, Swings J (2005) Phylogeny and identification of Enterococci using atpA gene sequence

analysis. J Clin Microbiol 43:2224-2230

16. O’Hara GW, Daniel RM (1985) Rhizobial denitrification: a review. Soil Biol Biochem 17:1-9


376

18. Qiu X-Y, Hurt RA, Wu L-Y, Chen C-H, Tiedje JM, Zhou J-Z (2004) Detection and quantification of

copper-denitrifying bacteria by quantitative competitive PCR. J Microbiol Meth 59:199-210

19. Radajewski S, Ineson P, Parekh NR, Murell JC (2000) Stable-isotope probing as a tool in microbial

ecology. Nature 403:646-649

20. Sasser,M (1990) Identification of bacteria by gas chromatography of cellular fatty acids. MIDI Technical

Note #101. www.midi-inc.com

21. Shapleig JP (2006) The denitrifying Prokaryotes. In: Dworkin M, Falkow S, Rosenberg E, Schleifer K-

H, Stackebrandt E (Eds), The prokaryotes, vol. 2, Springer-Verlag, New York, pp. 769-792

22. Smibert RM, Krieg NR (1994) Phenotypic characterization. In: Gerhardt P, Murray RGE, Wood WA,

Krieg NR (Eds.), Methods for general and molecular bacteriology, American Society for Microbiology,

Washington, pp. 623-624

23. Tiedje JM, (1988) Ecology of denitrification and dissimilatory nitrate reduction to ammonium. In: Zehnder

ABJ (Ed.), Environmental microbiology of anaerobes, John Wiley & Sons, New York, pp. 179-244

24. Weier KL, MacRae IC (1992) Denitrifying bacteria in the profile of a Brigalow clay soil beneath a

permanent pasture and a cultivated crop. Soil Biol Biochem 24:919-923

CHAPTER 2

63

25. Zumft, W.G. (1997) Cell biology and molecular basis of denitrification. Microbiol. Mol. Biol. Rev.

61:533-616


2.3 BACK & FORTH

Most cultivation-dependent studies deploy one commercial complex medium to isolate

and study the denitrifying diversity present in an environmental sample. However, these

media are probably biased towards certain bacteria and do not enable growth of all denitrifiers.

Therefore, this chapter reports on the development and evaluation of new defined growth

media for specific isolation of heterotrophic denitrifiers. A plate-based isolation procedure

tested a wide range of media, covering a variety of medium components selected to promote

growth of denitrifiers. The optimization procedure did not result in one optimal denitrifiers’

medium, but found several elective media suitable. More importantly, they allowed growth

of different denitrifying taxa compared to the commercial complex medium TSA, so that

their application in a multiple-media set covered a much wider culturable denitrifier diversity

than that found when only one growth medium was used. An overview of all denitrifiers

isolated in this thesis can be found in the Addendum Section.

Thus, the medium optimization procedure was a success. However, in hindsight, our strategy

had several shortcomings:

(i) The use of an evolutionary algorithm was probably not imperative. These

stoichastic search procedures work most optimal after many generations

and are therefore mostly used for solving theoretical problems. But the

work load per generation - the preparation of fifteen media, the

spreadplating, the incubation, and mostly the isolation, subculturing and

identification of twenty isolates per medium - did not allow more generations

in the time frame of this thesis. Statistical methods of medium optimization

would probably have resulted in similar optimal media. Nevertheless, the

EA evaluated the different medium parameter values without assumption

of the underlying fitness landscape, thus avoiding biased interpretations

of the results and obtaining a progressively improvement of the average

and maximal fitness. This objectivity may not be warranted in statistical

methods.

(ii) The variations in fitness values observed when testing a growth medium

(G4M3) in triplicate suggested uncorrect fitness determination. The cause

of these variations is probably the choice of cellular fatty acid analysis as

the fitness identification parameter expressing the cultured denitrifying

genus diversity, which was motivated by time limitations. Unfortunately,

the MIDI TSBA 50 database used for identification of bacteria through

66

fatty acid profiles does not cover all (proteo)bacterial taxa evenly. For

example, the family Rhodocyclaceae, from which several members were

retrieved from activated sludge based on 16S rRNA gene sequence analysis,

only contains one representative in the database. The same goes for the

genera Paracoccus and Ochrobactrum. When retrieving members of those

genera for which the cellular fatty acid content deviates from the single

genus representative in the database, the genus diversity will be

overestimated, leading to overestimating of the fitness value. On the other

hand, the isolation of bacteria from different genera, but with similar fatty

acid profiles, will lead to an underestimation of the diversity, and thus the

fitness value. To investigate the influence of this fatty acid based

identification, all fitness values for all growth media in the four generations

were re-assessed with a fitness identification parameter based on the

genus identification from 16S rRNA gene sequence analysis. The highest

scoring growth media - G1M1, G2M11, G3M12 and G4M3 - still scored

best, but the differences in fitness with the other growth media were less

pronounced. So, if identification based on 16S rRNA gene sequence

analysis was used to determine the fitness identification parameter, the

EA would have assigned different weights to the medium parameter values,

resulting in a different fitness evolution. An automated DNA extraction,

16S rRNA amplification and sequencing approach would have overcome

the time issue and would have resulted in a more reliable optimization

procedure.

(iii) Although only isolates from the highest dilution showing growth were

picked up, this dilution factor was not included as a parameter in the

fitness determination. Inclusion of such a parameter would have ensured

development of a growth medium suitable for isolation of the dominantly

present denitrifiers in the sample.

As an alternative for cultivation, recently described combinations of different molecular

tools now allow the identification of in situ important denitrifying bacteria. Two recent

papers report on the identification of the denitrifier community in activated sludge systems,

fed with methanol [1] and acetate [2], by using stable-isotope probing (SIP), full-cycle

rRNA analysis and fluorescence in situ hybridization-microautoradiography (FISH-MAR).

A 13C-labelled carbon source is fed to an activated sludge reactor exhibiting good

denitrification. The labeled DNA, biomarking the denitrifying community, was extracted

CHAPTER 2

67

and used for the construction of a 16S rRNA gene clone library. Probes for FISH were

designed for the dominant phylotypes and the correlation between their abundance and the

performance of the denitrification process was established. Subsequent FISH-MAR

independently confirmed that these dominant phylotypes were capable of anoxic 14C-

carbon uptake. The proof-of-principle of combined Raman-FISH [3], working on a single

cell level, is another exciting approach for structure-function analysis in complex microbial

communities. These combined methods have great potential and create new possibilities

for environmental monitoring. Yet, the need for expertise and expensive equipment for the

different techniques could be a major drawback for researchers. Also, other metabolic

guilds present in the environment that can utilize 13C-labeled carbon under anoxic conditions,

next to denitrifiers, are not taken into account. Nevertheless, the possible widespread

application of these combined methods may actually be the first attempt to really determine

the in situ important/dominant denitrifying bacteria.

REFERENCES1. Ginige MP, Hugenholtz P, Daims H, Wagner M, Keller J, Blackall LL (2004) Use of stable-isotope

probing, full-cycle rRNA analysis, and fluorescence in situ hybiridzation-microautoradiography to

study a methanol-fed denitrifying microbial community. Appl Environ Microbiol 70:588-596

2. Ginige MP, Keller J, Blackall LL (2005) Investigation of an acetate-fed denitrifying microbial

community by stable isotope probing, full-cycle rRNA analysis and fluorescent in situ hybridization-

microautoradiography. Appl Environ Microbiol 71:8683-8691

3. Huang WE, Stoecker K, Griffiths R, Newbold L, Daims H; Whiteley AS, Wagner M (2007) Raman-

FISH: combining stable-isotope Raman spectroscopy and fluorescence in situ hybridization for the

single cell analysis of identity and function. Environ Microbiol 9:1878-1889


FUNCTIONAL PHYLOGENETIC ANALYSIS OF PURE

CULTURE DENITRIFIERS

3

3.1 THE INCIDENCE OF NIRS AND NIRK AND THEIRGENETIC HETEROGENEITY IN CULTIVATEDDENITRIFIERS

Redrafted from: Heylen K, Gevers D, Vanparys B, Wittebolle L, Geets J, Boon N, De Vos

P (2006) The incidence of nirS and nirK and their genetic heterogeneity in cultivated

denitrifiers. Environ Microbiol 8:2012-2021

SUMMARY

Gene sequence analysis of nirS and nirK, both encoding nitrite reductases, was performed

on cultivated denitrifiers to assess their incidence in different bacterial taxa and their

taxonomical value. Almost half of the 227 investigated denitrifying strains did not render a

nir amplicon with any of five previously described primers. NirK and nirS were found to be

prevalent in Alphaproteobacteria and Betaproteobacteria respectively, nirK was detected

in the Firmicutes and Bacteroidetes, and nirS and nirK were present with equal frequency

in the Gammaproteobacteria. These observations deviated from the hitherto reported

incidence of nir genes in bacterial taxa. NirS gene phylogeny was congruent with the 16S

rRNA gene phylogeny on family or genus level, although some strains did group within

clusters of other bacterial classes. Phylogenetic nirK gene sequence analysis was incongruent

with the 16S rRNA gene phylogeny. NirK sequences were also found to be significantly

more similar to nirK sequences from the same habitat than to nirK sequences retrieved from

highly related taxa. These results support the hypothesis that horizontal gene transfer

events of denitrification genes have occurred and underlines that denitrification genes are

not suitable for assessing denitrifier diversity in the environment.

72

INTRODUCTION

Denitrification is a dissimilatory process in which nitrogenous oxides are used as alternative

electron acceptors for energy production when oxygen is limiting. As part of the global

nitrogen cycle, denitrification is responsible for the return of fixed nitrogen back to the

atmosphere. Although responsible for nutrient loss in agriculture and contribution to the

greenhouse effect and damage to the ozone layer, denitrification is also favourable in nutrient

removal from wastewater and bioremediation [20].

Denitrification is primarily a bacterial respiratory process and consists of four enzymatic

reaction steps, catalysed by four metalloproteins: nitrate reductase, nitrite reductase, nitric-

oxide reductase and nitrous oxide reductase. Nitrite reductase is the key enzyme and forms

the distinguishing feature between denitrifiers and nitrate reducers. The products of two

different nitrite reductase genes, containing either copper (the nirK product) or cytochrome

cd1 (the nirS product), can catalyse the reduction of nitrite to NO [27]. The two NiR types

are functionally and physiologically equivalent, which is indicated by the expression of the

Cu-NiR gene from Pseudomonas aureofaciens in a P. stutzeri mutant lacking the gene

encoding cd1-NiR [7]. It has also been demonstrated that the two reductases are mutually

exclusive in any given strain, although the NiR type may differ within the same genus and

even within the same species [5]. Whereas cd1-NiR reductase is assumed to dominate in

denitrifying bacteria, Cu-NiR shows greater variation in molecular weight and immunological

reactions and is present in more diverse taxa [5,27]. Why different classes of nitrite reductases

have evolved and what advantage one may provide over another is unknown.

Denitrifying bacteria are taxonomically very diverse [12, 20, 27]. Since denitrifiers are not

defined by a close phylogenetic relationship, an approach involving the 16S rRNA gene is

not suitable for general detection of this physiological group in the environment. Therefore,

during the last decade, the cultivation-independent research has focused on the study of the

functional genes nar, nir, nor and nos, coding for the four reductases involved in denitrification

[27]. Nir gene sequence diversity in the environment is sometimes taken as a measure for

the structural diversity of denitrifiers [3, 24], although it remains unclear to what extent –

if at all - nir genes contain phylogenetic information. As a result of the cultivation-

independent nature of most denitrification studies [3, 16, 22, 24, 25], the number of

functional gene sequences from cultivated and identified denitrifiers is limited; most

environmental studies produce partial sequences from unknown – because not cultivated –

bacteria. Several reports on plasmid-borne denitrification and indirect evidence of possible

horizontal gene transfer (HGT) of denitrifying genes [17, 21, 27] suggest that denitrification

CHAPTER 3

73

genes are not suitable for the study of organism diversity of denitrifiers in the environment.

More sequence data from cultivated denitrifiers can lead to an assessment of the level of

phylogenetic information carried by functional denitrification genes.

This paper reports on the culture-dependent study of nir genes. A large set of denitrifying

strains obtained in a previous study [11] was screened for the presence of nirS or nirK and

their prevalence in different taxonomical groups was determined. Nir gene sequence analysis

revealed a high genetic heterogeneity and suggested a significant influence of the environment

on the phylogeny of the nir gene sequence, regardless of the taxonomical position of the

denitrifier.

FUNCTIONAL PHYLOGENETIC ANALYSIS OF PURE CULTURE DENITRIFIERS

74

MATERIAL & METHODS

Bacterial strains and identificationA total number of 227 denitrifying strains were grown aerobically on tryptone soy agar at 28°C; Table 1 gives an

overview of all denitrifying bacteria included. A set of 198 denitrifying isolates from activated sludge was described

and identified previously [11]. Furthermore, eight denitrifying culture collection strains were included as reference:

Cupriavidus necator LMG 1201, Ochrobactrum anthropi LMG 2136, Paracoccus denitrificans LMG 4049,

Alcaligenes faecalis LMG 1229T, Achromobacter denitrificans LMG 1231T, Pseudomonas aeruginosa LMG

1242T, Pseudomonas stutzeri LMG 2243, Virgibacillus halodenitrificans LMG 9818T. An additional set of 21

denitrifying isolates from activated sludge was also included: nine Ochrobactrum sp. (R-24286, R-24289, R-

24290, R-24291, R-24343, R-24448, R-24467, R-24468, R-26465), three Rhizobium sp. (R-24260, R-24333, R-

26467), one Acidovorax sp. (R-24336), three Paracoccus sp. (R-24292, R-24342, R-26466), two Comamonas sp.

(R-24447, R-24451), one Thauera sp. (R-24450), one Pseudomonas sp. (R-24261) and one Pseudoxanthomonas

sp. (R-24339). The 21 strains were assigned at the genus level as described previously [11] by performing fatty acid

methyl ester analysis and 16S rRNA gene sequence analysis.

DNA extraction and nir PCRDNA was extracted from each denitrifying isolate using the guanidium-thiocyanate-EDTA-sarkosyl method

described by Pitcher et al. [15] for fast-growing strains and using alkaline lysis for slow-growing isolates. For

alkaline lysis, one colony was suspended in an eppendorf tube with 20 µl of lysis buffer (2.5 ml 10% SDS; 5 ml 1M

NaOH; 92.5 ml MilliQ water). After 15 min at 95°C, 180 ml MilliQ water was added, the tube was centrifuged for

5 min at 13,000 g and the supernatant was transferred to a new tube. DNA extracts were stored at –20°C until use.

For amplification and sequence analysis of nir genes, five primer sets previously described were used: for the nirK

gene nirK1F-nirK5R [2], F1aCu-R3Cu [10] and Cunir3-Cunir4 [4]; for the nirS gene nirS1F-nirS6R [2] and nir3-

nir4 [23]. Temperature-time profiles and protocols were adopted from the original description.

Nir gene sequence analysisThe PCR-amplified nir gene products were purified using the Nucleofast® 96 PCR system (Millipore). For each

sequence reaction a mixture was made using 3 µl purified and concentrated PCR product, 1 µl of BigDye™

Termination RR mix version 3.1 (Applied Biosystems), 1.5 µl of BigDye™ buffer (5x), 1.5 µl sterile milliQ water

and 3 µl (20 ng/µl) of one of the sequencing primers. The amplification primers were used as sequencing primers.

The temperature-time profile was as follows: 30 cycles of denaturation for 15 s at 96°C, primer annealing for 1 s at

35°C and extension for 4 min at 60°C. The sequencing products were cleaned up as described previously [13].

Sequence analysis of the nir gene was performed using an Applied Biosystems 3100 DNA Sequencer according

to protocols provided by the manufacturer.

Phylogenetic analysisForward and reverse strands of nirS and nirK were assembled with the BioNumerics 4.0 software and were submitted

to a BLAST search [1]. Amino acid sequences were deduced with Emboss Transeq (EMBL-EBI) and aligned with

sequences retrieved from the EMBL database using ClustalX [18]. Phylogenetic analyses were performed with

Treecon [26]. Trees were constructed with the neighbor-joining algorithm using the BLOSUM 62 substitution

matrix. Statistical evaluation of the tree topologies was performed by bootstrap analysis with 1000 resamplings.

Nucleotide sequence accession numbersThe 16S rRNA gene sequences have been submitted to the DDBJ/EMBL/GenBank databases under accession

number AM231040 through AM231060. The nirK gene sequences have been submitted to the DDBJ/EMBL/

GenBank databases under accession number AM230812 through AM230887. The nirS gene sequences have been

submitted to the DDBJ/EMBL/GenBank databases under accession number AM230888 through AM230922.

CHAPTER 3

75

RESULTS

Amplification of nirK and nirS from cultured denitrifiers

The nir genes were studied in a set of 227 denitrifying strains (Table 3.1). Eight

representatives of denitrifiers from Alpha-, Beta-, Gammaproteobacteria and Firmicutes

available in culture collections were included as reference strains. Other denitrifiers were

isolated in a previous cultivation-dependent study of denitrifiers from activated sludge of

a municipal wastewater treatment plant [11]: the bulk of these denitrifying isolates belonged

to the Alpha- and Betaproteobacteria , but also included representatives of

Gammaproteobacteria, Epsilonproteobacteria, Firmicutes and Bacteroidetes. Five previously

described primer sets were applied to amplify the two nir genes. The three primer sets used

to amplify nirK - nirK1F-5R, F1aCu-R3Cu, and Cunir3-4 - and the two primer sets used

to amplify nirS - nirS1F-6R and nir3-4.

Table 3.1 provides an overview of all tested denitrifiers, arranged according to their taxonomic

position, and the results for each primer set. Almost half of all included denitrifiers (109 out

of 227) did not render an amplicon with any of the five primer sets. NirK was detected in

76 denitrifiers. Of all nirK amplicons, 75% were found within the Alphaproteobacteria,

14.5% within the Betaproteobacteria, 5.3% within the Gammaproteobacteria, 3.9% within

the Firmicutes, and 1.3% within the Bacteroidetes. The primer set F1aCu-R3Cu clearly

scored best and provided an amplicon for all nirK sequences detected, even for denitrifiers

belonging to less-known denitrifying genera as Enterococcus, Staphylococcus and

Chryseobacterium. Cunir3-Cunir4 did not render any amplicons. NirS was detected in 42

denitrifiers. Of all nirS amplicons, 26.2% were found within the Alphaproteobacteria,

64.3% in the Betaproteobacteria, and 9.5% in the Gammaproteobacteria. For nirS

amplification, the primer set nirS1F-6R scored best. No pure culture denitrifier was found

carrying both nir genes.

Table 3.1. (Next two pages) An overview of the denitrifying strains arranged according to their taxonomicposition. Reliable species identification is given for reference strains from the LMG culture collection.a +, nir amplicon verified through sequence analysis; -, no amplification or unspecific amplification; ?, ampliconscould not be sequenced succesfully; b [2] c [10]; d [23]; e primer set Cunir3-Cunir4 [4] did not render any ampliconand was therefore not included in this overview.


76

CHAPTER 3

Id

entif

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ion

P

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nir

K a,

e P

rim

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nir

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Str

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, R-2

4638

, R-2

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, R-2

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, R-2

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, R-2

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, R-2

6465

, R-2

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, R-2

6825

, R-2

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,

R-2

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, R-2

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, R-2

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, R-2

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, R-2

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, R-2

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, R-2

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R-2

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, R-2

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, R-2

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, R-2

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-

-

+ +

R-2

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, R-2

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, R-2

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, R-2

6466

, R-2

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, R-2

7041

, R-2

8414

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-

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65, R

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39, R

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-

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43

-

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-

R-2

4292

, R-2

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, R-2

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, R-2

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, R-2

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, R-2

6844

, R-2

6888

, R-2

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,

R-2

6896

, R-2

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, R-2

7047

, R-2

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, R-2

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, R-2

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, R-2

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R-2

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, R-2

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4607

, R-2

4608

, R-2

5074

, R-2

6831

, R-2

6833

, R-2

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, R-2

6837

, R-2

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,

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, R-2

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, R-2

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77


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4660

, R-2

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, R-2

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, R-2

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, R-2

6812

, R-2

6817

, R-2

6829

, R-2

6886

,

R-2

6887

, R-2

6905

, R-2

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78

Sequence analysis of nir genes

Sequence similarity analysis of primary DNA sequences confirmed that PCR products of

the expected sizes from denitrifying strains were indeed nirK or nirS fragments. The applied

amplification and sequence analysis protocols were verified by including two reference

strains, Achromobacter denitrificans and Pseudomonas stutzeri, with a nirK sequence

(accession number AJ224905) and a nirS sequence (accession number X56813) already

published. For both strains, the generated nir sequence gave a perfect match with the

available sequence.

The genetic heterogeneity of nirS and nirK was assessed after pairwise comparison of all

retrieved sequences in this study with each other and with publicly available nir sequences

from cultivated and identified denitrifiers. The nirS sequence similarity ranged from 60.01%

to 100.00%, and the nirK sequence similarity ranged from 64.45% to 100.00%. For nirK, a

100% sequence similarity was found between strains from phylogenetically distinct taxa.

For example, the nirK sequences of Paracoccus sp. R-26822, Ochrobactrum sp. R-25203

belonging to the Alphaproteobacteria and Acidovorax sp. R-25075 belonging to the

Betaproteobacteria were identical.

Pairwise comparison showed that nirK sequences from pure culture denitrifiers isolated

from activated sludge of the same wastewater treatment plant were significantly more

similar (t test, P < 0.01) to each other, with an average identity of 88.2% (68.85 to 100%)

than to nirK sequences from denitrifying strains from culture collections or other studies

(nirK: <x> = 78.1%, range, 64.45 to 91.51%). The same observation could not be deduced

from a comparative analysis of nirS sequences.

Phylogenetic analysis

The phylogenetic analysis was performed on amino acid sequences derived from nirK and

nirS gene sequences. Amino acid sequences of cultured and identified strains from EMBL

were also included; environmental clones were omitted.

For the analysis of nirK, 114 sequences were included from Alphaproteobacteria (78),

Betaproteobacteria (23), Gammaproteobacteria (9), Firmicutes (3), and Bacteroidetes (1).

The tree topology of the nirK gene product showed three separate clusters (Figure 3.1).

The large cluster I contained mostly Alphaproteobacteria, but was interspersed with

sequences from Beta-, Gammaproteobacteria and Firmicutes. Three distinct subclusters

could be observed: subcluster Ia with highly similar sequences belonging to different genera

CHAPTER 3

79

(even from different classes), subcluster Ib with representatives of the Rhizobiaceae grouped

together with high bootstrap values, and subcluster Ic with highly related sequences from

representatives of Paracoccus. Cluster II grouped strains belonging to the Alpha-, Beta-,

Gammaproteobacteria and Firmicutes. All Nitrosomonas strains were situated in cluster

III showing distinct sequences, except for Nitrosomonas sp. TA-921i-NH4, which falls in

cluster II. In between cluster II and III, two Rhodobacter strains grouped separate from

other Alphaproteobacteria with a high bootstrap value.

For the analysis of nirS, 89 sequences were included from Alphaproteobacteria (15),

Betaproteobacteria (46), Gammaproteobacteria (24), Firmicutes (1), Actinobacteria (1)

and Bacteroidetes (2). The phylogenetic tree of the nirS gene product was more congruent

with their organism phylogeny. The tree topology showed four distinct clusters supported

with high bootstrap values (Figure 3.2). Cluster I contained the Betaproteobacteria, divided

over two subclusters. Strains in subcluster Ia mostly belonged to the Rhodocyclaceae,

while Comamonadaceae representatives grouped into subcluster Ib. Other strains of the

Rhodocyclaceae formed a separated cluster III, distinct from the other Betaproteobacteria

with high bootstrap values. Cluster II contained all Alphaproteobacteria, all but one of

which belonged to the genus Paracoccus. The Gammaproteobacteria were spread over

cluster IV and V. Cluster IV grouped all Pseudomonas representatives, while cluster V

contained all Marinobacter representatives. Spread over the tree, a number of sequences

did not follow this clear division in bacterial classes. Cluster I of Betaproteobacteria contained

interspersing sequences from representatives of Pseudomonas, Marinobacter,

Flavobacterium and Kocuria, while cluster IV and V of Gammaproteobacteria also contained

sequences from representatives of Burkholderia, Azoarcus, Bacillus and the family of the

Flavobacteriaceae.


80

Figure 3.1. Phylogenetic analysis of nirK. Unrooted neighbor joining tree was based on partial nirK gene products(114 amino acids; accession numbers between parentheses). Bootstrap values were generated from 1000 replicatesof neighbor joining. Bootstrap values higher than 50% are given. Sequences generated in this study are in bold.

III

Ib

Ia

II

Ic

CHAPTER 3

81

Figure 3.2. Phylogenetic analysis of nirS. Unrooted neighbor joining tree based on partial nirS gene products(253 amino acids; accession numbers between parentheses). Bootstrap values were generated from 1000 replicatesof neighbor joining. Bootstrap values higher than 50% are given. Sequences generated in this study are in bold.

II

III

IV

0.1 subst./site

I

Ib

V


82

DISCUSSION

Our study reports on the incongruence of nirS and nirK phylogenies with 16S rRNA gene

phylogeny. We recommend that functional nir gene sequence diversity should not be used

to estimate the structural denitrifier diversity but only functional gene diversity in the

environment.

NirK sequences did not form delineated taxon-based groups. The tree topology of the nirK

gene product showed that members of different bacterial classes grouped together, with the

exception of nirK sequences of the Rhizobiaceae and Nitrosomonas that did cluster according

to the taxonomic position of the strains, supported by high bootstrap values. The nirK

sequences of nitrifying genus Nitrosomonas, all except for one, grouped separate from

denitrifiers as was reported earlier [4]. The tree topology of the nirS gene product agreed

more with the 16S rRNA gene phylogeny. The distinct nirS clusters of Alpha-, Beta- and

Gammaproteobacteria were supported by high bootstrap values. Even within the large

clusters based on bacterial classes, further division onto family (Rhodocyclaceae and

Comamonadaceae in the Betaproteobacteria) or genus level (Pseudomonas and Marinobacter

in the Gammaproteobacteria) was quite reliable, not taken into account the few sequences

scattered over the tree and grouping within other bacterial classes. Thus, nir genes, especially

nirS, seemed to contain taxonomic information to some extent, but this appeared to be

taxon dependent and can certainly not be generalised in environmental studies.

NirK sequence analysis revealed two important features absent in nirS: (i) nirK sequences

from denitrifiers isolated from activated sludge of a municipal wastewater treatment plant

were significantly different from other publicly available nirK sequences, despite their

taxonomic position, and (ii) identical nirK sequences were retrieved from taxonomically

distinct strains. Both features suggest that nirK sequence similarity is to some degree

dependent on the habitat of the denitrifiers and not only on their phylogenetic affiliation.

Distinct separate clustering of nirK clones from the same environment was reported

previously for marine sediment and soil clones[16] and for clones retrieved from the

rhizosphere of legumes [22]. However, no taxonomic information was available to rule out

the grouping of strains belonging to the same taxon due to the culture-independent nature of

these studies.

Earlier nir gene studies on pure culture denitrifiers reported nir gene phylogeny incongruent

with the 16S rRNA gene phylogeny [4, 14, 23]. Recently, a denitrifying strain was found to

contain two nirS genes, from which one was probably acquired through HGT based on the

phylogenetic analysis [6]. The hypothesis that nir genes possibly were horizontally

CHAPTER 3

83

transferred between different taxa could explain several of our observations, besides aberrant

nir phylogenies. First, not all genes itinerate equally [9]; this could account for the different

degree in divergence of nirS and nirK phylogenies. Although the available data are limited

and possibly ambiguous, they suggest that, if HGT has occurred, nirK would have a higher

propensity for transfer than nirS. Second, not all groups of organisms experience HGT to

the same extent [9]. The Rhizobiaceae family and the genus Nitrosomonas would then be

less susceptible to HGT. Third, adaptation to the environment is one of the factors that

determines the occurrence and frequency of DNA exchange between taxa [8]. The significant

influence of the habitat on the nirK phylogeny could be correlated with the possibility to

exchange mobile DNA between strains within the same habitat. Denitrification genes are

probably advantageous for the studied strains in their original habitat, an activated sludge

system with periodic anoxic and aerobic conditions. However, the nir phylogenies were

based on partial gene sequences and phylogenetic examination of genes can give an indication

but is only ambiguous proof for HGT. Gene duplication followed by gene loss can also give

rise to different gene phylogenies, which are indistinguishable from conflicting phylogenetic

signals of HGT [9]. The indications of HGT presented in this study needs to be substantiated

with data on gene organisation and/or phylogenetic analysis of other denitrification genes.

It is generally accepted that nitrite reductase and nitric oxide reductase are genetically

linked [27] in certain denitrifiers, so supplemental sequence analysis on nor genes could

confirm possible HGT. Moreover, the study of denitrification genes should also focus on

the generation of complete gene sequences, to obtain a more complete picture of the

phylogeny of denitrification genes and possible HGT events.

Based on the available data of the nir genes of cultivated denitrifiers, Philippot [14] stated

that, when possible HGT is not taken into account, nirK mainly belonged to the Alpha- and

Betaproteobacteria and nirS to the Gammaproteobacteria. Our study confirmed the

prevalence of nirK in the Alphaproteobacteria but also found that nirS dominated in the

Betaproteobacteria. Both bacterial classes were well represented in the investigated strain

set and these results could be representative of the incidence of nir genes. Only a few

denitrifiers from the Gamma- and Epsilonproteobacteria, Firmicutes and Bacteroidetes

were included in our study, and therefore the detection of nirK in the Firmicutes and

Bacteroidetes and the equal occurrence of nirS and nirK in the Gammaproteobacteria can

give a wrong impression of nir gene incidence in these taxa. However, the basic idea that

nirS is dominantly present while nirK is present in more taxonomically unrelated strains

seems only partially true because nirK was prevalent in our pure culture study. More

cultivation-dependent studies are needed to broaden the information concerning nir gene

incidence and may confirm a correlation between the type of nir gene (and nar or nor gene)

and the bacterial taxon. This will improve our understanding of why different types of


84

nitrate, nitrite and nitric oxide reductases have evolved and what advantage one may provide

over another.

