Biology of the N2-fixing soil bacterium, Sinorhizobium meliloti

固氮土壤细菌苜蓿中华根瘤菌的生物学

• 批准号: RGPIN-2018-06481
• 负责人: Finan, Turlough
• 金额: $ 5.97万
• 依托单位: McMaster University
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2020
• 资助国家: 加拿大
• 起止时间: 2020-01-01 至 2021-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=714100
• 关键词: Biology; N2; fixing; soil; bacterium

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.

苜蓿中华根瘤菌（Sinorhizobium meliloti）在苜蓿的根部形成固氮根瘤。 这种共生关系是植物和细菌之间一系列复杂相互作用的结果。共生固氮（SNF）对农业具有巨大的意义，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S。苜蓿-苜蓿共生，特别是发生在根瘤中的细菌和土壤和根环境中的自由生活细胞中的事件。 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。 色葡萄苜蓿基因组由3.5Mb染色体和两个大的复制子组成，称为pSymA（1.34Mb）和pSymB（1.67Mb）。 在过去的融资期间，我们操纵了S。苜蓿属植物基因组中，并产生顺序缺失，其共同去除了pSymA和pSymB复制子上存在的约3000个基因。 我们确定了活性毒素/抗毒素系统，一个新的共生位点pSymB和两个基因（ArgtRNACCG和engA）是细胞活力所必需的。 这两个必需基因是pSymB上110 kb区域（称为ETR）的一部分，该区域在祖先菌株中从染色体易位到pSymB。 我们生成了S。缺乏pSymA和pSymB复制子的苜蓿属菌株，并且其中完整的ETR区域整合到染色体中。 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，也就是说，最小的基因互补，这是必要的和足够的苜蓿固氮根瘤的形成。 在过去的40年里，许多实验室的工作确定了许多与SNF有关的固氮基因（nod）、固氮基因（nif）和共生基因（fix）。 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联合收割机组合以产生最小的共生基因组，并通过靶向去除单个基因进一步减少该基因互补。 这将作为合成生物学方法操纵SNF的平台，也用于鉴定对特定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发现的smb 20752位点的分析。 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学更相关的项目中，我们建议分析pSymA复制子上的一个位点，该位点参与限制感染这些细菌的病毒的生长。 该研究计划解决了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。