Regulatory Circuits Controlling Development of Dormant Microbial Cysts

描述（由申请人提供）：本提案集中于控制革兰氏阴性菌代谢静止囊肿发育的调节因子的遗传和生化特征。许多为植物固定氮的有益细菌，以及有害的细菌病原体，都能够合成囊。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的囊肿形成，许多囊肿发育调节突变体已经被分离出来。一类突变体显著过量产生囊肿，而另一类突变体显著抑制囊肿形成。突变细胞系的分子遗传分析导致了一个令人惊讶的发现，囊肿形成的调节涉及cGMP的产生和排泄。cGMP是真核生物中一个众所周知的信使，它参与视觉、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很有争议。我们最近的研究明确地证明了cGMP参与百岁梭菌的囊肿发育，并暗示了cGMP在许多其他重要细菌物种中的产生，如巴西偶氮螺旋菌、豆科根瘤菌、loti中根瘤菌、Sinorhizobium medicae、Sinorhizobium meliloti、Sinorhizobium fredii、Delftia acidovorans （Comamonas acidovorans）、mallei Burkholderia （mallei假单胞菌）。cGMP生产的生化表征及其参与细菌信号转导是本提案的主要目标。我们还分离了许多额外的囊肿发育调节突变，这些突变在各种细菌信号转导成分和各种转录因子中包含遗传破坏。为了建立控制细菌囊肿形成的调控回路的详细模型，将对每一种突变细胞系进行全基因组基因表达的改变。

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.