Three nirK primer sets were used in the here presented work, nirK1F-nirK5R [2], F1aCu-

R3Cu [10], and Cunir3-Cunir4 [4]. All three nirK primer pairs are situated in the more

conserved 3’ region of the target gene. The primer nirK1F targets a region that includes a

three base insertion in some bacteria (located between nucleotides 528 and 529 in the nirK

gene of A. faecalis). This difference corresponds to a single amino acid codon present in

nitrifiers and some rhizobia, and maybe other denitrifiers. More upstream, the Cunir3

primer targets a highly conserved region encoding copper-binding sites. The other forward

primer, F1aCu, targets a more variable region downstream of nirK1F. All three reverse

primers focus on the same region. A re-assessment of these primers indicated F1aCu-R3Cu

as the most suitable primer pair [19], but all three were selected to cover a broad range of

sequence diversity. However, no nirK amplicons were retrieved with Cunir3-Cunir4 when

applied on our strain set, which corresponded with the results of the re-evaluation [19].

This re-assessment did not result in better nirK primers than applied in our study, but did

find more suitable nirS primers than the two sets that were applied here, nirS1F-nirS6R and

nir3-nir4. However, the former primer pair [2] was not re-evaluated [19] with the originally

described touchdown PCR amplification procedure, while the latter pair [23] was not

included in this re-evaluation. Both primer pairs target the 3’ region of the target gene.

Further, this cultivation-dependent study demonstrated the unsuitability of now available

nir primers as broad-range amplification primers. As reported [16], the applied primers

sets for nirS and nirK were developed based on only a few sequences available at the time

and may be biased towards more conserved nir genes. It should be stressed that reliable

primers are a prerequisite for microbial community surveys, since they ultimately determine

what is detected in environmental culture-independent studies. The fallibility of applied

primers in environmental studies should always be kept in mind. More complete nir gene

sequences, which are to date very limited but most needed for primer design, will be

rendered by new and ongoing genome projects. Furthermore, the challenge now is to obtain

the nir gene sequences from those cultivated denitrifiers that do not render an amplicon.

These aberrant sequences are most interesting for future work and for the development of

new primers.

In conclusion, it is becoming apparent that nir gene diversity is not (always) congruent

with 16S rRNA gene phylogeny. Nir gene sequence analysis reveals functional diversity in

pure cultures or environmental studies, but should not be used to deduce the structural

diversity of denitrifying bacteria in culture-independent studies. The hypothesis that

denitrification genes can be horizontally transferred is gathering more indirect evidence.

CHAPTER 3

85

However, substantiation is needed from more in-depth cultivation-dependent studies and

complete gene sequence data of denitrification genes.


86

REFERENCES1. Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ (1990) Basic local alignment search tool. J Mol

Biol 215:403-410


reductase genes (nirK and nirS) to detect denitrifying bacteria in environmental samples. Appl Environ


3. Braker G, Zhou K, Wu L, Devol AH, Tiedje JM (2000) Nitrite reductase genes (nirK and nirS) as functional

markers to investigate diversity of denitrifying bacteria in Pacific Northwest marine sediment communities.


4. Casciotti KL, Ward BB (2001) Dissimilatory nitrite reductase genes from autotrophic ammonia-oxidising


5. Coyne MS, Arunakumari A, Averill BA, Tiedje JM (1989) Immunological identification and distribution of

dissmiliratory heme cd1 and non-heme copper nitrite reductases in denitrifying bacteria. Appl Environ


6. Etchebehere C, Tiedje JM (2005) Presence of two different active nirS nitrite reductases genes in a denitrifying

Thauera sp. from a high nitrate-removal-rate reactor. Appl Environ Microbiol 71:5642-5645

7. Glockner AB, Jüngst A, Zumft WG (1993) Copper-containing nitrite reductases from Pseudomonas

aureofaciens is functional in a mutationally cytochrome cd1-free background (NirS-) of Pseudomonas

stutzeri. Arch Microbiol 160:2136-2141

8. Gogarten JP, Doolittle WF, Lawrence JG (2002) Prokaryotic evolution in light of gene transfer. Mol Biol

Evol 19:2226-2238

9. Gogarten JP, Townsend JP (2005) Horizontal gene transfer, genome evolution and evolution. Nat Microbiol

Rev 3:679-687

10. Hallin S, Lindgren P-E (1999) PCR detection of genes encoding nitrite reductase in denitrifying bacteria.



bacteria: optimization of isolation conditions and diversity study. Appl Environ Microbiol 72: 2637-2643


13. Naser S, Thompson FL, Hoste B, Gevers D, Vandemeulebroecke K, Cleenwerk I, Thompson CC, Vancanneyt

M, Swings J (2005) Phylogeny and identification of Enterococci using atpA gene sequence analysis. J Clin


14. Philippot L (2002) Denitrifying genes in bacterial and Archaeal genomes. Biochim Biophys 1577:355-376

15. Pitcher DG, Saunders LA, Owen NA (1989) Rapid extraction of bacterial genomic DNA with guanidium

thio-cyanate. Lett Appl Microbiol 8:151-156

16. Priemé A, Braker G, Tiedje JM (2002) Diversity of nitrite reductase (nirK and nirS) gene fragments in forested

upland and wetland soils. App. Environ Microbiol 68:1893-1900

17. Rees E, Siddiqui RA, Köster F, Schneider B, Friedrich B (1997) Structural gene (nirS) for the cytochrome cd1

nitrite reductases of Alcaligenes eutrophus H16. Appl Environ Microbiol 63:800-802

18. Thompson JD, Higgins DG, Gibson TJ (1994) CLUSTAL W: improving the sensitivity of progressive

multiple sequence alignment through sequence weighting, position specific gap penalties and weight matrix

choice. Nucleic Acids Res 22:4673-80

19. Throbäck IN, Enwall K, Jarvis Å, Hallin S (2004) Reassessing PCR primers targeting nirS, nirK and nosZ


20. Tiedje JM (1988) Ecology of denitrification and dissimilatory nitrate reduction to ammonium. In

Environmental microbiology of anaerobes, Zehnder AJB (ed.), John Wiley & Sons, New York, pp.179-

244

21. Toffanin A, Wu Q, Maskus M, Casella S, Abruna HD, Shapleigh JP (1996) Characterization of the gene

encoding nitrite reductase and the physiological consequence of its expression in the nondenitrifying

Rhizobium ‘hedysari’ strain HCNT1. Appl Environ Microbiol 62:4010-4025

22. Sharma S, Aneja MK, Mayer J, Munch JC, Schloter M (2005) Diversity of transcripts of nitrite reductases

genes (nirS and nirK) in rhizospheres of grain legumes. Appl Environ Microbiol 71:2001-2007

23. Song B, Ward BB (2003) Nitrite reductases genes in halobenzoate degrading denitrifying bacteria. FEMS

Microbial Ecol 43:349-357

24. Yan T, Fields MW, Wu L, Zu Y, Tiedje JM, Zhou J (2003) Molecular diversity and characterization of nitrite

reductase gene fragments (nirK and nirS) from nitrate- and uranium-contaminated groundwater. Environ

Microbiol 5:13-24

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87

25. Yoshie S, Nado N, Tsuneda S, Hirata A, Inamori Y (2004) Salinity decreases nitrite reductase gene diversity

in denitrifying bacteria of wastewater treatment systems. Appl Environ Microbiol 70:3152-3157

26. Van de Peer Y, De Wachter R (1994) TREECON for Windows: a software package for the construction and

drawing of evolutionary trees for the Microsoft Windows environment. Comput Applic Biosci 10:569-570

27. Zumft WG (1997) Cell biology and molecular basis of denitrification. Microbiol Mol Biol Rev 61:533-616


3.2 NITRIC OXIDE REDUCTASE (NORB) GENESEQUENCE ANALYSIS REVEALS DISCREPANCIESWITH NITRITE REDUCTASE (NIR) GENEPHYLOGENY IN CULTIVATED DENITRIFIERS

Redrafted from: Heylen K, Vanparys B, Gevers D, Wittebolle L, Boon N, De Vos P (2007)

Nitric oxide reductase (norB) gene sequence analysis reveals discrepancies with nitrite

reductase (nir) gene phylogeny in cultivated denitrifiers. Environ Microbiol 9:1072-1077

SUMMARY

Gene sequence analysis of cnorB and qnorB, both encoding nitric oxide reductases, was

performed on pure cultures of denitrifiers, for which previously nir genes were analyzed.

Only 30% of the 227 denitrifying strains rendered a norB amplicon. The cnorB gene was

dominant in Alphaproteobacteria, and dominantly coexisted with the nirK gene, coding for

the copper-containing nitrite reductase. Both norB genes were equally present in

Betaproteobacteria but no linked distributional pattern of nir and norB genes could be

observed. The overall cnorB phylogeny was not congruent with the widely accepted organism

phylogeny based on 16S rRNA gene sequence analysis, with strains from different bacterial

classes having identical cnorB sequences. Denitrifiers and non-denitrifiers could be

distinguished through qnorB gene phylogeny, without further grouping at a higher taxonomic

resolution. Comparison of nir and norB phylogeny revealed that genetic linkage of both

genes is not widespread among denitrifiers. Thus, independent evolution of the genes for

both nitrogen oxide reductases does also occur.

90

INTRODUCTION

Nitric oxide (NO) in excess is toxic for bacteria, fungi, microbial parasites, tumor cells and

viruses, because it can damage DNA, proteins and lipids. The detoxification of NO through

reduction is innate to denitrifiers, which catalyse the sequential dissimilatory reduction of

nitrate to nitrite, and further via NO to N2O and N

2 under oxygen-limited conditions. Non-

denitrifying, mostly pathogenic, strains can also contain a NO reductase, which is

advantageous for the survival in oxygen-limited environments and confers protection against

exogenous and endogenous nitrosative stress [18].

Three nitric oxide reductases have been described to date. The best described is the membrane-

bound dimer NORCB. All bacterial strains found with this nitric oxide reductase, designated

NORB, cNORB or short-chain NOR (scNOR), could perform denitrification [2, 4, 7]. A

second, single-component nitric oxide reductase has an N-terminal extension coding for

quinol as electron donor and two thirds of the catalytic region show homology with cNORB

[8]. This nitric oxide reductase is termed qNORB, NORZ or long-chain NOR (lcNOR).

The qnorB gene has been found to date in both denitrifiers and nondenitrifying strains [5,

9, 18]. A third nitric oxide reductase, purified from Bacillus azotoformans [23], uses

menaquinol or cytochrome c551

as electron acceptor [22]. The genes encoding this nitric

oxide reductase are hitherto unknown.

Since denitrifiers are not a monophyletic group of bacteria [13, 25], cultivation-independent

studies on the denitrifying communities in the environment use the genes targeting the key

enzymes of denitrification, nirS or nirK coding for nitrite reductase [3, 20, 21, 27, 28] and/

or norB coding for nitric oxide reductase [3, 5]. The incongruence between functional gene

and organism phylogeny [4, 11, 17] still makes cultivation-dependent denitrifier diversity

studies necessary because they can shed light on the occurrence and the co-existence of, and

the evolutionary relationship between functional denitrification genes.

This study aimed to broaden the knowledge on the taxonomic distribution of norB genes

and the co-incidence of nir and norB through the study of a set of cultivated and identified

strains. The norB gene phylogeny was also compared to the phylogeny of both 16S rRNA

gene and both nir genes, which was reported previously [11].

CHAPTER 3

91

MATERIALS & METHODS

Bacterial strains and identificationA total number of 227 denitrifying strains were grown aerobically on tryptone soy agar at 28°C; Table 1 gives an

overview of all denitrifying bacteria included. A set of 198 denitrifying isolates from activated sludge were described

and identified previously [12]. Furthermore, eight denitrifying culture collection strains were included as reference:

Ochrobactrum anthropi LMG 2136, Paracoccus denitrificans LMG 4049, Alcaligenes faecalis LMG 1229T,

Achromobacter denitrificans LMG 1231T, Pseudomonas aeruginosa LMG 1242T, Pseudomonas stutzeri LMG

2243, Virgibacillus halodenitrificans LMG 9818T. An additional set of 21 denitrifying isolates from activated

sludge was also included [11]: nine Ochrobactrum sp. (R-24286, R-24289, R-24290, R-24291, R-24343, R-

24448, R-24467, R-24468, R-26465), three Rhizobium sp. (R-24260, R-24333, R-26467), one Acidovorax sp. (R-

24336), three Paracoccus sp. (R-24292, R-24342, R-26466), two Comamonas sp. (R-24447, R-24451), one

Thauera sp. (R-24450), one Pseudomonas sp. (R-24261) and one Pseudoxanthomonas sp. (R-24339).

Identification onto genus level was based on fatty acid methyl ester analysis and 16S rRNA gene sequence analysis.

Reliable species identification is given for reference strains from the BCCM/LMG culture collection.

DNA extraction and nor PCRDNA was extracted from each denitrifying isolate using the guanidium-thiocyanate-EDTA-sarkosyl method

described by Pitcher et al. [19] for fast-growing strains and using alkaline lysis for slow-growing isolates. For

alkaline lysis, one colony was suspended in an eppendorf tube with 20 µl of lysis buffer (2.5 ml 10% SDS; 5 ml 1M

NaOH; 92.5 ml MilliQ water). After 15 min at 95°C, 180 ml MilliQ water was added, the tube was centrifuged for

5 min at 13,000 g and the supernatant was transferred to a new tube. DNA extracts were stored at –20°C until use.

For amplification and sequence analysis of nir genes, two primer sets previously described were used: cnorB2F-

cnorB6R, cnorB2F-cnorB7R and qnorB2F-qnorB7R [4]. Temperature-time profiles and protocols were adopted

from the original description.

Nor gene sequence analysisThe nor amplicons were purified using the Nucleofast® 96 PCR system (Millipore). For each sequence reaction a

mixture was made using 3 µl purified and concentrated PCR product, 1 µl of BigDye™ Termination RR mix version

3.1 (Applied Biosystems), 1.5 µl of BigDye™ buffer (5x), 1.5 µl sterile milliQ water and 3 µl (20 ng/µl) of one of

the sequencing primers. The amplification primers were used as sequencing primers. The temperature-time profile

was as follows: 30 cycles of denaturation for 15 s at 96°C, primer annealing for 1 s at 35°C and extension for 4 min

at 60°C. The sequencing products were cleaned up as described previously [15]. Sequence analysis of the nir gene

was performed using the Applied Biosystems 3100 DNA Sequencer according to protocols provided by the

manufacturer.

Phylogenetic analysisForward and reverse strands of cnorB and qnorB were assembled with the BioNumerics 4.0 software and were

submitted to a BLAST search [1]. Amino acid sequences were deduced with Emboss Transeq (EMBL-EBI) and

aligned with sequences retrieved from the EMBL database using Clustalx [24]. Phylogenetic analyses were performed

with Treecon [26]. Trees were constructed with the neighbor -joining algorithm using the BLOSUM 62 substitution


Nucleotide sequence accession numbersThe cnorB and qnorB gene sequences have been submitted to the DDBJ/EMBL/GenBank databases. Accession

numbers are given in Figure 3.3.


92


Taxonomic distribution of denitrification genes in cultivated denitrifiers

The cnorB and qnorB genes were studied in a set of 227 denitrifying strains (Table 3.2).

One primer pair per gene was used, qnorB2F-7R and cnorB2F-7R [4]. The qnorB primers

target the region homologous with cnorB. The qnorB2F primer has an insertion in qnorB

absent in cnorB. The cnorB7R primer targets a conserved site, coding for two putative

histidine ligands. Other primers were described but they target almost identical regions [6],

and thus were not included in this study.

Thirty percent of all included denitrifiers (69 out of 227) rendered an amplicon for the norB

genes (Table 3.2). The primers were designed with the limited number of norB sequences

available in the public databases at that time [4]. Therefore, this low amplification percentage

of norB genes could be expected. The cnorB gene was detected in 48 denitrifiers (Table 3.2).

Of all cnorB amplicons, 30 were retrieved from isolates assigned to the Alphaproteobacteria

and 18 from isolates assigned to the Betaproteobacteria. The number of cnorB gene sequences

available in public databases and the dominance of cnorB in the pure culture denitrifiers in

this study suggest that the majority of the denitrifiers contain the membrane-anchored

dimer nitric oxide reductase NORCB, as was previously suggested [17]. Nevertheless, the

qnorB gene was detected in 20 cultured denitrifiers of which 15 were found within the

Betaproteobacteria, which extended the current knowledge on the occurrence of this norB

type significantly. Table 3.2 also represents the nir gene results of the same set of isolates

[11]. Both a nir and a norB gene were detected in 48 denitrifying isolates. The observed

coincidence of nir and norB genes suggested the dominant coexistence of nirK and cnorB

within Alphaproteobacterial denitrifiers (28 out of 31), as was previously suggested [17].

No distributional pattern could be observed within the Beta- and Gammaproteobacteria.

CHAPTER 3

93

Phylogeny of cnorB and qnorB gene products from cultivated denitrifiers

The sequence analysis was performed on amino acid sequences deduced from cnorB and

qnorB nucleotide sequences. The different steps of the amplification and sequencing

approach were validated by including a reference strain, Alcaligenes faecalis LMG 1229T,

previously shown to contain a qnorB sequence (accession number AJ507329). The here

obtained qnorB sequence of this strain was identical to sequence available in the public

database.

Phylogenetic analysis of norB genes showed the distinct clustering of cnorB and qnorB

gene products (Figure 3.3). It is difficult to assess the level of phylogenetic information

contained by these functional genes. The overall phylogeny of cnorB genes did not agree

with the widely accepted organism phylogeny based on 16S rRNA gene sequence analysis,

as in subcluster I members of different bacterial classes show a 100% amino acid sequence

similarity for the cnorB gene product (Figure 3.3). Indeed, the cnorB sequences of Acidovorax

sp. R-27044, belonging to the Betaproteobacteria, and Paracoccus sp. R-26897 and R-

28245, belonging to the Alphaproteobacteria, were identical, and the cnorB amino acid

sequence of Pseudomonas R-25208, belonging to the Gammaproteobactera, was identical

to the cnorB sequence of several Ochrobactrum strains (R-24291, R-24653, R-26821, R-

25018, R-24289, R-24618, R-25054, R-27045), belonging to the Alphaproteobacteria.

Also, the qnorB sequences separated denitrifying (subcluster V) from non-denitrifying

strains (subcluster IV), but without an internal grouping of taxa. However, the discrepancy

between norB gene phylogeny and organism phylogeny seems to be taxon-dependent. The

cnorB sequences delineated the Rhodocyclaceae, the Brucellaceae and the genus Paracoccus

quite clearly (Figure 3.3). And the functional group of ammonium-oxidizing bacteria could

also be distinguished from denitrifiers, as was described previously[6].

Table 3.2. (next two pages) An overview of denitrifying isolates arranged according to their taxonomic position.a +, nir/norB amplicon verified through sequence analysis; -, no amplificationb Results described previously [11].


94

CHAPTER 3

Iden

tific

atio

n

nir

K a

,b

nir

S a

,b

cno

rB a

q

no

rB a

S

trai

n n

r.

Alp

hap

rote

obac

teri

a

B

ruce

lla s

p.

+ -

+

- R

-268

95

O

chro

bact

rum

ant

hrop

i +

-

- -

LMG

213

6

O

chro

bact

rum

sp.

+

-

+ -

R-2

4289

, R-2

4291

, R-2

4334

, R-2

4618

, R-2

4638

, R-2

4653

, R-2

5018

, R-2

5054

,

R-2

5203

, R-2

6821

, R-2

6825

, R-2

6826

, R-2

6891

, R-2

6889

, R-2

6894

, R-2

6898

,

R-2

6900

, R-2

7045

, R-2

7046

, R-2

8410

+ -

-

- R

-242

86, R

-242

90, R

-244

48, R

-244

67, R

- 244

68, R

-246

39, R

-250

55, R

-264

65,

R-2

6890

, R-2

6892

M

ethy

loba

cter

ium

sp.

+

-

+ -

R-2

5207

R

hizo

bium

sp.

+

-

+ -

R-2

4333

, R-2

4654

, R-2

4658

, R-2

6467

, R

-268

20

+ -

-

- R

-242

60, R

-246

63, R

-268

35

S

inor

hizo

bium

sp

+ -

+

- R

-250

78

+ -

-

- R

-246

05, R

-250

67

P

arac

occu

s de

nitri

fican

s -

+

- +

LMG

404

9

P

arac

occu

s sp

. +

-

- -

R-2

4342

, R-2

4623

, R-2

4649

, R-2

4650

, R-2

4652

, R-2

5058

, R-2

6822

, R-2

6823

,

R-2

6824

, R-2

6841

, R-2

6899

, R-2

6901

, R-2

7049

, R-2

8242

-

+ +

- R

-268

97, R

-270

41

-

+ -

- R

-246

15, R

-246

16, R

-246

17, R

-246

65, R

-264

66, R

-282

39, R

-282

41, R

-284

14

-

-

+ -

R

-282

44, R

-282

45

-

-

- -

R-2

4292

, R-2

4621

, R-2

5049

, R-2

5059

, R-2

6819

, R-2

6839

, R-2

6844

, R-2

6888

, R-2

6893

,

R-2

6896

, R-2

6902

, R-2

7043

, R-2

7047

, R-2

8237

,R-2

8238

, R-2

8243

, R

-282

94, R

-284

09

P

anno

niba

cter

sp.

-

-

-

-

R

-270

42

Bet

apro

teo

bact

eria

A

lcal

igen

es fa

ecal

is

+ -

- +

LMG

122

9T

A

chro

mob

acte

r de

nitri

fican

s +

-

- +

LMG

123

1T

C

upria

vidu

s ne

cato

r -

+

- +

LMG

120

1

A

cido

vora

x sp

. +

-

- +

R-2

5052

+ -

-

- R

-243

36, R

-246

13, R

-246

14, R

-250

75, R

-250

76

-

+ -

- R

-252

12

-

-

+ -

R-2

4667

, R-2

5074

, R-2

6831

, R-2

6833

, R

-270

44

-

-

- -

R-2

4607

, R-2

4608

, R-2

6834

, R-2

6837

, R-2

8240

, R-2

6842

, R-2

6843

, R-2

8416

A

licyc

liphi

lus

sp.

-

+ -

+ R

-246

04, R

-246

11

-

-

- +

R-2

4606

, R-2

6814

Com

amon

as s

p.

- +

+ -

R-2

8235

-

+ -

- R

-244

47, R

-244

51, R

-250

14, R

-250

48, R

-250

66, R

-282

10, R

-282

24, R

-282

27, R

-282

31,

R-2

8233

, R-2

8234

, R-2

8293

, R-2

8452

-

-

- -

R-2

4660

, R-2

5057

, R-2

5060

, R-2

6810

, R-2

6812

, R-2

6817

, R

-268

29, R

-268

86, R

-268

87,

R-2

6905

, R-2

7040

, R-2

8209

, R-2

8211

, R-2

8218

, R-2

8220

, R-2

8222

, R-2

8223

, R-2

8225

,

R-2

8226

, R-2

8228

, R-2

8229

, R-2

8230

, R-2

8232

, R-2

8236

, R-2

8413

, R-2

8449

95


D

iaph

orob

acte

r sp

. +

-

- +

R-2

4610

, R-2

4612

, R-2

6815

-

-

- +

R-2

4661

, R-2

5011

, R-2

6840

, R-2

8417

-

-

- -

R-2

5012

S

impl

icis

pira

sp.

-

-

-

- R

-284

08

A

zosp

ira s

p.

-

+ -

- R

-284

47

-

-

- -

R-2

5019

A

zovi

brio

sp.

-

-

+

- R

-250

62

D

echl

orom

onas

sp.

-

+

+ -

R-2

8407

-

+ -

- R

-284

00, R

-284

51

-

-

+ -

R-2

8401

-

-

- -

R-2

8215

, R-2

8291

, R-2

8317

, R

-284

12

T

haue

ra s

p.

-

+

+ -

R-2

5071

, R-2

6906

-

+

- -

R-2

4450

, R-2

5069

, R-2

6885

, R-2

8213

-

-

+ -

R-2

8205

, R-2

8207

, R-2

8289

, R-2

8403

, R-2

8404

-

-

- -

R-2

8206

, R-2

8216

, R-2

8217

, R-2

8292

, R-2

8312

, R-2

8402

, R-2

8405

, R-2

8406

, R-2

8448

Z

oogl

oea

sp.

-

-

- -

R-2

8315

Gam

map

rote

ob

acte

ria

P

seud

omon

as a

erug

inos

a -

+

- -

LMG

124

2

P

seud

omon

as s

tutz

eri

-

+ -

- LM

G 2

243

P

seud

omon

as s

p.

+ -

-

+ R

-242

61, R

-268

28

+ -

+

+ R

-252

08

+ -

-

- R

-246

09

-

+ -

- R

-250

61, R

-268

30

-

-

- +

R-2

5209

-

-

- -

R-2

4636

, R-2

5016

, R-2

5343

, R-2

8208

, R-2

8219

P

seud

oxan

thom

onas

sp.

-

-

-

-

R

-243

39

Ep

silo

nb

acte

ria

A

rcob

acte

r sp

. -

-

-

- R

-282

14, R

-283

13, R

-283

14, R

-283

16

Fir

mic

ute

s

V

irgib

acill

us h

alod

enitr

ifica

ns

-

-

-

-

LMG

981

8

B

acill

us s

p.

-

-

-

-

R-2

4620

, R-2

4666

, R-2

8399

T

richo

cocc

us s

p.

-

-

-

-

R-2

8212

E

nter

ococ

cus

sp.

+ -

-

+ R

-246

26

+ -

-

- R

-252

05

P

aeni

baci

llus

sp.

-

-

-

-

R-2

7048

S

taph

yloc

occu

s sp

. +

-

-

-

R-2

5050

Bac

tero

idet

es

C

hrys

eoba

cter

ium

sp.

+

-

-

-

R-2

5053

96

Figure 3.3. Phylogenetic analysis of norB gene products. Unrooted neighbor joining tree was based on partialnorB gene products (145 amino acids). Bootstrap values were generated from 1000 replicates of neighbor joining.•, bootstrap values higher than 70%; bootstrap values lower than 70% are omitted. Strains analyzed in this studyare in bold. The accession numbers are given between parentheses.

0.05 subst./site

Synechocystis sp. PCC 6803 (NC_000911)

Sinorhizobium sp. R-25078 (AM 284344)

Ochrobactrum sp. R-24653 (AM 284364)

Enterococcus sp. R-24626 (AM 284330)


Cupriavidus necator LMG 1201 (AM 284317)

Ochrobactrum sp. R-24289 (AM 284366) Ochrobactrum sp. R-24618 (AM 284367)

Pseudomonas sp. R-25208 (AM 284341) Ochrobactrum sp. R-27045 (AM 284374)

Colwellia psychrerythraea 34H (NC_003910)

Alcaligenes faecalis (AB031072)

Hahella chejuensis KCTC 2396 (NC_007645)


Acidovorax sp. R-25052 (AM 284333)

Brucella sp. R-26895 (AM 284354) Ochrobactrum sp. R-26825 (AM 284350)Ochrobactrum sp. R-26889 (AM 284375)Ochrobactrum sp. R-26891 (AM 284376)

Azoarcus tolulyticus (AJ507361)

Diaphorobacter sp. R-26815 (AM 284336)


Alicycliphilus sp. R-26814 (AM 284335)



Nitrosomonas europaea ATCC 19718 (NC_004757 )

Corynebacterium diphtheriae NCTC 13129 (NC_002935)


Comamonas sp. R-28235 (AM 284361)

Nitrosococcus oceani (AY139085)

Methylobacterium sp. R-25207 (AM 284347)

Agrobacterium tumefaciens str. C58 (NC_003305)


Shewanella sp. W3-18-1 (NZ_AALN01000035)


Nitrosomonas sp. TA-921i-NH4 (AY139087)

Pseudomonas aeruginosa (D38133)

Pseudomonas sp. R-26828 (AM284319)

Rhodopseudomonas palustris CGA009 (NC_005296)


Mannheimia succiniciproducens MBEL55E (NC_006300)

Diaphorobacter sp. R-26840 (AM284320)

Acidovorax sp. R-26831 (AM 284358)

Nitrosomonas marina (AY139084)

Thauera sp. R-28205 (AM 284378)


Azovibrio sp. R-25062 (AM 284370)

Pseudomonas fluorescens (AJ507356)

Achromobacter denitrificans LMG 1231T (AM 284322)

Paracoccus sp. R-27041 (AM 284372)

Roseobacter denitrificans (AB078896)

Blastobacter denitrificans (AJ507352 )

Pseudomonas sp. G-179 (AF083948)

Brucella abortus (NC_006933)

Rhizobium sp. R-24334 (AM 284337)

Ralstonia solanacearum GMI1000 (NC_003296)

Ochrobactrum anthropi (AJ507353) Corynebacterium nephridii (AJ507354)

Rhizobium sp. R-26820 (AM 284373) Rhizobium sp. R-24333 (AM 284365)

Brucella suis (NC_004311) Brucella melitensis (AE009732)

“Achromobacter cycloclastes” (AJ298324)Sinorhizobium meliloti (NC_003037) Rhizobium sp. R-24658 (AM 284340)

Rhizobium sp. R-24654 (AM 284339)

Cupriavidus necator (NC_005241) Bradyrhizobium japonicum USDA 110 (NC_004463)

Rhodobacter sphaeroides (AF000233) Rhodobacter sphaeroides 2.4.1 (NC_007493)

Paracoccus sp. R-28245 (AM 284363)

Paracoccus denitrificans Pd1222 (U28078)

Halomonas halodenitrificans (AB010889) Azospirillum brasilense (AJ507355)

Nitrosomonas sp. NO3W (AY139086 ) Nitrosomonas sp. URW (AY139088)

Acidovorax sp. R-24667 (AM 284368) Acidovorax sp. R-25074 (AM 284371)

Pseudomonas stutzeri (Z28384) Alcaligenes faecalis (AJ507358)

Thauera sp. R-26906 (AM 284377)

Dechloromonas sp. R-28407 (AM 284384)Dechloromonas sp. R-28401 (AM 284381)

Thauera aromatica (AJ507363) Azoarcus sp. EbN1 (NC_006513)

Thauera sp. R-28207 (AM 284380)Thauera sp. R-28404 (AM 284383)

Thauera sp. R-28403 (AM 284382)Thauera sp. R-28289 (AM 284379)

Burkholderia pseudomallei K96243 (NC_006350) Chromobacterium violaceum ATCC 12472 (AE016825)

Neisseria meningitidis Mc58 (NC_003112) Neisseria gonorrhoeae FA 1090 (NC_002946)

Alcaligenes faecalis (AJ507329) Alcaligenes faecalis LMG 1229T (AM 284323)

Pseudomonas sp. R-25208 (AM 284331)Pseudomonas sp. R-25209 (AM 284332)

Alcaligenes sp. DSM 30128 (AJ507330) Achromobacter xylosoxidans (AJ507331)

Alicycliphilus sp. R-24606 (AM 284326)Alicycliphilus sp. R-24604 (AM 284325)

Diaphorobacter sp. R-24610 (AM 284327)Diaphorobacter sp. R-28417 (AM 284321)

cNORB

qNORB

I

II

IV

V

CHAPTER 3

97

Linkage of nir and norB genes

The close clustering of the Alphaproteobacteria for both nirK and cnorB and the reliable

separate grouping of the Rhodocyclaceae and Paracoccus with both nirS and cnorB could

be explained by linkage of both genes, either on the chromosome [14, 16, 29] or on a

plasmid [7]. However, congruence of nir and norB phylogenies cannot be extrapolated to all

denitrifiers, as is demonstrated within the Rhizobiaceae, for which the nirK sequences [11],

but not the cnorB sequences, were closely correlated. Therefore, horizontal transfer of a

nir-norB gene cluster as an explanation for the aberrant functional gene phylogenies [11] is

difficult to substantiate. Denitrification genes dispersed over the chromosome, as is described

in Bradyrhizbium japonicum [14], or plasmides [8], resulting in independent evolutionary

trajectories for nitric oxide reductases and nitrite reductases, as was suggested by Zumft

[30], are therefore most probable for these denitrifiers. Independent evolution of different

denitrification genes is further supported by the high mobility of nir genes [30] and their

strong habitat specificity [2, 11, 17], which is absent for norB genes. Whether or not nir and

norB genes are genetically linked is probably taxon-dependent. However, further research

is necessary due to the lack of efficient primers for either of the investigated denitrification

genes.

