Bacterioplankton communities: single-cell characteristics and physiological structure

1. Bacterioplankton communities: single-cell characteristics and physiological structure Paul del Giorgio Université du Québec à Montrèal

2. Why study aquatic bacteria? • They are responsible for much of organic matter and nutrient transformation and mineralization • Bacteria are responsible for much of the aerobic respiration and all of anaerobic respiration in aquatic systems • Aquatic bacteria are one of the largest living reservoirs of carbon, P, N, Fe and other materials • Aquatic bacteria represent the largest surface in oceans and lakes • Bacterial biomass may be a significant food resource in aquatic food webs • Some bacteria pose sanitary or environmental problems

3. Ecosystem processes Carbon cycling Gas exchange Trophic interactions: Grazing (predation) Viral mortality Competition Bacterial community structure Bacterial processes: Production Respiration Nutrient cycling Resource supply: the nature and amount of organic matter and nutrients

4. What is community structure at the microbial level? • Bacterial biomass • Bacterial cell size and morphology • Attached versus free-living cells • The distribution of cells with different functions • Taxonomic (phylogenetic) composition • The distribution of cells with different growth and metabolic rates

7. 100 BP mgC m-3 d -1 10 1 10 100 1000 NPP mgC m-3 d -1 From Cole et al. (1988)

8. Bacterial response to changes in resources and conditions ? Δ bacterial community metabolism Δ Environment

9. Bacterial response to changes in resources and conditions ? Δ bacterial community metabolism Δ Environment

10. Bacterial response to changes in resources and conditions ? Δ bacterial community metabolism Δ Environment Changes in abundance

11. Ducklow 1999

12. Bacterial response to changes in resources and conditions ? Δ bacterial community metabolism Δ Environment Changes in composition of bacterial community Changes in abundance

13. Bacterial response to changes in resources and conditions ? Δ bacterial community metabolism Δ Environment Changes in composition of bacterial community Changes in composition of bacterial community Changes in abundance

14. Bacterial response to changes in resources and conditions ? Δ bacterial community metabolism Δ Environment Changes in composition of bacterial community ? Changes in composition of bacterial community Changes in abundance

15. Bacterioplankton black box

16. a Aerobic or CH quimioautotrophs 4 Large viruses or or fermenters small bacteria h O 2 O 2 With or w/o external structure O 2 z z z Attached z or Active free-living or dormant Small Alive or or large dead Phototrophic or heterotrophic Bacterioplankton black box Caja negra del picoplancton Caja negra del picoplancton

17. Starvation, dormancy, slow growth • Dormancy, starvation-survival, slow growth, and inactivity are often used interchangeably to denote low levels of cellular activity in marine bacteria, but these terms are not synonyms and refer to different states

18. Microbial bioenergetics: maintenance versus growth

19. Starvation survival • Under conditions of extreme substrate and energy deprivation, marine bacteria may undergo a “starvation” response • The starvation response is regulated by specific genes and involves cell miniaturization, and profound changes in macromolecular composition, with the synthesis of specialized protective proteins • Prolonged starvation may lead to cell “dormancy”, which is a state of complete metabolic arrest that allows long-term survival under unfavorable conditions. Cells in a dormant state are still more resistant to other environmental stresses • There are costs and benefits associated to entering dormancy as opposed to maintaining a slow level of metabolic activity and growth as a response to low substrate availability • Resource patchiness and temporal variability play a major role in shaping the survival strategies of marine bacteria, whether it is slow growth, starvation response or dormancy

20. The distribution of cells into different physiological categories is termed the “physiological structure” of bacterioplankton • Within a bacterial community there is a continuum of activity, from dead to highly active cells • The categories used to describe the physiological structure are operational and depend on the methods used • The physiological structure is related, albeit in complex ways, to the size structure of the community, as well as to the phylogenetic structure, i.e. the distribution of cells into operational taxonomical units • The physiological structure is dynamic, i.e. the proportions of cells in various physiological states may vary at short time scales and small spatial scales

21. Nyström et al. 1992

22. The starvation sequence Joux and Le Baron 2000

23. The reality of our disciplne: • Thomas Brock's classic microbial ecology text (Brock 1966) is prefaced by a quote attributed as a graduate student motto. The motto simply states, 'microbial ecology is microbial physiology under the worst possible conditions'.

24. “If I could do it all over again, I would be a microbial ecologist. Ten billion bacteria live in a gram of soil... They represent thousands of species, almost none of each are known to science” Wilson, E.O. 1994. Naturalist. Island Press

25. Approaches to measuring single-cell properties Abundance: Nucleic acid staining (SYTO13) Phylogenetic composition: Fluorescence In Situ Hybridization (FISH) Physiological state Highly active cells (CTC, HDNA) DNA Ribosomes ETS 3H 3H O2 CO2 Metabolism C respiration (O2 consumption) C production (3H-thym incorporation) Bacterial Growth Efficiency (BGE) Physiological state: Depolarized cells (DiBac) Physiological state: Altered membrane (BackLight)

26. Some approaches used to assess bacterial characteristics in situ that are culture independent • Microautoradiography to assess uptake of radiolabeled organic compounds • RNA (and other macromolecular) contents • Vital stains as indices of cell metabolism (Fluorescein, Calcein, INT, CTC) • Stains that reflect membrane polarization and integrity (PI, Oxonol, SYTOX, TOPRO) • Structural integrity under TEM

27. Heissenberger et al. 1996 Examples of cell and capsule structure observed by TEM in bacterioplankton samples

28. Zweifel & Hagström (1995) Site BT (106) NuCC (%) MPN (%) Baltic Sea, NB1 2.5 - 3.2 4 - 6 0.1 - 0.3 Baltic Sea, SR5 0.7 - 1.2 17 - 27 7 - 14 Baltic Sea, US5b 0.6 - 2.7 12 - 27 6 - 15 North Sea, Skagerrak-1 1.1 - 1.4 2 - 5 0.5 - 0.6 North Sea, Skagerrak-2 0.2 - 0.8 4 - 32 0.2 - 0.8 Mediterranean, Point B 0.5 20 16

31. Marie et al. 1997Marine picoplankton

32. Cytometric enumeration of in situ aquatic bacteria using green nucleic acid stains

33. Cytometric detection of dead or injured bacteria in situ using exclusion nucleic acid stains

34. Cytometric detection of in situ bacteria with depolarized membranes using the Oxonol DiBAC

35. Cytometric detection of in situ actively respiring bacteria using CTC

36. In situ hybridation visualized with epifluorescence microscopy

37. RNA probing of bacterioplankton using epifluorescence and cytometry

38. Intact cells Damaged cells Empty cells 60 50 40 % of bacterial community 30 20 10 0 1 2 3 4 5 6 Station in a gradient Figs. 1 y 2 from Heissenberger et al. (1996)

39. 0 20 40 60 80 100 From Hoppe (1976) Autoradiography 3H-AA 3H-thymidine 3H-aspartic 14C-glucose CFU Percentage of total cells

40. Smith and del Giorgio 2003

41. Bouvier et al. 2007

42. Bouvier et al. 2007

45. Bouver et al. 2007

47. Lebaron et al. 2001 River and coastal samples

48. Longnecker et al. 2006

49. del Giorgio and Gasol in press