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

现在已知海洋原核生物群落具有高度多样性，可能正在进行新型的新陈代谢，如果得到证实，可能从根本上改变海洋中的能量和物质流动模式。这些代谢包括光异养和化能无机营养途径，这些途径是全新的，或者被认为不会发生在海洋表层。问题是，我们不知道这一多样性群体中的哪一部分实际上在生物地球化学过程中是活跃的，也不知道代谢功能，特别是基因组数据提出的新功能，是否实际上是由海洋原核生物群体执行的。本研究将探讨以下问题与假设：1.在开放的海洋环境中，比如马尾藻海，什么细菌最活跃？ 研究人员假设，最丰富的浮游细菌群体也是最活跃的，而罕见的群体将不那么活跃。将使用四个活性指数来探索该假设：i）16 S rRNA与16 S rRNA基因的水平; ii）通过掺入胸苷类似物BrdU来测量的复制细胞; iii）如通过显微放射自显影结合荧光原位杂交所揭示的，大量细菌群掺入关键溶解化合物（Micro-FISH），和iv）除16 S rRNA以外的生长依赖性系统发育标记物（例如tuf、rpoB和dnaE）的转录水平。 研究人员特别感兴趣的是，稀有细菌是否是不活跃的，是否可能是“种子库”的一部分，作为未来社区的接种物。2.最常表达的基因代表了什么样的代谢过程？ 研究人员假设，最常表达的基因将是那些与溶解有机物的处理相关的基因，而不是其他能量产生机制，包括光异养和化能无机营养。将通过焦磷酸测序来自马尾藻海的mRNA（元转录组）来检查表达。 我们将映射的元转录组到宏基因组组装从马尾藻海，并探索哪些基因在宏基因组研究中称为是真实的，而不是生物信息学文物。该项目将使用焦磷酸测序和QPCR方法的组合来检查rRNA：rDNA比率，BrdU掺入细胞，以及马尾藻海表面水的元转录组中的转录物类型和数量。焦磷酸测序（454）避免了扩增和克隆伪影，并且具有成本效益。初步分析表明，454读段的序列长度和建议的序列数量对于解决这里提出的问题是理想的。研究者还将使用Micro-FISH检查胸苷、亮氨酸和PO 4的掺入。每年两次在春季浮游植物水华期间（异养细菌产量最低时）和夏季细菌产量高峰期采集样品。该项目将通过提供长期存在的问题的答案，关于细菌种群的活动和现存量的长期问题，以及将代谢过程与广泛的环境基因组数据联系起来，该项目将支持一名研究生，并让人数不足的本科生参与暑期研究项目，包括海上实地考察。该项目的结果将被纳入一个环境基因组学网站，并用于Kirchman教授的课程。Kirchman和Heidelberg实验室是向公众开放的实验室图尔斯之旅的特色（每年约1000名游客），坎贝尔和Kirchman也参与了海岸日，这是一个每年吸引约10，000名游客的开放日。最后，PI将参与K-12教师培训讲习班和其他特拉华州中心的关键区研究外展活动。

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.