Presentation at MOB-WUR sept-2011

Lessons from Microbial Eco(physio)logy: a few examples.

1. Behaviour of bacteria degrading PentaChloroPhenol (PCP) in soil ecosystems; (University of Helsinki, FIN).

2. Ecophysiological mechanisms involved in cyanobacterial bloom; (University of Amsterdam, NL).

3. Microbial identity within microbial ecology (WAU, NL & HU, FIN).

Presentation structure

Content of each part: Scientific and practical questions

leading to the research work; Aims, Experimental approach and

M&M; Results and Conclusions; Room for questions.

Lessons from Microbial Eco(physio)logy: few examples.

Behaviour of bacteria degrading PentaChloroPhenol (PCP) in soil ecosystems; (University of Helsinki, FIN).

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Place: University of Helsinki Department of General MicrobiologyParticipants: Prof. Dr. Ir. M. Salkinoja-Salonen Dr. M. Briglia Dr.Ir. P.J.M. Middeldorp, Dr. Ir. V. Kitunen, Dr.Ir.

R.Valo, and the technical supporting staff.

Why is it relevant to carry out pilot study on bacteria which are already known to attack and degrade PCP?

Because scientific evidence on their degrading performance and behaviour under field conditions would allow a more efficient and controllable use of the chosen bacterium besides gaining more knowledge.

Experimental Aims: To understand why externally introduced

xenobiotic degrading bacteria seldom perform well under field conditions;

To improve biodegradation of PCP in soils by using more effectively degrading bacteria;

To explore sustainable soil bioremediation possibilities.

Experimental approach: Study of the bacterial survival capacity in

different types of soils; Explore correlation/predictability between lab-

data knowledge versus natural ecosystems; Investigate the effect of contamination and

inoculums levels, and natural amendments on bacterial viability and activity.

M & M: Inocula of PCP-degrading bacteria: -

Flavobacterium and +Rhodococcus/(Mycobacterium) PCP-1;

Soil types: pristine sandy and peaty soils (Kettula farm, Suomusjärvi, FIN);

Natural amendments: Distillery Waste and Wood Chips;

Polyurethane (PUR) foam 90/16 type containing active carbon as bacterial carrier;

Pentachlorophenol crystals 97% purity and uniformly 14C-labelled PCP 96% radiochemical purity, 10.57 mCi mmol-1.

Electron micrographs of thin sections of PUR-foam containing active carbon before and after colonization with M. chlorophenolicus PCP-1. a) sterile, b) after colonization by a pure culture, … next …

Chemical structure of PCP

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Experimental set up:

Behaviour of bacteria degrading PCP in soil ecosystems: Experimental observations:

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… c) a similar PUR-foam pads as in b), but sampled after 290 days exposure to PCP contaminated soil w/o amendments, and d) sampled as in c) but in PCP contaminated soil added with DW. The arrows indicate presence of cells.

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Behaviour of bacteria degrading PCP in soil ecosystems ... Results:

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•Mycobacterium chl. PCP-1 remained viable and kept its PCP-degrading activity for up to 290 days regardless the different simulated field conditions i.e. ± PCP contaminant and ± natural amendments;•Flavobacterium lost its viability as well as its degrading activity within 60 days under the same simulated field conditions;•PCP was mineralized up to 97%;•The addition of natural amendments did not improve the bioremoval of PCP in the studied soils.

Behaviour of bacteria degrading PCP in soil ecosystems ...

Conclusions:

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•Field PCP biodegradation can be done effectively by using PUR-immobilized Mycobacterium chl. PCP-1 inoculum at cell density of ca. 108 cells/g.s.d.wt.; •Addition of natural amendments is not needed to improve the bacterial PCP-degrading activity;•Laboratory bacterial performance is not always equal to field performance.

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Questions

Eco-physiological mechanims involved in cyanobacterial bloom

Place: University of AmsterdamLaboratory of Microbiology

Participants: Prof. Dr. L. MurDr. H.J.M. MatthijsDr. M. BrigliaIr. J. Balke

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Eco-physiological mechanims involved in nitrogen-N stress cyanobacterial bloom

WhyNutrient stress?

(nitrate-N) Environmental factors

Intervention

Prevention

Cyanobacterial bloom

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HowCell behaviour

andProteomics of the cell

Why is it important to study nitrate-N stress?

