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Effect of viruses on bacteria-mediated C and Fe cycling M.G. Weinbauer CNRS-UPMC, UMR 7093 Villefranche-sur-mer The ‘viral shunt’ Wilhelm and Suttle (1999) Grazing food chain Primary Producers 100% Inorganic Nutrients Carnivores Grazers 1% 2-10% Dissolved Organic Matter 6-26% Viral shunt Viruses 3-15% Heterotrophic Prokaryotes Microbial loop Viral lysis influences biogeochemical cycles In situ samples Parameter Method Viral (and bacterial) abundance Flow cytomety Abundance of several viral (and bacterial) populations Flow cytomety Phage production Virus-dilution approach Frequency of lytically infected bacterial cells Virus-dilution approach Frequency of lysogenically infected bacterial cells Virus-dilution approach Viral diversity Pulsed-field gel electrophoresis DGGE? In situ samples Parameter No of samples Volume per depth per depth profile Viral (and bacterial) abundance Abundance of populations 6 4 ml Phage production Frequency of lytically infected bacterial cells Frequency of lysogenically infected bacterial cells 3-4 2-300ml Viral diversity 2(-3) 5000ml Viral and bacterial metagenomics 4-5 (total) 100-200L Effect of viruses on bacteria-mediated Fe dissolution Background: Viral lysis increases bacterial respiration and decreases growth efficiency by setting free the cell content during lysis. Bacteria are rich in Fe. 70 in out Fe fertilization stimulates viral infection of bacterioplankton FIC (%) 60 50 40 30 20 10 Hypothesis: 0 0 5 10 15 20 Time since first iron addition (days) Lysis should increase the pool of dissolved Fe and the high growth efficiency should increase dissolution of organically complexed Fe. This should have consequences for the distribution of Fe in different pools and for fluxes between pools and thus for carbon cycling. Effect of viruses on bacteria-mediated Fe dissolution (continued) Factorial approach: Bacterial communities with and without viruses could be ammended with and without Fe. Bacterial production and respiration could be measured, Fe in the LMW DOM, HMW DOM and bacterial pool could be measured, maybe hot Fe additions could be used to quantify Fe fluxes between pools. Collaboration: Geraldine Sarthou, others? An extension could be to add viral lysis products to natural communities to see whether dissolution of complexed iron stimulates primary production. Samples for 16S PCR DGGE will reveal potential influences of these mechanisms for bacterial species richness. Factorial approach Water sample Bacterial concentrate 1-µm Viral concentrate 0.2-µm -Viruses + Bacteria 100 kDa Virus-free water + Viruses + Bacteria +Fe/-Fe Viral diversity Pulsed-field gel electrophoresis separates viral genomes by size Bands can be excised and further analyzed by PCR-DGGE for specific groups Primers are available for cyanophages algal viruses Podoviridae? Q uic kTim e™ et un décom pr esseur TI FF ( non com pr essé) sont r equis pour vis ionner cet t e im age. Metagenomics (=Community genomics) Use of metagenomics: 1. Diversity estimates for various groups (viruses, bacteria,…18S rRNA) 2. Functional display: Detection of gene expression that differs between Fe-limited and Fe-replete stations and is thus likely linked to induction by presence/absence of Fe. Collaboration with Dirk Wenderoth, German Centre for Biotechnology From the same samples collected for metagenomics, an analysis of stable isotope composition of total proteins and lipids and specific Compounds could be performed for carbon and nitrogen. This may help to tease apart the flow of carbon and nitrogen through communities in Fe-limited and Fe-repleted stations. Collaboration with Wolf-Rainer Abraham, German Centre for Biotechnology Viral abundance (106 ml-1) A virus-reduction approach to estimate viral production, frequency of infected cells and prophage induction 0,8 y = 0,018x + 0,210 VAMCt1 Prophage induction 0,6 0,4 0,2 0 Frequency of infected cells (FIC): (VACt1 - VAt0)/BS/BA VACt1 VAt0 0 Mitomycin C Control 5 10 15 20 Lytic viral production Frequency of lysogenic cells (FLC): (VAMCt1 - VACt1)/BS/BA Viral production-slope method (VP-Slope): Slope of regression of viral abundance over time Viral production-FIC, BP, BS method: FICxBP (bacterial production) xBS Time (h) Dyfamed (French JGOFS station) NW Mediterranean Sea Weinbauer & Suttle 1996, Wilhelm et al. 2002 Weinbauer et al. 2002