The occurrence of cnorB and qnorB in one denitrifier

One Pseudomonas sp. (R-25208) was found to contain both a cnorB and a qnorB gene

(Table 3.2). The qnorB phylogeny matched the organism phylogeny, i.e. clustered together

with qnorB sequences of other Pseudomonas strains. However, the cnorB sequence was a

100% identical to the cnorB sequences of several Ochrobactrum strains. Interestingly, this

unexpected gene product phylogeny matches the nirK gene phylogeny described by Heylen

et al. [11]. Further research needs to assess whether one or more of these denitrification

genes is located on a plasmid and whether these genes are all functional. The presence of

two norB genes, one on the chromosome and one on the plasmid pHG1, was reported for

Cupriviavidus necator H16 (former Ralstonia eutropha) [8]. However, this concerned two

qnorB genes, whereby the plasmid-located gene had 90% sequence similarity with the gene

on the chromosome, and was probably a result of gene duplication event. Recently, two

active nitrite reductase (nirS) genes with different gene phylogenies were found in one

denitrifying Thauera sp. strain [10]. This indicates the possibility of the simultaneous

presence of two phylogenetically diverse functional denitrification genes, active in a single

denitrifier.


98

Conclusions

In conclusion, the norB gene sequence analysis of cultivated and identified denitrifiers

supports the general incongruence of denitrification gene phylogeny with the organism

phylogeny. Comparison with previous nir gene sequence analysis suggests that the presence

or absence of a nir-norB linkage is probably taxon-dependent. Further research should

assess whether these functional genes are located on plasmids or the chromosome.

CHAPTER 3

99

REFERENCES1. Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ (1990) Basic local alignment search tool. J Mol

Biol 215:403-410

2. Bartnikas TB, Tosques IE, Laratta WP, Shi J, Shapleigh JP (1997) Characterization of the nitric oxide

reductase-encoding region in Rhodobacter sphaeroides 2.4.3. J Bacteriol 179:3534:3540

3. Braker G, Zhou K, Wu L, Devol AH, Tiedje JM (2000) Nitrite reductase genes (nirK and nirS) as

functional markers to investigate diversity of denitrifying bacteria in Pacific Northwest marine sediment

communities. Appl Environ Microbiol 66:2096-2104

4. Braker G, Tiedje JM (2003) Nitric oxide reductase (norB) genes from pure cultures and environmental

sample. Appl Environ Microbiol 69:3476-3483


in the nondenitrifying Cyanobacterium Synechocystis sp. strain PCC6803. Appl Environ Mirobiol

68:668-672

6. Casciotti KL, Ward BB (2005) Phylogenetic analysis of nitric oxide reductase gene homologues from

aerobic ammonia-oxidizing bacteria. FEMS Microbiol Ecol 52:197-205

7. Chan Y-K, McCormick W (2004) Experimental evidence of plasmid-borne nor-nir genes in

Sinorhizobium meliloti JJ1c10. Can J Microbiol 50:657-667

8. Cramm R, Siddiqui RA, Friedrich B (1997) Two isofunctional nitric oxide reductases in Alcaligenes

eutrophus H16. J Bacteriol 179:6769-6777

9. Cramm R, Pohlmann A, Friedrich B (1999) Purification and characterisation of the single-component

nitric oxide reductase from Ralstonia eutropha H16. FEBS Lett 460:6-10

10. Etchebehere C, Tiedje JM (2005) Presence of two different active nirS nitrite reductases genes in a

denitrifying Thauera sp. from a high nitrate-removal-rate reactor. Appl Environ Microbiol 71:5642-

5645

11. Heylen K, Gevers D, Vanparys B, Wittebolle L, Geets J, Boon N, De Vos P (2006) The incidence of nirS

and nirK and their genetic heterogeneity in cultivated denitrifiers. Environ Microbiol 8:2012-2021


bacteria: optimization of isolation conditions and diversity study. Appl Environ Microbiol 72:2637-

2643


14. Mesa S, Göttfert M, Bedmar EJ (2001) The nir, nor, and nos denitrification genes are dispersed over

the Bradyrhizobium japonicum chromosome. Arch Microbiol 176:136-142

15. Naser S, Thompson FL, Hoste B, Gevers D, Vandemeulebroecke K, Cleenwerk I, Thompson CC,

Vancanneyt M, Swings J (2005) Phylogeny and identification of Enterococci using atpA gene sequence

analysis. J Clin Microbiol 43:2224-2230

16. Philippot L, Mirleau P, Mazurier S, Siblot S, Hartmann A, Lemanceau P, Germon JC (2001)

Characterisation and transcriptional analysis of Pseudomonas fluorescens denitrifying clusters

containing the nar, nir, nor and nos genes. Biochim Biophys Acta 1517:436-440

17. Philippot L (2002) Denitrifying genes in bacterial and Archaeal genomes. Biochim Biophys 1577:355-

376

18. Philippot L (2005) Denitrification in pathogenic bacteria: for better or worst? Trends Microbiol

13:191-192



20. Priemé A, Braker G, Tiedje JM (2002) Diversity of nitrite reductase (nirK and nirS) gene fragments in

forested upland and wetland soils. Appl Environ Microbiol 68:1893-1900

21. Sharma S, Aneja MK, Mayer J, Munch JC, Schloter M (2005) Diversity of transcripts of nitrite reductases

genes (nirS and nirK) in rhizospheres of grain legumes. Appl Environ Microbiol 71:2001-2007

22. Suharti, Heering HA, de Vries S (2004) NO reductase from Bacillus azotoformans is a bifunctional

enzyme accepting electrons from menaquinol and a specific endogenous membrane-bound cytochrome

c551

. Biochemistry 43:13487-13495



24. Thompson JD, Gibson TJ, Plewniak F, Jeanmougin F, Higgins DG (1997) The ClustalX windows

interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic

Acids Res 24:4876-4882


100


Environmental microbiology of anaerobes, Zehnder AJB (ed.), John Wiley & Sons, New York, pp.

179-244

26. Van de Peer Y, De Wachter R (1994) TREECON for Windows: a software package for the construction

and drawing of evolutionary trees for the Microsoft Windows environment. Comput Applic Biosci

10:569-570

27. Yan T, Fields MW, Wu L, Zu Y, Tiedje JM, Zhou J (2003) Molecular diversity and characterization of

nitrite reductase gene fragments (nirK and nirS) from nitrate- and uranium-contaminated groundwater.


28. Yoshie S, Nado N, Tsuneda S, Hirata A, Inamori Y (2004) Salinity decreases nitrite reductase gene

diversity in denitrifying bacteria of wastewater treatment systems. Appl Environ Microbiol 70:3152-

3157

29. Zumft WG (1997) Cell biology and molecular basis of denitrification. Microbiol. Mol Biol Rev 61:533-

616

30. Zumft W (2005) Nitric oxide reductase of prokaryotes with emphasis on the respiratory, heme-copper

oxidase type. J Inorg Biochem 99:194-215

CHAPTER 3

3.3 FUNCTIONAL GENE STUDY ON HETEROTROPHICDENITRIFIERS ISOLATED FROM SOIL

Redrafted from: Heylen K, Boon N, Verstraete W, De Vos P (2007) Functional

gene study on heterotrophic denitrifiers isolated fom soil. Microb Ecol, Submitted

SUMMARY

The key denitrification genes - that is nirS and nirK, encoding nitrite reductase, and cnorB

and qnorB, encoding nitric oxide reductase - were further investigated in a total of 112

heterotrophic soil denitrifiers. A very low number of the denitrifying isolates yielded a nir

or norB amplicon, partly due to the high percentage of bacilli. Isolates of Sinorhizobium,

Cupriavidus, and Pseudomonas, showing no genetic variation, gave nir or norB sequences

without polymorphisms. Detection of denitrification genes in Ensifer and Rhizobium

appeared to be strain-dependent. Unexpected norB sequences phylogeny was observed for

bacilli. These norB sequences clustered together with sequences from non-denitrifiers, and

were only very distantly related with the few norB sequences available from complete

genome projects of other bacilli. Interestingly, a Bacillus isolate was found containing both

a cnorB and qnorB gene, with only the latter showing the expected phylogeny. These

observations demonstrate the usefulness of the cultivation approach in the study of the

denitrification process and its ecology.

104

INTRODUCTION

Denitrification decreases the application efficiency of agricultural fertilizer, contributes to

global warming through N2O emission, but can also remove excess nitrogen from the

environment, counteracting nitrate pollution of ground- and drinking water [22]. It is

primarily a bacterial respiratory process in which four enzymatic reaction steps are catalyzed

by metalloproteins. True denitrifiers contain a nitrite reductase and a nitric oxide reductase,

because they are responsible for converting fixed into gaseous nitrogen. Nitrite can be

reduced to NO through the action of two different enzymes containing either copper or

cytochrome cd1, encoded by nirK and nirS respectively [47]. Three nitric oxide reductases,

which reduce NO to N2O, have been described to date: (i) a dimeric enzyme, only found in

bacterial strains that perform denitrification [3, 16] and encoded by cnorB, (ii) a monomeric

enzyme found in both denitrifying and non-denitrifying strains [8, 11, 25] and encoded by

qnorB, and (iii) a newly described qCuANOR, purified from Bacillus azotoformans [37],

for which the encoding genes are hitherto unknown.

Most denitrification studies qualitatively or quantitatively detect and analyse functional

genes in a cultivation-independent way. They thereby try to relate the occurrence of these

genes to environmental conditions [2, 21, 45, 46], the presence of other metabolic guilds

[23, 40] or other factors [37]. Unfortunately, this approach does not reveal information on

the denitrifier’s identity, because denitrification genes do not contain reliable taxonomic

information [24]. Also, the denitrifying cell is not collected, making further in depth studies

very difficult. In this context, the cultivation approach provides complementary information

for understanding the ecology of the denitrificationn process.

Here, we investigated the key functional genes from pure culture denitrifiers isolated from

soil. An in-depth discussion of the incongruence between nir and norB gene phylogenies

and organism phylogeny was reported previously [17, 18]. This work focuses on the

sequence heterogeneity of denitrifying strains of the same species or genus, and the

unexpected phylogeny of norB genes within the genus Bacillus. The results again confirmed

the unsuitability of available functional primers to detect all present gene sequence diversity.

CHAPTER 3

105

MATERIALS & METHODS

Biological materialThe isolation of heterotrophic denitrifiers from soil is described in Section 3.2 of this thesis. The majority of the

isolates belonged to Bacillus (71%) and the Rhizobiaceae (16%).

Repetitive element PCRBOX-PCR was performed with primer BoxA1R (5’-CTACGGCAAGGCGACGCTGACG-3’) as described

previously [26]. Gels were normalised and analysed with BioNumerics 4.6. Similarity matrices of densiometric

curves of the gel tracks were calculated with the Pearson’s product-moment correlation coefficient. Cluster analyses

of similarity matrices were performed by unweighted pair group method with arithmetic averages (UPGMA). Two

cluster significance tools were applied to express the stability of the error at each branching level and the overall

quality of the cluster analysis: (i) cluster cut-off method, which delimitates relevant clusters at different similarity

levels and corresponds to the lowest similarity value within the entries of a dendrogram; (ii) the cophenetic

correlation, which allows to distinguish reliable from unreliable subclusters.

Functional PCRs and sequence analysis of nir and norB genesFor amplification of nirS, primer pairs nirS1F-nirS6R [6] and nir3-nir4 [35] were used. For amplification of nirK,

primer pairs nirK1F-nirK5R [6], F1aCu-R3Cu [15] and Cunir3-Cunir4 [10] were used. Primer pairs cnorB2F-

cnorB7R and qnorB2F-qnorB7R were used to amplify cnorB and qnorB respectively [7]. Amplification of all

genes was performed as originally described. Because of low amplification percentages, all functional PCRs were

repeated two times on two different PCR machines, GeneAmp® PCR system 9600 (Applied Biosystems) and

MyCyclerTM thermal cycler (Bio-Rad). Further purification and sequencing protocol was described previously

[17]. The different steps of the amplification and sequencing protocol were validated by including reference strains

containing denitrification genes already published, i.e. Achromobacter denitrificans LMG 1231T for nirK

(accession number AJ224905), Pseudomonas stutzeri LMG 2243 for nirS (accession number X56813) and

Alcaligenes faecalis LMG 1229T for norB (accession number AJ507329).

Phylogenetic analysisForward and reverse strands of nirS and nirK were assembled with the BioNumerics 4.6 software and were submitted

to a BLAST search [1]. Amino acid sequences were deduced with Emboss Transeq (EMBL-EBI) and aligned with

sequences retrieved from the EMBL database using ClustalX [41]. Phylogenetic analyses were performed with

Treecon [43]. Trees were constructed with the neighbor-joining algorithm using the BLOSUM 62 substitution


Nucleotide sequence accession numbersThe sequence data generated in this study have been deposited in Genbank/EMBL/DDBJ. The accession numbers

of the 16S rRNA genes can be found in the supplementary table. The accession numbers of the nirS, nirK, cnorB and

qnorB genes are included in Figure 3.4, 3.5 and 3.6 respectively.


106


Denitrification genes in cultivated heterotrophic soil denitrifiers

All heterotrophic soil denitrifiers were subjected to five different nir PCRs, three for the

nirK gene and two for the nirS gene. Only 19.6% of the denitrifiers yielded a nir amplicon

(14 for nirK and 8 for nirS), and 12.5 % for norB (1 for cnorB and 13 for qnorB). These

results are significantly less than previously reported for both genes using the same primer

set (50% for the nir genes and 30% for the norB genes) [17, 18]. However, these very low

amplification percentages could be explained by the bulk of denitrifying Bacillus isolates in

the strain set. Until recently, almost no DNA sequences for denitrification genes from

bacilli were available in the public sequence databases, although a copper-containing nitrite

reductase was described in Virgibacillus halodenitrificans (previously Bacillus

halodenitrificans) [12] and a nirK and qnorB gene was found in the genome of the

nondenitrifying Geobacillus stearothermophilus (previously Bacillus stearothermophilus).

Therefore, available primers for the denitrification genes were designed based on functional

sequences from only Gram-negative denitrifiers [6, 7, 10, 15, 35]. Nevertheless, norB genes

were found in ten Bacillus isolates, one of which contained both cnorB and qnorB, and in

three Cupriavidus isolates. Suprisingly, no norB amplicons were detected in Pseudomonas

isolates or members of the Rhizobiaceae, although representatives of taxa closely related to

the latter, such as Bradyrhizobium japonicum and Paracoccus denitrificans, were included

in the primer design [7]. All nirK amplicons were found within the isolated

Alphaproteobacterial rhizobia. Primer set Cunir3-4 was included because of its potential

for amplification of a greater diversity [10], but no nirK amplicons were retrieved, as was

also reported previously [17]. NirS amplicons were retrieved from Cupriavidus

(Betaproteobacteria) and Pseudomonas (Gammaproteobacteria) isolates.

These observations again underline the need for broad range primers, covering the whole

sequence diversity. These primers ultimately determine what is detected in functional

microbial community surveys, and several studies have already reported the inability to

amplify nirS genes [34, 45] from the environment. More complete denitrification gene

sequences, which are to date very limited, will be rendered by new and ongoing genome

projects and become available for primer design.

Table 3.3. (opposite page) An overview of denitrifying isolates arranged according to their taxonomic position,with the PCR results for nir and norB genes. Isolation and identification of denitrifiers is described in Section 2.2.

CHAPTER 3

107

Iden

tifi

cati

on

nir

Kn

irS

cno

rBq

no

rBS

trai

n n

r.

Alp

hap

rote

ob

acte

ria

Ens

ifer

sp.

- -

- -

R-3

1757

+ -

- -

R-3

2544

Rhi

zobi

um s

p. -

- -

-R

-317

62, R

-325

39, R

-325

52 +

- -

-R

-315

49, R

-318

37, R

-318

57, R

-327

25S

inor

hizo

biu m

sp.

+ -

-

-R

-317

59, R

-317

63, R

-317

64, R

-318

16, R

-325

42, R

-325

46, R

-325

49, R

-327

3

Bet

apro

teo

bac

teri

aC

upria

vidu

s s

p. -

+ -

+R

-315

42, R

-315

43, R

-315

44

Gam

map

rote

ob

acte

ria

Pse

udom

onas

sp.

- -

- -

R-3

1765

, R-3

2541

, R-3

2726

- +

- -

R-3

1758

, R-3

1761

, R-3

1815

, R-3

1817

, R-3

2553

Fir

mic

ute

sB

acill

u s s

p. -

- -

-R

-327

05, R

-315

46, R

-318

48, R

-318

50, R

-318

55, R

-325

24, R

-325

25, R

-325

29,

RR

-325

74, R

-326

61, R

-326

99, R

-327

78, R

-327

79, R

-327

81, R

-328

48, R

-328

49, R

R-3

2518

, R-3

1554

, R-3

3774

, R-3

2784

, R-3

1852

, R-3

1541

, R-3

2663

, R-3

1849

, R-3

257

R-3

2706

, R-3

1835

, R-3

1769

, R-3

1830

, R-3

2700

, R-3

1547

, R-3

2707

, R-3

2695

, RR

-328

51, R

-318

34, R

-325

34, R

-327

87, R

-325

28, R

-326

96, R

-326

62, R

-325

17, R

R-3

2703

, R-3

2717

, R-3

3819

, R-3

2523

, R-3

1836

, R-3

1838

, R-3

3776

, R-3

2516

, RR

-328

45, R

-327

04, R

-327

89, R

-315

50, R

-318

45, R

-328

44, R

-315

53, R

-315

5 -

-

- +

R-3

1770

, R-3

1841

, R-3

2526

, R-3

2656

, R-3

2702

, R-3

2709

, R-3

2715

, R-3

377

- -

+

+R

-326

94S

taph

yloc

occu

s s

p. -

- -

-R

-337

71, R

-337

77, R

-341

81


108

Phylogeny of nir gene products

Sequence analysis was performed on amino acid sequences deduced from nirK and nirS

nucleotide sequences, after confirmation of the presence of their functional domains.

The isolated representatives of Sinorhizobium, all having a 100% 16S rRNA gene sequence

similarity with the type strain of S. morelense, yielded nirK sequences without any

polymorphisms. Four Rhizobium isolates gave a nirK amplicon, while three did not. Also,

only one of the two Ensifer isolates, both with a 100% 16S rRNA gene sequence similarity

to the type strain of E. medicae, gave a nirK amplicon. The phylogenetic tree of nirK gene

products is represented in Figure 3.4. In contrast to a previous report [17], nirK sequences

of members of the Rhizobiaceae did not form a separate cluster but grouped with nirK

sequences obtained from representatives of Ochrobactrum, Pseudomonas,

Chryseobacterium and Alcaligenes, while the nirK sequence of Bradyrhizobium japonicum

USDA 110 clustered separately.

Figure 3.4. Phylogenetic analysis of nirK gene products. Unrooted neighbor joining tree was based on partialnirK amplicons (130 amino acid positions; accession numbers in parentheses). Bootstrap values were generatedfrom 1000 replicates of neighbor joining. Bootstrap values higher than 70% are given. Sequences retrieved fromthis study are in bold.

0.1 subst./site

Nitrosomonas sp. C-45 (AF339047)

Bradyrhizobium japonicum USDA 110 (NC_004463 )

Sinorhizobium sp. R-32769 ( 778664) AM

Sinorhizobium meliloti 1021 (AE007256)

Ochrobactrum sp. 4FB14 (AY078253)

Alcaligenes faecalis faecalis subsp. LMG 1229 (AM230822)T

Pseudomonas chlororaphis ATCC 13985 (Z21945) T

Ensifer sp. R-32544 ( 403569) AM

Rhizobium sp. R-31837 ( 403566)AM

Sinorhizobium sp. R-31763( 778662) AMSinorhizobium sp. R-31759 ( 403563) AMSinorhizobium sp. R-31816 ( 403565) AMSinorhizobium sp. R-31764 ( 403564) AMSinorhizobium sp. R-32542 ( 403568) AMSinorhizobium sp. R-32737 ( 403572) AMSinorhizobium sp. R-32546(AM778663) Sinorhizobium sp. R-32549 ( 403570) AM

Ensifer sp. 4FB6 (AY078248) Ensifer sp. 2FB8 (AY078247)

Pseudomonas sp. R-25208 (AM230838) Chryseobacterium sp. R-25053 (AM230871)Ochrobactrum anthropi LMG 2136 (AJ224907) Alcaligenes faecalis (D13155)Sinorhizobium sp. R-25067 (AM230840)

Rhizobium sp. R-24663 (AM230832)

Rhizobium sp. R-31857 ( 403567) AMRhizobium sp. R-32725 ( 403571) AM

Rhizobium sp. R-31549 ( 403562)AMRhizobium radiobacter (NC_003305)

Achromobacter denitrificans LMG 1231 T

Achromobacter xylosoxidans NCIMB 11015 (AJ224905)

100

72

94

95

100

71

74

100

100

99

6996

9899

100

100

CHAPTER 3

109


Only eight nirS amplicons were found. All three Cupriavidus isolates, highly related to C.

necator LMG 1201T, and all five Pseudomonas isolates, highly related to P. kilonensis

LMG 1242T, gave a nirS amplicon, in which, for both taxa, no sequence polymorphisms

were observed. The phylogenetic tree of nirS gene products is represented in Figure 3.5.

The three Cupriavidus sequences clustered closely to the NiRS sequence of Cupriavidus

necator. The Pseudomonas sequences clustered closely to NiRS of the type strain of P.

aeruginosa. However, other Pseudomonas sequences were grouped in other clusters with

strains belonging to Betaproteobacteria.

Figure 3.5. Phylogenetic analysis of nirS gene products. Unrooted neighbor joining tree was based on partial nirSamplicons (189 amino acid position; accession numbers in parentheses). Bootstrap values were generated from1000 replicates of neighbor joining. Bootstrap values higher than 70% are given. Sequences retrieved from thisstudy are in bold.

0.1 subst./site

Colwellia psychrerythraea 34H (NC_003910)

Dechloromonas sp. R-28400 (AM230913)

Pseudomonas aeruginosa LMG 1242 (AM230891) TPseudomonas sp. R-31761 ( 403577)AM

Flavobacterium sp. BH12.12 (AJ440497)

Cupriavidus necator LMG 1201 (AM230890) Alicycliphilus sp. R-24611 (AM230896)


Thauera chlorobenzoica 4FB2 (AY078263)

Azoarcus toluvorans Td21 (AY078270)T

Comamonas sp. R-24451 (AM230894)

Cupriavidus sp. R-31542 ( 403573) AM

Pseudomonas sp. R-31815 ( 778666)AM

Pseudomonas sp. R-32553 ( 403578)AMPseudomonas sp. R-31758 ( 403576)AM

Flavobacteriaceae U43 (AJ626844)Marinobacter sp. NBC35 (AJ626837) Paracoccus sp. R-24615 (AM230906)

Paracoccus denitrificans LMG 4049 (AM230889)T

Pseudomonas sp. R-25061Acidovorax sp. R-25212 (AM230905)

Cupriavidus sp. R-31544 ( 403575) AM Cupriavidus sp. R-31543 ( 403574) AM

Thauera linaloolentis 47LolT (AY078265)

Marinobacter sp. NBC31 (AJ626831)Thauera aromatica K172 (AY078256) T

100

85

100

79

100100

99

100

100

100

100

87

100

100

93

110

CHAPTER 3

Cluster analysis of nirK and nirS gene products again confirmed the lack of taxonomic

information contained by these proteins. Repetitive sequence polymorphism analysis was

done for the denitrifying isolates (data not shown) to assess their genetic heterogeneity and

link these results to obtained nir sequence diversity or absence of nir data. This showed

that the Sinorhizobium, Pseudomonas and Cupriavidus isolates all represented one strain

in each taxon, explaining the lack of sequence polymorphisms in nirK and nirS respectively.

For Ensifer and Rhizobium, analysis of repetitive element revealed that the isolates with a

nirK amplicon were genetically different from those without an amplicon; they belonged to

different strains. Thus, not only the process itself [33], but also its detection is strain-

dependent, suggesting significant sequence divergence within these genera, as was previously

reported for the Rhodocyclaceae [35].

111


Phylogeny of nor gene products

Sequence analysis was performed on amino acid sequences deduced from cnorB and qnorB

nucleotide sequences, after confirmation of the presence of heme and iron binding sites.

Amino acid sequences from other cultured and named denitrifiers available in the EMBL

sequence database were also included. The NORB cluster was divided in the qNORB and

the cNORB cluster. The qnorB gene products fell into two distinct clusters, supported

with a bootstrap value of 100%. In the first qnorB gene product cluster, all Bacillus

sequences grouped together with sequences from non-denitrifying strains or strains for

which the denitrification capacity has not been unequivocally determined (Figure 3.6).

Figure 3.6. Phylogenetic analysis of cnorB and qnorB gene products. Unrooted neighbor joining tree was basedon partial norB gene products (138 amino acid positions; accession numbers in parentheses). Bootstrap valueswere generated from 1000 replicates of neighbor joining. Bootstrap values higher than 70% are given. Sequencesretrieved from this study are in bold.

0.1 subst./site

Synechocystis sp. PCC 6803 (NC_000911)

Acidobacterum sp. Ellin345 (YP_589443.1)Anaeromyxobacter dehalogenans 2CP-C (YP_466923.1)

Flavobacterium johnsoniae UW101 (YP_001194759) T

“Mannheimia succiniciproducens” MBEL55E (NC_006300)

Bacillus sp. R-31770 (AM778672)Bacillus sp. R-32715 ( 404295)AM

Alicycliphilus sp. R-24611

Acidovorax sp. R-26833

Nitrosomonas europaea ATCC 19718 (NC_004757) Bradyrhizobium japonicum USDA 110 (NC_004463 )

Cupriavidus necator (AF002661)

Rhodobacter sphaeroides (AF000233)Acidovorax sp. R-27044

Thiobacillus denitrificans ATCC 25259 (YP_314319)


“Achromobacter cycloclastes” (AJ298324)

Bacillus sp. R-32709 (AM778667)Bacillus sp. R-32694 (AM778668)Bacillus sp. R-33820 (AM778669)Bacillus sp. R-32702 (AM778670)Bacillus sp. R-32526 ( 403579)AMBacillus sp. R-31841 (AM778671)

Bacillus sp. R-32656 (AM778673)Bacillus sp. R-33773 ( 778674)AM

“Solibacter usitatus” Ellin6076 (YP_824206.1) Corynebacterium diphtheriae NCTC 13129 (NC_002935)

Neisseria meningitidis MC58 (NC_003112) Neisseria gonorrhoeae Fa1090 (NC_002946)

Alcaligenes faecalis faecalis subsp. LMG 1229T

Paracoccus denitrificans LMG 4049


Dechloromonas sp. R-28401 Thauera aromatica (AJ507363)

Pseudomonas stutzeri (AJ507357)Colwellia psychrerythraea 34H (NC_003910)

Bacillus sp. R-32694 (AM403581)Paracoccus denitrificans (AB014090 )

Corynebacterium nephridii (AJ507354)Ochrobactrum sp. R-24468

cNORB

qNORB

7270

100

85

89

91

100

90

86

91

96

100

100

100

100

100

100100

100

96

94

112

Flavobacterium johnsoniae UW101T is a Gram-negative, strict aerobe present in soil and

freshwater [39]; in the latter environment, it can be pathogenic to fish and cause skin

lesions [9]. As was previously mentioned by Horn et al. [19], there are conflicting

observations on the capacity to reduce nitrate [5, 28, 44], but denitrification of nitrate or

nitrite to N2O or N

2 has not been reported for this strain or this species. Anaeromyxobacter

dehalogenans is a Gram-negative facultative anaerobe found in soil and sediments that can

reduce nitrate to ammonium [32]. The model organism for Cyanobacteria, Synechocystis

sp. PCC6803, and the Gram-positive pathogen Corynebacterium diphtheriae are also known

as non-denitrifiers, although the genome of the latter organism also contains a gene encoding

a protein with homology to NiRK [16]. Acidobacterium sp. Ellin345 and Sollibacter usitatus

Ellin6076 belong to the recently cultivated Acidobacteria lineage [20, 31], for which the

metabolic properties are not yet well described. Rosch et al. [30] suggests that denitrification

is not a likely trait for Acidobacteria. The metabolic pathways reconstructed from their

complete genome sequences by the DOE Joint Genome Institute (currently unpublished)

suggest a putative ammonification pathway. The same is true for Shewanella sp. W3-18-1.

