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.

异养细菌在海洋中构成大量生物量，对生态系统功能具有重大影响。了解海洋浮游细菌的常备种群丰度和代谢活动的控制因素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且仍然相当大的争议，即通过标准落射荧光染色方法计算出的海洋细菌细胞的哪些部分是a）代谢活跃且正在生长，b）存活但不生长，或c）死亡。作为这一争议的简化，对于海洋细菌群落中的细胞代谢活动如何分布的反对观点可以表述为： 情景一？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群落中，或者场景 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的大部分新陈代谢和新细胞的产生。各种细胞特异性测定的结果产生了相互矛盾的结果，部分原因是这些测定不是非常定量，并且这些测定的活性检测阈值也不是很清楚。该项目的主要目标是确定场景 I 或场景 2 是否总体上更准确地描述了海水中细菌之间的代谢活性分布。第二个目标是确定一些基于荧光染料的测定对细菌代谢活性的意义。为此，我们将检查生理上可区分的细菌类别之间的细胞比活性水平。类别将基于： DNA 含量：含高 DNA 与低 DNA 的细菌（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）；电子传递系统的活性：高 ETS 活性与活性较低的细菌（CTC 氧化还原试剂）。我们建议通过以下方式量化特定活性：a）将细菌组合与放射性标记底物一起孵育，b）对放射性标记细菌进行染色和流式细胞术分选，然后c）确定各种类别的分选细胞的底物掺入率。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、切萨皮克湾到中观？到贫营养的俄勒冈州上升流系统。不同类别之间的窄范围的细胞特异性代谢率将支持第一种情况，而宽范围的细胞特异性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表示的传统方式兼容：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。然而，如果情景 2 更准确，那么我们仍然需要了解许多关于海洋细菌的知识，例如。非生长细胞和死亡细胞是如何产生、持续和消失的。该项目将朝着解决海洋中细菌活动分布的相互矛盾的观点迈出一步，这对于了解海洋生态系统中细菌的动态具有重要意义。我们的结果还将产生更广泛的影响，因为这项研究将在定量的基础上进行一些可用于微生物学许多领域的细胞特异性测定。