ORIGINAL ARTICLE

Isolation and characterization of lownucleic acid (LNA)-content bacteria

Yingying Wang1,2, Frederik Hammes1, Nico Boon3, Mohamed Chami4 and Thomas Egli1,2

1Department of Environmental Microbiology, Eawag, Swiss Federal Institute of Aquatic Science andTechnology, Dubendorf, Switzerland; 2Institute of Biogeochemistry and Pollutant Dynamics, ETH Zurich,Zurich, Switzerland; 3Ghent University, Faculty of Bioscience Engineering, Laboratory of Microbial Ecologyand Technology (LabMET), Gent, Belgium and 4ME Muller Institute for Structural Biology, Biozentrum,University of Basel, Basel, Switzerland

Most planktonic bacteria are ‘uncultivable’ with conventional methods. Flow cytometry (FCM) is oneapproach that has been taken to study these bacteria. In natural aquatic environments, bacteria withhigh nucleic acid (HNA) and low nucleic acid (LNA) content are commonly observed with FCM afterstaining with fluorescent dyes. Although several studies have focused on the relative abundanceand in situ activities of these two groups, knowledge on the growth of particularly LNA bacteria islargely limited. In this study, typical LNA bacteria were enriched from three different freshwatersources using extinction dilution (ED) and fluorescence-activated cell sorting (FACS). We haveshown for the first time that LNA bacteria can be isolated and cultivated by using sterile freshwateras a growth medium. During growth, the typical LNA characteristics (that is, low-fluorescenceintensity and sideward scatter (SSC)) remained distinct from those of typical HNA bacteria. ThreeLNA pure cultures that are closely affiliated to the Polynucleobacter cluster according to 16S rRNAsequencing results were isolated. Owing to their small size, cells of the isolates remained intactduring cryo-transmission electronic microscopy examination and showed a Gram-negative cell-wallstructure. The extremely small cell volume (0.05 lm3) observed for all three isolates indicates thatthey are among the smallest free-living heterotrophic organisms known in culture. Their isolationand cultivation allow further detailed investigation of this group of organisms under definedlaboratory conditions.The ISME Journal (2009) 3, 889–902; doi:10.1038/ismej.2009.46; published online 7 May 2009Subject Category: microbial population and community ecologyKeywords: AOC; cryo-TEM; DGGE; FACS; low nucleic acid (LNA) bacteria; extinction dilution

Introduction

Planktonic bacteria are ubiquitous in aquatic envir-onments and an average density of 106–107 cells ml–1

is typically observed in surface freshwater(Whitman et al., 1998; Wang et al., 2007). Not onlyare these organisms present in large numbers, butthey also play a critical role in the turnover oforganic matter and nutrient cycling in aquaticenvironments (Billen et al., 1990). However, a largefraction of these planktonic cells are difficult tostudy outside of their natural habitats. Owing to thelow-nutrient concentrations that prevail in fresh-water environments, these cells are typically smaller,lower in cell number and less active than cellsgrown under laboratory conditions, and hence pose

several difficulties to conventional cultivation andanalysis methods. Microbiologists have used var-ious different names and terminologies to classifyaquatic bacteria. Terms like ‘oligotrophs’, ‘viable-but-not-cultivable (VBNC) bacteria’, ‘ultramicrobac-teria’ and ‘uncultivable bacteria’ have all been usedto describe various bacterial groups (for example,Oliver, 1993; Morita, 1997; Schut et al., 1997;Kajander and Ciftcioglu, 1998). With the increasinguse of flow cytometry (FCM) as a tool in aquaticmicrobiology research, two new terms have beenassigned to planktonic bacteria visualized using thisparticular method. It started with the names of‘Group I cells’ and ‘Group II cells’ proposed by Liand co-workers (1995), which were then renamed to‘low-DNA (LDNA) bacteria’ and ‘high-DNA (HDNA)bacteria’, respectively, by Gasol et al. (1999), andagain later modified to ‘low-nucleic acid content(LNA) bacteria’, and ‘high-nucleic acid content(HNA) bacteria’ by Lebaron et al. (2001). The terms‘LNA’ and ‘HNA’ bacteria have since then been mostwidely used by researchers. This broad classifica-tion of the two groups is based on their distinctly

Received 12 November 2008; revised 2 April 2009; accepted 2April 2009; published online 7 May 2009

Correspondence: T Egli, Department of Environmental Micro-biology, EAWAG, Swiss Federal Institute of Aquatic Science andTechnology, Uberlandstrasse 133, PO Box 611, DubendorfCH-8600, Switzerland.E-mail: egli@eawag.ch

The ISME Journal (2009) 3, 889–902& 2009 International Society for Microbial Ecology All rights reserved 1751-7362/09 $32.00

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different fluorescence intensity and sideward scatter(SSC) signals detected by FCM in combination withnucleic-acid stains (see Figure 1a; Gasol et al., 1999;Lebaron et al., 2001). Fluorescence intensity, in thisrespect, is used as an indicator of apparent cellularnucleic-acid content (Gasol et al., 1999; Lebaronet al., 2001) and SSC signals have been applied as anindication of cellular size (Lebaron et al., 2001;Felip et al., 2007). This classification is essentiallymethod-specific; up to now, no other method hasspecifically recognized these two distinct groups.Nonetheless, numerous studies were published onthe topic of LNA and HNA bacteria, focussingmainly on the relative abundance of LNA andHNA bacteria in aquatic environments, their in situactivities and phylogenetic identity (for example,Jellett et al., 1996; Marie et al., 1997; Zubkov et al.,2001; Lebaron et al., 2002; Jochem et al., 2004;Longnecker et al., 2005).

A review of these studies shows clear contra-dictions and controversies in many crucial points.

High-nucleic acid bacteria are usually regarded asthe active part of the microbial metabolic group,whereas LNA bacteria are considered inactive(Lebaron et al., 2001; Lebaron et al., 2002; Servaiset al., 2003; Tadonleke et al., 2005). However, thereare contrasting reports showing that LNA bacteriaare an active part of microbial communities inseawater (Zubkov et al., 2001, 2005) and in fresh-water (Nishimura et al., 2005). This already posesthe essential question of whether LNA bacteriarepresent a unique group of organisms, or whetherthey are merely a different physiological state ofHNA cells, or even a combination of both (Bouvieret al., 2007). In addition, all attempts to describe thephylogenetic differences between the two groupshave so far resulted in contradictions. Someresearchers reported that both groups are composedof the same dominant species (Servais et al., 2003),whereas others have argued that they are phylogen-etically different (Zubkov et al., 2002). Furthermore,scenarios have been proposed in which bacterialcells may switch their phenotype from one group tothe other (Bouvier et al., 2007). These problems andcontradictions stem at least partially from the factthat no LNA bacteria have been isolated until now.Having such isolates would facilitate a better under-standing of the nature and importance of thesebacteria.

In this study, we tested the hypothesis that LNAbacteria represent a unique and viable fraction ofindigenous aquatic microbial communities. Thespecific objectives of this study were to (1) showthe universal presence of LNA bacteria in fresh-water; (2) show that LNA bacterial cells are able togrow under laboratory conditions without changingtheir unique LNA characteristics (that is, low-fluorescence intensity and low-SSC signal); and(3) isolate, cultivate and characterize bacterial commu-nities/strains that maintain unique LNA character-istics throughout cultivation and that can be usedfor further studies. The experimental approach andresults presented in this study may open a new pageon the investigation and understanding of indigen-ous planktonic bacteria in aquatic environments.