All these non-denitrifying bacteria could generate energy from nitric oxide reductase alone,

or may use this enzyme for detoxification of NO.

Unexpectedly, our amino acid sequences for qnorB from Bacillus strains have very low

similarity (between 44 and 48%) with the highly related putative qnorB amino acid sequences

from other bacilli, Oceanobacillus iheyensis [38], Geobacillus kaustophilus [39], Geobacillus

thermodenitrificans [14], Bacillus licheniformis [29], and Bacillus anthracis [27], determined

and annotated in complete genome projects, which were therefore discarded from the NorB

alignment. We checked the protein domain with blastp and found that these sequences

contained ATP-ase domains instead of the NORB domain, explaining the distant relation to

our norB sequences, which contained a NORB domain. Probably, these genome sequences

were not completely correctly annotated. In addition, little to no sequence polymorphisms

were detected within the qnorB gene products of Bacillus isolates associated with three

different species, B. drentensis, B. soli and B. bataviensis. However, their shared habitat

could be a possible explanation, as plasmid exchange can occur between bacilli [4]. As

mentioned above, the denitrification process in Bacillus is not well-studied and further

biochemical and molecular research is required to clarify the different phylogenetic positions

of NORB sequences from Bacillus and the observed lack of polymorphisms in our Bacillus

isolates.

CHAPTER 3

113

The qnorB gene product sequences of known denitrifiers grouped together in a second

qNORB cluster, also containing the nondenitrifying pathogenic Neisseria meningitidis and

N. gonorrhoeae. Within this cluster, the sequences of all three Cupriavidus isolates clustered

with the already published sequence of C. necator. As mentioned above, because all three

Cupriavidus isolates represent the same strain, both their DNA and AA sequences did not

show any polymorphisms.

Bacillus sp. R-32694 contained both a cnorB and qnorB gene. The qnorB gene product

grouped together with these of the other Bacillus sequences from this study, while the

cnorB gene product clustered closely with sequences from Paracoccus denitrificans and

Acidovorax sp. We made the same observation for a Pseudomonas strain from activated

sludge [18], with a qnorB gene product highly similar to sequences from other pseudomonads

and with a cnorB sequence identical to that from an Ochrobactrum isolate. Further research

needs to assess whether one or more of these denitrification genes is located on a plasmid

and whether these genes are all functional. The presence of two norB genes, located on the

chromosome and on plasmid pHG1, was reported for Cupriviavidus necator H16 (former

Ralstonia eutropha) [11]. However, this concerned two qnorB genes, whereby the plasmid-

located gene had 90% sequence similarity with the gene on the chromosome, and was

probably a result of gene duplication event. Recently, two active nitrite reductase (nirS)

genes with different gene phylogenies were found in one denitrifying Thauera sp. strain

[13]. This indicates the possibility of the simultaneous presence of two phylogenetically

diverse functional denitrification genes, active in a single denitrifier.

Conclusions

Denitrifiers were retrieved from soil through a qualitative isolation procedure and further

investigated for their denitrification genes. Very low amplification percentages were observed,

probably due to the predominance of Bacillus isolates, indicating that the use of these

primers for cultivation-independent research will not detect the whole present diversity.

Sequence polymorphisms were observed between strains of the same genus or species, but

not within strains. New norB sequences of Bacillus had very low similarity with those

from complete genome projects. Moreover, a denitrifier containing two different norB

genes was characterized. All these observations substantiate the value of cultivation-

dependent denitrification research to discover new aspects of the denitrification process,

and its ecology.


114

REFERENCES1. Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ (1990) Basic local alignment search tool. J Mol Biol

215:403-410

2. Avrahami S, Conrad R, Braker G (2002) Effect of soil ammonium concentration on N2O release and on the

community structure of ammonia oxidizers and denitrifiers. Appl Environ Microbiol 68:5685-5692

3. Bartnikas TB, Tosques IE, Laratta WP, Shi J, Shapleigh, JP (1997) Characterization of the nitric oxide reductase-

encoding region in Rhodobacter sphaeroides 2.4.3. J Bacteriol 179:3534:3540

4. Bavykin SG, Lysov YP, Zakhariev V, Kelly JJ, Jackman J, Stahl DA, Cherni A (2004) Use of 16S rRNA, 23S

rRNA, and gyrB gene sequence analysis to determine phylogenetic relationships of Bacillus cereus group

microorganisms. J Clin Microbiol 42:3711-3730

5. Bernardet J-F, Segers P, Vancanneyt M, Berthe F, Kersters K, Vandamme P (1996) Cutting a Gordian knot:

emended classification and description of the genus Flavobacterium, emended description of the family

Flavobacteriaceae, and proposol of Flavobacterium hydatis nom. nov. (basonym, Cytophaha aquatilis

Strohl and Tait 1978). Int J Syst Bacteriol 46:3711-3730


reductase genes (nirK and nirS) to detect denitrifying bacteria in environmental samples. Appl Environ


7. Braker G, Tiedje JM (2003). Nitric oxide reductase (norB) genes from pure cultures and environmental sample.



in the nondenitrifying Cyanobacterium Synechocystis sp. strain PCC6803. Appl Environ Mirobiol 68:668-

672

9. Carson J, Schmidtke LM, Munday BL (1993) Cytophaga johnsonae: a putative skin pathogen of juvenile

farmed barramundi, Lates calcarifer Bloch. J Fish Dis 16:209-218

10. Casciotti KL, Ward BB (2001) Dissmiliatory nitrite reductase genes from autotrophic ammonia-oxidizing


11. Cramm R, Pohlmann A, Friedrich B (1999) Purification and characterisation of the single-component nitric

oxide reductase from Ralstonia eutropha H16. FEBS Lett 460:6-10

12. Denariaz G, Payne WJ, LeGall J (1991) The denitrifying nitrite reductase of Bacillus halodenitrificans.

Biochim Biophys 1056:225-232

13. Etchebehere C, Tiedje JM (2005) Presence of two different active nirS nitrite reductases genes in a denitrifying

Thauera sp. from a high nitrate-removal-rate reactor. Appl Environ Microbiol 71:5642-5645

14. Feng L, Wang W, Cheng J, Ren Y, Zhao G, Gao C, Tang Y, Liu X, Han W, Peng X, Liu R, Wang L (2007) Genome

and proteome of lon-chain alkane degrading Geobacillus thermodenitrificans NG80-2 from a deep-subsurface

oil reservoir. PNAS, in press

15. Hallin S, Lindgren, P-E (1999) PCR detection of genes encoding nitrite reductase in denitrifying bacteria.


16. Hendriks J, Oubrie A, Castresana J, Urbani A, Gemeinhardt S, Saraste M (2000) Nitric oxide reductases in

bacteria. Biochim Biophys Acta 1459:266-273

17. Heylen K, Gevers D, Vanparys B, Wittebolle L, Geets J, Boon N, De Vos P (2006) The incidence of nirS and

nirK and their genetic heterogeneity in cultivated denitrifiers. Environ Microbiol 8:2012-2021

18. Heylen K, Vanparys B, Gevers D, Wittebolle L, Boon N, De Vos P (2007) Nitric oxide reductase (norB) gene

sequence analysis reveals discrepancies with nitrite reductase (nir) gene phylogeny in cultivated denitrifiers.


19. Horn MA, Ihssen J, Matthies C, Schramm A, Acker G, Drake H (2005) Dechloromonas denitrificans sp nov.,

Flavobacterium denitrificans sp. nov., Paenibacillus anaericanus sp. nov. and Paenibacillus terrae strain

MH72, N2O-producing bacteria isolated from the gut of the earthworm Aporrectodea caliginosa. Int J Syst

Evol Microbiol 55:1255:1265

20. Janssen P, Yates PS, Grinton BE, Taylor PM, Sait M (2002) Improved culturability of soil bacteria and

isolation in pure culture of novel members of the divisions Acidobacteria, Actinobacteria, Proteobacteria,

and Verrucomicrobia. Appl Environ Microbiol 68:2391-2396

21. Kandeler E, Deiglmayr K, Tscherko D, Bru D, Philippot L (2006) Abundance of narG, nirS, nirK, and nosZ

genes of denitrifying bacteria during primary successions of a glacier foreland. Appl Environ Microbiol

72:5957-5962


23. Mergel A, Schmitz O, Mallmann T Bothe H (2001) Relative abundance of denitrifying and dinitrogen-fixing

bateria in layers of a forest soil. FEMS Microbiol Ecol 36:33-42

CHAPTER 3

115


376

25. Philippot L (2005) Denitrification in pathogenic bacteria: for better or worst? Trends Microbiol 13:191-

192

26. Rademaker J L W, de Bruijn F J (1997) Characterization and classification of microbes by rep-PCR genomic

fingerprinting and computer assisted pattern analysis. In: Caetano-Anollés G, Gresshoff PM (eds) DNA

Markers: Protocols, Applications and Overviews. J. Wiley and Sons, Inc., New York, pp.1-26

27. Read TD, Peterson SN, Tourasse NJ, Baillie LW (2003) The genome sequence of Bacillus anthracis Ames

and comparison to closely related bacteria. Nature, 423:81-86

28. Reichenbach H (1989) Order I. Cytophagales Leadbetter 1974, 99AL. In: Staley JT, Bryant MP, Pfennig N,

Holt JG (eds) Bergey's Manual of Systematic Bacteriology, vol. 3, Williams & Wilkins, Baltimore, pp. 2011-

2082

29. Rey MW, Ramaiya P, Nelson BA, Brody-Karpin SD, Zareetsky E, Tang M, Lopez de Leon A, Xiang H, Gusti

V, Clausen IG, Olsen PB, Rasmussen MD et al. (2004) Complete genome sequence of the industrial bacterium

Bacillus licheniformis and comparison with closely related Bacillus species. Genome Biology 5:Art. No.

R77

30. Rosch C, Mergel A, Bothe H (2002) Biodiversity of denitrifying and dinitrogen-fixing bacteria in an acid


31. Sait M, Hugenholtz P, Janssen PH (2002) Cultivation of globally distributed soil bacteria from phylogenetic

lineages previously only detected in cultivation-independent surveys. Environ Microbiol 4:654-666

32. Sanford RA, Cole JR, Tiedje JM (2002) Characterization and description of Anaeromyxobacter dehalogenans

gen. nov., sp. nov., an aryl-halorespiring facultative anaerobic Myxobacterium. Appl Environ Microbiol

68:893-900

33. Shapleigh JP (2006) The denitrifying Prokaryotes. In: Dworkin M, Falkow S, Rosenberg E, Schleifer K-H,

Stackebrandt E (Eds), The Prokaryotes, vol. 2, Springer-Verlag, New York, pp. 769-792

34. Sharma S, Aneja MK, Mayer J, Munch JC, Schloter M (2005) Diversity of transcripts of nitrite reductase

genes (nirK and nirS) in rhizospheres of grain legumes. Appl Environ Microbiol 71:2001-2007

35. Song B, Ward BB (2003) Nitrite reductases genes in halobenzoate degrading denitrifying bacteria. FEMS

Microbial Ecol 43:349-357

36. Stanier R Y (1947) Studies on non-fruiting myxobacteria: I. Cytophaga johnsonae , n.sp., a chitin-

decomposing Myxobacterium. J Bacteriol 53: 297-315



38. Takami H, Takaki Y, Uchiyama I (2002) Genome sequence of Oceanobacillus iheyensis isolated from the

Iheya Ridge and its unexpected adaptive capabilities to extreme environments. Nucleic Acids Res 3927-

3935

39. Takami H, Takaki Y, Chee G-J, Nishi S, Shimamura S, Suzuki H, Matsui S, Uchiyama I (2004) Thermoadaptation

trait revealed by the genome sequence of thermophilic Geobacillus kaustophilus. Nucleic Acids Res 32:6292-

6303

40. Taroncher-Oldenburg G, Griner EM, Francis CA, Ward BB (2003) Oligonucleotide microarray for the study

of functional gene diversity in the nitrogen cycle in the environment. Appl Environ Microbiol 69:1159-

1171

41. Thompson JD, Gibson TJ, Plewniak F, Jeanmougin F, Higgins DG (1997) The ClustalX windows interface:

flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Res

24:4876-4882

42. Throbäck IN, Enwall K, Jarvis A, Hallin S (2004) Reassessing PCR primers targeting nirS, nirK and nosZ


43. Van de Peer Y, De Wachter R (1994) TREECON for Windows: a software package for the construction and

drawing of evolutionary trees for the Microsoft Windows environment. Comput Applic Biosci 10:569-570

44. Van der Meulen HJ, Harder W, Veldkamp H (1974) Isolation and characterization of Cytophaga flevensis sp.

nov., a new agarolytic flexibacterium. Antonie van Leeuwenhoek 40:329-346

45. Wolsing M, Priemé A (2004) Observation of high seasonal variation in community structure of denitrifying

bacteria in arable soil receiving artificial fertilizer and cattle manure by determining T-RFLP of nir gene

fragments. FEMS Microbiol Ecol 48:261-271

46. Yoshie S, Noda N, Tsuneda S, Hirata A, Inamori Y (2004) Salinity decreases nitrite reductase gene diversity

in denitrifying bacteria of wastewater treatment systems. Appl Environ Microbiol 70:3152-3157


116

47. Zumft WG (1992) The denitrifying Prokaryotes, In: Balows A, Trüper HG, Dworkin M, Harder W, Schleifer

K-H (Eds.), The Prokaryotes. A handbook on the biology of bacteria: ecophysiology, isolation, identification,

applications, 5nd ed., vol. 1. Springer-Verlag, New York, pp. 554-582

CHAPTER 3

3.4. BACK & FORTH

With the molecular tools at hand, denitrification research of the last decade has focused on the

environmental monitoring of the process. The functional genes were used as functional

markers of the process, but without actual assessment of their information content. The goal

of this chapter was to investigate the functional genes involved in the denitrification process

in a wide diversity of pure culture denitrifiers from two environmental samples. An overview

of all denitrifiers investigated in this thesis, and the detected denitrification genes are listed in

the Addendum. This chapter describes

* the incidence of both known types of nir and nor genes in the different

bacterial classes, and their co-existence;

* the unsuitability of functional genes to assess structural diversity due to

incongruence of their phylogeny with the organism phylogeny;

* the possible linkage of nir and nor in a taxon deduced from their phylogeny;

* the possible significant environmental influence on this phylogeny;

* indirect evidence for HGT;

* the occurence of both cnorB and qnorB in one denitrifier, reported for a

Pseudomonas and Bacillus strain;

* an unexpected phylogeny of Bacillus qnorB genes;

* the failure of now available PCR protocols as broad range detection methods;

* the strain-dependent detection of the functional genes.

All these observations were only possible through cultivation-dependent denitrification

research, thus underlining the continuous need for pure culture research. These results can

have significant influences on the interpretation of environmental monitoring studies.

Furthermore, this study was one of the first large-scale studies investigating functional gene

sequences in a diverse set of pure cultures. However, the conclusions on the incidence and

phylogeny of the denitrification genes will probably alter when more sequence information

becomes available.

To understand the denitrification process better, three topics deserve further attention in

future research: (i) the possible horizontal gene transfer of the denitrification genes, (ii) the

need for more full-gene sequences, and (iii) the failure to detect denitrification genes in

functional denitrifying bacteria. Some preliminary experiments were started on all three

subjects.

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CHAPTER 3

(i) The observed incongruence between organism and functional gene phylogeny, the

high gene sequence similarities among very distinct taxonomic groups, and the strain-

dependent nature of denitrification is best explained by wide spread horizontal gene transfer

events through out the whole prokaryotic kingdom. HGT would also account for the

sporadic records of denitrification as being unstable under laboratory conditions [1, 4]. If

the process were plasmid-borne, it could provide a limited rationale for these observations.

However, plasmid-dependent denitrification has only been substantiated for Cupriavidus

necator (previously Alcaligenes eutrophus) [2], with regulatory genes and a cnorB gene on

a megaplasmid, which is not easily transferred.

Therefore, a selection of seven pure cultures, exhibiting a deviant phylogeny or harboring

two different genes for the same function, were investigated for the presence of plasmids,

namely Pseudomonas sp. R-24609, Pseudomonas sp. R-25208, Pseudomonas sp. R-25061,

Diaphorobacter sp. R-24612, Enterococcus sp. R-25205, Bacillus sp. R-32694, and Bacillus

sp. R-31856. Using a commercially available Qiagen kit (Qiaprep Spin, miniprepKit cat.

no. 2704), a plasmid was retrieved only for Pseudomonas sp. R-25208. The negative result

for the other six strains does not imply that no plasmid is present, only that with the kit

used no plasmid could be found. Especially for the Gram-positive cultures, this result is

not surprising, as the commercial kit was developed for Escherichia coli.

Finding a plasmid specifically in R-25208 was very exciting, because this isolate contained

both a cnorB and a qnorB, as described in Section 3.3 of this thesis. The phylogeny of the

qnorB gene correlated well to its organism phylogeny, i.e. was highly similar to qnorB

genes of other Pseudomonas isolates. On the other hand, its cnorB gene was related to

sequences from Ochrobactrum isolates, as was its nirK gene. Functional PCR on the

extracted plasmid DNA showed that cnorB and nirK are located on the plasmid, making the

HGT of the denitrification capacity from an Ochrobactrum denitrifier to Pseudomonas

isolate R-25208 highly likely. However, these data are preliminary and need to be confirmed

be repeated tests and furter characterization of the plasmid.

(ii) To obtain a more complete picture of the phylogeny of denitrification genes and to

allow development of new molecular tools for their detection and analysis, more complete

gene sequences are needed. New and ongoing genome sequencing projects will provide

these complete sequences in the future. However, these efforts will focus on specific

organisms of interest. As an alternative, we tried to obtain complete nirS and nirK sequences

from our pure cultures, starting from the retrieved partial sequence in gene walking

experiments with “anchored PCR”. The trials were based on a linear amplification protocol,

followed by an adjusted protocol for the Invitrogen 5’ RACE System. The goal was to

amplify the nir regions upstream of the partial amplicon. A reverse primer was developed

121


close to the 5’ end of the amplicon and a linear amplification was performed. After

denaturation, the kit provided means to tail the ss DNA and target this tailed site with a

forward primer. After two nested PCRs, the upstream region of the gene should be amplified.

Unfortunately, several attempts did not result in an amplicon for either gene.

(iii) For the latter subject, further work is discussed in Chapter 4.

Next to the identification of denitrifying bacteria (see Section 2.3 of this thesis), also the

correlation of structural and functional bacterial diversity in the environment may in the

future become less dependent on cultivation studies. A recent paper describes a proof-of-

concept to match phylogeny and metabolism in individual bacteria cells from the environment

[3], using high-speed fluorescence-activated cell sorting, whole-genome multiple displacement

amplification and subsequent PCR screening. So, single amplified genomes are generated,

available for further DNA-based metabolic and phylogenetic analysis or whole-genome

sequencing. A high-throughput application of this approach would provide the genomic

information of bacteria from an environmental sample, and would enable denitrification

(and other) research on a whole new level.

REFERENCES1. Gamble TN, Betlach MR, Tiedje JM (1977) Numerically dominant denitrifying bacteria from world


2. Römermann D, Friedrich B (1985) denitrification by Alcaligenes eutrophus is plasmid dependent. J

Bacteriol 162:852-854

3. Stepanauskas R, Sieracki ME (2007) Matching phylogeny and metabolism in the uncultured marine

bacteria, one cell at a time. PNAS 104:9052-9057

4. Zumft WG (1992) The denitrifying Prokaryotes, In: Balows A, Trüper HG, Dworkin M, Harder W,

Schleifer K-H (Eds.), The Prokaryotes. A handbook on the biology of bacteria: ecophysiology,

isolation, identification, applications, 5nd ed., vol. 1. Springer-Verlag, New York, pp. 554-582

SCREENING FOR DENITRIFICATION GENES

UNDETECTABLE WITH PCR

4

4.1. SIMPLE SCREENING METHOD FOR NORB GENESSUGGESTS EXTRA ENZYMATIC REDUNDANCY FORTHE DENITRIFICATION PROCESS

Redrafted from: Heylen K, Lebbe L, De Vos P (2007) Simple screening method for norB

genes suggests extra enzymatic redundancy for the denitrification process. In preparation

SUMMARY

A previous study found that very few isolates from a large set of pure culture denitrifiers

from activated sludge and soil generated norB amplicons. A dot-blot hybridization protocol

was developed and optimized to detect norB genes in genomic DNA of functional denitrifying

bacteria. Screening of 80 pure culture denitrifiers, mostly belonging to the genera Paracoccus,

Bacillus and Comamonas, showed that low norB amplification percentages could be attributed

to both insufficient primers and PCR protocol, and unknown enzymatic redundancy for

nitric oxide reduction. The dot-blot revealed that denitrifiers from the genus Paracoccus

contained norB genes, although these could not be amplified with the currently available

primers. NorB genes were not detected in most of the included representatives of Bacillus

and Comamonas, using either PCR or the developed dot-blot procedure, which can possibly

be explained by the occurrence of qCuANOR or other new nitric oxide reductases. The

screening of a subset of denitrifiers unable to generate a norB amplicon with a newly

developed norB dot-blot procedure suggests the widespread presence of currently unknown

enzymatic redundancy for nitric oxide reduction.

126

INTRODUCTION

Denitrification is a redundant enzymatic process, reducing nitrate or nitrite over nitric

oxide to nitrous oxide and nitrogen gas. To date, several physiologically and structurally

different enzymes are described that can catalyze the same step in this process, namely

Nar or Nap for nitrate reduction, and NiRS or NiRK for nitrite reduction [23]. For nitric

oxide reduction, two different enzymes are known, a dimeric cNORB (alternatively named

NORB or scNOR) and a monomeric qNORB (alternatively named NORZ or lcNOR),

using c-type cytochrome and quinol respectively as electron donors and encoded by

orthologous genes [2, 27].

Previously, the authors reported on very low success rates of the amplification of cnorB

and qnorB genes in a large set of pure culture denitrifiers from activated sludge [11] and soil

[9]. Insufficiency of currently available primers and PCR protocols was raised as possible

explanation. However, considering the redundant nature of the denitrification process, it is

also possible that unknown NOR enzymes are responsible for the reduction of nitric oxide

in those denitrifiers not rendering a norB gene amplicon. This was thought to be especially

likely for Gram-positive bacteria [17], which lack a periplasmic compartment. Fairly recent,

unknown redundancy was discovered with a new NOR enzyme in Bacillus azotoformans

named qCuANOR [22], using c-type cytochrome and menaquinol as electron donors [21].

Unfortunately, to date the encoding genes are unknown.

Here, we aimed to determine the major explaining factor for the observed low norB

amplification percentage, either the PCR protocols or unknown enzymatic redundancy.

The rationale behind this study is that the use of a gene amplicon allows sequence homology

over a large fraction of the gene, as opposed to amplification for which a high degree of

homology in two 20bp regions is necessary. Thus, hybridization with a partial gene amplicon

could be less specific than amplification and probably detect more divergent sequences.

Therefore, a norB dot-blot hybridization protocol was developed and optimized to detect

the known norB genes in genomic DNA of functional denitrifying bacteria.

CHAPTER 4

127

MATERIAL & METHODS

Bacterial cultures80 pure cultures from activated sludge [12] and soil [9] were previously tested for their denitrifying capacity

through N2O measurements with acetylene inhibition of N

2O reduction. The isolates were reliably identified onto

genus level with fatty acid analysis and 16S rRNA gene sequence analysis. Isolates of Alpha-, Beta, and

Gammaproteobacteria and Firmicutes were included as well strain Virgibacillus halodenitrificans LMG 9818.

These functional denitrifiers did not render a norB amplicon with PCR protocol and primers previously published

[1].

Bacterial strains used for probe production were denitrifiers described by Heylen et al. [12] or obtained from the

BCCM/LMG culture collection. Escherichia coli LMG 2092T, Enterobacter cloacae LMG 2783T, and Klebsiella

pneumoniae subsp. pneumoniae LMG 2095T were used as negative controls.

Genomic target DNAGenomic target DNA was extracted using the guanidium-thiocyanate-EDTA-sarkosyl method described by Pitcher

et al. [18] and diluted to 50 ng/µl. Sheared DNA was prepared through sonication in a Branson 3200 sonication

bath for several lengths of time, from 30 seconds to 30 minutes. The heat ssDNA preparation protocol denaturated

DNA through boiling for 10 minutes and chilling on ice. For the alkali ssDNA protocol, 15µl 0.4 M NaOH, 15 µl

10mM EDTA and 15 µl sterile MilliQ was added to 5µl DNA (50ng/µl). The mixture was boiled for 10 minutes and

chilled on ice, after which 50 µl cold 2 M ammonium acetate (pH7) was added. A total of 250 ng DNA was manually

spotted onto a Hybond-N+ membrane (Amersham Biosciences), without pre-treatment of the membrane. The DNA

was fixed onto the membrane by incubation at 80°C for 2h.

ProbesSix partial gene amplicons were selected for probe use, based on sequence divergence and taxon assignment, after

a previously described phylogenetic analysis of norB genes [11]: Acidovorax sp. R-26831 (AM284358),

Sinorhizobium sp. R-25078 (AM284344) and Ochrobactrum sp. R-28410 (AM284385) for cnorB, and Alcaligenes

faecalis LMG 1229T (AM284323), Achromobacter denitrificans LMG 1231T (AM284322) and Diaphorobacter

sp. R-25011 (AM284334) for qnorB. Probes were prepared via amplification with primers cnorB2F-cnorB7R and

qnorB2F-qnorB7R as described previously [1], purification using the Nucleofast® 96 PCR system (Millipore),

dilution to 10 ng/µl and subsequent guidelines of the ‘ECL direct nucleic acid labelling and detection system’

(Amersham Biosciences). The GC content of the selected probes ranged between 58 and 60%. Individual probes and

combinations of either cnorB probes, qnorB probes or both were used.

Dot-blot hybridizationThe ‘ECL direct nucleic acid labelling and detection system’ (Amersham Biosciences) was used for dot-blot

hybridization. Optimization was performed on three positive and three negative controls, and sterile MilliQ as a

blanc. The hybridization temperature was dependent of the kit and set at 42°C. The hybridization stringency was

altered through a range of NaCl concentration, from 0.1 to 1 M. After hybridization, a primary wash for 10 minutes

at 42°C was repeated three times, followed by a secondary wash for 10 minutes at room temperature repeated two

times. Both post-hybridization washes were performed with different stringencies: from 0.1x to 5x SSC.

Autoradiography film was exposed to the hybridized membrane for 40 min and developed. The norB dot-blot of the

selected denitrifiers was repeated three times, with place re-arrangements to check for edge effects.

Phylogenetic analysis of 16S rRNA gene sequencesDNA extraction, 16S rRNA gene sequence analysis and taxon assignment of Paracoccus isolates were described

previously [10, 12]. Sequences were aligned with 16S rRNA gene sequences of closely related type strains of the

genus retrieved from from the EMBL databse using ClustalX [25]. Phylogenetic analyses were performed with

Treecon [26]. Trees were constructed with the neighbor-joining algorithm. Statistical evaluation of the tree

topologies was performed by bootstrap analysis with 1000 resamplings.

SCREENING FOR DENITRIFICATION GENES UNDETECTABLE WITH PCR

128


Optimization norB dot-blot hybridization

A dot-blot hybridization procedure was developed to screen for the presence of norB in

pure culture denitrifiers. Several dot-blot hybridization conditions were optimized: i) target

DNA preparation, ii) applied probe mixtures, iii) stringency of the hybridization buffer

through the concentration of NaCl, iv) stringency of primary and secondary washes. During

optimization, the denitrifying strains used for probe production were positive controls

(see further), while three non-denitrifying strains were chosen as negative controls, namely

Escherichia coli LMG 2092T, Enterobacter cloacae LMG 2783T, and Klebsiella pneumoniae

subsp. pneumoniae LMG 2095T. Optimal hybridization conditions resulted in clear positive

reactions for all positive controls and simultaneously negative (or very light positive)

reactions for all negative controls.

Both high-molecular genomic DNA and sheared DNA were used as target DNA. Shearing

was thought to ameliorate the target availability in the genomic DNA; however, the results

for both types of target DNA were identical. Six partial gene amplicons were selected for

probe use, three for the cnorB gene and three for the qnorB gene, based on sequence

divergence and taxon assignment. A combined strategy for both genes was chosen based on

their high sequence homology [2, 27]. Both single cnorB and qnorB probes were used, as

also probe mixtures of all three cnorB, all three qnorB and all six norB amplicons. The latter

probe mixture resulted in the best dot-blot reactions for all control strains, with the added

value of only one dot-blot test for both norB genes. The optimal hybridization buffer

contained 1M NaCl, allowing an average mismatch of at least 20% (calculated based on

norB sequences of the probes), necessary to cover the known divergence of norB genes.

Best results were obtained with a primary wash using 5x SSC, followed by secondary wash

using 2x SSC. Although the secondary wash had very low stringency, these results were

most optimal.

CHAPTER 4

129

Detection of norB through dot-blot hybridization

A subset of 80 isolates from activated sludge [12] and soil [9] was selected, for which the

denitrifying capacity was previously demonstrated and no norB genes could be amplified.

The three most represented genera were Bacillus (19 isolates), Comamonas (15 isolates)

and Paracoccus (14 isolates). Also, strain Virgibacillus halodenitrificans LMG 9818 was

included, for which to date no nitric oxide reductase genes have been sequenced. The

optimized dot-blot protocol allowed screening for norB in this subset; a blanc, two positive

and two negative controls were included (Figure 4.1). The results were scored positive after

visual comparison with the negative controls.

Twenty-two denitrifiers (or 27.50%) showed a distinct positive result, indicating the presence

of either cnorB or qnorB, which were previously undetected by PCR. Most included

Paracoccus isolates scored positive, as well as other members of the Alphaproteobacteria,

belonging to the genera Pannonibacter, Ochrobactrum, Rhizobium and Sinorhizobium, and

strains from the Betaproteobacterial Comamonadaeae family (genera Comamonas and

Acidovorax). The results of eleven denitrifiers (or 13.75%) were assessed as doubtful due

to a very light reaction, similar to the faint positive result of K. pneumoniae subsp.

pneumoniae (Figure 4.1). These observations could be attributed to (i) interference of the

norB hybridization with distantly related genes of the heme-copper oxidase superfamily, or

(ii) positive results but suboptimal hybridization conditions. Their positive or negative

character can only be confirmed by genomic sequencing.

