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

异养菌在海洋中构成了大量的生物量，并对生态系统的功能有重大影响。了解是什么控制了海洋浮游细菌的现存量和代谢活动是微生物海洋学的一个主要目标。这一目标的核心是解决长期而仍然相当大的争议，即通过标准的荧光染色方法计数的海洋细菌细胞的哪些部分是a)代谢活跃和生长的，b)活的但不生长的，或c)死亡的。作为这一争议的简化，关于海洋细菌群落中新陈代谢活动如何在细胞之间分配的对立观点可以表述为：情景I？大多数浮游细菌细胞是活的和活跃的，代谢过程广泛分布在群落中，还是情景2？细菌组合中只有一小部分是高度活跃的，正是这些细胞负责组合中大部分新陈代谢和新细胞的产生。各种特定细胞分析的结果产生了相互矛盾的结果，部分原因是这些分析不是非常定量的，而且这些分析的活性检测阈值也不是很清楚。这个项目的主要目标是确定总体上是情景I还是情景2更准确地描述海水中细菌之间的代谢活动分布。第二个目标是确定一些基于荧光色素的分析在细菌代谢活动方面的意义。为此，我们将检查生理上可区分的细菌类别中的细胞特定活动水平。分类将基于：DNA含量：高DNA与低DNA细菌(Syto染色)；细胞膜状态：膜电位(DiBAC染色)和完整膜与受损膜(BacLight染色)；以及电子传输系统的活性：高ETS活性与低活性细菌(CTC氧化还原试剂)。我们建议通过a)将细菌组合与放射性标记的底物孵育，b)放射性标记的细菌的染色和流式细胞仪分类，然后c)确定各种分类细胞的底物掺入率来量化特定的活性。我们将把这些结果与通过显微放射自显影检测为活跃的分选的放射性标记细胞的比例进行比较。细菌组合将在一系列营养状态的系统中进行研究，从富营养化的河口，切萨皮克湾，到中？低营养的俄勒冈州上升流系统。不同类别的细胞特定代谢率的狭窄范围将支持第一种情况，而广泛的细胞特定代谢率将支持第二种情况。情景一与浮游细菌在生态系统模型中的传统表现方式是一致的：作为具有统一呼吸速率和有机质利用的活细胞的连贯集合。然而，如果情景2更准确，那么关于海洋细菌，我们仍有许多需要了解的东西，例如，不生长的细胞和死亡细胞是如何产生、持续和丢失的。这个项目将是朝着解决关于海洋中细菌活动分布的相互矛盾的观点迈出的一步，这对理解海洋生态系统中细菌的动态具有重要意义。我们的结果也将具有更广泛的影响，因为这项研究将在定量的基础上进行一些细胞特异性的分析，这些分析可以用于微生物学的许多领域。

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.