Materials and methods

Source watersFreshwater samples were collected in autoclavedSchott bottles (0.5 l) from six different water sources:Chriesbach stream (Dubendorf, Switzerland), LakeGreifensee (Uster, Switzerland), a small alpinestream (Luzern, Switzerland), non-chlorinatedtap water (Dubendorf, Switzerland), groundwater(Vittel, France) and wastewater treatment planteffluent (Dubendorf, Switzerland). The temperatureof the source waters was in the range of 4–12 1C atthe time of sampling. All samples were kept below8 1C during transportation and were processedwithin 24 h after sampling. Samples from all source

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Figure 1 FCM dot-plots of the total bacterial community (a) andLNA bacterial enrichment culture (b) from stream water; the totalbacterial community (c) and LNA bacterial enrichment culture(d) from lake water; the total bacterial community (e) and LNAbacterial enrichment culture (f) from wastewater effluent. Thesolid line indicates LNA bacteria and the dashed line HNAbacteria.

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waters were analyzed using FCM to determine thetotal-cell concentration and specific-cell concentra-tions for LNA and HNA bacteria separately (asdescribed below).

Flow cytometryFlow cytometric measurements were carried out asdescribed by Hammes et al. (2008). Bacterial cellswere stained with 10 ml ml–1 SYBR Green I (1:100dilution in dimethyl sulfoxide; Molecular Probes,Carlsbad, CA, USA), and incubated in the dark for15 min at room temperature before measurement.Flow cytometry was carried out using a CyFlowSpace instrument (Partec, Hamburg, Germany)equipped with a 200 mW laser, emitting at a fixedwavelength of 488 nm, and volumetric countinghardware. Green fluorescence was collected at 520(±20) nm, red fluorescence was collected above630 nm and all data were analyzed with theFlowmax software (Partec). All samples were pro-cessed at a speed of 200 ml min–1. Where necessary,samples were diluted before measurements incell-free water, so that the concentration measuredin the flow cytometer was always o500 events sec–1.The specific instrumental gain settings for thesemeasurements were as follows: green fluores-cence¼ 450, red fluorescence¼ 550, SSC¼ 300. Thissetting was used for all experiments in this study.LNA and HNA cell populations were gated on thetwo-parameter dot-plot of green fluorescence(520 nm) against sideward scatter (SSC) and countedseparately (see Figure 1). Geometrical mean (GMean)values for green fluorescence (520 nm) and SSC forboth the LNA and HNA bacteria were recorded. Allsamples were measured in triplicate. The standardinstrument error on FCM measurement was alwaysbelow 3%. The detection limit of the instrumentused in this study was about 200 cells ml–1 (Hammeset al., 2008).

Continuous cultivation of an indigenous LNA bacterialcommunityIndigenous LNA and HNA bacterial communitiesfrom stream water (Chriesbach stream) were sepa-rated by 0.45 mm filtration (see Supplementaryinformation), and the resulting crude LNA fractionwas used to inoculate 0.2 l bioreactors for contin-uous cultivation (Figure 2). Autoclaved and filtered(0.22 mm) stream water (from the same source) was

supplied as growth medium (average dissolvedorganic carbon (DOC)¼ 3 mg l–1; assimilable organiccarbon (AOC)¼ 300 mg l–1), and the reactors wereoperated at 12.0±0.1 1C for 150 h at a dilution rate of0.08 h�1. The culture was continuously mixed at thespeed of 100 r.p.m. The theoretical washout line wascalculated as follows (Wick et al., 2002):

ct ¼ c0�e�D�t ð1Þwhere ct is cell concentration in the reactor at time t,c0 is the initial cell concentration at time 0 and D isthe dilution rate.

Samples from the reactor were taken at regulartime intervals (every 10–20 h) and analyzed withFCM as described above. No substantial wall growthwas observed in the reactor. The sterility of themedium was checked every 24 h with FCM.

Enrichment and isolation of LNA bacteriaLNA bacteria were enriched from three differentsource waters (Chriesbach stream, Lake Greifenseeand wastewater effluent). Source waters weresampled with a 1-litre Duran flask (Schott,Germany), pasteurized (30 min, 60 1C) and filteredthrough 0.1-mm-pore-size sterile syringe filters(PVDF, Millipore, Billerica, MA, USA) to removemost particles. The filters were washed with at least200 ml of carbon-free water before use to eliminateresidual organic carbon (Vital et al., 2008). Thissterile freshwater from each source was then used asthe growth medium for the enrichment experiments.LNA bacterial communities from the three sourcewaters were obtained by separation of LNA andHNA bacterial fractions with 0.45 mm filtration (seeSupplementary Figure 1). The cells passing throughthis filter were used to inoculate further enrichmentexperiments using either the extinction-dilution(ED) technique or fluorescence-activated cell sorting(FACS) (Figure 2). Extinction-dilution experimentswere carried out in carbon-free glass vials (asdescribed in Supplementary information) contain-ing 15 ml of sterile freshwater. The vials wereinoculated with the filtrate (after determinationof the cell concentration by FCM) to final cellconcentrations of 100 cells ml–1, 10 cells ml–1, and1 cell ml–1, respectively. For each dilution, 24 vialswere prepared (including three as blanks). Eachinoculum was cultivated in the source water fromwhere it originated. All the cultures were incubatedat 20 1C for 14 days, which was sufficient to attain

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Figure 2 Experimental flow chart, indicating the terminology used and experiments carried out in the current study. FACS;fluorescence-activated cell sorting; ED; extinction dilution.

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stationary phase, if growth occurred. After such agrowth cycle, 1 ml samples were withdrawn fromeach vial of all dilution series and analyzed usingFCM as described above. Cultures containing onlyLNA bacteria were selected for further ED andre-growth experiments. Such cycles were repeateduntil a stable LNA enrichment culture (that is,the culture that maintains the typical LNA char-acteristics) was obtained. In an alternative approachto obtain LNA enrichment cultures, cell sorting wasemployed using a BD FACS Aria instrument (BectonDickinson, Franklin Lakes, NJ, USA). Samples werestained with 10ml l–1 SYBR Green I (MolecularProbes). LNA bacteria from three different sources(Chriesbach stream, Lake Greifensee and wastewatereffluent) were sorted into 96-well plates (50 cellsper well) containing 150 ml sterile source water(as described above) in each well. For each watersource, three plates were used. After sorting, theplates were closed with their matching plastic coverand sealed with para-film to avoid evaporation. Allthe cultures were incubated at 20 1C for 14 days untilstationary phase was reached. After incubation, asample (50 ml) was withdrawn from each well andanalyzed with FCM. Cultures containing only typicalLNA bacteria were selected for further investigation.LNA enrichment cultures (obtained by either ED orFACS) were used for a first investigation of thegrowth properties of typical LNA bacteria and furtherisolation of LNA pure cultures (Figure 2). For thefinal step of achieving LNA pure cultures from theenrichment cultures, further ED experiments werecarried out with initial concentrations of 1 cell pervial (as described above). The terms used to describethe cultures in each step and the experimental designare illustrated in Figure 2.

Batch growth characterization of LNA enrichmentculturesThree LNA enrichment cultures, obtained fromthree separate sources (Chriesbach stream, LakeGreifensee and wastewater effluent), were cultivatedin sterile cell-free stream water (Chriesbach, Duben-dorf) (DOC¼ 2.6–3.3 mg l–1; AOC¼ 200–400mg l–1;pH¼ 7.7–7.9; conductivity¼ 402–502 mS). The watersample preparation procedure was described above.The initial cell concentration after inoculation was1� 104 cells ml–1 at all temperatures ranging from 12to 37 1C in 20 ml carbon-free vials (with 15 mlmedium; as described in Supplementary informa-tion). Aliquots (1 ml) were taken at regular timeintervals (t¼ 0, 15, 24, 39, 68, 89, 95, 112, 119 and138 h for cultivation at 12 1C and t¼ 0, 15, 23, 15, 39,41, 47 and 68 h for cultivation at 20, 25, 30 and37 1C) until stationary phase was reached. Sampleswere analyzed with FCM and for adenosine tri-phosphate (ATP) (as described in Supplementaryinformation). All experiments were carried out intriplicate and without shaking. The specific growthrate (m) based on cell number increase in each

sample was determined as follows (Equation 2)(Vital et al., 2007; Wang et al., 2007):

m ¼ ðLnðntÞ � Lnðn0ÞÞ=Dt ð2Þwhere nt and n0 are the cell concentrations mea-sured at two subsequent time points and Dt is thetime interval between these points.