All positive-scoring strains belonged to taxa known for their denitrifying capacities. The

detection of these norB genes previously undetected by PCR confirmed the unsuitability of

the available norB primers or PCR protocols [1] for broad-range application. Remarkable is

that, although the primers were designed with the limited norB sequences available at the

time, the positive-scoring strains were relatively closely related to the denitrifiers used for

primer and PCR development, such as Bradyrhizobium japonicum and Paracoccus

denitrificans [1].


130

Figure 4.1. NorB dot-blot hybridization of pure culture denitrifiers as target DNA and partial norB gene ampliconsas probes. Genus assignment and strain number are given in bold for positive results, and underlined for doubtfulresults. A blanc and positive and negative controls were included.

CHAPTER 4

Bac

illu

s sp

. R

-327

06

Bla

nc

Bac

illu

s sp

. R

-327

05A

cido

vora

x sp

. R

-246

13O

chro

bact

rum

sp.

R

-284

10T

haue

ra s

p.

R-2

5069

Tha

uera

sp.

R

-282

16T

haue

ra s

p.

R-2

8213

Com

amon

as s

p.

R-2

8228

Sin

orhi

zobi

um s

p.

R-3

2737

Rhi

zobi

um s

p.

R-3

1549

Com

amon

asR

-284

13

Bac

illu

s sp

. R

-327

79P

seud

omon

as s

p.

R-2

4609

Bac

illu

s sp

. R

-315

47S

inor

hizo

bium

sp.

R

-250

67D

echl

orom

onas

sp.

R

-282

15B

acil

lus

sp.

R-2

6824

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amon

as s

p.

R-2

8226

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zobi

um

sp.

R

-327

25P

arac

occu

s R

-284

09

Dia

phor

obac

ter

sp.

R-2

5011

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illu

s sp

. R

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49P

arac

occu

s sp

. R

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16B

acil

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sp.

R-3

1835

Aci

dovo

rax

sp.

R-2

5075

Dec

hlor

omon

as s

p.

R-2

8291

Com

amon

as s

p.

R-2

8224

Com

amon

as s

p.

R-2

8229

Virg

ibac

illus

ha

lode

nitr

ifica

ns

LM

G 9

818

Pse

udom

onas

sp.

R

-317

58Tr

icho

cocc

uR

-282

12

Esc

heri

chia

col

iL

MG

209

2T

Bac

illu

s sp

. R

-327

81P

arac

occu

s sp

. R

-246

23B

acil

lus

sp.

R-3

1550

Chr

yseo

-ba

cter

ium

sp.

R

-250

53T

haue

ra s

p.

R-2

8292

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amon

as s

p.

R-2

8227

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amon

as s

p.

R-2

8230

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udo-

xant

hom

onas

sp.

R

-243

39Si

norh

izob

ium

sp.

R

-317

59T

haue

ra s

p.R

-284

48

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bsie

lla p

neum

onia

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MG

209

5T

Bac

illu

s sp

. R

-327

89P

arac

occu

s sp

. R

-246

49B

acil

lus

sp.

R-3

1846

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roba

ctru

m s

p.

R-2

5055

Com

amon

as s

p.

R-2

8232

Com

amon

as s

p.

R-2

8231

Com

amon

as s

p.

R-2

8236

Aci

dovo

rax

sp.

R-2

4608

Sino

rhiz

obiu

m s

p.

R-3

1764

Com

amon

asR

-284

49

Och

roba

ctru

m s

p.

R-2

4286

Bac

illu

s sp

. R

-328

45P

arac

occu

s sp

. R

-246

50B

acil

lus

sp.

R-3

1541

Ent

eroo

ccus

sp.

R

-252

05B

acil

lus

sp.

R-3

1830

Par

acoc

cus

sp.

R-2

8241

Aci

dovo

rax

sp.

R-2

8416

Pan

noni

bact

er s

p.

R-2

7042

Sin

orhi

zobi

um s

p.

R-3

1816

Com

amon

as s

p.

R-2

8218

Och

roba

ctru

m s

p.

R-2

4290

Stap

hylo

cocc

us s

p.

R-3

3771

Par

acoc

cus

sp.

R-2

4652

Bac

illu

s sp

. R

-326

63P

arac

occu

s sp

. R

-270

49B

acil

lus

sp.

R-3

2700

Par

acoc

cus

sp.

R-2

8242

Par

acoc

cus

sp.

R-2

7043

Com

amon

as s

p.

R-2

6887

Sin

orhi

zobi

um s

p.

R-3

2542

Com

amon

as s

p.

R-2

8222

Par

acoc

cus

sp.

R-2

4342

Par

acoc

cus

sp.

R-2

4665

Par

acoc

cus

sp.

R-2

6822

Bac

illu

s sp

. R

-317

69T

haue

ra s

p.

R-2

8206

Par

acoc

cus

sp.

R-2

6888

Ens

ifer

sp.

R

-325

44C

omam

onas

sp.

R

-282

25

Pos

itiv

e co

ntro

ls

Neg

ativ

e c

ontr

ols

131

To assess whether strains with high 16S rRNA gene sequence similarity resulted in similar

norB results, a 16S rRNA gene sequence analysis was performed for members of the genus

Paracoccus. Paracoccus strains with a norB amplicon, without a norB amplicon, even after

repeated trails, but with a positive results for the norB dot blot, and without a norB

amplicon and with doubtful results for the dot-blot, were included (Figure 4.2). For Paracoccus

isolates affiliated with P. aminovorans, similar affiliation did not correlate with similar norB

detection results. Isolates for which amplicons were retrieved clustered together with isolates

giving positive and doubtful results with dot-blot screening. Six Paracoccus isolates (R-

28409, R-24623, R-24652, R-24650, R-26822, R-24649, R-28245) with positive dot-blot

results clustered together, as well as two isolates with a norB amplicon. Two isolates

affiliated with P. versutus gave different dot-blot results. Thus, high 16S rRNA gene sequence

similarities (>97%, data not shown) were found between Paracoccus isolates with different

results for norB detection. Thus, the discrepancy in norB detection between PCR and dot-

blot can only be explained by high inter- and intraspecies norB sequence divergence. This

was also concluded by Song and Ward [19], after failure to amplify nir genes in denitrifying

strains closely related to those in which nir genes were detected for Rhodocyclaceae.

The next step should be the retrieval of these norB sequences to assess their divergence

causing failure to amplify these genes and the origin of this divergence. Two strategies could

be followed, either norB dot-blot screening of a genomic clone libray and subsequent

sequencing, or genome sequencing of one or several Paracoccus isolates. Both approaches

can provide extra information on gene organization and regulation, which can be used to

substantiate possible horizontal gene transfer (HGT) [17], which was suggested previously

as cause for the high norB gene sequence divergence [1, 11, 14, 17, 27]. Newly gathered

sequence information could stimulate novel development of amplification protocols, possibly

focusing on group-specific strategies, with grouping based on taxon assignment or gene

phylogeny.


132

Figure 4.2. Phylogenetic dendrogram obtained by neighbour-joining of the 16S rRNA gene sequences ofParacoccus. The norB detection results are given for the denitrifying Paracoccus isolates. EMBL accessionnumbers are shown in parenthesis. Rhodobacter sphaeroides ATCC 17023T was used as outgroup. Bootstrapvalues (expressed as percentages of 1000 replicates) are shown at the branch points.

0.05

sub

stitu

tions

/site

Rho

doba

cter

sph

aero

ides

AT

CC

170

23 (

M55

498)

T

Par

acoc

cus

deni

trif

ican

s A

TC

C 1

7741

(Y

1692

7)

T

Par

acoc

cus

amin

ophi

lus

AT

CC

496

73 (

AY

0141

76)

T

Par

acoc

cus

yeei

CD

C G

1212

(A

Y01

4173

)T

Par

acoc

cus

caro

tinifa

cien

s E

-396

(A

B00

6899

)T

Par

acoc

cus

sp. R

-282

42 (A

M08

4137

)

Par

acoc

cus

amin

ovor

ans

JCM

768

5 (

D32

240)

T

Par

acoc

cus

pant

otro

phus

AT

CC

355

12 (

Y16

933)

T

Par

acoc

cus

vers

utus

AT

CC

253

64 (

AY

0141

74)

T

Par

acoc

cus

sp. R

-268

88 (

AM

0840

61)

Par

acoc

cus

sp. R

-243

42 (

AM

2310

51)

Par

acoc

cus

sp. R

-282

44 (

AM

0841

79)

Par

acoc

cus

sp. R

-282

41 (

AM

0841

63)

Par

acoc

cus

sp. R

-270

41 (A

M08

4095

)

Par

acoc

cus

sp. R

-270

43 (A

M08

4092

)

Par

acoc

cus

sp. R

-284

09 (

AM

0841

34)

Par

acoc

cus

sp. R

-246

23 (

AM

0840

03)

Par

acoc

cus

sp. R

-246

52 (

AM

0839

98)

Par

acoc

cus

sp. R

-246

50 (

AM

0840

45)

Par

acoc

cus

sp. R

-268

22 (

AM

0840

57)

Par

acoc

cus

sp. R

-246

49 (

AM

0840

02)

Par

acoc

cus

sp. R

-282

45 (

AM

0841

78)

Par

acoc

cus

sp. R

-268

97 (

AM

0841

00)

Par

acoc

cus

alca

liphi

lus

AT

CC

511

99 (

AY

0141

77)

T

Par

acoc

cus

sp. R

-246

16 (

AM

0840

41)

Par

acoc

cus

sp.

R-2

7049

(A

M08

4080

)

Par

acoc

cus

sp.

R-2

4665

(A

M08

4107

)

100

83

88

82

91

72

95

85

75 74 95

95

100

62

Dou

btfu

l dot

-blo

t res

ult

norB

det

ecti

on

Pos

itive

dot

-blo

t res

ult

norb

am

plic

on

Pos

itive

dot

-blo

t res

ult

norb

am

plic

on

Dou

btfu

l dot

-blo

t res

ult

Pos

itive

dot

-blo

t res

ult

Pos

itive

dot

-blo

t res

ult

norb

am

plic

on

Pos

itive

dot

-blo

t res

ult

Pos

itive

dot

-blo

t res

ult

Dou

btfu

l dot

-blo

t res

ult

CHAPTER 4

133

Absence of norB genes in functional denitrifiers

Unsuitable norB amplification conditions could only explain the undetectable genes for

nitric oxide reductase in less than a third of the studied denitrifiers. Surprisingly, more than

half of the tested denitrifying pure cultures (47 or 58.75%) did not give any reaction with

PCR or dot-blot, indicating the absence of known norB genes. These negative results were

mostly found in Bacillus and Comamonas. However, also Ochrobactrum and Sinorhizobium

strains gave negative results, genera of which other members gave positive results and were

therefore used for probe production. Again, these observations support high inter- and

intraspecies sequence divergence, possibly caused by HGT.

The absence of a norB gene in bacilli could be explained by the presence of qCuANOR. This

nitric oxide reductase was discovered in Bacillus azotoformans, thus far the only species in

which the enzyme is described [22]. Its occurence in other Bacillus strains is plausible, due

to its membrane-bound nature and the limited available periplasmic-like space. However,

confirmation of this hypothesis will only be possible with the discovery of the encoding

genes. Unfortunately, the denitrification process and the relevant enzymes in Gram-positive

bacteria are not well studied [5, 20], notwithstanding their great importance in the environment

[7, 16] and food production [4, 24].

The absence of norB genes in twelve out of fifteen strains (here the doubtful results is

conservatively considered positive) assigned to the well-known Gram-negative Comamonas

genus was unexpected, because of its well-known denitrifying capacity in wastewater

treatment [6, 8]. Surprisingly, the public sequence databases contain only one sequence for

a nitric oxide reductase from a member of this genus, Comamonas sp. R-28235, as published

previously by the authors [11]. The Comamonas isolates with a negative or doubtful dot-

blot result, and with an norB amplicon are all very highly related, showing more than 99%

16S rRNA gene sequence similarity to Comamonas denitrificans LMG 21602T (data not

shown). Thus, the norB detection results are not correlated with their 16S rRNA gene

sequence phylogeny. The qCuANOR could also be present in these denitrifiers, but its

advantages for denitrifying bacilli are unlikely to hold for Comamonas strains. These results

suggest the existence of one or more other undiscovered nitric oxide reductases, making the

denitrification process even more redundant than previously thought.

First of all, the genes encoding qCuANOR are necessary to develop molecular tools for its

detection and the assessment of its prevalence. Second, also more biochemical research and

whole genome projects are needed to confirm the presence of other unknown nitric oxide

reductases in pure culture denitrifiers. This search can be expedited by the availability of

the here presented pure culture denitrifiers potentially harbouring qCuANOR or other new


134

nitric oxide reductases. Tools for their detection are imperative, as the denitrification process

is monitored cultivation-independently through the study of its functional genes [3, 13,

15]. It could well be that an important subset of the denitrifying guild, containing qCuANOR

or other nitric oxide reductases, is now completely ignored.

Conclusions

A dot-blot protocol for the detection of norB genes in pure culture denitrifiers was developed.

Results showed both inadequate broad-range norB primers/PCR protocol and possible

large-scale occurrence of unknown enzymatic redundancy for nitric oxide reduction. The

developed screening method can be used when the available norB PCRs fail and may

provide insight into the prevalence of known norB genes in different bacterial taxa. The

norB detection results could not be correlated to the taxonomic affiliation of the denitrifiers,

and again demonstrate the strain-dependent nature of the denitrification trait. Hopefully,

this study is a step towards further in-depth investigation of the denitrification process in

diverse pure cultures, to complement the current knowledge from reference denitrifiers, for

which relevant biological material is provided here.

CHAPTER 4

135

REFERENCES1. Braker G, Tiedje JM (2003) Nitric oxide reductase (norB) genes from pure culture and environmental

samples. Appl Environ Microbiol 69:3476-3483

2. Cramm P, Pohlmann A, Friedrich B (1999) Purification and characterization of the single component

nitric oxide reductase from Ralstonia eutropha H16. FEBS Lett 460:6-10

3. Dandie CE, Miller MN, Burton DL, Zebarth BJ, Trevors JT, Goyer C (2007) Nitric oxide reductase-

targeted real-time PCR quantification of denitrifier populations in soil. Appl Environ Microbiol

73:4250-4258

4. De Clerck E, Vanhoutte T, Hebb T, Geerinck J, Devos J, De Vos P (2004) Isolation, characterization and

identification of bacterial contaminants in semifinal gelatine extracts. Appl Environ Microbiol 70:

3664-3672

5. Denariaz G, Payne WJ, LeGall J (1991) The denitrifying nitrite reductase of Bacillus halodenitrificans.

Biochim Biophys 1056: 225-232

6. Etchebehere C, Errazquin MI, Dabert P, Moletta R, Muxi L (2001) Comamonas nitrativorans sp. nov.,

a novel denitrifier isolated from a denitrifying reactor treating landfill leachate. Int J Syst Evol Microbiol,

51:977-983

7. Felske A, Wolterink A, Van Lis R, Akkermans ADL (1998) Phylogeny of the main bacterial 16S rRNA

sequences in Drentse A Grassland soils (The Netherlands). Appl Environ Microbiol, 64:871-879

8. Gumaelius L, Magnusson G, Pettersson B, Dalhammar G (2001) Comamonas denitrificans sp. nov.,

an efficient denitrifying bacterium isolated from activated sludge. Int J Syst Evol Microbiol 51:999-

1006

9. Heylen K, Boon N, Verstraete W, De Vos P (2007) Functional gene study on heterotrophic denitrifiers

isolated fom soil. Microb Ecol, Submitted

10. Heylen K, Gevers D, Vanparys B, Wittebolle L, Geets J, Boon N, De Vos P (2006) The incidence of nirS

and nirK and their genetic heterogeneity in cultivated denitrifiers. Environ Microbiol 8:2012-2021

11. Heylen K, Vanparys B, Gevers D, Wittebolle L, Boon N, De Vos P (2007) Nitric oxide reductase (norB)

genes sequence analysis reveals discrepancies with nitrite reductase (nir) gene phylogeny in cultivated

denitrifiers. Environ Microbiol 9:1072-1077


bacteria: optimization of isolation conditions and diversity study. Appl Environ Microbiol 72: 2637-

2643

13. Horn MA, Drake HL, Schramm A (2006) Nitrous oxide reductase genes (nosZ) of denitrifying microbial

populations in soil and the earthworm gut are phylogenetically similar. Appl Environ Microbiol

72:1019-1026

14. Ichiki H, Tanaka Y, Mochizuki K, Yoshimatsu K, Sakurai T, Fujiwara T (2001) Purification,

characterization and genetic analysis of Cu-containing dissimilatory nitrite reductase from a denitrifying

halophilic Archaeon, Haloarcula marismortui. J Bacteriol 183: 4149-4156

15. Kandeler E, Deiglmayr K, Tscherko D, Bru D, Philippot L (2006) Abundance of narG, nirS, nirK and

nosZ genes of denitrifying bacteria during primary successions of a glacier foreland. Appl Environ


16. Park SJ, Yoon JC, Shin K-S, Kim EH, Yim S, Cho Y-J, Sung GM, Lee D-G, Kim SB, Lee D-U, Woo S-

H, Koopman B (2007) Dominance of endospore-forming bacteria on a rotating activated Bacillus

contactor biofilm for advanced wastewater treatment. J Microbiol 45:113-121

17. Philippot L (2002) Denitrifying genes in bacterial and archaeal genomes. Biochim Biophys Acta

1577:355-376



19. Song B, Ward BB (2003) Nitrite reductases genes in halobenzoate degrading denitrifying bacteria.

FEMS Microbial Ecol 43:349-357

20. Suharti, de Vries S (2005) Membrane-bound denitrification in the Gram-positive bacterium Bacillus

azotoformans. Biochem Soc Transact 33:130-133

21. Suharti, Heering HA, de Vries S (2004) NO reductase from Bacillus azotoformans is a bifunctional

enzyme accepting electrons from menaquinol and a specific endogenous membrane-bound cytochrome

c551. Biochemistry 43:13487-13495

22. Suharti, Strampraad MJ, Schröder T, De Vries S (2001) A novel copper A containing menaquinol NO



136

23. Tavares P, Pereira AS, Moura JJG, Moura I (2006) Metalloenzymes of the denitrification pathway. J

Inorg Biochem 100:2087-2100

24. Ternström A, Lindberg A-M, Molin G (1993) Classification of the spoilage flora of raw and pasteurized

bovine milk, with special reference to Pseudomonas and Bacillus. J Appl Bacteriol 75:25-34



Acids Res 24:4876-4882

26. Van de Peer Y, De Wachter R (1994) TREECON for Windows: a software package for the construction


10:569-570

27. Zumft WG (2005) Nitric oxide reductases of prokaryotes with emphasis on the respiratory, heme-

copper oxidase type. J Inorg Biochem 99:194-215

CHAPTER 4

4.2. BACK & FORTH

The analysis of functional genes in pure culture denitrifiers described in Chapter 3 revealed

the failure of the available molecular tools to detect the denitrification genes in the high

numbers of denitrifiers. Because not many studies culture denitrifiers and analyze their

functional genes, such inadequacy of the frequently used nir and norB primers was not

reported previously. In this chapter, a norB dot-blot hybridization protocol showed that

the low norB amplification percentages were caused by both the unsuitability of the available

primers and PCR protocols, and the probable existence of previously undiscovered

redundancy for denitrification genes.

The latter is a bold statement, when only the here presented data are considered. However,

the existence of an unknown enzymatic redundancy for nitric oxide reduction is supported

by currently unpublished data from the University of Illinois, presented at the 107th General

Meeting of the Amercian Society of Microbiology in Toronto (Hemp & Gennis, unpublished).

The analysis of sequences from bacterial genomes and metagenomic projects identified over

2500 new members of the heme-copper superfamily, which also includes nitric oxide

reductases, from which several new families with unique properties in anaerobic respiration

were identified. They hypothesize that nitric oxide reductases have evolved at least four

times independently, and propose three additional NOR families. Future development of

molecular tools for these new families, as for qCuANOR, will allow the investigation of their

occurrence and importance in the denitrification process.

Similar failure to amplify nir genes in large numbers of the denitrifying isolates from activated

sludge and soil were also described in Chapter 3. Efforts were made to develop a comparable

dot-blot screening method for both nir genes. However, no optimal hybridization conditions

were found, for which the positive controls scored positive and the negative controles

scored negative, without crossreaction between nirS and nirK controls. No obvious reason

was found for these problems, as the GC content of the probes for both gene types was

similar and an identical optimization strategy was used. Because of time limitations, the

trials were stopped without results. However, it is very likely that also the failure to detect

nir genes is caused by a combination of unsuitable primers, although for these genes more

amplification tool are available than for norB genes, and currently unknown nitrite reductases.

DESCRIPTION OF NOVEL BACTERIAL SPECIES

INVOLVED IN THE NITROGEN CYCLE

5

5.1 STENOTROPHOMONAS TERRAE, SP. NOV. ANDSTENOTROPHOMONAS HUMI, SP. NOV., TWONOVELNITRATE-REDUCING STENOTROPHOMONASSPECIES ISOLATED FROM SOIL

Redrafted from: Heylen K, Vanparys B, Peirsegaele F, Lebbe L, De Vos P (2007)

Stenotrophomonas terrae, sp. nov. and Stenotrophomonas humi, sp. nov., two novel nitrate-

reducing Stenotrophomonas species isolated from soil. Int J Syst Evol Microbiol 57: 2056-

2061

SUMMARY

Three Gram-negative, rod-shaped, non-spore-forming, nitrate-reducing isolates were obtained

from soil. Analysis of repetitive sequence-based PCR showed that the three isolates belonged

to two different strains. The 16S rRNA gene sequence analysis and DNA-DNA

hybridization placed them within the genus Stenotrophomonas and showed the genotypic

differentiation from each other and from all other currently known Stenotrophomonas

species. Analysis of the fatty acid composition and physiological and biochemical tests

allowed differentiation from their closest phylogenetic neighbours and they therefore

represent two novel species, for which the names Stenotrophomonas terrae sp. nov. and

Stenotrophomonas humi sp. nov. are proposed, with strains R-32768T (= LMG 23958T =

DSM 18941T) and R-32729T (= LMG 23959T = DSM 18929T) respectively as type strains.

144

In the large-scale phenotypic study of Stanier et al. [18], Pseudomonas maltophilia was

regarded as an authentic member of the genus Pseudomonas. Subsequent allocation of the

species within the genus Xanthomonas as X. maltophilia was supported by rRNA

hybridization data [19], but finally resulted, already more than 10 years ago, in a

reclassification at the genus level into Stenotrophomonas Palleroni and Bradbury [14],

differentiating it by various taxonomic methods from both Pseudomonas and Xanthomonas.

At present, the genus Stenotrophomonas comprises six validly described species,

Stenotrophomonas maltophilia [14], Stenotrophomonas nitritireducens [7],

Stenotrophomonas acidaminiphila [1], Stenotrophomonas rhizophila [24],

Stenotrophomonas koreensis [25], and Stenotrophomonas dokdonensis [27].

A cultivation-dependent study on soil used selective isolation conditions to focus on the

microbial diversity involved in nitrogen removal. Isolates were screened for the removal of

nitrate and nitrite. Most nitrate reducers dominantly belonged to the Gammaproteobacteria,

based on fatty acid analysis. Seven isolates, retrieved from the defined isolation medium

G3M12 - with ethanol as carbon source and nitrate as nitrogen source [9] - could be

assigned to Stenotrophomonas. Three of these Stenotrophomonas isolates, R-32746, R-

32768T, and R-32729T, could possibly belong to new species, based on partial 16S rRNA

gene sequence similarities, and were analyzed further in a polyphasic study, in which they

were not, with any technique, identified as S. maltophilia. The type strains of all

Stenotrophomonas species, except for the phylogenetically most distant S. dokdonensis

(Figure 5.1), were re-examined for phenotyping, chemotaxonomy and biochemical analysis

to guarantee comparable data. In addition, S. maltophilia LMG 22072, which was the

proposed type strain of Stenotrophomonas africana was included [5] (Stenotrophomonas

africana was found to be a later heterotypic synonym of Stenotrophomonas maltophilia

[3].

In order to avoid studying duplicate isolates of the same strain, genotyping by Random

Amplified Polymorphism DNA PCR analysis [2] and repetitive sequence-based PCR

analysis with REP and BOX primers [10] was carried out. The three fingerprint methods

generated identical patterns for isolates R-32746 and R-32768T, but showed clear genetic

differences for isolate R-32729T (data not shown). Since R-32746 and R-32768T are most

likely members of a single strain, only R-32768T was subjected to further study. The DNA

G+C content of R-32768T and R-32729T, determined once by HPLC [13] was 65 mol% and

CHAPTER 5

145

64 mol%, respectively. The nearly complete 16S rRNA gene sequences of R-32768T and R-

32729T were determined as described previously [21]. Phylogenetic analysis was performed

using TreeCon [21] and the BioNumerics software version 4.6 after multiple alignment

with ClustalX [20]. Cluster analysis using the neighbour-joining algorithm with or without

corrections for evolutionary distances as described by Jukes & Cantor [11] and Kimura

[12] were in agreement with maximum-parsimony and maximum-likelihood methods. Strains

R-32729T and R-32768T clustered together with S. nitritireducens LMG 22074T and S.

acidaminiphila LMG 22073T (Figure 5.1). Therefore, DNA-DNA hybridization experiments

were performed within this cluster, using a modification of the microplate method of Ezaki

et al. [6] as described by Willems et al. [23]. A hybridization temperature of 45°C (calculated

with correction for the presence of 50% formamide) was used. R-32768T and R-32729T

showed DNA-DNA relatedness of 44.2% (± 2.8) with each other. R-32768T showed a

DNA-DNA relatedness of 41.3% (± 7) and 35.8% (± 4.7) with S. nitritireducens LMG

22074T and S. acidaminiphila LMG 22073T, respectively. R-32729T showed a DNA-DNA

relatedness of 37.2% (± 6.6) and 38.1% (± 5.1) with S. nitritireducens LMG 22074T and S.

acidaminiphila LMG 22073T, respectively. These results confirmed that R-32729T and R-

32768T belong to two novel genotypic species.

Figure 5.1. Unrooted phylogenetic dendrogram of the 16S rRNA gene sequences (1462 bp) shows the position ofR-32729T and R-32768T among the type strains of Stenotrophomonas species. Neighbour joining analysis wasperformed without corrections. Relevant bootstrap values (expressed as percentages of 1000 replicates) are shownat the branch points. EMBL accession numbers are shown in parenthesis.

Cell morphology, motility and possible sporulation was investigated by phase contrast

microscopy at a magnification of 1000x with cells grown on tryptone soya agar (TSA;

0.02 subst./site

Stenotrophomonas dokdonensis CIP 108839 (DQ178977)T

Stenotrophomonas rhizophila CCUG 47042 (AJ293463)T

Stenotrophomonas koreensis LMG 23369 (AB166885)T

Stenotrophomonas acidaminiphila LMG 22073 (AF273080)T

Stenotrophomonas humi sp. nov. R-32729 (AM403587)T

Stenotrophomonas nitritireducens LMG 22074 (AJ012229)T

Stenotrophomonas terrae sp. nov. R-32768 (AM403589)T

Stenotrophomonas maltophilia LMG 958 (AB008509)T

Stenotrophomonas maltophilia LMG 22072 (U62646)100

97

87

71

100

Description of novel bacterial species involved in the nitrogen cycle

146

Oxoid) for 48h at 28°C. Cells were Gram stained and examined for catalase and oxidase

activity. Utilization of carbon sources and enzyme production was tested with API 20NE

(48h, 28°C), API ZYM (4h, 28°C) and API 50CH (inoculated with AUX medium, 48h,

28°C) (BioMérieux) according to the manufacturer’s instruction. Strains R-32729T and R-

32768T were not identified as S. maltophilia with these conventional phenotypic taxonomic

tests. The temperature range (at 4, 15, 28, 37, and 52°C), the pH range (4.5 to 10.5 at 28°C)

and the salt range (0.5 to 5% at 28°C) for growth were recorded after incubation for 72h on

TSA. The ability to reduce nitrate was tested, as described by Smibert & Krieg [17], after

growth for two weeks on tryptone soya broth (TSB; Oxoid) supplemented with 10 mM

potassium nitrate at 37°C and on liquid G3M11 medium containing 18mM potassium

nitrate and 22.5mM ethanol [9] at 20°C - the solid variant of this medium was used as

isolation medium for the Stenotrophomonas strains. For all strains, these results were in

agreement with the nitrate reduction test in API 20NE. Lipolytic activity was tested by the

hydrolysis of Tween 80, as described by Sierra [16]. The phenotypic and biochemical

characteristics of all strains are given in Table 5.1.

Cells of all strains were identically incubated for exactly 24h at 28°C on TSA. A loopful of

cells was harvested, fatty acid methyl esters were prepared and extracted according to the

standardized protocol of the Microbial Identification System (MIS; Microbial ID Inc.).

The MIDI with the TSBA50 database was used for identification. All Stenotrophomonas

strains contained the characteristic fatty acids iso-C11:0

, isoC11:0

3OH and iso-C13:0

3OH [1,

26], and iso-C15:0

as dominant fatty acid. R-32768T and R-32729T showed highly similar

fatty acid profiles, only differing in the amount of specific fatty acids present, and mostly

containing iso-branched fatty acids. Characteristic fatty acids for R-32768T and R-32729T

were iso-C14:0

(14.2 & 15.7%), iso-C15:1

(4.6 & 2%), iso-C16:0

(8 &12.7%) and iso-C17:1

ω9c

(7.2 & 4.6%). Numerical analysis of the fatty acid profiles (Figure 5.2) showed that R-

32768T and R-32729T formed a distinct cluster, supported with a high cophenetic correlation,

and grouping closely with their phylogenetic nearest neighbours S. nitritireducens LMG

22074T and S. acidaminophila LMG 22073T. The MIDI fatty acid identification system

showed no relevant matches for strains R-32768T and R-32729T. However, the position of

these strains within the group Stenotrophomonas-Xanthomonas was further confirmed by

comparing the fatty acid profiles qualitatively and quantitatively with an in-house database

containing over 60000 fatty acid profiles. The failure of the MIDI identification system to

allocate these strains within the genus Stenotrophomonas was also observed for several

highly similar profiles of Xanthomonas members (unpublished data) and reported previously

for Stenotrophomonas acidaminiphila by Assih et al. [1].

