Collaborative Researchl: 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、死亡和代谢活性：不同营养状态海洋系统中细胞特异性活性水平的评估

• 批准号: 0002728
• 负责人: Thomas Malone
• 金额: $ 25.16万
• 依托单位: University of Maryland Center for Environmental Sciences
• 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=0002728&HistoricalAwards=false
• 关键词: Collaborative; Researchl; 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.

异养细菌构成大型生物量，并对海洋中的生态系统功能产生重大影响。了解是什么控制了海洋细菌的常规库存丰度和代谢活性，这是微生物海洋学的主要目标。这个目标的核心是解决长期存在但仍然相当大的争议，该争议是关于哪些海洋细菌细胞的分数，通过标准的表现荧光染色方法列举的，是a）代谢活跃且增长，b）活着，b）活着，或不增长或c）死亡。为了简化这一争议，可以将代谢活性如何分布在海洋细菌群落中的细胞之间的反对观点可以说为：方案i？大多数细菌细胞都活着且活跃，代谢过程在社区中广泛分布，还是方案2？只有一小部分细菌组合是高度活跃的，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。各种细胞？特定测定的结果产生了冲突的结果，部分是因为测定不是很定量，并且检测到该测定的活动的阈值尚不为人所知。该项目的主要目的是确定场景I或方案2是否是更准确的，是在海水中细菌活性分布的更准确描述。次要目标是确定基于细菌代谢活性的许多基于荧光色素的意思。为此，我们将检查细胞在生理上可区分的细菌类别之间的特定活性水平。类别将基于：DNA含量：高dNA与含有细菌的DNA（SYTO染色）；细胞膜状态：膜电位（DIBAC染色）和完整的膜与受损的膜（BACLIGHT污渍）；电子传输系统的活性：高度ETS？活性与活性细菌（CTC氧化还原试剂）。我们建议通过a）用放射性标记的底物孵育细菌组合，b）放射性标记细菌的染色和流式细胞术分选，然后c）确定各种类别的细胞的底物掺入速率。我们将将这些结果与通过微疗法被检测到的活性的分类放射性标记的细胞比较。将在整个营养状态的系统中检查细菌组合，从Chesapeake Bay到Meso？到贫营养的俄勒冈上升系统。各个类别之间的细胞特异性代谢率狭窄，将支持第一种情况，而细胞特异性代谢率的广泛范围将支持第二种情况。方案I与生态系统模型中的细菌浮游生物所代表的传统方式兼容：作为具有均匀呼吸速率和有机物利用率的活细胞的连贯组合。但是，如果方案2更准确，那么我们仍然需要了解海洋细菌，例如如何产生，持久并且丢失了非生长细胞和死细胞。该项目将是解决对海洋中细菌活动分布的矛盾观点的一步，这对了解海洋生态系统中细菌的动态具有重要意义。我们的结果也将具有更广泛的含义，因为这项研究将在定量基础上进行许多细胞？可以在许多微生物学领域中使用的特定测定。