Comparison of batch culture growthAn enrichment of an indigenous bacterial commu-nity (Chriesbach stream, Dubendorf) that is normallyused for the determination of AOC (Hammes andEgli, 2005; Vital et al., 2007) was used as thereference HNA enrichment culture (see Supplemen-tary Figure 2). A basic characterization of this HNAenrichment culture was documented earlier (Wanget al., 2007). The HNA enrichment culture was usedfor a comparison with the growth characteristics ofthe LNA enrichment cultures. Both HNA and LNAenrichment cultures were cultivated in sterile cell-free stream water (Chriesbach, Dubendorf) (asdescribed above). The initial cell concentrationwas 1� 104 cells ml–1 and the initial medium volumewas 15 ml. The comparison was carried out at 20 1Cwithout shaking. Samples (1 ml) were taken every2–10 h for FCM (as described above) and ATPdetermination (see Supplementary information).Average cell sizes were estimated from the GMeanSSC values recorded using FCM (as described inSupplementary information).

Microbial community analysisCatalyzed reporter deposition–fluorescence in situhybridization (CARD–FISH) was carried out on LNAenrichment cultures according to the modifiedpermeabilization protocol developed for freshwaterbacterioplankton (Sekar et al., 2003). The probesfor bacteria, b-proteobacteria, a-proteobacteria, cyto-phaga–flavobacterium–bacteroides, and actinobac-teria were used in the current study (Warnecke et al.,2005). Details of the procedure are documented byPernthaler and Pernthaler (2007). Counterstaining ofCARD–FISH preparations with 4,6-diamidino-2-phenylindole (DAPI) and mounting were carriedout as described earlier (Pernthaler et al., 2002).Total bacterial abundances and the fractions ofFISH-stained bacteria in at least 1000 DAPI-stainedcells per sample were quantified at a 1000-foldmagnification using an epi-fluorescence microscope(Leica, Wetzlar, Germany).

Total DNA was extracted from the three enrich-ment cultures and the corresponding source watersamples using the UltraClean Water DNA IsolationKit (Mo Bio, Carlsbad, CA, USA). Total bacterialcommunity analysis was carried out by PCR-DGGE,using general bacterial primers P338F and P518r(Boon et al., 2002). The processing of the DGGE gelswas carried out with the Bionumerics software 2.0(Applied Maths, Kortrijk, Belgium). The calculationof the dendrogram is based on the Pearson (product–

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moment) correlation coefficient and the clusteringalgorithm of Ward (1963). Selected bands from theDGGE gel for the LNA enrichment cultures were cutout with a clean scalpel and added in 50 ml of PCRwater. After 12 h of incubation at 4 1C, 1 ml of the PCRwater was re-amplified with primer sets P338F andP518r (Boon et al., 2002). Five ml of the PCR productwas loaded on a DGGE gel (see above) and if theDGGE pattern only showed one band, it was sent outfor sequencing. In case the DGGE band was not pureenough for sequencing, PCR fragments were clonedby using the TOPO TA cloning kit (Invitrogen,Carlsbad, CA, USA) according to the manufacturer’sinstructions. DNA sequencing of the ca. 180 bpfragments was carried out by ITT Biotech-Bioservice(Bielefeld, Germany).

Rep-PCR genomic fingerprinting was carried outto discriminate the different isolates on a strainlevel, using the BOX-primers reported by Versalovicet al. (1994), and the BOX patterns were analyzed byTBionumerics software 2.0 (Applied Maths) asdescribed above. Genomic DNA of LNA pure cul-tures was extracted (as described above), amplifiedand sequenced (see Supplementary information).

Scanning/transmission electron microscopyLNA pure cultures were examined using both scan-ning electron microscopy (SEM) (see Supplementaryinformation) and cryo-transmission electron micro-scopy (cryo-TEM). For cryo-TEM, a 4ml aliquot ofconcentrated LNA pure culture was adsorbed ontoan entirely carbon-coated grid (quantifoil, Jena,Germany), blotted with Whatman 4 filter paper andvitrified in liquid ethane at �178 1C. Frozen gridswere transferred onto a Philips CM200-FEG electronmicroscope (Philips, FEI, Eindhoven, The Nether-lands) using a Gatan 626 cryo-holder. Electronmicrographs were recorded at an accelerating voltageof 200 kV and a magnification of � 50 000, using alow-dose system (10e� A–2) and keeping the sample at�175 1C. Defocus values were �3mm. Micrographswere recorded on Kodak SO-163 films and thendigitized with Heidelberg Primescan 7100 (Heidel-berg Primescan, Heidelberg, Germany) at 4 A per pixelresolution at the specimen level.

The cell volume of the individual bacterial cell wascalculated based on the measurements from cryo-TEMpictures (Equation 3, Wang et al., 2008) on theprinciple of a rod-shaped particle with spherical ends.

Cell volume ðmm3Þ ¼ 4

3�p�r3 þ p�r2�ðl � 2rÞ ð3Þ

where r represents half of the smallest width and lrepresents the length of the bacterial cell.

Results

Presence of LNA bacterial communities in differentaquatic environmentsTypical LNA and HNA bacterial communities wereobserved in all aquatic samples tested, ranging from

an extremely oligotrophic (alpine stream,DOC¼ 0.5 mg l–1) to a relatively copiotrophic envir-onment (wastewater effluent, DOC¼ 7.0 mg l–1). Thetwo communities are clearly distinguishable onFCM dot-plots of green fluorescence against side-ward scatter (SSC) (Figure 1). LNA bacteria werenumerically dominant (450%) in the planktonicmicrobial communities from most sampled fresh-waters, and there was no relation between theirrelative abundance and either the total bacterialabundance or the DOC content of the water (Table 1).

Continuous cultivation of an indigenous LNAbacterial communityA rough separation of HNA/LNA bacterial commu-nities was achieved through 0.45 mm filtration,which retained about 90% of the HNA bacteria,while allowing most of the LNA bacteria to pass(Figure 2, Supplementary Figure 1). In a firstexperiment to test the viability of these separatedLNA bacterial communities, we applied the above-mentioned filtration step and inoculated the filtrateinto sterile river water. Growth of an indigenousLNA bacterial community was tested using contin-uous cultivation at a low dilution rate in sterilestream water at 12 1C, selected because of the lowtemperature (10–12 1C) recorded in the source water.The LNA bacterial community established in thecontinuous culture at a ‘steady-state’ level of1.2� 105 cells per ml over 150 h at a dilution rateof 0.08 h�1 (Figure 3). If all the cells in the LNAbacterial group were inactive or dead, they wouldhave washed-out from the reactor under the con-tinuous cultivation conditions used (Figure 3). Dur-ing the entire period of continuous cultivation, theLNA bacterial community maintained its typicalLNA characteristics, that is, low fluorescence in-tensity and low SSC signal (Figures 3b and c).

