Regulatory Circuits Controlling Development of Dormant Microbial Cysts

描述（由申请方提供）：该提案集中于控制革兰氏阴性菌中代谢静止包囊发育的调节因子的遗传和生化表征。许多为植物固氮的有益细菌以及有害的细菌病原体都能够合成孢囊。囊肿是代谢休眠的静止细胞，对干燥和生长抑制剂如抗生素具有极强的抵抗力。本研究分析了革兰氏阴性模式生物R. centenum，已经分离出许多孢囊发育调节突变体。一类突变体显着过量产生孢囊，而第二类突变体显着抑制孢囊形成。突变细胞系的分子遗传学分析导致了一个令人惊讶的发现，即囊肿形成的调节涉及cGMP的产生和排泄。cGMP是真核生物中众所周知的信使，其参与视觉、肌肉收缩、血小板聚集和发育。然而，到目前为止，它与细菌的关系一直存在争议。我们最近的明确研究明确证明了环鸟苷酸与R. Centenum孢囊发育，并且涉及许多其它重要细菌物种中的cGMP产生，例如巴西固氮菌、豆科根瘤菌、Loti中根瘤菌、Sinorhizobium medicae、苜蓿中华根瘤菌、费氏中华根瘤菌、Delftia acidovorans（Comamonas acidovorans）、鼻疽伯克霍尔德氏菌（Burkholderia mallei）（鼻疽假单胞菌））。cGMP产生的生物化学表征及其在细菌信号转导中的参与是该提议的主要目标。我们还分离出了许多额外的囊肿发育调控突变，这些突变包含各种细菌信号转导成分和各种转录因子的遗传破坏。全基因组基因表达的变化将与这些突变体细胞系中的每一个进行，以开发参与控制细菌包囊形成的调控回路的详细模型。

DESCRIPTION (provided by applicant): This proposal is centered on genetic and biochemical characterization of regulatory factors that control development of metabolically quiescent cysts in gram-negative bacteria. Numerous beneficial bacteria that fix nitrogen for plants, as well as harmful bacterial pathogens, are capable of synthesizing cysts. Cysts are metabolically dormant resting cells that are extremely resistant to desiccation as well as to growth inhibitors such as to antibiotics. This study analyzes cyst formation in the gram-negative model organism R. centenum for which numerous cyst developmental regulatory mutants have been isolated. One class of mutants dramatically overproduces cysts while a second class of mutants dramatically inhibits cyst formation. Molecular genetic analysis of mutant cell lines led to a surprising discovery that regulation of cyst formation involved production and excretion of cGMP. cGMP is a well known messenger in eukaryotes where it is involved in vision, muscle contraction, platelet aggregation, and development. However, its involvement in bacteria has until now been quite controversial. Our recent definitive studies have unequivocally demonstrated the involvement of cGMP in R. centenum cyst development and has implicated cGMP production in many other important bacterial species such as Azospirillum brasilense, Rhizobium leguminosarum, Mesorhizobium loti, Sinorhizobium medicae, Sinorhizobium meliloti, Sinorhizobium fredii, Delftia acidovorans (Comamonas acidovorans), Burkholderia mallei (Pseudomonas mallei)). Biochemical characterization of cGMP production, and its involvement in bacterial signal transduction, is a major goal of this proposal. We have also isolated numerous additional cyst developmental regulatory mutations that contain genetic disruptions in a variety of bacterial signal transduction components and in various transcription factors. Changes in genome wide gene expression will be undertaken with each of these mutants cell lines in order to develop detailed models of the regulatory circuits that are involved in controlling bacterial cyst formation.