Collaborative Research : Determining Growth Rates of Specific Bacterioplankton

微生物海洋学见证了一场革命，分子和最近的基因组方法在海洋微生物研究中的应用。虽然对环境有重要意义的生物的多样性和代谢能力现在有了改善的图景，但系统发育多样性和异养海洋浮游细菌活动之间的直接联系仍然难以捉摸。该项目将通过一系列实验室和实地实验来弥补这一差距，其最终目标是通过直接测量参与基本细胞过程(例如细胞分裂、DNA复制等)的基因的表达来测量浮游细菌特定成员的原位生长率。这种方法的一个优点是直接测量瞬时人口参数，而不需要不稳定的DOC修正或孵化。此外，将监测特定细菌种群的活动，而不是整个群落的活动，从而更好地了解群落结构对生态系统功能的重要性。这项研究将集中在海洋细菌的玫瑰杆菌谱系上。这一分支的成员普遍存在于海洋浮游生物中，并经常丰富，与特定和重要的生物地球化学作用有关，是最近全基因组测序工作的主要焦点。在不同参数(如稳态和非稳态)下培养的玫瑰杆菌的基因表达谱将通过微阵列分析和定量PCR进行分析；这些研究的结果将指导环境研究中目标基因的选择(如FtsZ)。诊断基因将直接从北大西洋沿岸水域自然丰富的罗氏杆菌谱系种群中克隆和测序，并通过定量逆转录聚合酶链式反应监测环境样本中的DIEL表达谱。最后，基于对基因丰度(基因拷贝数/毫升)随时间增加的测量的增长率估计将与基因表达水平(基因拷贝数/基因拷贝数)相关。拟议工作的更广泛的科学影响包括与科学家和整个科学界的接触。该项目的发现将为研究特定浮游细菌生态型的原位活动奠定基础。结果将通过出版物和全国会议情况介绍进行传播。此外，还将开发专门用于环境样本定量聚合酶链式反应的网页，包括详细的协议和与环境有关的引物组和探针组的储存库。该项目的成果也将传达到更广泛的教育结构中。PIS将把这一项目的成果纳入其本国机构的本科和研究生课程，并将通过外联计划(如课堂讲座、课程建议、科学竞赛)为当地学校系统的小学和中学教育做出贡献。最直接的影响将是两名研究生和多名本科生的教育，他们将接受微生物生理学、定量分子生态学和生物海洋学的综合培训。

Microbial Oceanography has witnessed a revolution with the application of molecular, and more recently genomic, approaches to the study of marine microbes. While an improved picture of the diversity and metabolic capabilities of environmentally significant organisms now exists, direct links between phylogenetic diversity and activity of heterotrophic marine bacterioplankton remain elusive. This project will address this gap with a series of laboratory and field experiments designed with the ultimate goal of measuring in situ growth rates of specific members of the bacterioplankton by direct measurement of the expression of genes involved in fundamental cellular processes (e.g. cellular division, DNA replication, etc.). An advantage of this approach is that instantaneous population parameters are measured directly without labile DOC amendment or incubation. Also, the activity of specific bacterial populations, rather than entire communities, will be monitored and thus provide an improved understanding of the significance of community structure to ecosystem function. The research will focus on the Roseobacter lineage of marine bacteria. Members of this clade are ubiquitous and often abundant in marine plankton, have been linked to specific and significant biogeochemical roles and are a main focus of recent whole genome sequencing efforts. The gene expression profiles of cultured Roseobacters grown under different parameters (e.g. steady and non-steady state) will be analyzed by microarray analysis and quantitative PCR; results from these studies will guide the selection of genes to be targeted in environmental studies (e.g. ftsZ). Diagnostic genes will be directly cloned and sequenced from naturally abundant Roseobacter lineage populations in North Atlantic coastal waters and diel expression profiles monitored in environmental samples by quantitiative reverse transcription PCR. Finally, growth rate estimates based on measurements of increases in gene abundance (gene copies/ml) with time will be correlated to gene expression levels (cDNA/gene copies). The broader scientific impact of the proposed work encompasses outreach to scientists and the scientific community at large. The project's findings will lay the foundation for studies of in situ activity of specific bacterioplankton ecotypes. Results will be disseminated through publications and national meeting presentations. In addition, web pages dedicated to the application of quantitative PCR to environmental samples will be developed, including detailed protocols and a repository for environmentally relevant primer and probe sets. Results from this project will also be conveyed to a broader educational structure. The PIs will integrate results from this project into both undergraduate and graduate curricula at their home institutions and will contribute to primary and secondary education in local school systems through outreach programs (i.e. classroom lectures, advising in curricula, science competitions). The most direct impact will be the education of two graduate and multiple undergraduate students, who will receive combined training in microbial physiology, quantitative molecular ecology and biological oceanography.