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Microbial loop and nutrient cycling

Microbial loop and nutrient cycling. International Short-Course Series Bioremediation and Phytoremediation of Organics and Nutrients. University of Ljubljana Biotechnical faculty Vecna pot 111, SI-1000 Ljubljana, Slovenia. David Stopar. October, 2001 Nova Gorica. O 2 , CO 2, other gases.

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Microbial loop and nutrient cycling

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  1. Microbial loop and nutrient cycling International Short-Course Series Bioremediation and Phytoremediation of Organics and Nutrients University of Ljubljana Biotechnical faculty Vecna pot 111, SI-1000 Ljubljana, Slovenia David Stopar October, 2001 Nova Gorica

  2. O2, CO2, other gases organic material hn GRAZING CH4 DMS Phytoplankton Zooplankton Fish POM C, N, P, S, Fe,... aggregates 0-200 m Protozoa DOM MICROBIAL LOOP SEDIMENTATION Bacteria Viruses bentos solubilization 200-11000 m

  3. primary producer protozoa 20 % 50 % DNA, RNA, sugars, ions 10 % DNA, RNA, sugars, ions Microbial loop

  4. CFU time (h) Why bacteria die? • predation • lethal environment • starvation • disease (phages) • programmed cell death

  5. Fatty acids acetate propionate butyrate caprate Polymers gelatin DNA cellobiose peptone yeast extract Organic acids succinate DL-malate DL-lactate citrate a-ketoglutarate piruvate Amino acids L-serine L-glutamat L-proline L-aspartate Alcoholes D-manitol D-sorbitol glycerol Vibrio gazogenes organic carbon sources Sugars glucose D-fructose D-mannose maltose D-xylose sucrose trehalose L-arabinose D-galactose D-ribose

  6. CFU lysate 9.6 x 108 lysate + mN +mN 7.3 x 108 PYE 8.4 x 108 initial 8.2 x 105 no growth growth Out of 26 different natural bacterial isolates tested, 20 bacterial isolates were able to use bacteria lysate as a source of organic carbon. Vibrio lysate as a source of organic carbon for a bacterial community Natural bacterial community is able to grow on bacterial lysate

  7. Why bacteria die? • starvation • disease (phages) • programmed cell death • predation • lethal environment

  8. Phage life cycle

  9. Phage abundances phages are probably the most abundant living entities in the ecosystem sea water 106 - 108/ml fresh water 106 - 108/ml sediments 108 - 109/g soil ND

  10. Phage role in the ecosystem • phages mediate horizontal gene exchange • phages mediate community structure • phages influence the flow of energy and carbon

  11. OD No phages with TEM t (min) OD Phages with TEM Impact of lysogenic viruses on nutrient cycling Bacteriophage induction experiment control OD660 = 0.5 mitomycin C t (min)

  12. In vitro phage induction from bacterial isolates • 75 % of all tested strains were lysogenic • 51 % of all tested strains were polylysogenic

  13. Aerobic incubation Anaerobic incubation to control Mit-C to control Mit-C 58 % of bacterial community induced 32 % of bacterial community induced In situ induction of phages from a sea water samples

  14. phage titer tG MFT E B R E L-E t (min) L Impact of lytic viruses on nutrient cycling

  15. Simulating phage production with and withoutmean free time simplification

  16. Phage growth as a function of host density:theoretical versus experimental  host density o phage titer  exponential decay  MFT function  MFT function, Eqn2

  17. Impact of host density on phage latent-period optima

  18. acetate glucose glycerol Impact of host quality on latent period optima  high quality host, control  E-varied  k-varied  R-varied  E + R + k varied

  19. developmental processes (i.e. sporulation) altruistic suicide ageing antibiotics or stress related factors Why bacteria die? • starvation • disease (phages) • programmed cell death • predation • lethal environment

  20. What is the benefit for unicellular organism of committing a suicide? • no obvious reason unless we consider a unicellular organism as being part of a complex microbial community • better use of resources • reduced mutation rate (elimination of DNA damaged cells) • reducing the impact of infection by pathogens • lowering the probability of take over mutants • facilitating genetic exchange

  21. Population of Vibrio committing a suicide after entering a stationary phase At high cell density in a rich medium a sub population of cells commit suicide. In the lysate viruses are present. At low host density cell in a poor medium there are no viruses present. PYE 5 PYE 2

  22. Survival of rare cells in a population • sensitivity of the whole population to a programme cell death could eliminate the whole clonal population (a contraproductive strategy) • experimentally it is known that the entire population is not sensitive to the external damaging effect (i.e. UV, antbiotics) • a random variation of regulator molecules can induce or prevent a suicide program

  23. Survival of rare cells after induction with mitomycin C rich growth conditions poor growth conditions

  24. cells attracted by pheromone cells aggregate Pheromones and quorum sensing(a coordinated response to stress environment) cell producing pheromone

  25. % competence Cell density time (h) Genetic competence inBacillus subtilis • develops during stationary phase, when 1-10% cells become competent and ready to uptake foreign DNA • genetic competence is under nutritional control and cell density control i.e. quorum sensing • it is cell last chance to avoid sporulation

  26. Pheromone precursor Response regulator Receptor kinase Modification maturation comX comQ comP comA ComX ComP kinase domain ComQ ATP pre-ComX P ComA DNA Quorum sensing players in Bacillus subtilis

  27. tester strain comX comP comQXP srfA-lacZ lacZ activity Pheromone comX specificity test producer strain

  28. comQX comPA producer Tester strain strain 168 RO-C2 RO-FF1 RO-E2 RO-H1 RO-B2 NAF4 168 ++ ++ + RO-C2 + ++ RO-FF1 ++ RO-E2 + ++ + RO-A4 + + + RO-PP2 + + RO-H1 ++ RO-B1 ++ + RO-DD2 ++ + RO-B2 + ++ NAF4 ++ Quorum-sensing specificities * Strains are grouped according to phylogenetic relationship

  29. ComX(s) purification and characterization 1- comQ and comX cloning and expression in E. coli 2- Purification by reverse phase chromatography srfA-lacZ

  30. StrainSequence Δ Mass 168 (A)DPITRQWGD + 206 RO-C-2 TREWDG + 206 RO-E-2 GIFWEQ + 136 RS-B-1 (M)MDWHY + 120 RO-H-1 (M)LDWKY + 120 RO-B-2 (Y)TNGNWVPS + 136 *Δ Mass = obtained mass - calculated mass ComX(s) characteristics Modification masses are consistent with farnesylation or geranylation of com Xin addition ComQ resembles a farnesyl-geranyl transferase

  31. Why bacteria die? • starvation • disease (phages) • programmed cell death • predation • lethal environment

  32. DNA, RNA, sugars, ions DNA, RNA, sugars, ions Bacterial and viral loop facilitate nutrient cycling

  33. Acknowledgements Ivan Mahne Ines Mandič-Mulec Kaja Gnezda Aleša Černe Andrej Žagar Duško Odič Dave Dubnaw, New York University, USA Valentina Turk, National Institute of Biology, SI Mateja Poljšak-Prijatelj, Institute of Microbiology and Immunology, SI Stephen T. Abedon, Ohio State University, USA

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