CHAPTER 5

147

Table 5.1. Physiologic characteristics of the novel species and their closest phylogenetic neighbours.

Strains: 1, S. terrae R-32768T; 2, S. humi R-32729T; 3, S. nitritireducens LMG 22074T; 4, S. acidaminiphila LMG

22073T; 5, S. koreensis LMG 23369T; 6, S. maltophilia LMG 958T; 7, S. maltophilia LMG 22072; 8, S. rhizophila

CCUG 47042T. The type strain of S. dokdonensis is not included. Data are from this study unless indicated. +,

Positive; w, weakly positive; -, negative; v, variable.*, LMG 22073T reduces nitrate when grown on TSA supplemented with 10mM nitrate, but does not reduce nitrate

when grown on defined growth medium G3M11; §, LMG 22074T cannot denitrify starting from nitrate, only from

nitrite; †, data from [1]; ‡, data from [5]; #, data from [7]; $, data from [24]; °, data from [27].

All strains characterized in this study were positive for catalase, alkaline phosphatase, esterase, esterase lipase,

trypsin, acid phosphatase, glucose fermentation. All strains characterized in this study were negative for lipase,

cystine arylamidase, chymotrypsin, α-galactosidase, β-galactosidase, β-glucuronidase, α-glucosidase, α-

mannosidase, α-fucosidase, indole production, arginine dihydrolase, urease, β-galactosidase, assimilation of caprate,

adipate, phenyl-acetate, glycerol, erythritol, D-arabinose, L-arabinose, D-ribose, L-xylose, D-adonitol, methyl-

βD-xylopyranisode, D-galactose, L-sorbose, L-rhamnose, dulcitol, inositol, D-mannitol, D-sorbitol, methyl-αD-

mannopyranoside, methyl-αD-glucopyranoside, inulin, D-melezitose, D-raffinose, starch, xylitol, D-lyxose, D-

tagatose, D-fucose, L-fucose, D-arabitol, L-arabitol, potassium gluconate, potassium 2-ketogluconate, potassium

5-ketogluconate.

Characteristic 1 2 3 4 5 6 7 8 Enzymatic activities hydrolysis of Tween 80 (lipolytic activity) + - -,+# -,+† + + - + nitrate reduction + + - v* - + - + denitrification - - v§ -, +† - - - - oxidase - - + + -, +° - - + leucine arylamidase + - - - - - + + valine arylamidase - - - - - - + + Naphthol-AS-BI-phosphohydrolase + + + + - + + + b-glucosidase - - - - - w + + N-acetyl-b-glucosaminidase - + + + - - - - b-glucosidase (aesculin hydrolysis) w - - + - + + + protease (gelatin hydrolysis) + w - - + + + + Assimilation of D-cellobiose - - - - - + + + D-fructose + + + + - + + + gentiobiose - - - - - + + -,+$ D-glucose + - - + - + + + D-lactose - - - - - -, +‡ w, +‡ - D-maltose + + - + - + + + D-mannose + + - + - + + + D-melibiose - - - - - - + - D-saccharose (sucrose) - - - - - + + - D-trehalose - - - - - -, +‡ + -,+$ D-turanose - - - - - - + -,+$ D-xylose - - - - - - - -,+$ citrate + + + - - + + + malate - + - - - + + + N-acetyl-glucosamine + + + + - + + + inositol w - - - - - - - N-acetylglucosamine + + + + - + + + amygdalin - - - - - + v - arbutin - - - - - + + - esculin - - - - - + + + salicin - - - - - + - - glycogen - - - - - - - -,+$

DESCRIPTION OF NOVEL BACTERIAL SPECIES INVOLVED IN THE NITROGEN CYCLE

148

Coenye et al. [4] and Hauben et al. [8] suggested that, prior to their description, the

relationship of novel species within the genus Stenotrophomonas with the heterogeneous

species S. maltophilia should be investigated. GyrB-RFLP analysis of S. maltophilia strains

has proven to be in accordance with DNA-DNA hybridization results [4]. Therefore, the

gyrB-RFLP profiles of strains R-32768T and R-32729T were analyzed as described previously

[4], together with an additional set of Stenotrophomonas strains representing gyrB-RFLP

clusters A, B, C, E, F, G and I. Strains R-32768T and R-32729T did not render a gyrB

amplicon, even after several repeats. The same observation was made by Coenye et al. for

two Stenotrophomonas spp. strains (personal communication). It was concluded that this

approach was not suitable for the new strains, which, therefore, must differ from the

strains that did render gyrB amplicons.

Figure 5.2. Numerical comparison of the obtained fatty acid profiles. UPGMA clustering with Pearson’s correlationsimilarity coefficients was performed using BioNumerics version 4.6. The cophenetic correlation tool, whichdistinguishes reliable from unreliable subclusters, was used for cluster significance analysis.

To further substantiate that R-32729T and R-32768T do not belong to S. maltophilia, SDS-

PAGE analysis of whole-cell proteins was performed on all strains included in the gyrB-

RFLP trials. Aerobically grown cells were harvested after incubation at 28°C for 24h on

phosphate-buffered nutrient agar (pH 6.8). A SDS-PAGE banding pattern for all strains

was generated according to previously described standardized protocol [15]. Pearson’s

correlation similarity coefficients were clustered with UPGMA and analyzed with the

cophenetic correlation method in BioNumerics version 4.6 (Figure 5.3). The grouping of

the whole-cell proteins profiles was supported by high cophenetic correlation values but

did not correlate with the gyrB-RFLP grouping, except for gyrB-RFLP cluster G. R-

32729T and R-32768T did not group together. Their phylogenetically closest neighbors, S.

nitritireducens LMG 22074T and S. acidaminophila LMG 22073T, together with strain R-

12772, grouped separate from all other Stenotrophomonas strains.

Pearson correlation coefficient (% similarity)

100

90807060

100

100

88

100

79

85

98

.

.

.

.

.

.

.

.

Stenotrophomonas terrae sp. nov.

Stenotrophomonas acidaminiphila

Stenotrophomonas nitritireducens

Stenotrophomonas rhizophila

Stenotrophomonas maltophilia

Stenotrophomonas maltophilia

Stenotrophomons koreensis

R-32729T

R-32768T

LMG 22073T

LMG 22074T

CCUG 47042T

LMG 22072

LMG 958T

LMG 22369T

CHAPTER 5

149

Figure 5.3. Grouping of normalized digitized SDS-PAGE patterns. UPGMA clustering with Pearson’s correlationsimilarity coefficients was performed using BioNumerics version 4.6. The cophenetic correlation tool, whichdistinguishes reliable from unreliable subclusters, was used for cluster significance analysis. The identification ofnon-type strains and the assignment to gyrB-RFLP clusters were taken from [4].


150

Based on the polyphasic data presented, the strains R-32768T and R-32729T represent two

novel species in the genus Stenotrophomonas, for which the names Stenotrophomonas

terrae sp. nov. and Stenotrophomonas humi sp. nov. are proposed.

Description of Stenotrophomonas terrae sp. nov.

Stenotrophomonas terrae (ter.ra’e. L. gen. n. terrae, of/from soil)

After 24h incubation at 28°C on TSA, colonies are irregular shaped and light yellow. Cells

are motile, non-spore-forming, rod-shaped, Gram-negative, catalase-positive, oxidase-

positive. Growth is observed at 15-37°C but not at 4°C or 52°C, at a pH of 5-10.5, but not

at 4.5, and at salt concentration of 0.5% to 5%. Anaerobic growth is possible through

nitrate reduction. Enzyme activities and carbon utilization are given in Table 1. Can be

differentiated from the type strains of its closest phylogenetic neighbours, S. humi, S.

nitritireducens and S. acidaminiphila by SDS-PAGE analysis and the presence of leucine

arylamidase, protease, the assimilation of glucose and the absence of N-acetyl-β-

glucosaminidase.

The type strain is R-32768T (= LMG 23958T = DSM 18941T), which has a DNA G+C

content of 65 mol% and was isolated from soil from a university test field in Ghent,

Belgium.

The Genbank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of R-

32768T is AM403589.

Description of Stenotrophomonas humi sp. nov.

Stenotrophomonas humi (hu’mi. L. gen. n. humi, of/from soil)

After 24h incubation at 28°C on TSA, colonies are round, smooth and beige. Cells are

motile, non-spore-forming, rod-shaped, Gram-negative, catalase-positive, oxidase-positive.

Growth is observed at 15-37°C but not at 4°C or 52°C, at a pH of 5-10.5, but not at 4.5,

and at salt concentration of 0.5% to 4% but not at 5%. Anaerobic growth is possible

through nitrate reduction. Enzyme activities and carbon utilization are given in Table 1. Can

be differentiated from the type strains of its closest phylogenetic neighbour, S. terra, S.

nitritireducens and S. acidaminiphila by SDS-PAGE analysis and the assimilation of malate.

The type strain is R-32729T (= LMG 23959T = DSM 18929T), which has a DNA G+C

content of 64 mol% and was isolated from soil from a university test field in Ghent,

Belgium.


32729T is AM403587.

CHAPTER 5

151

REFERENCES1. Assih EA, Ouattara AS, Thierry S, Cayol J-L, Labat M, Macarie H (2002) Stenotrophomonas

acidaminiphila sp. nov., a strictly aerobic bacterium isolated from an upflow anaerobic sludge blanket

(UASB) reactor. Int J Syst Evol Microbiol 52:559-568

2. Coenye T, Spilker T, Martin A, LiPuma JJ (2002) Comparative assessment of genotyping methods for

epidemiologic study of Burkholderia cepacia Genomovar III. J Clin Microbiol 40:3300-3307

3. Coenye T, Vanlaere E, Falsen E, Vandamme P (2004) Stenotrophomonas africana Drancourt et al.

1997 is a later synonym of Stenotrophomonas maltophilia (Hugh 1981) Palleroni and Bradbury

1993. Int J Syst Evol Microbiol 54:1235-1237

4. Coenye T, Vanlaere E, LiPuma JJ, Vandamme P (2004) Identification of genomic groups in the genus

Stenotrophomonas using gyrB RFLP analysis. FEMS Immun Med Microbiol 40:181-185

5. Drancourt M, Raoult D (1997) Stenotrophomonas africana sp. nov., an opportunistic human pathogen

in Africa. Int J Syst Bacteriol 47:160-163

6. Ezaki T, Hashimoto Y, Yabuuchi E (1989) Fluorometric deoxyribonucleic acid-deoxyribonucleic acid

hybridization in microdilution wells as an alternative to membrane filter hybridization in which

radioisotopes are used to determine genetic relatedness among bacterial strains. Int J Syst Bacteriol

39:224-229

7. Finkman W, Altendorf K, Stackebrandt E, Lipski A (2000) Characterization of N2O-producing

Xanthomonas-like isolates from biofilters as Stenotrophomonas nitritireducens sp. nov, Luteimonas

mephitis gen. nov., sp. nov. and Pseudoxanthomonas broegbernensis gen. nov., sp. nov. Int J Syst

Evol Microbiol 50:273-282

8. Hauben L, Vauterin L, Moore ERB, Hoste B, Swings J (1999) Genomic diversity of the genus

Stenotrophomonas. Int J Syst Bacteriol 49:1749-1760


bacteria: optimisation of isolation conditions and diversity study. Appl Environ Microbiol 72:2637-

2643

10. Heyrman J, Verbeeren J, Schumann P, Swings J, De Vos P (2005) Six novel Arthrobacter species isolated

from deteriorated mural paintings. Int J Syst Evol Microbiol 55:1457-1464

11. Jukes TH, Cantor CR (1969) Evolution of protein molecules, In: Muruo HN (ed.) Mammalian Protein

Metabolism, New York : Academic Press, pp 21-132

12. Kimura M (1980) A simple method for estimating evolutionary rates of base substitutions through

comparative studies of nucleotide sequences. J Mol Evol 16:111-120

13. Mesbah M, Premachandran U, Whitman WB (1989) Precise measurement of the G + C content of

deoxyribonucleic acid by high-performance liquid chromatography. Int J Syst Bacteriol 39:159-167

14. Palleroni NJ, Bradbury JF (1993) Stenotrophomonas, a new bacterial genus for Xanthomonas

maltophilia (Hugh 1980) Swings et al., 1983. Int J Syst Bacteriol 43:606-609

15. Pot B, Vandamme P, Kersters K (1994) Analysis of electrophoretic whole organism protein fingerprints,

In: Goodfellow M, O’Donnel AG (Eds.), Chemical Methods in Prokaryotic Systematics, Chichester:

Wiley, pp. 493-521

16. Sierra G (1957) A simple method for the detection of lipolytic activity of micro-organisms and some

observations on the influence of the contact between cells and fatty substrates. Antonie van

Leeuwenhoek 23:15-22

17. Smibert RM, Krieg NR (1994) Phenotypic characterization. In: Gerhardt P, Murray RGE, Wood WA,

Krieg NR (Eds.), Methods for General and Molecular Bacteriology, Washington: American Society for

Microbiology, pp. 623-624

18. Stanier RY, Palleroni NJ, Doudoroff M (1966) The aerobic pseudomonads : a taxonomic study. J Gen


19. Swings J, De Vos P, Van den Mooter M, De Ley J (1983) Transfer of Pseudomonas maltophilia Hugh

1981 to the genus Xanthomonas as Xanthomonas maltophilia (Hugh 1981) comb. nov. Int J Syst




Acids Res 24:4876-4882

21. Van de Peer Y., De Wachter R (1994) TREECON for Windows: a software package for the construction


10:569-570


152

22. Vanparys B, Heylen K, Lebbe L, De Vos P (2005) Pedobacter caeni sp. nov., a novel species isolated

from a nitrifying inoculum. Int J Syst Evol Microbiol 55:1315-1318

23. Willems A, Doignon-Bourcier F, Goris J, Coopman R, de Lajudie P, De Vos P, Gillis M (2001) DNA-

DNA hybridization study of Bradyrhizobium strains. Int J Syst Evol Microbiol 51:1315-1322

24. Wolf A, Fritze A, Hagemann M, Berg G (2002) Stenotrophomonas rhizophila sp. nov., a novel plant-

associated bacterium with antifungal properties. Int J Syst Evol Microbiol 52:1937-1944

25. Yang H-C, Im W-T, Kang MS, Shin D-Y, Lee S-T (2006) Stenotrophomonas koreensis sp. nov., isolated

from compost in South Korea. Int J Syst Evol Microbiol 56:81-84

26. Yang P, Vauterin L, Vancanneyt M, Swings J, Kersters K (1993) Application of fatty acid methyl esters

for the taxonomic analysis of the genus Xanthomonas. Syst Appl Microbiol 16:47-71

27. Yoon J-H, Kang S-J, Oh HW, Oh T-K (2006) Stenotrophomonas dokdonensis sp. nov., isolated from

soil. Int J Syst Evol Microbiol 56:1363-1367

CHAPTER 5

5.2 ACIDOVORAX CAENI SP. NOV., A NOVELDENITRIFYING SPECIES WITH GENETICALLYDIVERSE ISOLATES FROM ACTIVATED SLUDGE

Redrafted from: Heylen K, Lebbe L, De Vos P (2007) Acidovorax caeni sp. nov., a novel

denitrifying species with genetically diverse isolates from activated sludge. Int J Syst Evol

Microbiol, Accepted

SUMMARY

Four Gram-negative, rod-shaped, non-spore-forming, denitrifying isolates were obtained

from the activated sludge of an aerobic-anaerobic wastewater treatment plant in Belgium.

Analysis of repetitive sequence-based PCR showed that the four isolates were genetically

different. 16S rRNA gene sequence analysis and DNA-DNA hybridization placed them

within the genus Acidovorax and showed their genotypic differentiation from all other

currently known Acidovorax species. Analysis of the whole cell proteins, and physiological

and biochemical tests allowed differentiation from their closest phylogenetic neighbours

and they therefore represent a novel species, for which the name Acidovorax caeni sp. nov.

is proposed, with strains R-24608T (= LMG 24103T = DSM 19327T) as type strain.

156

The genus Acidovorax contains eight species, which can be separated into soil and water

inhabitants, A. facilis, A. delafieldii, A. temperans [22] and A. defluvii [15], and the

phytopathogenic species A. avenae with its three subspecies, A. konjaci [24], A. anthurii

[4] and A. valerianell1a [5]. This separation in occurrence and habitat is reflected in the 16S

rRNA gene sequence phylogeny of these organisms, although separate phylogenetic clustering

of the genus within the Comamonadaceae is confirmed [23].

A previous cultivation-dependent study on activated sludge from an aerobic-anaerobic

wastewater treatment plant used different defined growth media for elective isolation of

denitrifying bacteria [7]. Nineteen denitrifiers were assigned to the genus Acidovorax,

based on partial 16S rRNA gene sequence analysis. The role of members of the

Comamonadaceae in the removal of nitrogen in wastewater treatment plants has been

recognized and described previously [2, 6, 9, 12]. Four Acidovorax isolates, R-24607, R-

24608T, R-24613, R-24614, were retrieved from G1M1, a mineral medium containing 15mM

sodium succinate, 3mM potassium nitrite and different vitamins. These isolates could

possibly belong to a new species within the environmental cluster of Acidovorax, based on

partial 16S rRNA gene sequence similarities and were analyzed further in a polyphasic

study. The type strain and a second representative of each described environmental

Acidovorax species, i.e. A. facilis, A. delafieldii, A. defluvii and A. temperans, were re-

examined for phenotyping, chemotaxonomy and biochemical analysis to guarantee

comparable results.

To avoid studying duplicate isolates of the same strain, genotyping by Random Amplified

Polymorphism DNA PCR analysis [1] and repetitive sequence-based PCR analysis with

REP and BOX primers [8] was carried out. The three fingerprint methods generated different

patterns for isolates R-24607, 24608T, R-24613, and R-24614, indicating genetic differences

between all four isolates (data not shown). The average DNA G+C content of the four

isolates, determined once by HPLC [13] was 64.3 mol% (± 0.8). The nearly complete 16S

rRNA gene sequences of R-24607, 24608T, R-24613, and R-24614 were determined as

described previously [20]. Phylogenetic analysis was performed using TreeCon [19] and

the BioNumerics software version 4.6 after multiple alignment with ClustalX [18]. Cluster

analysis using the neighbour-joining algorithm with or without corrections for evolutionary

distances as described by Jukes & Cantor [10] and Kimura [11] were in agreement with

maximum-parsimony and maximum-likelihood methods. Strains R-24607, 24608T, R-24613,

and R-24614 clustered together with A. temperans LMG 7169T, A. delafieldii LMG 5943T,

CHAPTER 5

157

A. defluvii DSM 12644T and A. facilis LMG 2193T, but clearly formed a separate group,

supported by high bootstrap values (Figure 5.4). Therefore, DNA-DNA hybridization

experiments were performed within this cluster, using a modification of the microplate

method of Ezaki et al. [3] as described by Willems et al. [21]. A hybridization temperature

of 45°C (calculated with correction for 50% formamide) was used. First, R-24607, R-

24608T, R-24613, and R-24614 were hybridized among themselves, to substantiate the

hypothesis of their relatedness on species level. The DNA-DNA similarity ranged between

78.5-88.5% (± 1.3-10.9), thus forming one species, but also confirming the genetic diversity

within the strains. R-24608T was further hybridized with A. temperans LMG 7169T (26.7%

± 5.6), A. delafieldii LMG 5943T (23.9% ± 5.9), A. defluvii DSM 12644T (26.0% ± 0.6) and

A. facilis LMG 2193T (18.2% ± 4.8). These results confirmed that R-24607, R-24608T, R-

24613, R-24614 belong to a novel genospecies.

Figure 5.4. Phylogenetic dendrogram obtained by neighbour-joining clustering of the 16S rRNA gene sequences(without correction), showing the position of the Acidovorax isolates R-24608T, R-24607, R-24613, R-24614among the type strains of Acidovorax species. EMBL accession numbers are shown in parenthesis. Variovoraxparadoxus LMG 1797T was used as outgroup. Relevant bootstrap values (expressed as percentages of 1000replicates) are shown at the branch points. The scale bar represents 0.02 changes per sequence position.


Acidovorax caen i sp. nov. R-24614 ( 084008)AM

Acidovorax dela fieldii LMG 5943 ( 078764)T AF

Acidovorax avenae citru lli subsp. LMG 5376 ( 078761) T AF

Acidovorax caeni sp. nov. R-24608 ( 084006)T AM

Acidovorax caeni sp. nov. R-24607 ( 084011)AM

Acidovorax caeni sp. nov. R-24613 ( 084007)AM

Acidovorax defluvii DSM 12644 (Y18616) T

Acidovorax avenae cattleyae subsp. LMG 5286 ( 078762)T AF

Acidovorax avenae venae subsp. a LMG 2117 ( 078759)T AF

0.02 subst./site

Variovorax paradoxus LMG 1797 (D30793)T

Acidovorax valerianellae CFBP 4730 ( )T AJ431731

Acidovorax temperans LMG 7169 ( 078766) T AF

Acidovorax facilis LMG 2193 ( 078765) T AF

Acidovorax anthurii CIP 107058 ( 007013)T AJ

Acidovorax konjaci LMG 5691 ( 078760)T AF100

89

53

100

100

86

73

60

57

158

Cell morphology and motility was investigated by electron microscopy (Figure 5.5.) and

phase contrast microscopy (at a magnification of 1000x) respectively, for cells grown on

Tryptone Soy Agar (TSA; Oxoid) for 48h at 28°C. Cells were Gram stained and examined

with light microscopy, and catalase and oxidase activity was determined. Utilization of

carbon sources and enzyme production was tested with API 20NE (48h, 28°C), API ZYM

(4h, 28°C) (BioMérieux) and BIOLOG (24h, 28°C) according to the manufacturer’s

instruction. The temperature range (at 4, 15, 28, 37, 45 and 52°C), the pH range (4.5 to 10.5

at 28°C) and the salt range (0.5 to 5% NaCl w/v at 28°C) for growth were recorded after

incubation for 48h in Tryptone Soy Broth (TSB; Oxoid). The ability to denitrify was

tested, as described by Smibert & Krieg [17], after growth for one week on TSB supplemented

with 10 mM potassium nitrate at 37°C and on liquid isolation medium G1M1 at 37°C, and

confirmed with N2O measurements, as described by Heylen et al. [7]. Lipolytic activity

was determined after 72h by the hydrolysis of Tween 80, as described by Sierra [16].

Biochemical characteristics of all strains are given in Table 5.2.

Figure 5.5. Electron microscopic picture of R-24608T, showing peritrichous rods (about 0.9 x 1.8µm).

CHAPTER 5

159

After a pre-culture, all strains were identically incubated for exactly 48h at 28°C on

TSA. A loopful of well-grown cells was harvested and fatty acid methyl esters were

prepared and extracted according to the standardized protocol of the Microbial

Identification System (MIS; Microbial ID Inc.), and identified using MIDI with the

TSBA database version 5.0. All Acidovorax strains contained the characteristic fatty

acids 3-hydroxyoctanoic acid (C8:0

3OH) and 3-hydroxydecanoic acid (C10:0

3OH) [23].

The dominant fatty acids for strains R-24607, R-24608T, R-24613 and R-24614 were

Summed Feature 3 (38-41%), C18:1

ω7c (22-32%) and C16:0

(25-26.5%). Unfortunately,

the Sherlock MIS software could not clearly resolve Summed Feature 3, referring to the

peaks of C16:1

ω7c and/or isoC15:0

2OH. However, Sherlock lists the closest to the

observed ECL first, which was C16:1

ω7c. And also, comparison fatty acid data in

literature of the already described type strains [15, 22] and our data on the same strains

suggests C16:1

ω7c as the major fatty acid for this peak. No characteristic fatty acids for

the new genospecies were detected (Table 5.3).

SDS-PAGE analysis of whole-cell proteins was performed on aerobically grown cells after

incubation at 28°C for 40h on phosphate-buffered Nutrient Agar (pH 6.8). A SDS-PAGE

banding pattern for all strains was generated according to previously described standardized

protocol [14]. Pearson’s correlation similarity coefficients were clustered with UPGMA

and analyzed with the cophenetic correlation method in BioNumerics version 4.6 (Figure

5.6). The different strains of all species grouped together, supported with high cophenetic

correlation values. Although strains R-24607, R-24608T, R-24613 and R-24614 of the new

genospecies demonstrated significant variation in the whole protein profiles, they formed a

distinct group, separate from the other Acidovorax species.

All four strains were able to denitrify. Their denitrification genes were investigated with PCR

for nir and norB genes and with a dot-blot protocol for norB. R-24613 and R-24614, did

render a nirK sequence (see Section 3.1.). R-24608 and R-24607 did not render any sequence

for all four tested denitrification genes. Only R-24613 and R-24608 were included in the norB

dot-blot screening, including one strain with a nirK sequence and one without any detected

denitrification gene. Both tested positive for the presence of either cnorB or qnorB. Thus, the

nir genes show high sequence divergent within this species, resulting in strain-dependent

detection of nirK. Probably all four strains have a cnorB or qnorB gene that can only be

detected via dot-blot. The results for the functional gene analysis within this novel Acidovorax

species substantiate previous conclusions on strain-dependent detection of denitrification

genes.


160

Table 5.2. (Opposite page) Physiological characteristics of the novel species and his closest phylogeneticneighbours.Strains: 1, A. caeni sp. nov. R-24607, R-24608T, R-24613, R-24614; 2, A. defluvii DSM 12644T, DSM 12578; 3,A. delafieldii LMG 5943T, LMG 8909; 4, A. facilis LMG 2193T, LMG 6598; 5, A. temperans LMG 7169T, LMG7163. Data are from this study unless indicated. +, Positive; w, weakly positive; -, negative; v, variable with thetest result for the type strain given between parenthesis. #, In contrast to DSM 12578, DSM 12644T does not reducenitrate to nitrite or further (denitrification) in the tested growth conditions (in G1M1 and in supplemented TSA).With the API 20NE gallery, both strains tested positive for nitrate reduction and denitrification; |, DSM 12644Tscored negative for Tween 80 hydrolysis in the Biolog screening (after 24h), but positive when grown on Sierramedium for 72h; °, data from [23]; *, data from [15].All Acidovorax species are positive for assimilation of leucine arylamidase, methyl pyruvate, mono-methyl succinate,β-hydroxybutyric acid, α-keto valeric acid, and D,L lactic acid. All Acidovorax species are negative for argininedihydrolase, urease, lipase (C14), valine arylamidase, cystine arylamidase, α-chymotrypsin, acid phosphatase, α-galactosidase, β-galactosidase, β-glucuronidase, α-glucosidase, β-glucosidase (aesculin hydrolysis), N-acetyl-D-glucosamidase, α-mannosidase, α-fucosidase, indole production, glucose fermentation, and assimilation of L-mannose, phenylacetic acid, N-acetyl-D-galactosamine, adonitol, cellobiose, L-fucose, gentiobiose, m-inositol,α-D-lactose, lactulose, D-melibiose, β-methyl-glucoside, D-raffinose, sucrose, D-trehalose, turanose, xylitol, citricacid, D-galactonic acid lactone, D-galacturonic acid, D-glucosaminic acid, D-glucuronic acid, saccharic acid,glucuronamide, L-histidine, inosine, uridine, thymidine, phenyl ethylamine, putrescine, 2-amino ethanol, 2,3-butanediol, D,L-α-glycerol phosphate, glucose-1-phosphate.

CHAPTER 5

161


Characteristic 1 2 3 4 5 oxidase + + + + + catalase + v (+) -, v° + + hydrolysis of tween 40 + v (-) + v (-) + hydrolysis of tween 80 + +| + v (-) + nitrate reduction + v#, +* + + + denitrification + v# - - + alkaline phosphatase w - + w + esterase (C4) w + - + w esterase lipase (C8) + w + + + trypsin + - - - - naphthol-AS-BI-phosphohydrolase + - - - - Indole production + - - - - gelatinase - - -, v° v (-), +° - assimilation of glucose - - + -, +° + L-arabinose - - + -, +° - D-mannose - - + -, +° - D-mannitol - - + -, +° + N-acetyl-glucosamine - - - + - maltose - - - + - potassium gluconate - - + -, +° - capric acid - - -, v° - -, v° adipic acid - + - - - malate + - v - + trisodium citrate - - -, v° - - α-cyclodextrin v (-) - v (-) - - dextrin - - v (-) - - glycogen + - - - +, -° D-arabitol - - + - v (-) i-eritritol v (-) - - - - D-fructose - - + v (-) + D-galactose - v (+) + - - α-D-glucose - - v (-) - + D-psicose - - v (-) v (-) + D-sorbitol - - + - + acetic acid v (+) - - - - cis-aconitic acid v (+) - v (+) - - formic acid + - v (+) - - D-gluconic acid - - + - - α -hydroxybutyric acid + v (-) + - + γ-hydroxybutyric acid + v (-) + - + p-hydroxy phenylacetic acid - - + - - itaconic acid - - - - - α -keto butyric acid + v (-) + v (-) + α -keto glutaric acid + v (+) + v (-) + malonic acid v (-) - - v (-) - propionic acid + - v (+) - v (-) quinic acid - - + - - sebacic acid v (+) + + v (-) + succinic acid + + + v (-) + bromo succinic acid + - v (+) v (-) + succinamic acid v (-) - v (+) v (-) + alaninamide v (-) v (+) + v (-) + D-alanine + - + v (-) + L-alanine + + + v (-) + L-alanyl-glycine + v (+) + + + L-asparagine v (-) - + - + L-aspartic acid + - + - + L-glutamic acid + - + - + glycyl-L-aspartic acid - - + v (-) - glycyl-L-glutamic acid - - + - v (+) hydroxy-L-proline - - + - - L-leucine + - + v (-) + L-ornithine - - v (+) - v (-) L-phenylalanine - - v (+) + + L-proline + - + + + L-pyroglutamic acid + - v (+) + + D-serine v (-) - - - - L-serine + - + -, +° - L-threonine + v (-) + - v (-) D,L-carnithine - - v (+) v (+) - γ-amino butyric acid - - + v (+) v (-) urocanic acid - - + - - glycerol + v (+) + - + glucose-6-phosphate v (-) - v (+) - -

162

Table 5.3. Fatty acid content of the novel species and his closest phylogenetic neighbours.Values are percentages of the total fatty acid content. Strains: 1, A. caeni sp. nov. R-24607, R-24608T, R-24613, R-24614; 2, A. defluvii DSM 12644T, DSM 12578; 3, A. delafieldii LMG 5943T, LMG 8909; 4, A. facilis LMG 2193T,LMG 6598; 5, A. temperans LMG 7169T, LMG 7163. Summed Feature 3 contains C16:1ω7c and/or iso-C15:0 2-OH.Summed feature 7 contains C

19:1 ω6c and C

19-cyclo. All data are obtained in this study. /, not present; tr, trace elements

< 1%

Figure 5.6. Grouping of normalized digitized SDS-PAGE patterns. UPGMA clustering with Pearson’s correlationsimilarity coefficients was performed using BioNumerics version 4.6. The cophenetic correlation tool was used forcluster significance analysis.

