Collaborative Researchl: Life, Death and Metabolic Activity in Marine Bacteria: Assessment of Cell-Specific Activity Levels in Marine Systems of Differing Trophic States

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Heterotrophic bacteria constitute a large biomass, and have a significant impact on ecosystem function, in the sea. Understanding what controls the standing stock abundance and metabolic activity of marine bacterioplankton is a main goal of microbial oceanography. Central to this goal is resolution of the longstanding, and still considerable, controversy concerning what fractions of marine bacterial cells, enumerated by standard epifluorescence staining methods, are a) metabolically active and growing, b) alive but not growing, or c) dead. As a simplification of this controversy, opposing views for how metabolic activity is distributed among cells in marine bacterial communities can be stated as: Scenario I? most bacterioplankton cells are alive and active, with metabolic processes broadly distributed over the community, or Scenario 2 ? only a small fraction of the bacterial assemblage is highly active, and it is these cells that are responsible for the bulk of metabolism and production of new cells in the assemblage. Results of various cell?specific assays have yielded conflicting results, in part because the assays are not very quantitative, and thresholds of detection of activity for the assays are not well known.The main goal of this project is to determine whether Scenario I or Scenario 2 is, in general, a more accurate depiction of the distribution of metabolic activity among bacteria in seawater. A secondary goal is to establish what a number of fluorochrome?based assays mean in terms of bacterial metabolic activity. To do this, we will examine cell?specific activity levels among physiologically distinguishable categories of bacteria. The categories will be based on: DNA content: high?DNA versus low?DNA containing bacteria (SYTO stain); state of the cell membrane: membrane potential (DiBAC stain) and intact membrane versus damaged membrane (BacLight stains); and activity of the electron transport system: highly ETS?active versus less active bacteria (CTC redox reagent). We propose to quantify specific activities by a) incubating bacterial assemblages with radiolabeled substrates, b) staining and flow cytometric sorting of the radiolabeled bacteria, and then c) determining substrate incorporation rates for the various categories of sorted cells. We will compare these results with proportion of sorted radiolabeled cells detected as active via microautoradiography. Bacterial assemblages will be examined in systems over a range of trophic states, from a eutrophic estuary, Chesapeake Bay, to the meso? to oligotrophic Oregon upwelling system. A narrow range of cell?specific metabolic rates among the various categories would support the first scenario, while wide ranges of cell-specific metabolic rates would support the second one. Scenario I is compatible with the traditional way that bacterioplankton are represented in ecosystem models: as a coherent assemblage of living cells with uniform rates of respiration and utilization of organic matter. If, however, Scenario 2 is more accurate, then there would be much that we still need to understand about marine bacteria, e.g. how non?growing cells and dead cells are produced, persist, and are lost. This project will be a step toward resolving the conflicting views of distribution of bacterial activity in the sea, which has significant implications for understanding the dynamics of bacteria in marine ecosystems. Our results will also have broader implications, as this study will put on a quantitative basis a number of cell?specific assays that can be used in many fields of microbiology.