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Study on Iron Regulon in Pseudomonas aeruginosa

铜绿假单胞菌铁调节子的研究

基本信息

批准号：
11670257
负责人：
TSUDA Masataka
金额：
$ 2.3万
依托单位：
Tohoku University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
1999
资助国家：
日本
起止时间：
1999 至 2000
项目状态：
已结题

项目摘要

The Pseudomonas aeruginosa genes for production of several pathogenic factors (represented by pyoverdin, pyochelin, alkaline proteinase, and exotoxin A) belong to the iron regulon. The transcription of these genes under iron-rich conditions is repressed by an activated from of Fur, a global repressor of the regulon. This regulator often controls the transcription of some genes by repressing the transcription of pvdS, a gene encoding an alternative sigma factor of RNA polymease that is at least required for transcription of the structural genes for pyoverdin and alkaline proteinase. In this study, the fur gene was analyzed and two methods were devised to identify novel genes in the iron regulon. A 14-kb chromosomal region covering the fur gene was cloned. Its sequence analysis demonstrated that the Fur box, a consensus sequence for binding of Fur, was not found at the upstream region of the fur gene, suggesting that the fur transcription is not auto-regulated by Fur. Genetic attempts we … More re unsuccessful to isolate the chromosomal null mutations of the fur gene as well as the grpE and dnaJ genes that are located downstream of fur, unless the wild-type alleles of these three genes were supplied in trans. These results indicated that the latter two genes are essential and that Fur regulates positively the expression of the unidentified gene(s) whose functions are essential under normal growth conditions. A number of mutants were isolated in each of which a promoter-probe transposon carrying a promoterless gfp gene was inserted into the chromosome. Among such mutants, three mutants expressed the gfp gene preferentially only under iron-rich conditions and three other mutants preferentially only under iron-limiting conditions. The chromosomal regions flanking these six mutation sites were isolated. It has been well known that the iron regulon includes the genes whose protein products are localized to the membrane fraction, and we established a biochemical method by which such products are easily detected by the two-dimension gel electrophoresis. Less

用于产生几种致病因子（以绿脓菌荧光素、绿脓菌螯铁蛋白、碱性蛋白酶和外毒素A为代表）的铜绿假单胞菌基因属于铁调节子。富铁条件下这些基因的转录被调节子的全局阻遏物Fur的激活形式抑制。该调节子通常通过抑制pvdS的转录来控制一些基因的转录，pvdS是编码RNA聚合酶的替代σ因子的基因，其至少是绿脓菌荧光素和碱性蛋白酶的结构基因转录所需的。在这项研究中，毛皮基因进行了分析，并设计了两种方法来确定新的基因在铁调节子。一个14 kb的染色体区域覆盖的毛皮基因进行了克隆。序列分析表明，在fur基因的上游区域没有发现与Fur结合的共有序列Fur box，表明fur基因的转录不受Fur的自动调节。基因的尝试， ...更多信息我们未能成功地分离出毛发基因以及位于毛发下游的grpE和dnaJ基因的染色体无效突变，除非这三个基因的野生型等位基因被反式提供。这些结果表明，后两个基因是必不可少的，并且毛发正调控未鉴定基因的表达，这些基因的功能在正常生长条件下是必不可少的。分离了许多突变体，其中每个突变体中携带无启动子gfp基因的启动子-探针转座子被插入染色体。在这些突变体中，三个突变体仅在富铁条件下优先表达gfp基因，另外三个突变体仅在铁限制条件下优先表达gfp基因。分离这六个突变位点侧翼的染色体区域。铁调节子包括蛋白质产物定位于膜组分的基因，我们建立了一种生物化学方法，通过这种方法可以很容易地通过二维凝胶电泳检测这些产物。少