Enrichment and cultivation of LNA bacteriaTypical LNA bacteria were enriched and cultivatedfrom three different freshwater samples with a batchgrowth assay using natural AOC from sterile fresh-

Table 1 Percentage of LNA bacteria in total bacterial commu-nities from different aquatic environments

Source DOC(mg l–1)

Total cellconcentration(106 cells ml–1)

Percentageof LNA

Chriesbach stream 3.0±0.3 3.21±0.20 68%±2%Alpine stream 0.5±0.1 0.10±0.00 74%±5%Tap water 0.7±0.1 0.15±0.01 53%±1%Groundwater 0.5±0.2 0.31±0.02 75%±3%Lake Greifensee 3.2±0.3 2.41±0.23 23%±5%Wastewater effluent 7.0±0.5 10.6 ±1.20 58%±2%

Results are shown in the format of average±standard deviationcalculated from at least three samples for each source location.

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water (Hammes and Egli, 2005; Vital et al., 2007;Wang et al., 2007) in combination with FACS or theED technique. The FACS/ED steps were essential, aswe have observed that during a direct batchcultivation of indigenous bacterial communities,HNA bacteria always overgrew LNA bacteria (datanot shown). Using FACS, 1 out of 24 samples fromthe Chriesbach stream showed positive growth ofLNA bacteria after 14 days incubation (at least105 cells ml–1). However, no positive results wereobtained for the samples from Lake Greifensee andthe wastewater effluent. With the ED approach, allwaters gave about three positive results out of 21assays when inoculated with 10 cells ml–1. However,

all samples inoculated with 100 cells ml–1 showedeither HNA bacterial growth or a mixture of LNAand HNA bacteria. The positive LNA samples werere-inoculated (10 cells ml–1) into sterile naturalfreshwater and incubated for another 14 days. Suchcycles were repeated until a stable LNA bacterialenrichment was achieved. Three enrichment cul-tures were obtained (Figure 1) from different aquaticenvironments. They are referred to as LNA enrich-ment culture A (enriched from the Chriesbachstream by FACS; Figure 1b), LNA enrichmentculture B (enriched from Lake Greifensee by ED;Figure 1d) and LNA enrichment culture C (enrichedfrom wastewater effluent by ED; Figure 1f). Theirpositions on FCM dot-plots correspond to theoriginal LNA fraction in the water from which theywere enriched (Figure 1 and Table 2).

After successful enrichment, the growth proper-ties of the different LNA enrichment cultures werefurther characterized during batch cultivation. Allthree LNA enrichment cultures were capable ofgrowing on natural AOC at different temperatures,while maintaining their typical LNA characteristics.Growth of LNA enrichment cultures was observedfrom 12 to 30 1C and a typical example for culture Ais shown in Figure 4. Generally, a longer lag phasewas observed for the growth at 12 1C compared withthat at higher temperatures. The maximum specificgrowth rate (mmax) increased with ascending incuba-tion temperature up to 30 1C, whereas no growth wasdetected at 37 1C. The recorded mmax values were asfollows: 0.10 h�1 (12 1C); 0.23 h�1 (20 1C); 0.31 h�1

(25 1C); and 0.37 h�1 (30 1C). Irrespective of thecultivation temperature, all LNA enrichment cul-tures reached similar final cell concentrations whencultivated in the same water (Figure 4).

Comparison between batch growth of LNA and HNAenrichment culturesDuring batch growth on sterile stream water, LNAand HNA enrichment cultures exhibited distinctcharacteristics. LNA and HNA enrichment culturesobtained from the same source water (Chriesbachstream) were compared for their growth propertieswith natural AOC at 20 1C. Similar lag phases wereobserved for both LNA and HNA enrichment

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Table 2 Growth characteristics of three separately isolated LNA enrichment cultures and a HNA enrichment culture in sterile freshwater(see Materials and methods)

mmax Green fluorescence SSC Estimated cell volume Cellular ATP ATP per volume(h�1) (GMean) (GMean) (mm3) (�10�17 gATP per cell) (�10�15 gATP per mm3)

LNA A 0.23±0.06 7.4±0.6 4.6±0.3 0.01±0.01 1.2±0.3 1.2±0.2LNA B 0.19±0.05 5.9±0.6 5.1±0.5 0.02±0.02 3.2±0.4 1.6±0.3LNA C 0.24±0.06 8.2±0.6 4.1±0.4 0.01±0 1.5±0.3 1.5±0.3HNA 0.31±0.08 40.7±1.7 11.6±0.9 0.13±0.02 15.5±2.6 1.2±2.6

GMean values of green fluorescence and SSC are data of stationary phase cultures. Results are shown in the format of average±standard deviation,calculated from triplicate samples (n¼ 3) from each culture. Cell volume was estimated from the SSC value of the enrichment cultures (Equation S1).

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cultures (Figure 5a). The lag phase was followed bya short phase of exponential growth where the cellsdivided at a maximum rate for 2–3 generations, andthen the rate gradually decreased until stationaryphase was reached (Figure 5b). The maximumspecific growth rate (mmax) was higher for the HNAenrichment cultures (0.31 h�1) than that of the LNAenrichment cultures (0.23 h�1) (Figure 5b, Table 2).The HNA enrichment culture grew to a finalconcentration of 3� 106 cells ml–1 and attained sta-tionary phase after 60 h. The LNA enrichmentculture grew only to half this density (1.6� 106

cells ml–1) and reached stationary phase alreadyafter 42 h (Figure 5a). The average cell size estimatedfrom SSC values for the HNA enrichment cultureshowed an 18-fold change during the batch cultiva-tion. In comparison, the change in cell size for theLNA enrichment cultures was much smaller (only4-fold); however, a similar trend was observed(Figure 5a). During batch cultivation, the patternsin the specific growth rates were similar for LNAand HNA enrichment cultures (Figure 5b). The totalATP concentrations of both the LNA and HNAcultures increased concurrent to the total cellconcentrations (Figure 5b). In general, the ATPconcentration of LNA enrichment cultures wasmuch lower than that of HNA enrichment cultures(0.04 vs 0.75 nM ATP at stationary phase), which canbe directly attributed to the lower cell density andsmaller apparent cell volume (Figure 5b and Table 2).

Phylogenetic diversity of LNA enrichment culturesUsing a dual approach of DGGE and CARD-FISH, wewere able to pinpoint the identity of the three LNAenrichment cultures. The DGGE analysis showedthe presence of multiple bands, with one or twodominant bands in all three LNA enrichmentcultures (Figure 6a). However, the dominant bands

in the LNA enrichment cultures (circled inFigure 6a) do not match the dominant ones in thetotal bacterial community, from where they origi-nated (Figure 6a). Such results make sense in viewof the highly selective procedures we carried out toenrich the LNA cultures. The dominant bands fromeach enrichment culture were further sequencedand compared with the sequences obtained fromLNA isolates (see below). The CARD-FISH resultscorroborated the DGGE results. Most cells of theLNA enrichment cultures were affiliated to theb-proteobacteria cluster (80% for enrichment cul-ture A, 72% for B and 88% for C). No a-proteobac-teria were observed in all LNA enrichment cultures,but about 6% of enrichment culture B belonged tothe Gram-positive domain Actinobacter and 3% ofenrichment culture C hybridized with the Cytopha-ga–Flavobacterium probe. These results, especiallythe DGGE pattern, indicate that the enrichmentcultures are dominated by one or two species.Hence, further purification steps were carried outto obtain LNA pure cultures.

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Figure 5 Growth comparison between LNA and HNA enrich-ment cultures. Cell concentration and size changes (a), andchanges in specific growth rate and ATP concentration (b) duringbatch cultivation in sterile stream water at 20 1C. Error barsrepresent the s.d. for triplicate samples. Cell volume wasestimated from the SSC value of the enrichment cultures(Equation S1).

