REGULATION OF ALGINATE GENE EXPRESSION

海藻酸盐基因表达的调控

• 批准号: 3146563
• 负责人: Ananda Mohan Chakrabarty
• 金额: $ 18.99万
• 依托单位: UNIVERSITY OF ILLINOIS AT CHICAGO
• 依托单位国家: 美国
• 财政年份: 1993
• 资助国家: 美国
• 起止时间: 1993-07-01 至 1998-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3146563
• 关键词: DNA binding protein; DNA directed RNA polymerase; DNA footprinting; Escherichia coli; Pseudomonas aeruginosa; adenylate kinase; alginates; bacterial genetics; binding proteins; biological signal transduction; bronchial mucus; cystic fibrosis; enteric bacteria; gene expression; genetic manipulation; genetic promoter element; genetic transcription; guanosine triphosphate; infection; microorganism immunology; mutant; nucleic acid sequence; phosphorylation; site directed mutagenesis; structural genes; transcription factor

One of the most intriguing examples of bacterial pathogenesis is the infection of the Cystic Fibrosis (CF) patients by Pseudomonas aeruginosa. The CF patients accumulate in their lungs a viscous, dehydrated mucus high in salts, which predisposes the patients to infection by P. aeruginosa. The initial infecting cells are typically nonmucoid, but on prolonged infection, they become highly mucoid due to the production of an exopolysaccharide called alginate, which encapsulates the infecting P. aeruginosa cells and is believed to protect them from phagocytosis and antibiotic treatment. In other infections such as eye, urinary tract or burn, they are seldom mucoid (i.e. alginate producer). We have recently demonstrated that many of the alginate structural genes are clustered at 34 min on the P. aeruginosa chromosome, and the expression of one of the terminally located genes, algD, is controlled by a promoter PalgD. This promoter must be activated by a set of regulatory proteins AlgR1, AlgR2 and AlgR3 under conditions of high osmolarity and ethanol-induced dehydration -- two conditions unique to the CF lung mucus environment. Supercoiling of the PalgD region by DNA gyrase is essential for transcription from PalgD. In addition, we have purified the positive regulatory protein AlgR1 and demonstrated its binding at two far upstream sites of the algD promoter. In this proposal, we want to determine how important the two binding sites are, and whether the orientation or the spacing of the binding sites may be important for algD promoter activation. We also want to determine if other alginate genes such as AlgA, Alg8, Alg44, etc. are also regulated. Recently, we have purified AlgR2 and demonstrated autophosphorylation of the AlgR2 protein with either ATP or GTP and subsequent transfer of the phosphate to algR1. It would be important to determine the mechanism of phosphorylation, the nature and extent of phosphorylated amino acids in the two proteins and the role phosphorylation plays in the activation of the algD promoter. AlgR1 is also phosphorylated in Escherichia coli by an AlgR2-analog protein which undergoes phosphorylation in presence of ATP or GTP. This raises interesting questions about the role of this intermediate phosphorylating agent in E. coli. In addition, another gene encoding a starvation induced protein SspA allows complementation of the algR2 mutation, raising the question of the role of AlgR2 as an RNA polymerase contacting protein.

细菌致病机理最有趣的例子之一是 囊性纤维化（CF）患者感染铜绿假单胞菌。 CF患者在肺部积聚了一种粘稠的脱水粘液 盐含量高，容易使患者感染疟原虫。 铜绿。 最初的感染细胞通常是非粘液样的，但 长期感染，由于产生 一种叫做藻酸盐的胞外多糖， P.铜绿假单胞菌细胞，并被认为是保护他们免受吞噬作用， 抗生素治疗 在其他感染，如眼睛，泌尿道或 烧伤，他们很少粘液（即藻酸盐生产者）。 我们最近 证明了许多藻酸盐结构基因聚集在 在铜绿假单胞菌染色体上的34分钟，并且其中一种表达的表达， 位于末端的基因algD由启动子PalgD控制。 这 启动子必须由一组调节蛋白AlgR 1、AlgR 2 和AlgR 3在高渗透压和乙醇诱导的条件下 脱水--CF肺粘液环境特有的两种情况。 DNA促旋酶对PalgD区域的超螺旋化是必需的， 来自PalgD的转录。 此外，我们还纯化了阳性 调节蛋白AlgR 1，并证明其结合在两个远上游 algD启动子的位点。 在本提案中，我们希望确定如何 重要的是这两个结合位点，无论是方向或 结合位点的间隔对于algD启动子可能是重要的 activation. 我们还想确定其他藻酸盐基因， AlgA、Alg 8、Alg 44等也受到调节。 最近，我们净化了 AlgR 2的表达，并证明了AlgR 2蛋白的自磷酸化， ATP或GTP，随后将磷酸盐转移到algR 1。 它 对于确定磷酸化的机制很重要， 两种蛋白质中磷酸化氨基酸的性质和程度， 磷酸化在algD启动子的激活中起作用。 AlgR 1在大肠杆菌中也被AlgR 2类似物磷酸化 在ATP或GTP存在下进行磷酸化的蛋白质。 这 提出了一些有趣的问题， 磷酸化试剂在E.杆菌 此外，另一个编码a 饥饿诱导蛋白SspA允许algR 2的互补 突变，提出了AlgR 2作为RNA聚合酶的作用的问题 接触蛋白质。