Molecular Genetics of Rhizobium Nodulation Plasmids

描述（由申请人提供）：本研究的长期目标是发展一个详细的、综合的观点，即墨氏中华根瘤菌如何建立共生基础的慢性细胞内感染，并利用这些发现获得哺乳动物慢性细胞内病原体的分子机制的新见解。这项研究为布鲁氏菌（一种严重的、难以治疗的病原体和生物恐怖主义威胁）提供了可能的新药物靶点，为脂多糖和外多糖在细菌与真核宿主相互作用中的作用提供了详细的见解，也有助于阐明维生素B12生物合成的未知部分。我们将通过分析5,6-二甲基苯并咪唑的生物合成途径，以及研究哪些B12依赖酶在木犀草中具有重要的共生作用，来进一步研究木犀草中维生素B12的生物合成及其功能。我们将继续研究CbrA，这一以前未被认识的共生主调控因子，包括通过微阵列分析确定调控靶点，确定CbrA突变体对洗涤剂敏感性的基础，以及确定布鲁氏菌CbrA同源物在发病机制中的作用。我们将利用M. truncatula微阵列鉴定受丁二聚糖调控的植物基因，并利用RNAi敲低和其他方法解剖这些基因的功能，从而确定共生活性根瘤菌外多糖与植物宿主之间相互作用的分子基础。我们将继续研究脂多糖修饰对共生的重要性，并以BacA为重点，包括比较植物对BacA突变体和琥珀聚糖缺乏突变体的反应，确定BacA蛋白的拓扑结构，以及筛选与BacA相互作用的蛋白。我们将通过进一步表征锰对超氧化物歧化酶水平的控制，研究超氧化物敏感性的其他因素，测试其他Mn2+依赖性酶可能的共生重要性，以及测试非同源末端连接对共生的重要性，继续研究锰在氧化应激保护和共生中的作用。我们将确定pmh的生化和生理功能，pmh是一种高度保守的细菌基因，也是一种可能的新药物靶点，它的失活极大地使细胞对许多抗生素敏感，这些抗生素由于获得性耐药而在临床上不再有用。

DESCRIPTION (provided by applicant): The long-term goal of this research is to develop a detailed, integrated view of how Sinorhizobium meliloti establishes the chronic intracellular infection that underlies symbiosis and to use these findings to gain new insights into molecular mechanisms used by chronic intracellular pathogens of mammals. This research is suggesting possible new drug targets for Brucella, a serious, hard-to-treat pathogen and a bioterrorism threat, offering detailed insights into the roles of lipopolysaccharides and exopolysaccharides in bacterial interactions with their eukaryotic hosts, and also helping elucidate the unknown part of vitamin B12 biosynthesis. We will continue to investigate the biosynthesis and function of vitamin B12 in S. meliloti by analyzing the 5,6-dimethylbenzimidazole biosynthetic pathway and investigating which B12-dependent enzymes are of symbiotic importantance in S. meliloti. We will continue to investigate CbrA, a previously unrecognized master regulator of symbiosis, including identifying regulatory targets through microarray analysis, determining the basis of detergent-sensitivity of cbrA mutants, and determining the role of the Brucella CbrA homolog in pathogenesis. We will determine the molecular basis of the interactions between symbiotically active rhizobial exopolysaccharides and the plant host by utilizing M. truncatula microarrays to identify plant genes regulated in response to succinoglycan and then dissecting the function of these genes using RNAi knockdowns and other approaches. We will continue to investigate the importance of lipopolysaccharide modifications for symbiosis and with a focus on BacA, including comparing plant responses to bacA mutants to that of a succinoglycan-deficient mutant, determining the topology of BacA protein, and screening for proteins that interact with BacA. We will continue to investigate the role of manganese in oxidative stress protection and symbiosis by further characterizing control of superoxide dismutase levels by manganese, investigating additional factors in superoxide sensitivity, testing the possible symbiotic importance of other Mn2+dependent enzymes, and testing the importance of non-homologous end-joining for symbiosis. We will determine the biochemical and physiological function of pmh, a highly conserved bacterial gene and a possible new drug target whose inactivation greatly sensitizes cells to numerous antibiotics that are no longer clinically useful because of acquired drug resistance.