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Isolation, cultivation, identification and morphologicalcharacterization of LNA pure culturesFurther ED (on average 1 cell per vial) was carriedout using all three LNA enrichment cultures. Threeisolates were obtained by this approach, that is,isolate CB (from enrichment A), isolate GS (fromenrichment B) and isolate WW (from enrichment C).The 16S rRNA gene of each isolate was sequencedand the sequences were deposited in GenBankunder the accession numbers EU780139, EU780140and EU780141. The sequences matched 100% withthose obtained from DGGE analysis of the dominantbands from the corresponding LNA enrichmentcultures (Figure 6a), which confirms that the isolateswere also the most dominant in the respectiveenrichment cultures. All three isolates were shownto be closely affiliated to the Polynucleobactercluster (Figure 6b). Furthermore, repetitive extra-genic palindromic-PCR (Rep-PCR) genomic finger-printing of the three isolates indicated that theywere different genotypes (Figure 6c).

The three pure LNA cultures were further exam-ined with both SEM and cryo-TEM (Figure 7).Different cell shapes were observed for the differentisolates. Isolate CB and WW appeared mainly as rod-shaped cells (Figures 7a, c, d and f), whereas thosefrom isolate GS showed typically a vibriod mor-phology (Figures 7b and e). Cells of all isolates

possess a cytoplasm full of ribosome particles andcontain a diffuse nucleoid. The cell envelope wasclearly seen around all cells. The excellent cellpreservation of vitrified sample allowed visualizingthe lipid bilayer of the plasma membrane (PM) andthe outer membrane (OM) (inset in Figure 7f),showing that the cell envelope of these LNA isolatesis structurally analogous to that of Gram-negativebacteria. The space between the PM and the OM(periplasmic space) is 20 nm wide and contains alayered structure similar to the peptidoglycanobserved by cryo-sectioning of Gram-negative bac-teria (Matias et al., 2003). Furthermore, cells ofisolate CB at different cellular cycle stages wereimaged by cryo-TEM, documenting a clear celldivision process (Figure 8).

During the sample preparation for SEM, thebacterial cells probably shrunk due to the dehydra-tion. In contrast, the cells for imaging with cryo-TEM were frozen-hydrated in their native state.Owing to the well-kept shape and the volumepreservation of the frozen-hydrated cells, the cellvolume determination was carried out from thecryo-TEM micrographs. Cells of all isolates were inthe range of 300–400 nm in width and 500–600 nmin length. The biovolume for the cells was onaverage below 0.05 mm3. The periplasmic volume isrelatively big for the cells. For isolate CB (Figure 7d)

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Polynucleobacter necessarius (T)Ralstonia solanacearum (T) ; ATCC 11696

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Figure 6 Phylogenetic analysis of LNA bacteria. (a) DGGE and clustering analysis of the water samples and the related LNA enrichmentcultures. The distance matrix of all the possible gel tracks within the DGGE pattern was calculated by using the Pearson correlation.Bands that were further sequenced are encircled. (b) Phylogenetic tree of the three LNA isolates. (c) Rep-PCR of the LNA isolates fromthree different aquatic environments.

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it accounts for ca. 34% of the total cell volume, ca.37% for isolate GS (Figure 7e) and ca. 23% forisolate WW (Figure 7f). The cell membranes arebetween 4–5 nm thick (inset in Figure 7f). Thecondensed dark region inside the cell is believedto be the chromosome DNA of the cell, whichoccupies 450% of the cytoplasmic area.

Discussion

Omnipresence of LNA bacteria in freshwaterThe separation of natural aquatic microbiota intoLNA and HNA bacterial groups is a typical FCMphenomenon based on cellular properties observed

by FCM analysis after nucleic acid staining. Thisobservation has been reported for virtually allaquatic environments, including sea water (that is,Li et al., 1995; Gasol and Moran, 1999; Lebaronet al., 2001; Jochem et al., 2004), lakes and rivers(Nishimura et al., 2005; Bouvier et al., 2007),groundwater, drinking water and wastewater treat-ment plant effluents (Figure 1 and Table 1). Based oncell concentration, LNA bacteria and HNA bacteriaare equally abundant in most aquatic microbialcommunities sampled in the current study (Table 1).Nonetheless, LNA bacteria are mostly reported to bethe ‘inactive’ or even ‘ghost’ part of the microbialcommunity (for example, Lebaron et al., 2001;Servais et al., 2003). In the present study, we

Figure 7 Scanning electron micrograph of LNA isolate CB (a), isolate GS (b) and isolate WW (c); and transmission electron micrographof frozen-hydrated LNA isolate CB (d), isolate GS (e) and isolate WW (f). Inset in F is an enlarged micrograph to show the cell envelopestructure of isolate WW.

Figure 8 Transmission electron micrographs of four frozen-hydrated from LNA isolate CB at different stages of the division cycle (a–d).

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showed that LNA bacteria constitute a viablefraction of indigenous freshwater communities,and that bacteria that maintain unique LNA char-acteristics can be isolated and cultivated.

Cultivation of LNA bacteriaDespite the omnipresence of LNA bacteria in aquaticenvironments, there is little information on theirgrowth and characteristics in the literature. To ourknowledge, no LNA bacteria have been isolated orcultivated earlier. One of the main reasons for this isthat LNA bacteria, like the majority of freshwaterbacteria, cannot be cultivated in conventionalgrowth media that employ high-nutrient concentra-tions. To circumvent this problem, we have usedsterile river water as a growth medium. Sterilefreshwater provides an undefined complex mixtureof carbon compounds at low concentrations (in therange of mgC l–1), similar to the natural habitat ofmany bacteria (Munster, 1993). Even though heatsterilization (either autoclaving or pasteurization)might alter the chemical properties of the water to acertain degree, sterile fresh/marine water hasbeen proven to be a proper medium to cultivate‘uncultivable’ bacteria from the natural aquaticenvironments (Rappe et al., 2002; Vital et al., 2007;Wang et al., 2007). We have shown that anindigenous LNA community can be maintainedthrough continuous cultivation at low temperaturein such sterile freshwater (Figure 3). The ability ofthe LNA bacteria to sustain a cell density of about105 cells per ml over an extended period of time incontinuous culture proved that a dominant fractionof the indigenous LNA bacterial community isactively multiplying without changing their LNAcharacteristics when provided with the correctcultivation conditions.

Enrichment of LNA bacteria through FACS or EDwas a prerequisite for batch cultivation. All LNAenrichment cultures obtained in this mannershowed their ability to grow in batch culture atvarious temperatures and maintain the typical LNAcharacteristics (Figures 4 and 5). Their growth rateswere comparable to those of bacterial communitiesand other bacterial strains growing in carbon-limitedfreshwater (Hammes and Egli, 2005; Vital et al.,2007; Wang et al., 2007). It is interesting to noticethat their optimal growth temperature was between20 and 30 1C, which is much higher than the watertemperature where they originated from (ca. 12 1C).This suggested that most of the planktonic bacteriain aquatic environments are not necessarily living attheir optimum growth temperature (Morita, 1997;White et al., 1991; Rosenberg and Ben-Haim, 2002).

The successful cultivation of LNA bacteria in-dicates that the cultivation approach applied in thecurrent study may also be used for cultivation ofother so-called ‘uncultivable’ bacteria, which repre-sent 499% of the natural microbial communities(Rappe and Giovannoni, 2003; Wang et al., 2007).

Bouvier et al. (2007) proposed four scenarios for therelationship between LNA and HNA bacteria basedon FCM parameters for each group, and suggestedthat each group consists of cells that are intrinsic toa group, and cells that may exchange betweengroups. Assuming the scenarios proposed by Bou-vier and co-workers (2007) are right, we have foundand cultivated strains that are intrinsic members ofthe LNA group bacteria and do not exchange withHNA bacteria.

