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

海洋原核生物群落现在已知高度多样化，可能正在进行新型新陈代谢，如果得到证实，可能会从根本上改变海洋中能量和物质流动的模式。这些代谢作用包括光异养和化学异养途径，这些途径都是全新的或被认为不会发生在海洋表层。问题是，我们不知道这个多样性群落中的哪一部分实际上活跃在生物地球化学过程中，也不知道代谢功能，特别是基因组数据所表明的新功能，是否实际上是由海洋原核生物群落执行的。这个项目将解决以下问题和假设：1.在像马尾藻海这样的开阔海洋环境中，哪些细菌最活跃？研究人员推测，最丰富的浮游细菌群也是最活跃的，而稀有的浮游细菌群将不那么活跃。这一假说将使用四个活性指数来探索：i)16S rRNA与16S rRNA基因的水平；ii)胸苷类似物BrdU掺入的复制细胞；iii)显微放射自显影结合荧光原位杂交(Micro-FISH)显示的大量细菌群掺入关键溶解化合物；以及iv)除16S rRNA外的生长依赖的系统发育标记的转录水平(例如TUF、rpoB和dna E)。研究人员特别感兴趣的是，稀有细菌是否处于不活跃状态，是否可能是作为未来社区接种剂的“种子库”的一部分。2.最常见的表达基因代表哪些代谢过程？研究人员假设，最常见的表达基因将是那些与处理溶解的有机物有关的基因，而不是其他能量产生机制，包括光异质营养和化学营养。表达将通过来自马尾藻的焦测序mRNA(偏转录组)进行检测。我们将从马尾藻海将超基因组映射到超基因组组合上，并探索在超基因组研究中所称的哪些基因是真实的，而不是生物信息学的人工制品。该项目将使用焦磷酸测序和定量聚合酶链式反应相结合的方法来检测rRNA：rDNA比率、掺入BrdU的细胞以及马尾藻海表水中转录体的类型和数量。焦磷酸测序(454)避免了扩增和克隆人工产物，并且具有成本效益。初步分析表明，454个阅读片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。研究人员还将使用微型鱼类来检查胸苷、亮氨酸和PO4的掺入情况。每年将在异养细菌产量最低的春季浮游植物繁殖期和夏季细菌生产高峰期采集两次样本。这个项目将极大地改变我们对寡营养海洋中微生物过程的看法，因为它回答了关于细菌种群活动和常备库存的长期问题，并将代谢过程与目前可用的广泛环境基因组数据联系起来。该项目将支持一名研究生，并让未获代表的本科生参与夏季研究项目，包括海上实地工作。该项目的结果将被纳入环境基因组学网站，并用于Kirchman教授的课程。Kirchman和Heidelberg实验室向公众开放(每年约1000名游客)，Campbell和Kirchman还参加了每年吸引约10,000名游客的海岸日开放日。最后，专业人员将参加K-12教师培训讲习班和特拉华州关键区域研究中心的其他外联活动。

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.