Microbial Genome Sequencing: Phylogeny to Function: Genomic Analyses of Dominant, Uncultured Soil Bacteria

土壤是一个巨大的微生物库，其活动对全球变暖潜力、作物生产力、土壤肥力和生物地球化学产生深远影响。 然而，绝大多数土壤微生物仍未培养，因此对它们的了解很少。 通过独立于培养的分子方法鉴定出的主要且一致存在但尚未培养的微生物群包括酸杆菌门、疣微菌门、浮霉菌门和α-变形菌门的成员。为了超越这些群体的系统发育识别和计数，了解其功能，正在采用基因组学方法。设计了一种新方法来从土壤中创建大型（100-150 kb）基因组 DNA 插入文库，以便每个插入片段的一端具有系统发育信息，并且两侧是大部分基因组序列。从这样的文库中，正在选择捕获每个目标微生物群（上图）的系统发育多样性和广度的克隆，并且正在对插入片段进行测序，以识别与独特的营养和代谢模式相关的基因，例如，自养、固氮、反硝化。可以从 22 种不同的尚未培养的土壤微生物中获得 250-750 kb 的基因组信息（覆盖率 * 3 倍），其工作量与对已培养的细菌的完整 5 MB 基因组进行测序所需的工作量相同。根据这些信息，可以了解目标生物的代谢潜力及其在土壤生态系统中的可能作用。新兴的序列数据库将有助于指导未来对这些生物体的研究，以及在纯培养物中获得它们的持续努力。 序列数据可通过核糖体数据库项目 (RDP) 和 GenBank 提供。除了提供用于额外分析的序列之外，RDP 服务器还将通过将序列数据链接到有机体系统发育来在进化背景下呈现数据。 这是一项微生物基因组测序奖，由美国国家科学基金会和农业部未来农业和粮食系统倡议之间的合作项目资助。

Soils constitute a huge reservoir of microbes whose activities have a profound impact on global warming potential, on crop productivity, and on soil fertility and biogeochemistry. However, the vast majority of soil microbes remain uncultured, so knowledge about them is meager. Among the major and consistently present, but not-yet-cultured, microbial groups identified by cultivation-independent molecular methods are members of the Acidobacteria, Verrucomicrobia, Planctomycetes and alpha-Proteobacteria. In an effort to move beyond phylogenetic recognition and enumeration of these groups to their functionality, a genomics approach is being conducted. A novel way was devised to create large (100-150 kb) insert libraries of genomic DNA from soil such that one end of each insert is phylogenetically informative and is flanked by a large portion of genomic sequence. From such libraries, clones are being selected that capture the phylogenetic diversity and breadth of each target microbial group (above) and the inserts are being sequenced to identify genes associated with distinctive modes of nutrition and metabolism, e.g. autotrophy, diazotrophy, denitrification. Between 250-750 kb of genomic information (at * 3X coverage) can be obtained from each of 22 different not-yet-cultured soil microbes with the same effort required to sequence a complete 5 MB genome of a bacterium already in culture. From such information, the metabolic potential of the target organisms and their probable role(s) in soil ecosystems can be realized. The emerging database of sequences will help guide future work on these organisms, as well as ongoing efforts to obtain them in pure culture. Sequence data is being made available through the Ribosomal Database Project (RDP) and through GenBank. In addition to providing the sequences for additional analyses, the RDP server will present the data in a evolutionary context by linkage of sequence data to an organismal phylogeny. This is a Microbial Genome Sequencing Award funded through a collaborative program between the National Science Foundation and the Department of Agriculture Initiative for Future Agriculture and Food Systems.