Collaborative Proposal: Are abundant bacteria more active than rare bacteria in the Sargasso Sea?

合作提案：马尾藻海中丰富的细菌是否比稀有细菌更活跃？

• 批准号: 0824981
• 负责人: John Heidelberg
• 金额: $ 33.74万
• 依托单位: University of Southern California
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-10-01 至 2013-09-30
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=0824981&HistoricalAwards=false
• 关键词: Collaborative; Proposal; abundant; bacteria; more

Marine prokaryotic communities are now known to be highly diverse and may be carrying out new types of metabolisms that, if confirmed, could fundamentally alter models of energy and material flow through the oceans. These metabolisms include photoheterotrophic and chemolithotrophic pathways that are entirely novel or were not thought to be occurring in the surface layer of the oceans. The problem is, we do not know which fraction of this diverse community is actually active in biogeochemical processes and whether the metabolic functions, especially the new ones suggested by genomic data, are actually being carried out by marine prokaryotic communities. This project will address the following questions and hypotheses: 1. What bacteria are most active in open oceanic environments like the Sargasso Sea? The investigators hypothesize that the most abundant bacterioplankton groups are also the most active whereas the rare groups will be less active. This hypothesis will be explored using four indices of ?activity?: i) levels of 16S rRNA vs. 16S rRNA genes; ii) replicating cells as measured by the incorporation of the thymidine analog, BrdU; iii) incorporation of key dissolved compounds by abundant bacterial groups as revealed by microautoradiography combined with fluorescence in situ hybridization (Micro-FISH), and iv) transcript levels of growth-dependent phylogenetic markers other than 16S rRNA (e.g. tuf, rpoB and dnaE). The investigators are especially interested in whether rare bacteria are inactive and are potentially part of a ?seed bank? that serves as the inoculum for future communities. 2. What metabolic processes are represented by the most commonly expressed genes? The investigators hypothesize that the most commonly expressed genes will be those associated with the processing of dissolved organic matter rather than other energy generating mechanisms, including photoheterotrophy and chemolithotrophy. Expression will be examined by pyrosequencing mRNA (?metatranscriptome?) from the Sargasso Sea. We will map the metatranscriptome onto metagenomic assemblies from the Sargasso Sea and explore which genes called in metagenomic studies are real rather than bioinformatic artifacts. The project will use a combination of pyrosequencing and QPCR approaches to examine rRNA:rDNA ratios, BrdU incorporating cells, and transcript types and amounts in the metatranscriptome of Sargasso Sea surface water. Pyrosequencing (454) avoids amplification and cloning artifacts and it is cost effective. Preliminary analyses indicate that the sequence length of 454 reads and the proposed number of sequences are ideal for addressing the questions raised here. The investigators will also use Micro-FISH to examine incorporation of thymidine, leucine, and PO4. Samples will be collected twice yearly during the spring phytoplankton bloom when heterotrophic bacterial production is lowest and during the peak of bacterial production in summer.This project will do much to alter our perception of microbial processes in the oligotrophic ocean by providing answers to long-standing questions about activity and standing stocks of bacterial populations and by linking metabolic processes to the extensive environmental genomic data now becoming available.The project will support a graduate student and involve underrepresented undergraduates in summer research projects, including at sea field work. The results from this project will be incorporated into an environmental genomics web site and used in courses taught by Kirchman. The Kirchman and Heidelberg labs are featured in lab tours open to the public (~ 1000 visitors per year) and Campbell and Kirchman are also involved in Coast Day, an annual open house that attracts about 10,000 visitors. Finally, the PIs will be involved in K-12 teacher training workshops and other Delaware Center for Critical Zone Research outreach activities.

现在众所周知，海洋原核生物群落是高度多样的，可能正在进行新型的代谢，如果得到确认，可以从根本上改变能量和物质流过海洋的模型。这些新陈代谢包括完全新颖或不认为在海洋表面层中发生的光旋转和化学养分途径。问题是，我们不知道这个多样化社区的哪一部分实际上在生物地球化学过程中活跃，以及代谢功能（尤其是基因组数据建议的新作用）是否实际上是由海洋原核生物群落执行的。该项目将解决以下问题和假设：1。在萨尔加索海等开放海洋环境中，哪些细菌最活跃？ 研究人员假设最丰富的细菌组组也是最活跃的，而稀有组的活性较小。该假设将使用“活动的四个指数？：i）16S rRNA与16S rRNA基因的水平； ii）复制细胞，如通过胸苷类似物BRDU掺入而测量的； iii）微型摄影术揭示了丰富的细菌基团结合了关键的溶解化合物，并结合了荧光原位杂交（微生物）和IV）生长依赖性系统发育标记物的转录水平以外的16S rRNA（例如TUF，RPOB和DNAE） 。 研究人员对稀有细菌是否不活跃并且可能是“种子库”的一部分特别感兴趣吗？这是对未来社区的接种物。 2。哪些代谢过程由最常见的基因表示？ 研究者假设最常见的基因将是与溶解有机物的处理相关的基因，而不是其他能源生成机制，包括光旋杂体和化学硫代营养。表达将通过从Sargasso Sea的焦磷酸测序mRNA（？metatranscriptome？）来检查。 我们将将元文字组映射到来自萨尔加索海的宏基因组组件上，并探索元基因组研究中所谓的哪些基因是真实的，而不是生物信息学的伪影。该项目将结合使用焦磷酸测序和qPCR方法来检查rRNA：rDNA比，brdu掺入细胞的rRNA，以及萨尔加索海地地表水的元转录组中的转录类型和量。焦磷酸测序（454）避免了放大和克隆伪像，并且具有成本效益。初步分析表明，454个读取的序列长度和提出的序列数是解决此处提出的问题的理想选择。研究人员还将使用微生物检查胸苷，亮氨酸和PO4的掺入。当异养细菌生产最低，在夏季的细菌产生期间，每年将在春季浮游植物开花期间每年两次收集样品。这个项目将对我们对少亲营养海洋中微生物过程的看法作出很大的作用关于细菌种群的活动和站立库存的问题，以及通过将代谢过程与现在可用的广泛环境基因组数据联系起来。该项目将支持研究生，并参与夏季研究项目（包括在海上现场工作）中代表不足的本科生。该项目的结果将纳入环境基因组学网站，并在Kirchman教授的课程中使用。 Kirchman和Heidelberg Labs在向公众开放（每年约1000名游客）的实验室旅行中，坎贝尔和Kirchman也参与了Coast Day，这是一年一度的开放日，吸引了约10,000名游客。最后，PI将参与K-12教师培训研讨会和其他特拉华州关键区研究外展活动中心。