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: 2378246
• 负责人: PETER ZUBER
• 金额: $ 21.23万
• 依托单位: LOUISIANA STATE UNIV HSC SHREVEPORT
• 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 2000-02-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2378246
• 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来控制 细胞对环境变化的反应。在枯草芽孢杆菌中，这样的 网络控制在增长限制时激活的进程 遇到环境。感测高电池状况的系统 密度和饥饿汇聚在一起激活 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、表面活性素和编码遗传调节因子 能力，ComS，这是基因表达所必需的 在 DNA 输入中发挥作用。 srf 调控和功能的研究将 增强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， 是细菌获得耐药基因的一种方式。突变 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 在较高浓度的 sigma-H 中，由 spoOH 编码，并且是 信息素 Csf（能力刺激因子）的产生 激活 srf 转录。 ggr 突变还赋予 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和细胞密度中的作用 srf表达的营养控制。 comS 基因，位于 和 与 srf 操纵子的 srfB 基因超出框架，受到 与控制 srf 转录的调控分开的调控 引发。该调节需要 sinR，它也在 细胞决定进行孢子形成或能力发展。这 在 sinR 相关控制中起作用的 srf 区域将是 确定。其他特异性影响 comS 表达的突变将 通过核苷酸序列测定来分离和表征，并且通过 将突变引入 sinR 突变体或过度表达的细胞中 罪R。细胞内位置和细胞类型分布（感受态 ComS 的（或非感受态细胞）将使用抗 ComS 来确定 抗体。 ComS 的结构及其在控制中的可能作用 DegU（一种类似反应调节蛋白，在 降解酶产生和能力的控制器），ComK（ 能力基因的转录激活因子）或 MBA 和 B（同源物 ATP 依赖性蛋白酶和热休克蛋白以及负调节因子 ComK）将通过突变分析和蛋白质-蛋白质进行研究 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。