Because:*N is necessary for growth and metabolism of the cell;

*nitrate-N is one of the inorganic sources for N;*prior to its reduction it can be used for different cell purposes:

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*... it can be used prior to its reduction for different cell purposes:

Assimilatory = NO3-

NO2- NH4

+ GlnGlu

respirationdissimilation

Dissimilatory =

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1. Elucidate cyanobacterial behaviour under nitrate-N stress;

2. Determine whether the cyanobacterial cell wall responds specifically to nitrate stress;

3. Develop molecular tools to monitor bloom-warning signals (multiprobe array: identity + activity monitoring).

Experimental Aims:

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Experimental approach:

1- Growth under nitrate-N stress conditions; (Cellular behaviour)

2- Molecular structure changes at the cell wall level; (Proteomics of the cell wall).

* Batch culture system (rich and depleted nitrate conditions);* Continuous culture system (nitrate inputs 0.5 and 0.05 mM, d=0.015, NH4

+ input 0.05 mM);* Cyanobacterium strain model Synechocistys PCC 6803.

M & M:

Cellular behaviour

Proteomics of the cell wallApproach:

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Effect of nitrate-N stress on the behaviour of Synechocystis cells

Under nitrate depletion Synechocystis cells undergo a quick loss of pigments (bleaching);

They keep dividing though at almost undetectable level;

Experimental observations (batch culture):

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0 2 4 6 8 10 12 14

Time (day)

control

NH4+ limitation

NO3- limitation

light limitation

Fig. 1

modulation phase

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Experimental observations (continuous culture):

Low nitrate input slows down the growth;

The type of nitrogen source influences the growth of Synechocystis PCC 6803;

The modulation phase of nitrate limited cells is shorter than that of ammonium limited cells.

Results:

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Elucidate cyanobacterial behaviour under nitrate-N stress;

Determine whether the cyanobacterial cell wall responds specifically to nitrate-N stress;

Develop molecular tools to monitor bloom-warning signal/s (multiprobe array: identity + activity).

Experimental Aims:

Cellular behaviour

Proteomics of the cell wall

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Approach:

Effect of nitrate-N stress on strain PCC 6803 cell wall: study of the protein pattern.

* Isolation of the cell wall fraction (by floatation ultracentrifugation on discontinuous sucrose density gradient);

* Analysis of the cell wall fraction (by SDS-PAGE and polypeptide molecular weight determination);

M & M:

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1. Cell disruption by shearing forces (bead beating);

2. Preparation of discontinuous sucrose density gradient;

10%30%45%

48%55%90%

Beforecentrifuging

Aftercentrifuging

Cytoplasmic membrane

Cell wall

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Isolation of the cell wall fractionM & M:

Analysis of the cell wall protein patternExperimental observations:

1) SDS-PAGE of the cell wall protein pattern of PCC 6803 cells submitted to rich (+) and depleted (-) nitrate treatment.

+ + +- - -

66,2 Kb

2.2ųg 1.8ųg 1.4ųg 1.2ųg 0.7ųg 0.6ųg

21,5 Kb

97,4 Kb116,2 Kb

200 Kb

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2) SDS-PAGE of the cell wall protein pattern of PCC 6803 cells submitted to sufficient (+), limited (-) nitrate and ammonium (NH) treatment.200 Kb

97,4 Kb116,2 Kb66,2 Kb

31 Kb 21,5 Kb

+N -N -NH7ųl

10ųl 10ųl 10ųl7ųl 7ųl15ųl 15ųl 15ųlDr.

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Analysis of the cell wall protein pattern Experimental observations:

Effect of nitrate-N stress on strain PCC 6803 cell wall: study of the protein pattern.

* Depletion of nitrate-N induces synthesis of new polypetides in the cell wall of strain PCC 6803 as shown by SDS-PAGE analysis;* Induction of the synthesis of these proteins occurs already at low nitrate-N concentration (0,05 mM);* Low concentration (0,05 mM) of ammonium-N induces also synthesis of new protein.

Results:

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Molecular ecophysiology of strain PCC 6803/cyanobacteria under nitrate-N stress.

Conclusions:1) Indeed strain PCC 6803 responds

specifically to the stress of different Nitrogen sources.

2) In strain PCC 6803 nutrient stress (N) induces a specific adaptation of the cell wall rather than a non-specific increase of its permeability.

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1. Elucidate cyanobacterial behaviour under

nitrate-N stress;2. Determine whether the cyanobacterial cell

wall responds specifically to nitrate stress;3. ... Future study: develop molecular

tools to monitor bloom-warning signals (multiprobe array: identity + activity).

