Phylogenetic diversity of bacterioplankton in relation to distribution of cell-specific physiological properties and environmental conditions in an upwelling ecosystem

海洋浮游细菌是海洋生态系统的核心和数量上重要的组成部分；它们在确定海洋系统将大气二氧化碳隔离到深海的能力方面发挥着重要作用。目前为研究这些问题而构建的模型认为，海洋浮游细菌是一个整体活动的隔间。这在很大程度上是因为缺乏对特定的系统发育或生理上可识别的细菌群体是否具有最佳生长的独特条件的理解，或者是否如模型假设的那样，不同的浮游细菌群体实际上对不断变化的环境条件做出了一致的反应。用核酸特异性染色对海洋浮游细菌进行的流式细胞术计数表明，根据细胞特异性核酸(NA，即DNA和RNA)的含量，常规存在两种不同的细菌细胞群。对于这些不同的细菌集群，有人提出了相反的观点：1)大多数细菌的活性是由于较高核酸含量(高NA)的细胞，而较低核酸含量的细胞(Low-NA)要么生长缓慢，要么死亡，或2)高NA和低NA细胞都具有相似的生物量比活跃率。这项研究将集中在俄勒冈州上升流生态系统中细菌群落中存在的高NA和低NA细胞。两个可供选择的假说将被检验：HO 1.高NA和低NA细菌细胞由系统发育上不同的细菌组合组成，这些细菌组合显示出不同的生理活性。如果高NA和低NA细胞倾向于落入适应不同生长策略的不同细胞组合，就会出现这种情况。HO-2-高-NA和低-NA细胞往往具有相似的系统发育组成和相似的生物量比活动率。该方法将涉及基于细胞特异性核酸或蛋白质含量的细菌的流式细胞仪计数和分类。分选的细菌将通过事先用放射性底物标记来检测细胞特异性活性，并通过从分选的细胞中提取的DNA进行聚合酶链式反应扩增，然后进行DGGE分析、选定的凝胶条带测序和系统发育鉴定来分析系统发育多样性。同时，将在过滤器上收集放射性标记的亚样本，并使用区域特异性探针和更特异的探针通过显微放射自显影术和荧光原位杂交(FISH)相结合的方法进行分析，以确定底物活性系统发育群对细菌总丰度的相对贡献。该项目的一个主要目标是将结果放在生态背景下，即将高NA和低NA浮游细菌之间的活动和多样性的变异性与可能影响海洋细菌生长的环境参数(例如，潜在的自下而上的控制，如浮游植物生物量和河流DOC流入)联系起来。知识价值：这项研究项目将探讨海洋浮游细菌的生理多样性和系统发育多样性之间的联系，以及这种联系可能如何影响海洋系统中有机物的细菌加工。这一结果将是理解浮游细菌群落的特定系统发生成分在全球生物地球化学循环和碳通量中的作用的重要一步。更广泛的影响：该项目将支持一名女研究生的博士论文研究，并为该学生提供博士后培训；它还将加强P.I.S的教学努力，并支持海洋与大气科学学院常用的流式细胞仪设备和分子遗传学能力。

Marine bacterioplankton are central and quantitatively significant components of marine ecosystems; their role looms large in determining the capacity of oceanic systems to sequester atmospheric CO2 into the deep ocean. Current models constructed to examine these questions consider marine bacterioplankton as a monolithically active compartment. This is in large part due to the lack of understanding regarding whether specific phylogenetic or physiologically identifiable groups of bacteria have distinct conditions for optimal growth, or whether diverse groups of bacterioplankton do in fact respond uniformly to changing environmental conditions, as the models assume. Flow cytometric enumeration of marine bacterioplankton using nucleic-acid specific stains has shown the routine presence of two distinct clusters of bacterial cells based on cell-specific nucleic acid (NA, i.e. DNA and RNA) content. Opposing ideas have been proposed concerning these distinct clusters of bacteria: 1) most bacterial activity is due to cells with higher nucleic acid content (high-NA), while lower nucleic acid content cells (low-NA) are either slow-growing or dead, or 2) both high-NA and low-NA cells have similar biomass-specific rates of activity. This research will focus on the high-NA and low-NA cells present in bacterial communities in the Oregon upwelling ecosystem. Two alternative hypotheses will be examined: Ho 1. High-NA and low-NA bacterial cells are composed of phylogenetically distinct bacterial assemblages that show differences in physiological activity. This would be the case if high NA and low-NA cells tend to fall into separate assemblages of cells that are adapted to different growth strategies. Ho 2- High-NA and low-NA cells tend to have similar phylogenetic compositions and similar biomass-specific rates of activity. The approach will involve flow cytometric enumeration and sorting of bacteria based on cell-specific nucleic acid or protein content. Sorted bacteria will be assayed for cell-specific activity via prior labeling with radioactive substrates, and assayed for phylogenetic diversity via PCR amplification of DNA extracted from sorted cells followed by DGGE analysis, sequencing of selected gel bands, and phylogenetic identification. At the same time, radiolabeled subsamples will be collected on filters and analyzed by combined microautoradiography and fluorescence in situ hybridization (FISH) using domain-specific probes, as well as more specific probes, in order to determine the relative contribution of substrate-active phylogenetic groups to total bacterial abundance. A main goal of the project is to put the results in ecological context, i.e. to relate variability in activity and diversity among high-NA and low-NA bacterioplankton to environmental parameters likely to affect the growth of marine bacteria (e.g. potential bottom-up controls such as phytoplankton biomass and riverine DOC influx). Intellectual merit: This research project will address the linkages between physiological and phylogenetic diversity in marine bacterioplankton, and how such linkages might affect bacterial processing of organic matter in marine systems. The results would be a significant step in understanding the role of specific phylogenetic components of the bacterioplankton community in global biogeochemical cycles and carbon flux. Broader impacts: This project will support the Ph.D. thesis research of a female graduate student and provide the student with post-doctoral training; it will also enhance the teaching efforts of the P.I..s and support the common use Flow Cytometry Facility and molecular genetics capabilities in the College of Oceanography and Atmospheric Sciences.