Comparison between batch growth of LNA and HNAenrichment culturesDuring the batch growth on sterile freshwater at thesame temperature, the LNA enrichment cultures hadlower-specific growth rates, smaller changes inaverage cell size and lower production of cellnumbers in comparison with the HNA enrichmentculture (Figure 5a and Table 2). The key question isthen how these organisms can compete with HNAbacteria in natural environments. If the small cellsize of LNA bacteria is taken into consideration, thebiomass-specific production of LNA bacteria can besimilar to that of HNA bacteria (Jochem et al., 2004).It has been reported that grazing pressure is heavieron middle-size class bacteria than on those with asmall cell volume (Bernard et al., 2000; Grossartet al., 2008). Hence, the resistance of LNA bacteria tograzing may also partially explain why LNA bacter-ia, although having lower-specific growth rates, stillpersist in high numbers in most natural aquaticenvironments. Laboratory studies showed thatCyclidium species selectively graze on HNA com-munities (Tadonleke et al., 2005), and althoughsmall bacteria are also vulnerable to grazing, thegrazing-related mortality was reported to be muchlower (Boenigk et al., 2004).

During batch cultivation, LNA bacteria exhibit aless prominent size variation than HNA bacteria(Figure 5a). Still, distinct cell division and cell sizechange was observed by cryo-TEM (Figure 8),indicating a normal cell growth cycle. This phenom-enon is again explainable by their extremely smallcell size; changes within this size range are noteasily detectable by FCM. In this study, flowcytometric SSC signals were used as an estimationof bacterial cell size (Felip et al., 2007; Vital et al.,2008). However, these estimations are used mainlyfor size comparison between LNA and HNA bacter-ia, and not for absolute cell size measurements.

The small cell volume of LNA bacteria is alsoclosely linked to their low ATP content (Figure 5band Table 2), which may explain why they are oftenregarded as an inactive or dead part of the microbialcommunity (Berney et al., 2008). LNA bacteria havean apparently 10-fold lower ATP-per-cell contentthan HNA bacteria (Table 2). However, when thedata are normalized based on cell volume, the ATP/biovolume for the LNA and HNA bacteria is quitesimilar (1.2–1.6� 10�15 gATP mm–3). Our data suggest

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that the ATP per microbial biovolume in active cellsis a relatively constant value in the range of1–2� 10�15 gATPmm–3 for both HNA and LNAbacteria when grown under similar conditions,which is similar to data reported recently (Eydaland Pedersen, 2007). Therefore, the failure to detectactivity in LNA bacteria may be because of thelimited sensitivity of the applied methods ratherthan their actual activity status.

Phylogenetic identity of LNA enrichment culturesObserving that LNA enrichment cultures can growactively with natural AOC and retain their LNAcharacteristics, the question ‘what are they?’ natu-rally follows. It has been documented earlier that themain bacterial population in freshwater environ-ments consists of members of the b-proteobacterialgroup (Glockner et al., 1999; Bruns et al., 2003).Therefore, our finding that the cells of LNA enrich-ment cultures are affiliated mainly with b-proteo-bacteria is not surprising. The phylogeneticcomposition of LNA enrichment cultures, however,changes certainly with location (Figure 6a) andprobably also with time, suggesting that also LNAbacterial communities in different aquatic environ-ments may exhibit considerable diversity, which isconsistent with earlier reports (Servais et al., 2003;Longnecker et al., 2005; Zubkov et al., 2002).

Characterization of LNA pure culturesOne LNA pure culture was isolated from each of theLNA enrichment cultures in the current study,which corresponds to the dominant band shownby DGGE in the enrichment cultures (Figure 6a). Thethree isolates exhibit very similar growth propertiesas the enrichment cultures (Supplementary Figure3). According to their 16S rRNA gene sequences andthe REP-fingerprints, the three isolates are comple-tely different strains, but are all closely related to thePolynucleobacter cluster (Figures 6b and c).Although the original Polynucleobacter type strain(P. necessarius) is a large and long obligate endo-symbiotic bacterium having multiple nucleiods(Heckmann and Schmidt, 1987), several species ofthis genus were recently reported to be dominant infreshwater habitats (Page et al., 2004; Hahn et al.,2005). Recently, members of the Polynucleobacterlinage were isolated from various sites by graduallyenriching nutritional conditions during cultivation(Hahn, 2003). Although our isolates are phylogene-tical by closely related to the Polynucleobacter spp.isolated by Hahn (2003), they have clearly differentgrowth properties. Whereas the former grow (so far)at low-nutrient concentration only (SupplementaryFigure 4), the latter form visible colonies onconventional high-nutrient agar. Further physiolo-gical and genetic data are required to confirm theexact phylogenetic position of these isolates and therelationship with their closest neighbors.

Cultivation and isolation of LNA bacteria not onlyfacilitated investigation of their growth properties,but also gave us the chance to obtain their trueportrait other than the cluster image on FCM dot-plots. To date, no microscopic images have beenpublished for pure LNA cultures. Here, for the firsttime, we report morphological characteristics ofpure LNA cultures from freshwater (Figure 7).Owing to their extremely small cell size, there maybe limited options of cell shape. Still, the cell shapeof the three isolates covers quite diverse shapes,including a normal rod (Figure 7d), a vibrio shape(Figure 7e) and a short rod (Figure 7f). It is believedthat surface-to-volume ratio is one of the reasonswhy most bacteria are rod-, filamentous- or vibrio-shaped, as non-spherical shape increases this ratiofor the volume enclosed (Koch, 1996). Cell sizeestimated by electron microscopy indicates that theisolates obtained are among the smallest planktonicbacteria known in culture (Schut et al., 1993; Hahnet al., 2003). So far, a marine isolate affiliated withthe SAR11 clade has been reported to have thesmallest cell size (0.01 mm3 estimated after glutar-aldehyde fixation with TEM (Rappe et al., 2002)). Incomparison, our isolates from freshwater environ-ments have a similar cell size (on average 0.05 mm,3

with the smallest value of 0.01 mm3 estimated withcryo-TEM). It is known that cells can shrink to acertain extent during the SEM sample preparationand therefore are smaller than the actual size. On theother hand, cells may slightly expand when trappedin very thin ice during cryo-TEM examination. Ourresults showed that the cell size estimated by SEM isup to five times smaller than that estimated bycryo-TEM (Figure 7). Hence, the accurate cell sizeprobably lies between the estimation carried out bySEM and cryo-TEM. In this study, the cell sizes ofLNA isolates were reported according to cryo-TEM,as it preserved a much better cell structure than SEM(Figure 7). Despite their small size, all LNA isolateshave a proportionally larger periplasmic space(20–40% of the total cell volume) than that ofEscherichia. coli (20% on average) (Graham et al.,1991). Owing to their extremely small size, LNAbacteria only represent a minor part (5–10%) of thetotal biomass of the microbial community despitethe fact that they have an equal share of cellabundance with HNA bacteria (Table 1).

Cryo-TEM is an optimal method for preservingbiological structures (Dubochet et al., 1988). Cryo-TEM of thin vitreous films has become a routinehigh-resolution technique for the study of isolatedsmall particles such as viruses, liposomes, proteinsand other macromolecular assemblies. This methodis not suitable for larger objects (41 mm in diameter)such as the normal-sized HNA bacteria. The mostaccurate cryo-technique for viewing prokaryoticultrastructure involves the use of frozen-hydratedthin sections (Matias et al., 2003; Al-Amoudiet al., 2004). Owing to their extremely small cellsize (o500 nm diameter), it was possible to obtain

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cryo-TEM pictures of the frozen-hydrated wholeLNA bacteria without thin sectioning, showingcell envelope structure and cytoplasmic content(Figure 7). To our knowledge, this is the first studyshowing the fine structure of frozen-hydrated wholebacteria. Our results show that cryo-TEM is asuitable tool to characterize the cell structure ofbacteria with minute cell volumes.

