SRF OPERON--REGULATION AND ROLE IN GENETIC COMPETENCE

SRF 操纵子——遗传能力的调节和作用

• 批准号: 2883006
• 负责人: PETER ZUBER
• 金额: $ 23.67万
• 依托单位: OREGON GRADUATE INSTITUTE SCIENCE & TECH
• 依托单位国家: 美国
• 财政年份: 1992
• 资助国家: 美国
• 起止时间: 1992-02-01 至 2000-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2883006
• 关键词: Bacillus; Escherichia coli; bacterial genetics; fusion gene; gene mutation; genetic regulation; genetic transcription; genetic translation; immunoelectron microscopy; molecular cloning; nucleic acid sequence; operon

Bacteria possess complex signal transduction networks that govern the cells' response to environmental changes. In Bacillus subtilis, such a network controls processes that are activated when a growth-limiting environment is encountered. Systems which sense conditions of high cell density and starvation converge to activate the expression of the srf operon, encoding the enzyme complex that catalyzes the biosynthesis of the lipopeptide antibiotic, surfactin, and encoding a regulator of genetic competence, ComS, which is required for the expression of genes that function in DNA import. Studies of srf regulation and function will enhance our understanding of how bacteria regulate the production of toxins and antibiotics, and the process of genetic transformation, which is one way bacteria acquire genes that confer drug resistance. Mutations that alter the nutrition-dependent regulation of srf expression and sporulation, called ggr (glucose-glutamine-dependent regulation), result in an elevated concentration of sigma-H, encoded by spoOH and required for the production of the pheromone Csf (competence stimulating factor) which activates srf transcription. The ggr mutations also confer a Crs (catabolite resistant sporulation) phenotype. The ggr loci will be isolated and characterized to determine their roles in cell density- and nutritional control of srf expression. The comS gene, located within and out-of-frame with the srfB gene of the srf operon, is subject to regulation that is separate from that which controls srf transcription initiation. This regulation requires sinR, which also functions in the cell's decision to undergo sporulation or competence development. The region(s) of srf that functions in sinR- dependent control will be identified. Other mutations that effect specifically comS expression will be isolated and characterized by nucleotide sequence determination, and by introducing the mutations into sinR mutants or cells that overexpress sinR. The intracellular location and the cell type distribution (competent or non-competent cell) of ComS will be determined using anti-ComS antibody. The structure of ComS and its possible role in controlling the activity of DegU (a response regulator-like protein that functions in the contor of degradative enzyme production and competence), ComK (the transcriptional activator of competence genes) or MBA and B (homologues of ATP-dependent proteases and heat shock proteins, and negative regulators of ComK) will be investigated by mutational analysis and protein-protein crosslinking experiments. The identification of genes that encode ComS- binding proteins will be isolated using the yeast two-hybrid system.

细菌拥有复杂的信号转导网络， 细胞对环境变化的反应。在枯草芽孢杆菌中，这样的 网络控制的过程是激活时，增长限制 环境遇到了。感应高压电池条件的系统 密度和饥饿汇聚以激活SRF的表达 操纵子，编码催化生物合成的酶复合物， 脂肽抗生素，表面活性素，并编码遗传调节因子 能力，ComS，这是表达基因所必需的， 导入DNA的功能。对SRF调控和功能的研究将 提高我们对细菌如何调节生产的理解， 毒素和抗生素，以及基因转化的过程， 是细菌获得耐药性基因的一种方式。突变 其改变SRF表达的营养依赖性调节， 孢子形成，称为ggr（葡萄糖-谷氨酰胺依赖性调节），结果 在高浓度的sigma-H中，由spoOH编码， 信息素Csf（感受态刺激因子）的产生， 激活SRF转录。ggr突变也赋予Crs （分解代谢物抗性孢子形成）表型。ggr基因座将是 分离并表征以确定它们在细胞密度中的作用， SRF表达的营养控制。comS基因位于 与srf操纵子的srfB基因框外， 与控制SRF转录的调节分开的调节 初始化。这种调节需要sinR，sinR也在 细胞决定进行孢子形成或感受态发育。的 在sinR相关控制中起作用的srf的区域将是 鉴定其他特异性影响comS表达的突变将 通过核苷酸序列测定进行分离和表征， 将突变引入sinR突变体或过表达的细胞中 sinR。细胞内位置和细胞类型分布（感受态） 或非感受态细胞）的ComS将使用抗ComS 抗体的ComS的结构及其在控制 DegU（一种在细胞中起作用的反应调节剂样蛋白）的活性 降解酶的生产和能力的控制器），ComK（ 感受态基因的转录激活因子）或MBA和B（ ATP依赖性蛋白酶和热休克蛋白，以及负调节因子 将通过突变分析和蛋白质-蛋白质 交联实验编码ComS的基因的鉴定- 将使用酵母双杂交系统分离结合蛋白。