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

合作研究：海洋细菌的生命、死亡和代谢活动：不同营养状态海洋系统中细胞特异性活动水平的评估

• 批准号: 0002236
• 负责人: Barry Sherr
• 金额: $ 37.86万
• 依托单位: Oregon State University
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-09-01 至 2004-08-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=0002236&HistoricalAwards=false
• 关键词: Collaborative; Research; Life; Death; Metabolic

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.

海洋中的异养细菌构成了大量的生物量，对海洋生态系统功能有着重要的影响。了解是什么控制海洋浮游细菌的现存量、丰度和代谢活动是微生物海洋学的主要目标。这一目标的核心是解决长期存在的、仍然相当大的争议，即通过标准落射荧光染色方法计数的海洋细菌细胞的哪些部分是a）代谢活性和生长的，B）存活但不生长的，或c）死亡的。作为这场争论的简化，关于海洋细菌群落中细胞间代谢活动如何分布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整个社区，或情景2？只有一小部分细菌集合体是高度活跃的，正是这些细胞负责集合体中大量的新陈代谢和新细胞的产生。各种细胞的结果？特定的化验产生了相互矛盾的结果，部分原因是化验不是非常定量，和检测的活性测定的阈值是不是众所周知的。这个项目的主要目标是要确定是否场景I或场景2是，一般来说，更准确地描绘了分布的代谢活动在海水中的细菌。第二个目标是确定荧光染料的数量是多少？根据细菌代谢活性的测定。为了做到这一点，我们将检查细胞？生理上可区分的细菌类别之间的比活性水平。这些类别将基于：DNA含量：高？DNA对比低？含DNA的细菌（SYTO染色）;细胞膜的状态：膜电位（DiBAC染色）和完整膜与受损膜（BacLight染色）;和电子传递系统的活性：高度ETS？活性与活性较低的细菌（CTC氧化还原试剂）。我们建议通过a）将细菌集合体与放射性标记的底物一起孵育，B）对放射性标记的细菌进行染色和流式细胞术分选，然后c）确定各种类别分选细胞的底物掺入率来定量比活性。我们将这些结果与通过显微放射自显影检测为活性的分选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检查，从富营养化河口，切萨皮克湾，中观？到贫营养的俄勒冈州上升流系统。狭窄的细胞范围？各种类别中的特定代谢率将支持第一种情况，而细胞特定代谢率的广泛范围将支持第二种情况。情景一是兼容的传统方式，浮游细菌在生态系统模型中表示：作为一个连贯的集合的活细胞的呼吸和利用有机物质的统一率。然而，如果情景2更准确，那么我们仍然需要了解海洋细菌，例如，如何不？生长细胞和死亡细胞产生、存留和丢失。该项目将朝着解决海洋中细菌活动分布的相互冲突的观点迈出一步，这对了解海洋生态系统中细菌的动态具有重要意义。我们的研究结果也将有更广泛的影响，因为这项研究将把定量的基础上，一些细胞？可用于许多微生物学领域的特定测定。