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

海洋原核生物群落现在被认为是高度多样化的，并且可能正在进行新的代谢类型，如果得到证实，可能从根本上改变海洋中能量和物质流动的模式。这些代谢包括光异养和化学营养途径，这些途径是完全新颖的，或者不被认为发生在海洋的表层。问题是，我们不知道这个多样性群落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，特别是基因组数据所提示的新功能，是否实际上是由海洋原核生物群落进行的。本项目将解决以下问题和假设：什么细菌在开放的海洋环境中最活跃，比如马尾藻海？研究人员假设，数量最多的浮游细菌群也是最活跃的，而数量较少的浮游细菌群则不那么活跃。这一假设将使用四个活性指标进行探讨：i) 16S rRNA与16S rRNA基因的水平；ii)通过胸腺嘧啶类似物BrdU的掺入来测量复制细胞；iii)显微放射自显影结合荧光原位杂交（Micro-FISH）显示，大量细菌群中存在关键溶解化合物；iv)除16S rRNA外的生长依赖性系统发育标记物（如tuf、rpoB和dnaE）的转录水平。研究人员特别感兴趣的是，稀有细菌是否不活跃，是否可能成为“种子库”的一部分，作为未来群落的接种物。2. 哪些代谢过程由最常表达的基因所代表？研究人员推测，最常表达的基因将是那些与溶解有机物处理相关的基因，而不是其他能量产生机制，包括光异养和趋化养石。通过对马尾藻海mRNA（亚转录组）的焦磷酸测序来检测其表达。我们将把超转录组映射到马尾藻海的宏基因组集合上，并探索在宏基因组研究中哪些基因是真实的，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QPCR相结合的方法来检测马尾藻海表层水的rRNA:rDNA比率、BrdU结合细胞以及转录物类型和数量。焦磷酸测序（454）避免了扩增和克隆产物，并且具有成本效益。初步分析表明，454个reads的序列长度和建议的序列数量是解决本文提出的问题的理想选择。研究人员还将使用Micro-FISH检查胸苷、亮氨酸和PO4的结合。在异养细菌产量最低的春季浮游植物繁殖期和夏季细菌产量高峰期间，每年采集两次样本。该项目将通过提供关于细菌种群活动和存量的长期问题的答案，并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，大大改变我们对寡营养海洋微生物过程的看法。该项目将支持一名研究生，并让代表性不足的本科生参与夏季研究项目，包括海上实地工作。这个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之旅的特色（每年约有1000名游客），Campbell和Kirchman还参与了海岸日（Coast Day），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关键区域研究中心的外展活动。

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.