Biology of the N2-fixing soil bacterium, Sinorhizobium meliloti

这种细菌名为紫花苜蓿中华根瘤菌，在紫花苜蓿的根部形成固氮结节。这种共生关系代表了植物和细菌之间一系列复杂相互作用的高潮。共生固氮对农业具有重大意义，因为它绕过了与氮肥相关的经济和环境成本。我的NSERC计划旨在了解紫花苜蓿与紫花苜蓿的共生，特别是在根瘤中的细菌以及土壤和根环境中的自由细胞中发生的事件。 苜蓿中华根瘤菌是研究共生固氮、土壤微生物生物学和多组分基因组生物学的模式生物。苜蓿链霉菌基因组由一条3.5Mb的染色体和两个被称为pSymA(1.34Mb)和pSymB(1.67Mb)的大复制子组成。在过去的资助期间，我们操纵了苜蓿链霉菌的基因组，并产生了连续的缺失，总共移除了存在于pSymA和pSymB复制子上的~3000个基因。我们鉴定了活性毒素/抗毒素系统，pSymB上一个新的共生基因和两个对细胞活力至关重要的基因(ArgtRNACCG和EngA)。这两个必需基因是pSymB上110kb区域(指定为ETR)的一部分，在祖先株中，该区域从染色体转移到pSymB。我们已经产生了缺乏pSymA和pSymB复制子的苜蓿链霉菌株，其中一个完整的ETR区域整合到了染色体中。 我们现在建议使用该菌株作为切入点或基本的遗传底盘，我们希望在其上识别最小的共生基因组，即形成紫花苜蓿上固定氮气的根瘤所必需的和足够的基因的最小补充。在过去的40年里，许多实验室的工作确定了与SNF有关的许多结瘤(NOD)、固氮(NIF)和共生基因(FIX)基因。到目前为止，我们已经确定了SNF所需的pSymA和pSymB区域，在我们未来的工作中，我们建议将这些区域结合起来，以产生最小的共生基因组，并通过有针对性地移除单个基因来进一步减少该基因的补体。这将作为合成生物学方法操纵SNF的平台，也将用于识别对特定SNF植物共生和特定细菌宿主重要的其他基因。 在相关项目中，我们建议完成我们最近确定的smb20752基因座的分析。我们的目标是确定有缺陷的SNF表型的分子基础。此外，在一个与土壤中细菌生态更相关的项目中，我们建议分析pSymA复制子上的一个基因座，该基因与限制感染这些细菌的病毒的生长有关。 这项研究计划涉及广泛关注的基本问题，它已经并将继续为本科生、研究生和博士后人员提供高质量的培训。

The bacterium, Sinorhizobium meliloti, forms N2-fixing nodules on the roots of alfalfa. This symbiotic association represents the culmination of a series of complex interactions between plant and bacteria. Symbiotic N2-fixation (SNF) is of huge significance to agriculture as it by-passes the economic and environmental costs associated with nitrogen fertilizer. My NSERC program is directed towards understanding the S. meliloti-alfalfa symbiosis, particularly events occurring within bacteria in nodules and in free-living cells in the soil and root environment. Sinorhizobium meliloti is a model organism for studying symbiotic N2-fixation (SNF), soil-microbe biology and multipartite genome biology. The S. meliloti genome consists of a 3.5 Mb chromosome and two large replicons referred to as pSymA (1.34 Mb) and pSymB (1.67 Mb). During the past funding period, we manipulated the S. meliloti genome and generated sequential deletions that collectively removed the ~ 3000 genes present on the pSymA and pSymB replicons. We identified active toxin/antitoxin systems, a new symbiotic locus on pSymB and two genes (ArgtRNACCG and engA) that are essential for cell viability. These two essential genes are part of a 110 kb region (designated ETR) on the pSymB that translocated from the chromosome to pSymB in an ancestral strain. We have generated S. meliloti strains that lack the pSymA and pSymB replicons, and in which a complete ETR region is integrated into the chromosome. We now propose using this strain as the entry point or a basic genetic chassis onto which we wish to identify the minimal symbiotic genome that is, the minimal complement of genes that are necessary and sufficient for the formation of N2-fixing nodules on alfalfa. Work from many labs over the last 40 years identified many of the nodulation (nod), N2-fixation (nif) and symbiotic genes (fix) genes involved in SNF. Thus far, we have identified pSymA and pSymB regions that are required for SNF and in our future work, we propose to combine these regions to generate a minimal symbiotic genome and further reduce that gene complement through targeted removal of individual genes. This will serve as a platform for synthetic biology approaches to manipulating SNF and also for identification of other genes that are important to the specific SNF plant symbiosis and specific bacterial hosts. In related projects we propose to complete the analysis of the smb20752 locus that we recently identified. Our goal to determine the molecular bases for the defective SNF phenotype. In addition, in a project more related to the ecology of the bacteria in soil, we propose to analyze a locus on the pSymA replicon that is involved in restricting the growth of viruses that infect these bacteria. This research program addresses fundamental questions of broad interest and it has and will continue to generate high quality training for undergraduate, graduate and post-doctoral personnel.