LNA bacteria, ‘oligotrophs’ and ‘ultramicrobacteria’Until now, the concept of ‘LNA bacteria’ hasremained confined to the field of FCM. However,the enrichment cultures and isolates obtained andcharacterized in the present study are not onlyrepresentatives of the group of ‘LNA bacteria’, butalso possess the properties of so-called ‘obligateoligotrophs’ (that is, they are unable to grow in richmedia) (Supplementary Figure 4) and ‘ultramicro-bacteria/ nanobacteria’ (that is, a cell size o0.1 mm3).Formerly attributed to the class of ‘uncultivablebacteria’ we have shown that they are perfectlycultivable in natural freshwater. All these nameswere created by researchers, based on isolatedaspects of bacteria (for example, physiologicalproperties, morphological properties) or differentinvestigation techniques (for example, FCM, plat-ing). Unfortunately, most of these names are oftenonly vaguely defined and also used differentlyunder various circumstances (Morita, 1997; Schutet al., 1997). This may lead to confusion in under-standing the true nature of the organisms studiedand hamper the interpretation and exchange ofknowledge among scientists.

Conclusions

In summary, we have documented the cultivation,isolation, morphological characterization and phy-logenetic analysis of the LNA bacteria from differentfreshwater environments. We have shown that theseorganisms are alive, cultivable, extremely small andmaintain unique LNA characteristics irrespective ofgrowth phase. Future research will focus on thecharacterization of the growth properties of the LNApure cultures isolated herein. The cultivationapproach reported here is a valuable complementarymethod to the traditional assays applied in micro-bial ecological studies.

Acknowledgements

We are grateful to the financial support from Eawaginternal funding (Wave21), the EU project TECHNEAU(018320), a research grant from the Flemish Fund forScientific Research (FWO-Vlaanderen, GP. 005.09N) andelectron microscope facility support from the Biozentrumof the University of Basel. We also thank Andreas Engel(Biozentrum, University of Basel) for his support andfruitful discussions, and Eva Siebel, Diederik VanDriessche and Oralea Buchi for their technical support.

References

Al-Amoudi A, Norlen LP, Dubochet J. (2004). Cryo-electron microscopy of vitreous sections of nativebiological cells and tissues. J Struct Biol 148: 131–135.

Bernard L, Courties C, Servais P, Troussellier M, Petit M,Lebaron P. (2000). Relationships among bacterial cellsize, productivity, and genetic diversity in aquaticenvironments using cell sorting and flow cytometry.Microb Ecol 40: 148–158.

Berney M, Vital M, Hulshoff I, Weilenmann H-U, Egli T,Hammes F. (2008). Rapid, cultivation-independentassessment of microbial viability in drinking water.Water Res 42: 4010–4018.

Billen G, Joiris C, Meyer-Reil L, Lindeboom H. (1990). Roleof bacteria in the North Sea ecosystem. Neth J Sea Res26: 265–293.

Boenigk J, Stadler P, Wiedlroither A, Hahn MW. (2004).Strain-specific differences in the grazing sensitivitiesof closely related ultramicrobacteria affiliated with thePolynucleobacter cluster. Appl Environ Microbiol 70:5787–5793.

Boon N, Windt W, Verstraete W, Top EM. (2002).Evaluation of nested PCR-DGGE (denaturing gradientgel electrophoresis) with group-specific 16S rRNAprimers for the analysis of bacterial communities fromdifferent wastewater treatment plants. FEMS MicrobiolEcol 39: 101–112.

Bouvier T, del Giorgio PA, Gasol JM. (2007). A compara-tive study of the cytometric characteristics of high andlow nucleic-acid bacterioplankton cells from differentaquatic ecosystems. Environ Microbiol 9: 2050–2066.

Bruns A, Nubel U, Cypionka H, Overmann J. (2003). Effectof signal compounds and incubation conditions on theculturability of freshwater bacterioplankton. ApplEnviron Microbiol 69: 1980–1989.

Dubochet J, Adrian M, Chang JJ, Homo JC, Lepault J,McDowall AW et al. (1988). Cryo-electronmicroscopy of vitrified specimens. Q Rev Biophys21: 129–228.

Eydal HSC, Pedersen K. (2007). Use of an ATP assay todetermine viable microbial biomass in FennoscandianShield groundwater from depths of 3–1000 m.J Microbiol Meth 70: 363–373.

Felip M, Andreatta S, Sommaruga R, Straskrabova V,Catalan J. (2007). Suitability of flow cytometry forestimating bacterial biovolume in natural planktonsamples: comparison with microscopy data. ApplEnviron Microbiol 73: 4508–4514.

Gasol JM, Moran XAG. (1999). Effects of filtration onbacterial activity and picoplankton community struc-ture as assessed by flow cytometry. Aquat Microb Ecol16: 251–264.

Gasol JM, Zweifel UL, Peters F, Fuhrman JA, Hagstrom A.(1999). Significance of size and nucleic acid contentheterogeneity as measured by flow cytometry innatural planktonic bacteria. Appl Environ Microbiol65: 4475–4483.

Glockner FO, Fuchs BM, Amann R. (1999). Bacterioplank-ton compositions of lakes and oceans: a first compar-ison based on fluorescence in situ hybridization. ApplEnviron Microbiol 65: 3721–3726.

Graham LL, Beveridge TJ, Nanninga N. (1991). Periplas-mic space and the concept of the periplasm. TrendsBiochem Sci 16: 328–329.

Grossart H-P, Jezbera J, Hornak K, Hutalle KML, Buck U,Simek K. (2008). Top-down and bottom-up induced

Low nucleic acid-content bacteria from freshwaterY Wang et al

900

The ISME Journal

shifts in bacterial abundance, production andcommunity composition in an experimentally dividedhumic lake. Environ Microbiol 10: 635–652.

Hahn MW. (2003). Isolation of strains belonging to thecosmopolitan Polynucleobacter necessarius clusterfrom freshwater habitats located in three climaticzones. Appl Environ Microbiol 69: 5248–5254.

Hahn MW, Lunsdorf H, Wu Q, Schauer M, Hofle MG,Boenigk J et al. (2003). Isolation of novel ultramicro-bacteria classified as actinobacteria from five fresh-water habitats in Europe and Asia. Appl EnvironMicrobiol 69: 1442–1451.

Hahn MW, Pockl M, Wu QL. (2005). Low intraspecificdiversity in a Polynucleobacter subcluster populationnumerically dominating bacterioplankton of afreshwater pond. Appl Environ Microbiol 71:4539–4547.

Hammes F, Berney M, Wang Y, Vital M, Koster O,Egli T. (2008). Flow-cytometric total bacterial cellcounts as a descriptive microbiological parameter fordrinking water treatment processes. Water Res 42:269–277.

Hammes FA, Egli T. (2005). New method for assimilableorganic carbon determination using flow-cytometricenumeration and a natural microbial consortium asinoculum. Environ Sci Technol 39: 3289–3294.

Heckmann K, Schmidt HJ. (1987). Polynucleobacternecessarius gen. nov., sp. nov., an obligately endo-symbiotic bacterium living in the cytoplasm ofEuplotes aediculatus. Int J Syst Bacteriol 37: 456–457.

Jellett JF, Li W, Dickie P, Boraie A, Kepkay P. (1996).Metabolic activity of bacterioplankton communitiesassessed by flow cytometry and single carbon sub-strate utilization. Mar Ecol Prog Ser 136: 213–225.

