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REGULATION OF ALGINATE GENE EXPRESSION

海藻酸盐基因表达的调控

基本信息

批准号：
2442494
负责人：
Ananda Mohan Chakrabarty
金额：
$ 21.09万
依托单位：
UNIVERSITY OF ILLINOIS AT CHICAGO
依托单位国家：
美国
项目类别：
财政年份：
1993
资助国家：
美国
起止时间：
1993-07-01 至 1999-06-30
项目状态：
已结题

项目摘要

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.

细菌致病的最耐人寻味的例子之一是囊性纤维化患者铜绿假单胞菌感染的研究慢性萎缩性肺病患者的肺部会积聚一种粘稠的脱水粘液。盐分含量高，易感染P。铜绿假单胞菌。最初的感染细胞通常是非粘液样的，但在长时间感染，它们会由于产生一种叫做海藻酸盐的胞外多糖，它包裹着感染病毒铜绿假单胞菌细胞，据信可以保护它们免受吞噬和抗生素治疗。在其他感染中，如眼睛、尿路或烧伤时，它们很少是粘液状(即藻酸盐产生者)。我们最近做了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定位34min，并表达了其中一种末端定位基因海藻D由启动子PalgD控制。这启动子必须被一组调控蛋白阿尔法1、阿尔法2激活在高渗透压和乙醇诱导的条件下脱水--CF肺粘液环境所特有的两种情况。 DNA旋转酶对PalgD区的超卷曲是必不可少的由PalgD转录而成。此外，我们还提纯了阳性的并证明了其与远上游两个位点的结合藻类D启动子的位置。在这项提案中，我们希望确定如何重要的是两个结合位点，以及方向或结合位点的间距对藻类D启动子可能很重要激活。我们还想确定其他藻酸盐基因，如海藻、海藻8、海藻44等也受到调控。最近，我们提纯了 ···并证明了通过 ATP或GTP，并随后将磷酸盐转移到algR1。它对于确定磷酸化的机制是很重要的，两种蛋白质中磷酸化氨基酸的性质和程度磷酸化在藻D启动子激活中的作用。在大肠埃希氏菌中，也可以通过一种alg2-类似物将alg1磷酸化。在三磷酸腺苷或三磷酸鸟苷存在下发生磷酸化的蛋白质。这提出了关于这个中间人的角色的有趣的问题大肠埃希菌中的磷酸化试剂。此外，另一个编码a的基因饥饿诱导蛋白SSPA允许与algR2互补突变，引发了阿尔法R2作为RNA聚合酶的作用的问题接触蛋白质。