Regulatory Circuits Controlling Development of Dormant Microbial Cysts

描述(由申请人提供)：这项建议的中心是控制革兰氏阴性细菌中代谢静止的包囊发展的调控因素的遗传和生化特征。许多为植物固定氮素的有益细菌，以及有害的细菌病原体，都能够合成包囊。包囊是新陈代谢休眠的休眠细胞，对干燥和抗生素等生长抑制物具有极强的抵抗力。这项研究分析了革兰氏阴性模式生物中的包囊形成，已分离出大量包囊发育调控突变体。一类突变体极大地繁殖包囊，而第二类突变体极大地抑制包囊的形成。对突变细胞系的分子遗传分析导致了一个令人惊讶的发现，即对包囊形成的调控涉及cGMP的产生和排泄。CGMP是真核生物中众所周知的信使，参与视觉、肌肉收缩、血小板聚集和发育。然而，到目前为止，它对细菌的参与一直存在相当大的争议。我们最近的权威研究已经明确地证明了cGMP参与了百合根瘤菌的包囊发育，并涉及到许多其他重要细菌物种中cGMP的产生，如巴西固氮螺杆菌、豆类根瘤菌、中生根瘤菌、医用中华根瘤菌、苜蓿中华根瘤菌、费氏中华根瘤菌、酸性德尔夫氏菌(Comamonas Acidovans)、马来伯克霍尔德氏菌(Burkholderia Mallei)。CGMP产生的生化特征及其参与细菌信号转导，是这一提议的主要目标。我们还分离出了许多额外的孢子发育调控突变，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基因中断。将对每个突变细胞系进行全基因组基因表达的变化，以开发参与控制细菌包囊形成的调控电路的详细模型。

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.