Jochem FJ, Lavrentyev PJ, First MR. (2004). Growth andgrazing rates of bacterial groups with different appar-ent DNA content in the Gulf of Mexico. Mar Biol 145:1213–1225.

Kajander EO, Ciftcioglu N. (1998). Nanobacteria: analternative mechanism for pathogenic intra- andextracellular calcification and stone formation. PNAS95: 8274–8279.

Koch AL. (1996). What size should a bacterium be? Aquestion of scale. Annu Rev Microbiol 50: 317–348.

Lebaron P, Servais P, Agogue H, Courties C, Joux F. (2001).Does the high nucleic acid content of individualbacterial cells allow us to discriminate between activecells and inactive cells in aquatic systems? ApplEnviron Microbiol 67: 1775–1782.

Lebaron P, Servais P, Baudoux A-C, Bourrain M, CourtiesC, Parthuisot N. (2002). Variations of bacterial-specificactivity with cell size and nucleic acid contentassessed by flow cytometry. Aquat Microb Ecol 28:131–140.

Li WKW, Jellett J, Dickie PM. (1995). DNA distributions inplanktonic bacteria stained with TOTO or TO-PRO.Limnol Oceanogr 40: 1485–1495.

Longnecker K, Sherr BF, Sherr EB. (2005). Activity andphylogenetic diversity of bacterial cells with high andlow nucleic acid content and electron transportsystem activity in an upwelling ecosystem. ApplEnviron Microbiol 71: 7737–7749.

Marie D, Partensky F, Jacquet S, Vaulot D. (1997).Enumeration and cell cycle analysis of naturalpopulations of marine picoplankton by flow cytometryusing the stain SYBR Green I. Appl Environ Microbiol63: 186–193.

Matias VRF, Al-Amoudi A, Dubochet J, Beveridge TJ.(2003). Cryo-transmission electron microscopy offrozen-hydrated sections of Escherichia coli andPseudomonas aeruginosa. J Bacteriol 185: 6112–6118.

Morita RY. (1997). Starved bacteria in oligotrophicenvironments. In: Morita RY (ed.) Bacteria in oligo-trophic environments: starvation-survival life style.Chapman & Hall: NY, USA, pp 193–245.

Munster U. (1993). Concentrations and fluxes of organiccarbon substrates in the aquatic environment. AntonieVan Leeuwenhoek 63: 243–274.

Nishimura Y, Kim C, Nagata T. (2005). Vertical andseasonal variations of bacterioplankton subgroupswith different nucleic acid contents: possibleregulation by phosphorus. Appl Environ Microbiol71: 5828–5836.

Oliver JD. (1993). Formation of viable but nonculturablecells. In: Kjelleberg S. (ed.). Starvation in bacteria.New York: Plenum Press, pp 239–272.

Page KA, Connon SA, Giovannoni SJ. (2004). Representa-tive freshwater bacterioplankton isolated from CraterLake, Oregon. Appl Environ Microbiol 70: 6542–6550.

Pernthaler A, Pernthaler J. (2007). Fluorescence in situhybridization for the identification of environmentalmicrobes. In: Hilario, E., and Mackay, JF (eds).Methods in Molecular Biology, vol. 353: Humana PressInc., NJ, pp 153–164.

Pernthaler A, Pernthaler J, Amann R. (2002). Fluorescencein situ hybridization and catalyzed reporter depositionfor the identification of marine bacteria. Appl EnvironMicrobiol 68: 3094–3101.

Rappe M, Connon SA, Vergin KL, Giovannoni SJ. (2002).Cultivation of the ubiquitous SAR11 marine bacter-ioplankton clade. Nature 418: 630–633.

Rappe M, Giovannoni S. (2003). The uncultured microbialmajority. Annu Rev Microbiol 57: 369–394.

Rosenberg E, Ben-Haim Y. (2002). Microbial diseasesof corals and global warming. Environ Microbiol 4:318–326.

Sekar R, Pernthaler A, Pernthaler J, Warnecke F, Posch T,Amann R. (2003). An improved protocol for quantifi-cation of freshwater Actinobacteria by fluorescencein situ hybridization. Appl Environ Microbiol 69:2928–2935.

Schut F, Prins R, Gottschal J. (1997). Oligotrophy andpelagic marine bacteria: facts and fiction. AquatMicrob Ecol 12: 177–202.

Schut F, de Vries EJ, Gottschal JC, Robertson BR, HarderW, Prins RA et al. (1993). Isolation of typical marinebacteria by dilution culture: growth, maintenance, andcharacteristics of isolates under laboratory conditions.Appl Environ Microbiol 59: 2150–2160.

Servais P, Casamayor EO, Courties C, Catala P, ParthuisotN, Lebaron P. (2003). Activity and diversity ofbacterial cells with high and low nucleic acid content.Aquat Microb Ecol 33: 41–51.

Tadonleke RD, Planas D, Lucotte M. (2005). Microbial foodwebs in boreal humic lakes and reservoirs: ciliates as amajor factor related to the dynamics of the most activebacteria. Microb Ecol 49: 325–341.

Versalovic J, Schneider M, de Bruijn FJ, Lupksi JR. (1994).Genomic fingerprinting of bacteria using repetitivesequence-based polymerase chain reaction. MethodsMol Cell Biol 5: 25–40.

Vital M, Fuchslin HP, Hammes F, Egli T. (2007). Growth ofVibrio cholerae O1 Ogawa Eltor in freshwater. Micro-biology 153: 1993–2001.

Low nucleic acid-content bacteria from freshwaterY Wang et al

901

The ISME Journal

Vital M, Hammes F, Egli T. (2008). Escherichia coli O157can grow in natural freshwater at low carbon concen-trations. Environ Microbiol 10: 2387–2396.

Wang Y, Hammes F, Boon N, Egli T. (2007). Quantificationof the filterability of freshwater bacteria through 0.45,0.22, and 0.1mm pore size filters and shape-dependentenrichment of filterable bacterial communities. Envir-on Sci Technol 41: 7080–7086.

Wang Y, Hammes F, Duggelin M, Egli T. (2008). Influenceof size, shape, and flexibility on bacterial passagethrough micropore membrane filters. Environ SciTechnol 42: 6749–6754.

Ward JH. (1963). Hierarchical grouping to optimize anobjective function. J Am Stat Assoc 58: 236–244.

Warnecke F, Sommaruga R, Sekar R, Hofer JS, Pernthaler J.(2005). Abundances, identity, and growth state ofActinobacteria in mountain lakes of different UVtransparency. Appl Environ Microbiol 71: 5551–5559.

White PA, Kalff J, Rasmussen JB, Gasol JM. (1991). Theeffect of temperature and algal biomass on bacterial

production and specific growth rate in freshwater andmarine habitats. Microb Ecol 21: 99–118.

Whitman WB, Coleman DC, Wiebe WJ. (1998).Prokaryotes: The unseen majority. PNAS 95:6578–6583.

Wick LM, Weilenmann H, Egli T. (2002). The apparentclock-like evolution of Escherichia coli inglucose-limited chemostats is reproducible at largebut not at small population sizes and can beexplained with Monod kinetics. Microbiol 148:2889–2902.

Zubkov MV, Fuchs BM, Burkill PH, Amann R. (2001).Comparison of cellular and biomass specific activitiesof dominant bacterioplankton groups in stratifiedwaters of the Celtic Sea. Appl Environ Microbiol 67:5210–5218.

Zubkov MV, Fuchs BM, Tarran GA, Burkill PH, Amann R.(2002). Mesoscale distribution of dominant bacterio-plankton groups in the northern North Sea in earlysummer. Aquat Microb Ecol 29: 135–144.

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