CHAPTER 5

Fatty acid 1 2 3 4 5 C8:0 3OH 0,78-1,19 tr tr 0,88-1,87 1,04-1,27 C9:0 3OH / 0,32-1,14 / / tr C11:0 / tr / / tr C10:0 3OH 2,81-4,32 3,13-3,90 3,16-3,59 2,78-6,78 3,96-4,25 unknown 11,799 / / / tr / C12:0 2,64-3,24 2,18-2,39 2,77-2,89 2,91-5,48 5,52-5,72 C11:0 iso 3OH / tr / / / C11:0 3OH / tr / / / C13:0 / tr / / 1,08-3,46 C14:0 tr 1,57-1,63 3,25-3,37 3,09-,374 0,99-3,53 C15:1 w6c / 1,15-11,97 / / 3,23-4,51 C15:0 iso / tr / / / Summed Feature 3 38,24-41,16 40,57-48,05 39,35-41,32 43,68-43,71 46,92-47,15 C16:0 24,90-26,41 17,70-22,78 26,33-27,99 25,51-29,24 19,35-20,99 iso C17:1 w9c / tr / / / C17:0 iso tr tr / / / C17:0 cyclo / / tr tr / C17:1 w8c / tr / / / C17:1 w6c / 1,41-2,09 / / tr C17:0 tr 4,87-6,27 tr tr 2,10-3,7 C18:1 w7c 22,24-31,96 8,07-11,04 19,42-22,73 11,78-16,56 9,12-9,64 C18:0 tr / tr tr / methyl C18:1 w7c tr tr tr / / sum in feature 7 tr / / / /

Pearson correlation (Opt:2.00%) [2.4%-80.6%]

100

908070

10059

93

100

100

79

100

71

100

80

87

.

.

.

.

.

.

.

.

.

.

.

.

Acidovorax caeni sp. nov.




Acidovorax temperans


Acidovorax facilis

Acidovorax facilis

Acidovorax delafi eldiiAcidovorax delafi eldii

Acidovorax defluvii

Acidovorax defluvii

R-24614

R-24608T

R-24607

R-24613

LMG 7169T

LMG 7163

LMG 2193T

LMG 6598

LMG 5943T

LMG 8909

DSM 12644T

DSM 12578

Pearson correlation (Opt:2.00%) [2.4%-80.6%]

100

908070

10059

93

100

100

79

100

71

100

80

87

.

.

.

.

.

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.







Acidovorax facilis

Acidovorax facilis

Acidovorax delafi eldiiAcidovorax delafi eldii

Acidovorax defluvii

Acidovorax defluvii

R-24614

R-24608T

R-24607

R-24613

LMG 7169T

LMG 7163

LMG 2193T

LMG 6598

LMG 5943T

LMG 8909

DSM 12644T

DSM 12578

163


Based on the polyphasic data presented, the strains R-24607, R-24608T, R-24613 and R-

24614 represent a novel species in the genus Acidovorax, for which the name Acidovorax

caeni sp. nov. is proposed.

Description of Acidovorax caeni sp. nov.

Acidovorax caeni (ca.e’ni. L. gen. neut. n. caeni of sludge).

After 48h, colonies are round, smooth and yellow-brown. Cells are motile, non-spore

forming, rods (0.9 x 1.8 µm). The strains are Gram-negative, and catalase and oxidase

positive. Growth is observed at 15-37°C but not at 4°C or 45-52°C, at a pH of 5.5-10.5,

but not at 4.5-5, and at salt concentration of 0.5% to 2%, but not at 3-5% (NaCl, w/v).

Anaerobic respiration and growth is possible through denitrification. Following enzyme

activities were detected: hydrolysis of Tween 40 and 80, esterase lipase, trypsin, naphthol-

AS-BI-phosphohydrolase and production of indole. Malate, glycogen, formic acid, α-

hydroxybutyric acid, β- hydroxybutyric acid, α-keto butyric acid, α-keto glutaric acid,

propionic acid, succinic acid, bromo succinic acid, D-alanine, L-alanine, L-alanyl-glycine,

L-aspartic acid, L-glutamic acid, L-leucine, L-proline, L-pyroglutamic acid, L-serine, L-

threonine, methyl pyruvate, mono-methyl succinate, β-hydroxybutyric acid, α-keto valeric

acid, and D,L lactic acid and glycerol can be used as carbon source. Can be differentiated

from the type strains of its closest phylogenetic neighbours, A. defluvii, A. delafieldii, A.

facilis and A. temperans, through SDS-PAGE analysis of whole cell proteins, by the ability

to produce indole and by the presence of trypsin and naphthol-AS-BI-phosphohydrolase.

The type strain R-24608T (= LMG 24103T = DSM 19327T) has a DNA G+C content of

65.7 mol% and was isolated from activated sludge from an aerobic-anaerobic wastewater

treatment plant (Bourgoyen-Ossemeersen) in Gent, Belgium. Due to the genetic variation

within this new species, R-24607, R-24613 and R-24614 were also deposited in the BCCM/

LMG collection with strain numbers LMG24104, LMG 24105 and LMG 24106,

respectively.


24608T is AM084006.

164

CHAPTER 5

REFERENCES1. Coenye T, Spilker T, Martin A, LiPuma JJ (2002) Comparative assessment of genotyping methods

for epidemiologic study of Burkholderia cepacia Genomovar III. J Clin Microbiol 40:3300-3307

2. Etchebehere C, Errazquin I, Barrandeguy E, Dabert P, Moletta R, Muxi L (2001) Evaluation of the

denitrifying microbiota of anoxic reactors. FEMS Microbiol Ecol 35:259-265

3. Ezaki T, Hashimoto Y, Yabuuchi E (1989) Fluorometric deoxyribonucleic acid-deoxyribonucleic

acid hybridization in microdilution wells as an alternative to membrane filter hybridization in

which radioisotopes are used to determine genetic relatedness among bacterial strains. Int J Syst


4. Gardan L, Dauga C, Prior P, Gillis M, Saddler GS (2000) Acidovorax anthurii sp. nov., a new

phytopathogenic bacterium which causes bacterial leaf-spot of anthurium. Int J Syst Evol Microbiol

50:235-246

5. Gardan L, Stead DE, Dauga C, Gillis M (2003) Acidovorax valerianellae sp. nov., a novel

pathogen of lamb's lettuce [Valerianella locusta (L.) Laterr.]. Int J Syst Evol Microbiol 53:795-

800

6. Gumaelius L, Magnusson G, Pettersson B, Dalhammar G (2001) Comamonas denitrificans sp.

nov., an efficient denitrifying bacterium from activated sludge. Int J Syst Evol Microbiol 51:999-

1006

7. Heylen K, Vanparys B, Wittebolle L, Verstraete W, Boon N, De Vos P (2006) Cultivation of

denitrifying bacteria: optimisation of isolation conditions and diversity study. Appl Environ


8. Heyrman J, Verbeeren J, Schumann P, Swings J, De Vos P (2005) Six novel Arthrobacter species

isolated from deteriorated mural paintings. Int J Syst Evol Microbiol 55:1457-1464

9. Hoshino T, Terahara T, Tsuneda S, Hirata A, Inamori Y (2005) Molecular analysis of microbial

population transition associated with the start of denitrification in a wastewater treatment

process. J Appl Microbiol 99:1165-1175

10. Jukes TH, Cantor CR (1969) Evolution of protein molecules. In Mammalian Protein Metabolism,

Edited by H.N. Munro. New York : Academic Press, pp 21-132

11. Kimura M (1980) A simple method for estimating evolutionary rates of base substitutions through

comparative studies of nucleotide sequences. J Mol Evol 16:111-120

12. Mechichi T, Stackebrandt E, Fuchs G (2003) Alicycliphilus denitrificans gen. nov., sp. nov., a

cyclohexanol-degrading, nitrate-reducing B-proteobacterium. Int J Syst Evol Microbiol 53:147-

152

13. Mesbah M, Premachandran U, Whitman WB (1989) Precise measurement of the G + C content of

deoxyribonucleic acid by high-performance liquid chromatography. Int J Syst Bacteriol 39:159-

167

14. Pot B, Vandamme P, Kerstens K (1994) Analysis of electrophoretic whole organism protein

fingerprints. In: Goodfellow M, O'Donnel AG (Eds.) Chemical Methods in Prokaryotic Sytematics,

Chichester: Whiley, pp. 493-521

15. Schulze R, Spring S, Amann R, Huber I, Ludwig W, Schleifer K-H, Kämpfer P (1999) Genotypic

diversity of Acidovorax strains isolated from activated sludge and description of Acidovorax

defluvii sp. nov. Syst Appl Microbiol 22:205-214

16. Sierra G (1957) A simple method for the detection of lipolytic activity of micro-organisms and

some observations on the influence of the contact between cells and fatty substrates. Antonie van

Leeuwenhoek 23:15-22

17. Smibert RM, Krieg NR (1994) Phenotypic characterization. In: Gerhardt P, Murray RGE, Wood

WA, Krieg NR (Eds.), Methods for General and Molecular Bacteriology, Washington: American

Society for Microbiology, pp. 623-624


interface: flexible strategies for multiple sequence alignment aided by quality analysis tools.

Nucleic Acids Res 24:4876-4882

19. Van de Peer Y., De Wachter R (1994) TREECON for Windows: a software package for the

construction and drawing of evolutionary trees for the Microsoft Windows environment. Comput

Applic Biosci 10:569-570

20. Vanparys B, Heylen K, Lebbe L, De Vos P (2005) Pedobacter caeni sp. nov., a novel species

isolated from a nitrifying inoculum. Int J Syst Evol Microbiol 55:1315-1318

165

21. Willems A, Doignon-Bourcier F, Goris J, Coopman R, de Lajudie P, DeVos P, Gillis M (2001)

DNA-DNA hybridization study of Bradyrhizobium strains. Int J Syst Evol Microbiol 51:1315-

1322

22. Willems A, Falsen E, Pot B, Jantzen E, Hoste B, Vandamme P, Gillis M, Kersters K, De Ley J (1990)

Acidovorax, a new genus for Pseudomonas facilis, Pseudomonas delafieldii, E. Falsen (EF)

Group 13, EF Group 16, and several clinical isolates, with the species Acidovorax facilis comb.

nov., Acidovorax delafieldii comb. nov., and Acidovorax temperans sp. nov. Int J Syst Bacteriol

40:384-398

23. Willems A, Gillis M (2005) Acidovorax. In: Garrity GM, Brenner DJ, Krieg NR, Staley JT (Eds.),

Bergey's Manual of Systematic Bacteriology vol.2 (2nd ed.), New York: Springer-Verlag, pp. 689-

703

24. Willems A, Goor M, Thielemans S, Gillis M, Kersters K, De Ley J (1992) Transfer of several

phytopathogenic Pseudomonas species to Acidovorax avenae subsp. avenae subsp. nov., comb.

nov., Acidovorax avenae supsp. citrulli, Acidovorax avenae supsp. cattleyae, and Acidovorax

konjaci. Int J Syst Bacteriol 42:107-119


5.3. BACK & FORTH

The isolation studies described in Chapter 2 yieled a large set of isolates from both activated

sludge and soil, containing bacteria possibly belonging to novel species or genera. For

example, representatives of possibly new species of the genera Stenotrophomonas,

Acidovorax, Paracoccus, Ochrobactrum, Flavobacterium, Rhizobium, and Dechloromonas,

and possibly new genera of the Rhodocyclaceae and Flavobacteriaceae were found. Time

limitations forced us to select certain taxa for further polyphasic characterization, based on

in-house expertise, available biological reference material, and the number of isolates retrieved.

Novel species of Stenotrophomonas and Acidovorax are discussed here, isolates from the

genus Paracoccus are still under study.

Literature research on the already described denitrifying capacities of the cultured denitrifying

taxa retrieved in this PhD study revealed a shortage of information concerning the nitrogen

reduction characteristics in novel species descriptions. And when included, their nature and

quality is often lamentably poor, for example describing only the results of miniaturized

API tests. However, nitrogen reduction tests of all novel described taxa would enable

researchers to develop a better understanding of the widespread occurence of denitrification,

nitrate reduction to nitrite or further to ammonium. Denitrification should preferably be

tested through N2O measurements as described by Mahne & Tiedje [6], but, if not possible,

the conventional colorometric tests coupled to pH determination are also indicative. In

addition, the conditions used to detect phenotypic denitrification are also important. With

the truncated denitrification pathways described in Chapter 1 in mind, it is logic that some

strains can only start denitrification from nitrite, for which the denitrification capacity will

not be detected when using nitrate as nitrogen source. Also, some bacteria will prefer

certain carbons sources for denitrifying growth, or will not be able to use complex growth

media. Thus, it is not surprising that our isolation studies, described in Chapter 2, found

denitrifying members of genera for which the genus or species descriptions did not recognize

denitrifying capacities. A few examples:

(i) Nitrate reduction is common in the genus Bacillus [2] and denitrification is

known to be present in some strains [3, 8]. However, the most recent genus

overview [2] mentioned ‘anaerobic growth’ and ‘nitrate reduced to nitrite’

as characteristics but did not further specify possible denitrification

capacities.

(ii) Some Staphylococcus species can reduce nitrate to nitrite, with sometimes

further dissimilatory reduction to ammonium [5]. The species description

168

CHAPTER 5

of Staphylococcus hyicus [4] mentioned ‘nitrate reduction beyond nitrite’,

but it is not clear which process is referred to, either dissimilatory nitrite

reduction to ammonium or denitrification.

(iii) Enterococci usually do not reduce nitrate [7].

(iv) The genus description of Arcobacter reports the ability to reduce nitrate

but not nitrite.

(v) And so far, the possibility to reduce nitrate further than nitrite is not

described for Flavobacteriaceae [1].

Summarizing, future denitrifier cultivation studies and novel taxon description should

consider that phenotypic denitrification is dependent on the test conditions. This means

that miniaturized API tests, testing denitrification starting from nitrate reduction in

microaerophilic conditions without growth, will only provide limited detection of the

denitrification trait. Optimally, different condition should be tested and clearly described.

REFERENCES1. Bernardet J-F, Nakagawa Y, Holmes B (2002) Proposed minimal standards fro describing new taxa of

the family Flavobacteriaceae and emended description of the family. Int J Syst Evol Microbial 52:1049-

1070

2. Claus D, Berkeley RCW (1986) ‘Bacillus Cohn 1872, 174AL In: Sneath PHA, Holt JC (Eds.), Bergey’s

Manual of Systematic Bacteriology, vol 2, Williams & Wilkins, Baltimore, Md. p.p 1105-1138

3. Denariaz G, Payne WJ, Legall J (1991) The denitrifying nitrite reductases of Bacillus halodenitrificans.


4. Devriese LA, Hajek V, Oeding P, Meyer SA, Schleifer KH (1978) Staphylococcus hyicus (Sompolinsky

1953) comb. nov. and Staphylococcus hyicus subsp. chromogenes subsp. nov. Int J Syst Evol Microbial

28:482-490

5. Kloos WE, Schleifer KH (1986) ‘Staphylococcus, Rosenbach 1884, 18AL’, In: Sneath PHA, Holt JC

(Eds.), Bergey’s Manual of Systematic Bacteriology, vol. 2. Williams & Wilkins, Baltimore, Md, pp.

1013-1035

6. Mahne I, Tiedje J.M (1995) Criteria and methodology for identifying respiratory denitrifiers. Appl

Environ Microbiol 6: 1110-1115

7. Mundt O (1986) ‘Enterococci’, In: Sneath PHA, Holt JC (Eds.), Bergey’s Manual of Systematic

Bacteriology, vol.2, Williams & Wilkins, Baltimore, Md., pp. 1063-1065



9. Vandamme P, Vancanneyt M, Pot N, Mels L, Hoste B, Dewettinck D, Vlaes L, Van Den Borre C, Higgins

R, Hommez J, Kerstens K, Butzler J-P, Goossens H (1992) Polyphasic taxonomic study of the emended

genus Arcobacter with Arcobacter butzleri comb. nov. and Arcobacter skirrowii sp. nov., an

aerotolerant bacterium isolated from veterinary specimens. Int J Syst Bact 42:344-356

CONCLUDING REMARKS

6

The denitrification process has been known for over a century, and its biochemistry,

molecular biology, ecology and involved organisms were studied extensively. Still, classic

cultivation methods were used to culture denitrifiers, the informational content of the

involved functional genes was unclear and the denitrification research focused on

environmental monitoring of the process, without knowledge on the interpretation of this

data. The concept of this PhD was to investigate the denitrification process starting from

the functional bacterium. Therefore, new cultivation approaches were developed, yielding

unknown or previously not cultured denitrifiers. Subsequently, these denitrifying bacteria

were used to further examine the genetic basis of the denitrification process through the

analysis of their functional genes. Different aspects of the research, and specific conclusions

and future perspectives per topic were addressed in each chapter.

Working in a taxonomic research lab, it seemed logical to investigate a bacterial process

starting from the functional bacterium. Of course, every researcher starts from its own

expertise, and nowadays most denitrification research is performed by microbial ecologists,

resulting in mostly culture-independent research. Nevertheless, the study of the denitrifying

ability in a bacterium, species or genus can lead to unexpected observations. Here, pure

culture research led to the discovery of two different genes encoding the same function in

one bacterium, the general unsuitability of detection tools, or the possible existence of new

denitrification enzymes, all of which will hopefully trigger new biochemical and molecular

work.

As is mentioned throughout this thesis, little information is available on denitrification in

Gram-positive bacteria. Because almost no nitrogen reduction tests were performed with

novel species descriptions, the prevalence of the denitrification capacity within the genus

Bacillus is unknown. Most Bacillus strains can reduce nitrate under fermentative growth,

which is frequently called denitrification in outdated literature, but also some Bacillus

species are known to denitrify [2, 4, 5]. Bacilli are the dominant biota in advanced wastewater

treatments such as the Korean ‘B3 (Bio Best Bacillus) process’ (Korean Patent No. 151928)

or the ‘Rotating Activated Bacillus Contactor Biofilm’ [9]. And next to the implications of

its growth and toxins produced in food products, such as gelatin [1] and milk [6, 7], its

vigorous nitrate reduction and possible denitrification can diminish the anti-clostridial

effect of added nitrate/nitrite in cheesemaking [11]. It seems strange that a group of bacteria,

which is known for so many years and has great potential in wastewater treatment and

impact on the food industry, is somewhat neglected in the denitrification research. Especially

since (i) bacilli are capable of aerobic denitrification [3] and heterotrophic nitrification [8,

12], (ii) their enzymatic pathway is membrane-bound, which could have consequences for

CONCLUDING REMARKS

173

the enzymes involved, and (iii) some bacilli harbor a new class of nitric oxide reductases

[10]. Therefore, I believe that thorough investigation of the denitrification process in pure

culture bacilli will be most interesting for future work and possibly could reveal novel

aspects of the process, unknown enzymes and further biotechnological applications.

REFERENCES1. De Clerck E, Vanhoutte T, Hebb T, Geerinck J, Devos J, De Vos P (2004) Isolation, characterization and

identification of bacterial contaminants in semifinal gelatine extracts. Appl Environ Microbiol 70, 3664-

3672

2. Denariaz G, Payne WJ, LaGall J (1991) The denitrifying nitrite reductase of Bacillus halodenitrificans.


3. Kim JK, Park KJ, Cho KS, Nam S-W, Park T-J, Baipai R (2005) Aerobic nitrification-denitrification by

heterotrophic Bacillus strains. Biores Technol 96:1897-1906

4. Manachini PL, Mora D, Nicastro G, Parini C, Stackebrandt E, Pukall R, Fortina MG (2000) Bacillus

thermodenitrificans sp. nov., nom. rev. Int J Syst Evol Microbiol 50:1331-1337

5. Mahne I, Tiedje J.M (1995) Criteria and methodology for identifying respiratory denitrifiers. Appl Environ

Microbiol 6: 1110-1115

6. McKillip JL (2000) Prevalence and expression of enetrotoxins in Bacillus cereus and other Bacillus spp.,

a literature review. Antonie van Leeuwenhoek 77:393-399

7. Meer RR, Baker J, Bodyfelt FW, Griffiths MW (1991) Psychrotrophic Bacillus spp in fluid milk-products –

a review. J Food Protec 54:969-979

8. Mevel G, Prieur D (2000) Heterotrophic nitrification by a thermophilic Bacillus species as influenced by

different culture conditions. Can J Microbiol 46:465-473

9. Park SJ, Yoon JC, Shin K-S, Kim EH, Yim S, Cho Y-J, Sung GM, Lee D-G, Kim SB, Lee D-U, Woo S-H,

Koopman B (2007) Dominance of endospore-forming bacteria on a rotating activated Bacillus contactor

biofilm for advanced wastewater treatment. J Microbiol 45:113-121

10. Suharti, Strampraad MJ, Schröder T, De Vries S (2001) A novel copper A containing menaquinol NO reductase

from Bacillus azotoformans. Biochemistry 40:2632-2639

11. Ternström A, Lindberg A-M, Molin G (1993) Classification of the spoilage flora of raw and pasteurized

bovine milk, with special reference to Pseudomonas and Bacillus. J Appl Bacteriol 75:25-34

12. Yan L, He YL, Kong HN, Tanaka S, Lin Y (2006) Isolation of a new heterotrophic nitrifying Bacillus sp.

strain. J Environ Biol 27:323-326

CHAPTER 6

174

ADDENDUM

Overview of all isolates and strains investigated in this thesis. Strains are sorted according to their taxonomic positionand strain number. Identification is based on cellular fatty acid analysis and 16S rRNA gene sequence analysis.x, gene detected; ?, doubtful detection

177

Identificatie Strain nr. origin

nirK nirS cnorB qnorB included norB

AlphaproteobacteriaBrucella sp. R-26895 activated sludge x xEnsifer sp. R-31757 soilEnsifer sp. R-32544 soil x x ?

Methylobacterium sp. R-25207 activated sludge x x

Ochrobactrum anthropi LMG 2136 reference strain x xOchrobactrum sp R-25018 activated sludge x xOchrobactrum sp R-28410 activated sludge x xOchrobactrum sp. R- 24468 activated sludge xOchrobactrum sp. R-24286 activated sludge x x xOchrobactrum sp. R-24289 activated sludge x xOchrobactrum sp. R-24290 activated sludge x xOchrobactrum sp. R-24291 activated sludge x xOchrobactrum sp. R-24334 activated sludge xOchrobactrum sp. R-24343 activated sludge x xOchrobactrum sp. R-24448 activated sludge x xOchrobactrum sp. R-24467 activated sludge xOchrobactrum sp. R-24618 activated sludge x xOchrobactrum sp. R-24638 activated sludge x xOchrobactrum sp. R-24639 activated sludge xOchrobactrum sp. R-24653 activated sludge x xOchrobactrum sp. R-25054 activated sludge x xOchrobactrum sp. R-25055 activated sludge x x ?Ochrobactrum sp. R-25203 activated sludge x xOchrobactrum sp. R-26465 activated sludge xOchrobactrum sp. R-26821 activated sludge x xOchrobactrum sp. R-26825 activated sludge x xOchrobactrum sp. R-26826 activated sludge x xOchrobactrum sp. R-26889 activated sludge x xOchrobactrum sp. R-26890 activated sludge x xOchrobactrum sp. R-26891 activated sludge x xOchrobactrum sp. R-26892 activated sludge x xOchrobactrum sp. R-26894 activated sludge x xOchrobactrum sp. R-26898 activated sludge x xOchrobactrum sp. R-26900 activated sludge x xOchrobactrum sp. R-27045 activated sludge x xOchrobactrum sp. R-27046 activated sludge x xPannonibacter sp. R-27042 activated sludge x x

Paracoccus denitrificans LMG 4049 reference strain x x

Paracoccus pantotrophus LMG 4218Treference strain

Paracoccus sp. R-24292 activated sludge xParacoccus sp. R-24342 activated sludge x x xParacoccus sp. R-24615 activated sludge xParacoccus sp. R-24616 activated sludge x x xParacoccus sp. R-24617 activated sludge xParacoccus sp. R-24621 activated sludgeParacoccus sp. R-24623 activated sludge x x xParacoccus sp. R-24649 activated sludge x x xParacoccus sp. R-24650 activated sludge x x xParacoccus sp. R-24652 activated sludge x x xParacoccus sp. R-24665 activated sludge x x ?Paracoccus sp. R-25049 activated sludgeParacoccus sp. R-25058 activated sludge xParacoccus sp. R-25059 activated sludgeParacoccus sp. R-26466 activated sludge xParacoccus sp. R-26819 activated sludgeParacoccus sp. R-26822 activated sludge x x xParacoccus sp. R-26823 activated sludge xParacoccus sp. R-26824 activated sludge x x ?Paracoccus sp. R-26839 activated sludgeParacoccus sp. R-26841 activated sludge xParacoccus sp. R-26844 activated sludgeParacoccus sp. R-26888 activated sludge x ?Paracoccus sp. R-26893 activated sludgeParacoccus sp. R-26896 activated sludge

Detection via PCR Detection via dot-blot

ADDENDUM

178


nirK nirS cnorB qnorB included norBParacoccus sp. R-26897 activated sludge x xParacoccus sp. R-26899 activated sludge xParacoccus sp. R-26901 activated sludge xParacoccus sp. R-26902 activated sludgeParacoccus sp. R-27041 activated sludge x xParacoccus sp. R-27043 activated sludge x ?Paracoccus sp. R-27047 activated sludge xParacoccus sp. R-27049 activated sludge x x xParacoccus sp. R-28237 activated sludgeParacoccus sp. R-28238 activated sludgeParacoccus sp. R-28239 activated sludgeParacoccus sp. R-28241 activated sludge x x xParacoccus sp. R-28242 activated sludge x x xParacoccus sp. R-28243 activated sludgeParacoccus sp. R-28244 activated sludge xParacoccus sp. R-28245 activated sludge xParacoccus sp. R-28294 activated sludgeParacoccus sp. R-28409 activated sludge x xParacoccus sp. R-28414 activated sludge xRhizobium sp . R-26835 activated sludge xRhizobium sp. R-24261 activated sludge xRhizobium sp. R-24333 activated sludge x xRhizobium sp. R-24654 activated sludge x xRhizobium sp. R-24658 activated sludge x xRhizobium sp. R-24663 activated sludge xRhizobium sp. R-26467 activated sludge x xRhizobium sp. R-26820 activated sludge x xRhizobium sp. R-31549 soil x x ?Rhizobium sp. R-31762 soilRhizobium sp. R-31837 soil xRhizobium sp. R-31857 soil xRhizobium sp. R-32539 soilRhizobium sp. R-32552 soilRhizobium sp. R-32725 soil x x x

Sinorhizobium sp R-24605 activated sludge xSinorhizobium sp R-25067 activated sludge x x xSinorhizobium sp R-25078 activated sludge x xSinorhizobium sp. R-31759 soil x xSinorhizobium sp. R-31763 soil xSinorhizobium sp. R-31764 soil x xSinorhizobium sp. R-31816 soil x x xSinorhizobium sp. R-32542 soil x x xSinorhizobium sp. R-32546 soil xSinorhizobium sp. R-32549 soil xSinorhizobium sp. R-32737 soil x x xSinorhizobium sp. R-32769 soil x

Betaproteobacteria

Achromobacter denitrificans LMG 1231Treference strain x x

Alcaligenes faecalis LMG 1229Treference strain x x

Acidovorax sp. R-24336 activated sludge xAcidovorax sp. R-24607 activated sludgeAcidovorax sp. R-24608 activated sludge x xAcidovorax sp. R-24613 activated sludge x x xAcidovorax sp. R-24614 activated sludge xAcidovorax sp. R-24667 activated sludge xAcidovorax sp. R-25052 activated sludge x xAcidovorax sp. R-25074 activated sludge xAcidovorax sp. R-25075 activated sludge x xAcidovorax sp. R-25076 activated sludge x xAcidovorax sp. R-25212 activated sludge xAcidovorax sp. R-26831 activated sludge xAcidovorax sp. R-26833 activated sludge xAcidovorax sp. R-26834 activated sludgeAcidovorax sp. R-26837 activated sludge xAcidovorax sp. R-26842 activated sludgeAcidovorax sp. R-26843 activated sludgeAcidovorax sp. R-27044 activated sludge x

Acidovorax sp. R-28240 activated sludge

Acidovorax sp. R-28416 activated sludge xAlicycliphilus sp. R-24604 activated sludge x xAlicycliphilus sp. R-24606 activated sludge xAlicycliphilus sp. R-24611 activated sludge x x


ADDENDUM

179


nirK nirS cnorB qnorB included norBAlicycliphilus sp. R-26814 activated sludge x

Azospira oryzae R-28447 activated sludgeAzospira sp. R-25019 activated sludgeAzovibrio sp. R-25062 activated sludge x