Experimental Aims:

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Questions

Microbial identity within microbial ecology (WAU, NL & HU, FIN).

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Places: Wageningen Agricultural University, Laboratory of Microbiology

& Helsinki University,

Department of General MicrobiologyParticipants: Prof. Dr. Ir. W. De Vos (NL)

Prof. Dr. Ir. M. Salkinoja-Salonen (FIN) Dr.Ir. G. Schraa (NL) Dr. M.Sc. M. Briglia (FIN-IT-NL)and the technical supporting staff (FIN-NL)

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Why study microbial identity?

Because it helps to gather information needed to place the studied microbe somewhere in the culture collection and mainly to give it a name so that it is no more an unknown “identity” and when you need it you can find it back;

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When to study microbial indentity?

If: you need another paper to finish your Ph.D. or you do not have much work to do (lacking

brilliant ideas period), and by accident you have just isolated a

bacterium that resembles anything else but itself,

etc. etc.

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Experimental aims: To create another original scientific article; To define the identity of a bacterium that

appeared to do a nasty job that other known bacteria did not want to do, and that especially seems to have a strange look;

To check whether it might have ancestors that are harbouring similar behaviour.

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Experimental approaches: Determine the xenobiotic degrading and

physiological properties, morphology, size, and ultrastructure that are relevant to its taxonomical classification;

Define the sequence of 16S rDNA and perform phylogenetic inference;

Design DNA probe to specifically detect the microbial target;

Develop protocol for the isolation of DNA in the ecosystem under study.

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M & M: One PCP-degrading bacterium known as

Rhodococcus chlorophenolicus strain PCP-1; One 2,4,6-TCP and DCP-degrading newly

isolated bacterium; Pristine and contaminated soil samples.

Microbial identity within microbial ecology

(WAU, NL & HU, FIN).

Experimental observation:

Experimental observations:

1. The genus specific helix of the 16S rRNA of strain PCP-1 strongly resembles that of the genus Mycobacterium;

2. The inferred phylogenetic relationship to all of the species belonging to the genus Mycobacterium examined indicated similarity values greater than 95%;

Microbial identity within microbial

ecology (WAU, NL & HU, FIN).

1. Along the 16SrRNA molecule three nucleotides streches could be identified for designing a specific detection probe;

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1. The designed probes were tested for specific detection of strain PCP-1 in inoculated soils and showed specificity to the target A), B) and C);

2. The sensitivity of the detection method was tested at different inoculums densities and detection was possible down to 3x102 cells p.g. soil.

Results:1. The taxonomical data and genetic

determination based on the sequence analysis of 16S rDNA gene showed that Rhodococcus chlorophenolicus strain PCP-1 belongs no longer to the genus Rhodococcus but to the genus Mycobacterium.

2. The nucleic acid probe allowed specific detection of strain PCP-1 in soil and enhanced detection of PCP-1 down to 3x102 cells p.g. soil d.wt.

Electron micrographs of 2,4,6-TCP and DCP-degrading newly isolated bacterium;

Results:The taxonomical data, genetic

determination based on the sequence analysis of 16S rDNA gene, and phylogenetic inference allowed the classification of the 2,4,6-TCP degrading bacterium as Rhodococcus percolatus sp. nov. strain MBS1.

Conclusions: Molecular genetics and phylogenetic inference

ensure an accurate microbial classification; Nucleotidic probes allow a more sensitive,

specific, and quicker microbial identification in environmental samples;

Thorough taxonomic and phylogenetic characterization of microbes can support understanding of microbial behavior in natural ecosystems.

... And much more ...

Microbial identity whithin microbial ecology

Recent developments:Metagenomic Analyses: past and future trends, Minireview by: C. Simon and R. Daniel, AEM, Feb. 2011, p. 1153-1161.

... A few extracts:Metagenomic bypasses the need for isolation or cultivation of microorganisms. Metagenomic

approaches based on direct isolation of nucleic acids from the environmental samples have proven to be powerful tools for comparing and exploring the ecology and metabolic profiling of complex environmental microbial communities, as well as for identifying novel biomolecules by use of libraries constructed from isolated nucleic acids ...

Metatranscriptomics provides information on the actual metabolic activity ... Metatranscriptomic studies of microbial assemblages in situ are rare. ....

Metaproteomic analysis of mixed microbial communities ia a new emerging research area which aims at assessing the immediate catalytic potential of a microbial community. ...

Presentation at MOB-WUR sept-2011

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