Developmental Asymmetry in Agrobacterium tumefaciens

描述(申请人提供)：许多细菌，包括根癌农杆菌，在与潜在宿主及其环境相互作用时，依赖于特定附属物的不对称定位、分布和定向。发育不对称性的研究在模式a-新月形杆菌中得到了很好的发展，其中连锁的Plec/DivJ-DivK和Cck A-ChpT-CtrA/CpdR His-Asp磷酸传递系统起着核心作用。尽管包括根癌农杆菌在内的a-变形杆菌之间高度保守，但人们对这些调控蛋白在这些其他生物中的确切作用知之甚少。两个相关的蛋白质，Plec-DivJ同源传感蛋白PdhS1和PdhS2，被限制在与真核宿主共生或致病关系频繁的a-变形杆菌的一个子集上。这项研究考察了这些调节蛋白在根癌农杆菌中的作用。特别是，这项应用研究了PdhS1和PdhS2如何对根癌农杆菌的形态、生理和时间发育做出贡献，以及这些过程是否有助于宿主和非宿主生态位之间的转换。这将结合遗传、生化和细胞学分析进行。PdhS1和PdhS2都将被生化解剖，以确定单个催化和调节结构域对其活性的贡献，并确定潜在的相互作用伙伴。根癌农杆菌以极向结合在非生物和生物表面，并以极向鞭毛、菌毛和IV型分泌系统而闻名。此外，根癌农杆菌和许多其他a-变形杆菌通过不对称的萌芽过程进行分裂，通过附着的非运动型进入形态上不同的运动型。表型分析将评估PdhS1和PdhS2对极地发育、运动性、生物膜形成和毒力的贡献。通过结合转座子突变和新型高通量显微镜筛选，将鉴定影响根癌农杆菌极性形态发生、运动性和生物膜形成的协调的其他基因和调控相互作用。

DESCRIPTION (provided by applicant): Many bacteria, including Agrobacterium tumefaciens, rely on an asymmetric localization, distribution, and orientation of specific appendages when interacting with potential hosts and with their environment. The study of developmental asymmetry is well advanced in the model a-Proteobacterial organism Caulobacter crescentus where the interlinked PleC/DivJ-DivK and CckA-ChpT-CtrA/CpdR His-Asp phosphorelays play a central role. Despite a high degree of conservation among the a-Proteobacteria, including A. tumefaciens little is known about the precise role of these regulatory proteins in these other organisms. Two related proteins, the PleC-DivJ homologue sensor kinases PdhS1 and PdhS2, are restricted to a subset of a-Proteobacteria that frequently associate with a eukaryotic host in a symbiotic or pathogenic relationship. This study examines the role of these regulatory proteins in A. tumefaciens. In particular, this application addresses how PdhS1 and PdhS2 contribute to the morphological, physiological, and temporal development of A. tumefaciens and whether these processes contribute to the transition between host and non-host niches. This will be performed using a combination of genetic, biochemical, and cytological analyses. PdhS1 and PdhS2 will both be biochemically dissected to determine the contribution of individual catalytic and regulatory domains to their activity and to identify potential interacting partners. A. tumefaciens binds to abiotic and biotic surfaces in a polar orientation and is known to elaborate polar flagella, pili, and a type IV secretion system. In addition, A. tumefaciens and many other a-Proteobacteria divide by an asymmetric process of budding, proceeding through an attached, non-motile form to a morphologically distinct motile form. Phenotypic analysis will evaluate the contribution of PdhS1 and PdhS2 to polar development, motility, biofilm formation, and virulence. Additional genes and regulatory interactions that influence coordination of polar morphogenesis, motility, and biofilm formation in A. tumefaciens will be identified using a combination of transposon mutagenesis and a novel high-throughput microscopy screen.