Comamonas sp. R-24447 activated sludge xComamonas sp. R-24451 activated sludge xComamonas sp. R-24660 activated sludgeComamonas sp. R-25014 activated sludge xComamonas sp. R-25048 activated sludge xComamonas sp. R-25057 activated sludgeComamonas sp. R-25060 activated sludgeComamonas sp. R-25066 activated sludge xComamonas sp. R-26810 activated sludgeComamonas sp. R-26812 activated sludgeComamonas sp. R-26817 activated sludgeComamonas sp. R-26829 activated sludgeComamonas sp. R-26886 activated sludgeComamonas sp. R-26887 activated sludge xComamonas sp. R-26905 activated sludgeComamonas sp. R-27040 activated sludgeComamonas sp. R-28209 activated sludgeComamonas sp. R-28210 activated sludge xComamonas sp. R-28211 activated sludgeComamonas sp. R-28218 activated sludge xComamonas sp. R-28220 activated sludge xComamonas sp. R-28222 activated sludge x ?Comamonas sp. R-28223 activated sludgeComamonas sp. R-28224 activated sludge x x xComamonas sp. R-28225 activated sludge xComamonas sp. R-28226 activated sludge xComamonas sp. R-28227 activated sludge x xComamonas sp. R-28228 activated sludge xComamonas sp. R-28229 activated sludge xComamonas sp. R-28230 activated sludge xComamonas sp. R-28231 activated sludge x x xComamonas sp. R-28232 activated sludge xComamonas sp. R-28233 activated sludge xComamonas sp. R-28234 activated sludge xComamonas sp. R-28235 activated sludge xComamonas sp. R-28236 activated sludge xComamonas sp. R-28293 activated sludgeComamonas sp. R-28413 activated sludge xComamonas sp. R-28449 activated sludge xComamonas sp. R-28452 activated sludge x

Cupriavidus necator LMG 1201 reference strain xCupriavidus sp. R-31542 soil x xCupriavidus sp. R-31543 soil x xCupriavidus sp. R-31544 soil x x

Dechloromonas sp. R-28215 activated sludge xDechloromonas sp. R-28291 activated sludge xDechloromonas sp. R-28317 activated sludgeDechloromonas sp. R-28400 activated sludge xDechloromonas sp. R-28401 activated sludge xDechloromonas sp. R-28407 activated sludge x xDechloromonas sp. R-28412 activated sludgeDechloromonas sp. R-28451 activated sludge xDiaphorobacter sp. R-24610 activated sludge x xDiaphorobacter sp. R-24612 activated sludge x xDiaphorobacter sp. R-24661 activated sludge xDiaphorobacter sp. R-25011 activated sludge xDiaphorobacter sp. R-25012 activated sludgeDiaphorobacter sp. R-26815 activated sludge x xDiaphorobacter sp. R-26840 activated sludge xDiaphorobacter sp. R-28417 activated sludge x

Simplicispira sp R-28408 activated sludgeThauera sp R-25069 activated sludge x x ?Thauera sp R-25071 activated sludge x xThauera sp. R-24450 activated sludge xThauera sp. R-26885 activated sludge xThauera sp. R-26906 activated sludge x xThauera sp. R-28205 activated sludge xThauera sp. R-28206 activated sludge xThauera sp. R-28207 activated sludge x


ADDENDUM

180

Identificatie Strain nr. origin Detection via dot-blot

nirK nirS cnorB qnorB included norBThauera sp. R-28213 activated sludge x xThauera sp. R-28216 activated sludge xThauera sp. R-28217 activated sludgeThauera sp. R-28289 activated sludge xThauera sp. R-28292 activated sludge xThauera sp. R-28312 activated sludgeThauera sp. R-28402 activated sludge xThauera sp. R-28403 activated sludge xThauera sp. R-28404 activated sludge xThauera sp. R-28405 activated sludge xThauera sp. R-28406 activated sludgeThauera sp. R-28448 activated sludge xZoogloea sp. R-28315 activated sludge

Gammaproteobacteria

Pseudomonas aeruginosa LMG 1242Treference strain x

Pseudomonas sp. R-24261 activated sludge x xPseudomonas sp. R-24609 activated sludge x xPseudomonas sp. R-24636 activated sludgePseudomonas sp. R-25016 activated sludgePseudomonas sp. R-25061 activated sludge xPseudomonas sp. R-25208 activated sludge x x xPseudomonas sp. R-25209 activated sludge x xPseudomonas sp. R-25343 activated sludgePseudomonas sp. R-26828 activated sludge x xPseudomonas sp. R-26830 activated sludge x xPseudomonas sp. R-28208 activated sludgePseudomonas sp. R-28219 activated sludgePseudomonas sp. R-31758 soil x x ?Pseudomonas sp. R-31761 soil xPseudomonas sp. R-31765 soilPseudomonas sp. R-31815 soil xPseudomonas sp. R-31817 soil xPseudomonas sp. R-32541 soilPseudomonas sp. R-32553 soil xPseudomonas sp. R-32726 soil

Pseudomonas stutzeri LMG 2243 reference strain xPseudoxanthomonas sp. R-24339 activated sludge x

EpsilonproteobacteriaArcobacter sp. R-28214 activated sludgeArcobacter sp. R-28313 activated sludgeArcobacter sp. R-28314 activated sludgeArcobacter sp. R-28316 activated sludge

FirmicutesBacillus sp. R-24620 activated sludgeBacillus sp. R-24666 activated sludgeBacillus sp. R-28399 activated sludgeBacillus sp. R-31541 soil xBacillus sp. R-31546 soilBacillus sp. R-31547 soil xBacillus sp. R-31550 soil xBacillus sp. R-31552 soilBacillus sp. R-31553 soilBacillus sp. R-31553 soilBacillus sp. R-31554 soilBacillus sp. R-31769 soil xBacillus sp. R-31770 soil xBacillus sp. R-31830 soil xBacillus sp. R-31832 soilBacillus sp. R-31834 soilBacillus sp. R-31835 soil xBacillus sp. R-31836 soilBacillus sp. R-31838 soilBacillus sp. R-31841 soil xBacillus sp. R-31845 soilBacillus sp. R-31846 soil xBacillus sp. R-31848 soilBacillus sp. R-31849 soilBacillus sp. R-31850 soilBacillus sp. R-31852 soil

Detection via PCR

ADDENDUM

Identificatie Strain nr. origin Detection via dot-blot

nirK nirS cnorB qnorB included norBBacillus sp. R-31855 soilBacillus sp. R-31856 soilBacillus sp. R-32516 soilBacillus sp. R-32517 soilBacillus sp. R-32518 soilBacillus sp. R-32519 soilBacillus sp. R-32521 soilBacillus sp. R-32522 soilBacillus sp. R-32523 soilBacillus sp. R-32524 soilBacillus sp. R-32525 soilBacillus sp. R-32526 soil xBacillus sp. R-32528 soilBacillus sp. R-32529 soilBacillus sp. R-32530 soilBacillus sp. R-32531 soilBacillus sp. R-32533 soilBacillus sp. R-32534 soilBacillus sp. R-32535 soilBacillus sp. R-32574 soilBacillus sp. R-32575 soilBacillus sp. R-32656 soil xBacillus sp. R-32661 soilBacillus sp. R-32662 soilBacillus sp. R-32663 soil xBacillus sp. R-32665 soilBacillus sp. R-32694 soil x xBacillus sp. R-32695 soilBacillus sp. R-32696 soilBacillus sp. R-32699 soilBacillus sp. R-32700 soil xBacillus sp. R-32702 soil xBacillus sp. R-32703 soilBacillus sp. R-32704 soilBacillus sp. R-32705 soil xBacillus sp. R-32706 soil xBacillus sp. R-32707 soilBacillus sp. R-32708 soilBacillus sp. R-32709 soil xBacillus sp. R-32713 soilBacillus sp. R-32715 soil xBacillus sp. R-32717 soilBacillus sp. R-32778 soilBacillus sp. R-32779 soil xBacillus sp. R-32781 soil xBacillus sp. R-32784 soilBacillus sp. R-32787 soilBacillus sp. R-32789 soil xBacillus sp. R-32844 soilBacillus sp. R-32845 soil xBacillus sp. R-32848 soilBacillus sp. R-32849 soil xBacillus sp. R-32851 soilBacillus sp. R-33773 soil xBacillus sp. R-33774 soilBacillus sp. R-33776 soilBacillus sp. R-33819 soilBacillus sp. R-33820 soil x

Enterococcus sp R-25205 activated sludge x xEnterococcus sp. R-24626 activated sludge x xPaenibacillus sp R-27048 activated sludge

Staphylococcus sp. R-25050 activated sludge xStaphylococcus sp. R-33771 soil xStaphylococcus sp. R-33777 soilStaphylococcus sp. R-34181 soil x ?Trichococcus sp. R-28212 activated sludge x

Virgibacillus halodenitrificans LMG 9818Treference strain x

BacteroidetesChryseobacterium sp. R-25053 activated sludge x

Detection via PCR

181

SUMMARY

Denitrification is a step-wise dissimilatory reduction of nitrate and/or nitrite over nitric

oxide and dinitric oxide, also named nitrous oxide, to nitrogen gas, coupled to electron

transport phosphorylation. This modular process is accomplished in four enzymatic steps,

catalyzed by four metalloproteins: nitrate reductase, nitrite reductase, nitric oxide reductase

and nitrous oxide reductase. The denitrification process is of major importance because it

causes fertilizer loss in agriculture, contributes to global warming through release of N2O,

and has an unclear role in bacterial pathogenicity, but it is also responsible for removal of

excess nitrogen from natural and wastewater systems, and can be useful for destruction of

other pollutants such as hydrocarbons.

With the advent of molecular techniques, focus of denitrification research shifted from

physiological and biochemical research on pure cultures to environmental monitoring of

the whole denitrification process in situ. However, the correlation between structural and

functional diversity remains a challenge, because denitrification is a phylogenetically

dispersed trait. Denitrification genes can be used as functional markers, but their taxonomic

information content is not clear, after several reports of differences between functional

gene phylogeny and organism phylogeny. Aim of this thesis was to investigate denitrification

starting from the functional bacterium, with focus on the genetic basis of the process. The

taxonomic value of the genes encoding the key enzymes was assessed, and their detection,

occurrence, prevalence and phylogeny were investigated.

Because most denitrification research was been performed on reference strains, new isolation

procedures from activated sludge and soil were performed. Therefore, sixty different defined

growth media were screened for their elective isolation of denitrifiers from activated sludge.

The influence of eleven medium parameters with different values and their combinations

on the number and the diversity of isolated denitrifiers were examined. All media were

investigated and scored for their selection of a high number and a high diversity of

denitrifying bacteria. The three best scoring media, G2M11, G3M12 and G4M3, together

with trypticase soy agar, were used to isolate denitrifiers from soil. From both inocula, a

higher cultivable denitrifying diversity was retrieved than previously reported in literature.

Also, both studies clearly demonstrated that use of a multiple-media set generated more

information than individual growth media.

The functional genes encoding the key enzymes nitrite and nitric oxide reductase, namely

nirS/nirK and cnorB/qnorB respectively, were investigated in a total number of 227 pure

culture denitrifiers from activated sludge and 112 pure culture denitrifiers from soil. The

183

functional genes were amplified in only a very low fraction of the pure cultures, 50% for

the nir genes and 30% for the norB genes in denitrifiers from activated sludge and even

less for both genes in cultures from soil. Both nir and norB genes showed high genetic

heterogeneity. Based on our data, Alphaproteobacteria mostly contained nirK and cnorB,

while nirS and both cnorB and qnorB were present in the Betaproteobacteria. Too few

representatives of the other bacterial classes were retrieved in the isolation studies to draw

conclusions on the functional gene prevalence.

For both gene types, overall gene phylogeny was not congruent with the widely accepted

organism phylogeny based on 16S rRNA gene sequence analysis. Tree topologies showed

that members of different bacterial classes grouped together. Nevertheless, dependent on

gene type and taxon, functional genes do contain limited taxonomic information. Grouping

based on nirS allowed rough delineation of bacterial taxa, while both norB genes did not

show any congruence with the organism phylogeny. The qnorB genes retrieved from several

denitrifying Bacillus isolates from soil grouped together with sequences from non-

denitrifiers, and clustered distant from qnorB sequences of other bacilli. This data indicates

that functional gene data cannot be generally used for deducing structural diversity

information in environmental studies. Also, comparative analysis of both nir and norB

genes showed that congruence between both gene types couldn’t be extrapolated to all

denitrifiers. Thus, linkage of both genes, either on the chromosome or on a plasmid, is not

a general feature in denitrifiers. Both gene types can be dispersed on the bacterial DNA,

and thus can cover independent evolutionary trajectories.

Next to the discussed incongruence between gene and organism phylogeny, some unexpected

observations were made. First, identical nirK and cnorB sequences were found in

representatives of different bacterial classes. Second, two isolates, Pseudomonas sp. R-

25208 and Bacillus sp. R-32694, contained both a cnorB and a qnorB gene. These are the

first reports of the presence of two different norB genes in one bacterium. Worth noting is

that the qnorB gene phylogeny matched the organism phylogeny in both organisms, while

the cnorB gene phylogeny did not. Third, the environment had a significant influence on

the nirK phylogeny, regardless of the taxonomical position of the denitrifiers. Fourth, the

detection of the functional genes within some taxa was strain-specific, suggesting significant

sequence divergence. All above-mentioned observations can be explained through horizontal

gene transfer (HGT) events of nir and norB between different taxa. However, these data

are only indirect evidence and further substantiation of the HGT theory is necessary.

Therefore, as a first step, we have been able to demonstrate that the cnorB and the nirK

SUMMARY

184

gene of the above-mentioned Pseudomonas sp. R-25208, which both did not match the

organism phylogeny, are located on a plasmid.

The functional genes were amplified in only a very low fraction of the pure cultures. A

possible explanation would be that applied primers and PCR protocols were developed

based on very limited sequences available at the time. Their use in a large strain set showed

their fallibility as broad-range amplification and detection tools. To assess whether the

very low detection percentage was only the result of non-optimal amplification, a dot-blot

method was developed to detect norB. Screening of 80 pure culture denitrifiers showed

that low norB amplification percentages could only be partially attributed to insufficient

primers and PCR protocols. More than half of the functional denitrifiers tested did not

give a positive result, which suggested the presence of unknown enzymatic redundancy

for nitric oxide reduction. Thus, denitrifiers can use enzymes other than those currently

known to reduce nitric oxide to nitrous oxide. More genome analysis and molecular work

is needed to identify those new genes/enzymes and to develop suitable detection tools.

The isolation campaign on activated sludge and soil rendered a large set of isolates,

containing bacteria possibly belonging to novel species or genera. Two new nitrate-reducing

Stenotrophomonas species and one new denitrifying Acidovorax species were described.

Although the denitrification trait is studied extensively, predominantly cultivation-

independently, little information was available on the ecology of the functional genes.

This study provides this much needed, scrutinized ecological information on the

denitrification genes, as well as new insights in the cultivation of denitrifiers, and can be

used as a starting point for further in-depth research on the denitrification process.

SUMMARY

185

SAMENVATTING

Denitrificatie is de stapsgewijze reductie van nitraat en/of nitriet over stikstofoxide en

distikstofoxide tot stikstofgas, gekoppeld aan electron transport fosforylatie. Dit modulair

proces wordt uitgevoerd door vier enzymatische stappen en gekatalyseerd door vier

metaalproteïnen, namelijk nitraatreductase, nitrietreductase, stikstofmonoxidereductase en

distikstofmonoxidereductase. Vanuit een antropogeen standpunt is denitrificatie

tegelijkertijd een negatief en positief proces. Het is namelijk verantwoordelijk voor verlies

van meststoffen in de landbouw, de productie van het broeikasgas N2O en heeft mogelijks

een rol in de bacteriële pathogeniciteit, maar kan ook zowel overtollig stikstof als andere

polluenten verwijderen uit natuurlijke en afvalwatersystemen.

Denitrificatie is sinds de ontdekking van het proces in 1882 reeds uitvoering onderzocht.

En met de ontwikkeling van moleculaire technieken is de focus van denitrificatieonderzoek

verschoven van fysiologisch en biochemisch onderzoek op reinculturen naar opvolging

van het volledige denitrificatieproces in situ. Desondanks blijft het correleren van structurele

en functionele diversiteit een grote uitdaging, meer bepaald door de wijdverspreidheid van

de denitrificatie capaciteit in de bacteriële wereld. Denitrificatiegenen zijn zeker nuttig als

functionele merkers, maar, na verscheidene berichten van incongruentie tussen functionele

gen fylogenie en organisme fylogenie, is het niveau van taxonomische informatie vervat in

deze genen onduidelijk. Daarom heeft deze thesis tot doel de genetische basis van

denitrificatie te onderzoeken, vertrekkend van de functionele bacteriële reincultuur. De

taxonomische waarde van de genen coderend voor de sleutelenzymen en hun detectie,

voorkomen, dominantie en fylogenie werden onderzocht.

Aangezien het meeste onderzoek op denitrificatie in het verleden is uitgevoerd op

referentiestammen, werden nieuwe cultivatiecampagnes opgestart met actief slib en bodem

als inoculum. Hiervoor werden zestig verschillende gedefinieerde groeimedia gescreend

voor de electieve isolatie van denitrificeerders uit actief slib. De invloed van elf

mediumparameters met verschillende waarden en hun combinaties werd nagegaan. Elk

medium kreeg een score op basis van het aantal denitrificeerders en hun diversiteit. In een

volgende studie werden de drie bestscorende media - G2M11, G3M12 en G4M3 – samen

met ‘trypticase soy agar’ gebruikt om denitrificerende bacteriën te isoleren uit bodem. De

denitrificerende isolaten omvatten een grotere diversiteit dan voordien vermeld in de

literatuur. Ook tonen beide studies duidelijk aan dat het gebruik van een multiple-media

set meer informatie genereert dan de applicatie van één medium.

De functionele genen coderend voor de sleutelenzymen nitrietreductase en

stikstofmonoxidereductase, namelijk nirS/nirK en cnorB/qnorB respectievelijk, werden

187

onderzocht in 227 denitrificerende reinculturen uit actief slib en 112 uit bodem. Deze

functionele genen konden slechts geamplificeerd worden in een lage fractie van de

stammenset, meer bepaald in 50% voor de nir genen en in 30% voor de norB genen in

denitrificeerders uit actief slib, en nog minder in reinculturen uit bodem. Beide gentypes

vertoonden hoge genetische heterogeniteit. Alphaproteobacteria bevatten vooral nirK en

cnorB, terwijl in Betaproteobacteria hoofdzakelijk nirS en zowel cnorB als qnorB aanwezig

waren. Onze stammenset bevatte te weinig representatieven van de andere bacteriële klassen

om conclusies te trekken rond het voorkomen van de functionele genen in deze taxa.

Voor beide gentypes was de totale gen fylogenie niet congruent met de algemeen aanvaarde

organisme fylogenie op basis van 16S rRNA gen sequentieanalyse. De clusteringen tonen

aan dat leden van verschillende bacteriële klassen samen groepeerden. Niettegenstaande

blijken de functionele genen wel beperkte taxonomische informatie te bevatten, afhankelijk

van het gen en het taxon. Groepering op basis van nirS laat toe om bacteriële taxa ruw af te

lijnen, terwijl beide norB genen geen enkele overeenstemming vertonen met de organisme

fylogenie. De qnorB genen van verscheidene denitrificerende Bacillus isolaten uit bodem

clusteren samen met sequenties van niet-denitrificeerders, en ver van de qnorB sequenties

van andere bacilli. Deze data tonen aan dat informatie rond functionele diversiteit uit

milieustudies niet algemeen kan gebruikt worden om de structurele diversiteit af te leiden.

Daarnaast blijkt uit vergelijkende analyses van nir en norB genen dat overeenstemming

tussen fylogenie van beide gentypes niet kan geëxtrapoleerd worden naar alle

denitrificeerders. Een koppeling tussen beide genen, of op het chromosoom of op een

plasmide, is dus geen algemeen kenmerk in denitrificeerders. Beide gentypes kunnen

verspreid voorkomen in het bacterieel DNA, waardoor ze onafhankelijke evolutionele

trajecten kunnen doorlopen.

Naast de bediscussieerde incongruentie tussen genen organisme fylogenie werden volgende

observaties gemaakt.

(i) Identieke nirK en cnorB sequenties werden teruggevonden in

representatieven van verschillende bacteriële klassen.

(ii) Twee isolaten, Pseudomonas sp. R-25208 en Bacillus sp. R-32694, bevatten

elk een cnorB en een qnorB gen. Dit is de eerste melding van een bacterie

met twee verschillende norB genen. Opvallend was dat de qnorB fylogenie

overeenstemde met de organisme fylogenie, wat niet het geval was voor

cnorB.

(iii) Het habitat had een significante invloed op de nirK gen fylogenie,

onafhankelijk van de taxonomische positie van de denitrificeerders.

SAMENVATTING

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(iv) De detectie van de functionele genen in sommige taxa was stamafhankelijk,

wat een significante sequentiedivergentie suggereert.

Het frequent voorkomen van horizontale gen transfers (HGT) van functionele genen tussen

verschillende taxa zou bovenstaande observaties verklaren. Echter, deze data zijn slechts

een indicatie en verdere bewijzen voor de HGT theorie zijn noodzakelijk. In een eerste

stap naar een bewijs hebben we kunnen aantonen dat cnorB en nirK van de hierboven

vernoemde Pseudomonas sp. R-25208, waarvan de fylogenie niet overeenstemde met de

organisme fylogenie, gelegen zijn op een plasmide.

Zoals reeds eerder vermeld, werden de functionele genen slechts geamplificeerd in een

kleine fractie van de stammenset. Een mogelijke verklaring hiervoor zijn de gebruikte

primers en PCR protocols, ontwikkeld op basis van de enkele gensequenties toen

beschikbaar. Hun gebruik in een grote stammenset toont nu aan dat ze als amplificatie- en

detectiemechanismen een beperkt bereik hebben. Om te bepalen of de lage

detectiepercentages enkel het gevolg zijn van niet-optimale amplificatie werd een dot-blot

methode ontwikkeld om norB te detecteren. Een screening van 80 denitrificerende

reinculturen toonde aan dat de lage norB amplificatie percentages slechts partieel te wijten

zijn aan ontoereikende primers en PCR protocols. Meer dan de helft van de functionele

denitrificeerders gaven geen positief resultaat, wat wijst op de aanwezigheid van extra,

ongekende enzymen voor stikstofmonoxidereductie. Dus, denitrificeerders kunnen

mogelijks andere dan de nu gekende enzymen gebruiken voor de reductie van

stikstofmonoxide naar distikstofmonoxide. Meer genoomanalyses en moleculair werk is

noodzakelijk om deze nieuwe genen/enzymen te identificeren en geschikte

detectietechnieken te ontwikkelen.

De grote stammenset, gegenereerd door de isolatiecampagnes met actief slib en bodem als

inoculum, bevat bacteriën behorende tot potentieel nieuwe species of genera. Twee nieuwe,

nitraatreducerende Stenotrophomonas species en één nieuwe denitrificerende Acidovorax

species werden reeds beschreven.

Hoewel denitrificatie al uitgebreid bestudeerd werd, vooral dan cultuuronafhankelijk, is er

weinig informatie beschikbaar over de ecologie van de betrokken functionele genen. Deze

PhD studie verschaft de hoogstnoodzakelijke ecologische informatie over

denitrificatiegenen, alsook nieuwe inzichten over de cultivatie van denitrificeerders, en

kan gebruikt worden als startpunt voor verder diepgaand onderzoek van het

denitrificatieproces.

SAMENVATTING

189

CURRICULUM VITAE

PERSONALIAFull name: Kim HeylenAddress: Haanstraat 11, Gent, BelgiumDate of birth: December 29th 1979Place of birth: Borgerhout, Belgium

EDUCATIONAL BACKGROUND2004 – present: Ghent University, Gent, BelgiumPhD studies at the Laboratory of Microbiology, Department of Physiology, Biochemistryand Microbiology (WE10), Faculty of Sciences

2002-2003: Ghent University, Gent, BelgiumMaster of Science in ‘Applied Microbial Systematics’ (graduated magna cum laude)Dissertation: ‘Characterization of chloramphenicol-resistant heterotrophic bacteria fromSoutheast Asian aquaculture environment’

1997-2001: University of Antwerp, Antwerp, BelgiumLicentiate Biology, option physiology (graduated cum laude)Dissertation: ‘Removal of dyes from textile waste water: isolation of dye-degrading bacteriaand their microbial ecology’, in collaboration with VITO (Flemisch Institute ofTechnological Research)

WORK EXPERIENCEFebruary 2002 – June 2002Fulltime Highschool teacher Biology/Maths in second grade at College O-L-V. Ten Doorn,Eeklo, Belgium

SCIENTIFIC OUTPUTA1 PublicationsVanparys B, Heylen K, Lebbe L, De Vos P (2005) Pedobacter caeni sp. nov., a novelspecies isolated from a nitrifying inoculum. Int J Syst Evol Microbiol 55:1315-1318

Vanparys B, Heylen K, Lebbe L, De Vos P (2005) Devosia limi sp. nov., a novel speciesisolated from a nitrifying inoculum. Int J Syst Evol Microbiol 55:1997–2000

Wittebolle L, Boon N, Vanparys B, Heylen K, De Vos P, Verstraete W (2005) Failure ofthe ammonia oxidation process in two pharmaceutical wastewater treatment plantsis linkedto shifts in the bacterial communities. J Appl Microbiol 99:997-1006

Heylen K, Gevers D, Vanparys B, Wittebolle L, Geets J, Boon N, De Vos P (2006) Theincidence of nirS and nirK and their genetic heterogeneity in cultivated denitrifiers. EnvironMicrobiol 8:2012-2021

Heylen K, Vanparys B, Wittebolle L, Verstraete W, Boon N, De Vos P (2006) Cultivationof denitrifying bacteria: optimization of isolation conditions and diversity study. ApplEnviron Microbiol 72:2637-2643

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Vanparys B, Heylen K, Lebbe L, De Vos P (2006) Pseudomonas peli sp. nov. andPseudomonas borbori sp. nov., isolated from a nitrifying inoculum. Int J Syst EvolMicrobiol 56:1875-1881

Heylen K, Vanparys B, Gevers D, Wittebolle L, Boon N, De Vos P. (2007) Nitric oxidereductase (norB) gene sequence analysis reveals discrepancies with nitrite reductase (nir)gene phylogeny in cultivated denitrifiers. Environ Microbiol 9:1072-1077

Vanparys B, Spieck E, Heylen K, Wittebolle L, Geets J, Boon N, De Vos P (2007) Thephylogeny of Nitrobacter based on comparative rep-PCR, 16S rRNA and nitriteoxidoreductase gene sequence analysis. Syst Appl Microbiol 30:297-308

Verstraete W, Wittebolle L, Heylen K, Vanparys B, De Vos P, Van de Wiele T, Boon N(2007) Microbial Resource Management: The Road to go for Environmental Biotechnology.Engineering in Life Sciences 7:117-126

Geets J, de Cooman M, Wittebolle L, Heylen K, Vanparys B, De Vos P, Verstraete W,Boon N (2007) Real-time PCR assay for the simultaneous quantification of nitrifying anddenitrifying bacteria in activated sludge. Appl Microbiol Biotechnol 75:211-221

Heylen K, Vanparys B, Peirsegaele F, Lebbe L, De Vos P (2007) Stenotrophomonas terrae,sp. nov. and Stenotrophomonas humi , sp. nov., two novel nitrate-reducingStenotrophomonas species isolated from soil. Int J Syst Evol Microbiol 57: 2056-2061

Heylen K, Lebbe L, De Vos P (2007) Acidovorax caeni sp. nov., a novel denitrifyingspecies with genetically diverse isolates from activated sludge. Int J Syst Evol Microbiol,Accepted.

Heylen K, Boon N, Verstraete W, De Vos P (2007) Functional gene study on heterotrophicsoil denitrifiers isolated through a cultivation strategy on soil. Microb Ecol, submitted.

Oral presentationsHeylen K, Vanparys B, Wittebolle L, Boon N, Verstraete W, De Vos P (2005) Diversitystudy of denitrifying bacteria in activated sludge: a culture-dependent approach. COSTAction 856 – Ecological aspects of denitrification, with emphasis on agriculture, April6th-10th, Legnaro (Padova), Italy.

Heylen K, Lebbe L, De Vos P (2007) New insights in the genetic basis of denitrification.BSM symposium – Evolution in the Microbial world, November 23rd, Brussels, Belgium.

Heylen K, Lebbe L, De Vos P (2007) New insights in the genetic basis of denitrification.COST Action 856 - Denitrification ans related aspects, December 5th - 8th, Uppsala,Sweden.

Poster presentationsHeylen K, Vanparys B, Boon N, Verstraete W, De Vos P (2004) Culture-dependent studyon the diversity of the denitrifying microbial community in activated sludge. BSMSymposium – Crossroads of microbiology and informatics, December 3rd, Brussels,Belgium.

Heylen K, Vanparys B, Wittebolle L, Boon N, Verstraete W, De Vos P (2005) Developmentof selective growth media for denitrifying bacteria using an evolutionary algorithm: astrategy outline. ASPD4 – Microbial population dynamics in biological wastewatertreatment, July 17th-20th, Gold Coast, Queensland, Australia.

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Heylen K, Vanparys B, Wittebolle L, Verstraete W, Boon N, De Vos P (2005) Developmentof selective growth media for denitrifying bacteria using an evolutionary algorithm. BSMsymposium – Imaging technology in microbiology cytometric and molecular approaches,November 18th, Brussels, Belgium.

Heylen K, Gevers D, Vanparys B, Wittebolle L, Geets J, Boon N, De Vos P (2006) Theincidence of nirS and nirK and their genetic heterogeneity in cultivated denitrifiers. ISME-11 – The hidden powers – Microbial communities in action, August 20th-25th, Vienna,Austria.

Heylen K, Vanparys B, Boon N, Verstraete W, De Vos P (2006) Bacterial diversity in soilinvolved in the removal of nitrogen demonstrates a high dependence of nitrate and/ornitrite reduction on growth conditions. BSM symposium – Novel compounds and strategiesto combat pathogenic microorganisms, November 24th, Brussels, Belgium. Second nominee.

Heylen K, Boon N, Verstraete W, De Vos P (2007) Validation of previously developedgrowth media for isolation of the denitrifying bacterial diversity from soil.Doctoraatssymposium UGent, April 24th, Gent, Belgium. Second nominee.

Heylen K, De Vos P (2007) Development of a Detection Procedure for DenitrificationGenes Not Amplified by Currently Available PCR Approaches. The 107th General Meetingof the Amercian Society of Microbiology, May 21th - 25th , Toronto, Canada.

Study of the genetic basis of denitrification in pure culture denitrifiers ...

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