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BACTERIAL CHEMOSENSORY TRANSDUCTION AND DEVELOPMENT OF A RAPID ASSAY TECHNIQUE FOR CHEMOATTRACTANTS

细菌化学感应转导和化学引诱剂快速测定技术的开发

基本信息

批准号：
03650793
负责人：
OHTAKE Hisao
金额：
$ 1.22万
依托单位：
HIROSHIMA UNIVERSITY
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (C)
财政年份：
1991
资助国家：
日本
起止时间：
1991 至 1992
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-03650793/
关键词：
Bacteria Chemotaxis Transducer Attractant Sensory 微生物刺激感知機能行動活性物質

项目摘要

We investigated the sensory transduction in Pseudomonas aeruginosa. A widely used method for measuring chemotactic responses in bacteria is the capillary assay technique. Although this assay technique provides quantitative information on chemotaxis, it is time-consuming and tends to show great variability from trial to trial. We developed, therefore, the computer-assisted version of the capillary assay technique so that the bacterial response to an attractant could be assessed within a few minutes.With this quick technique, we found that P. aeruginosa strain PAOI exhibited phosphate taxis when grown under phosphate limitation. In parallel with alkaline phosphatase activity, the strength of phosphate taxis was observed to increase during growth under phosphate limitation. We isolated mutants constitutive for alkaline phosphatase. All of the mutants showed the response to phosphate, regardless of whether the cells were starved for phosphate. This result suggests that phosphate taxis in P … More .aeruginosa is under the control of the same set of regulatory genes as phoA. To study further this point, we isolated a phoB mutant of P. aeruginosa and examined its ability to respond to phosphate. However, the phosphate taxis of the phoB mutant was still inducible under phosphate limitation, showing the phosphate taxis is not under the regulatory control of phoB. We further attempted to clone P.aeruginosa genes that are involved in its sensory transduction. A hybridization probe was prepared using the S. typhimurium tar transducer gene. The probe hybridized well with the EcoRI-digested genomic DNA of P. aeruginosa strain PAOI, and 5.4-kb EcoRI DNA fragment, to which the probe strongly hybridized, was cloned into a high-copy-number plasmid pUC18. After subcloning, we determined the nucleotide sequence of a 1.98-kb region of the cloned fragment. Nucleotide sequence analysis showed that this fragment contained an open reading frame which shared a high degree of homology with that of S.typhimurium tar. Less

我们研究了铜绿假单胞菌的感觉传导。一个广泛使用的方法来测量细菌中的趋化反应是毛细管测定技术。虽然这种测定技术提供了关于趋化性的定量信息，但它很耗时，并且往往在试验之间显示出很大的变化性。因此，我们开发了毛细管测定技术的计算机辅助版本，以便细菌对引诱剂的反应可以在几分钟内进行评估。通过这种快速技术，我们发现铜绿假单胞菌菌株PAOI在磷酸盐限制下生长时表现出磷酸盐趋药性。在平行的碱性磷酸酶活性，磷酸盐的的士的强度被观察到增加在磷限制下的生长过程中。我们分离出碱性磷酸酶的组成型突变体。所有的突变体都表现出对磷酸盐的反应，无论细胞是否缺乏磷酸盐。这一结果表明，磷的趋化性在P ...更多信息铜绿假单胞菌受与phoA相同的一组调控基因控制。为了进一步研究这一点，我们分离了铜绿假单胞菌的phoB突变体，并检查了其对磷酸盐的反应能力。然而，phoB突变体的磷酸盐趋化性在磷酸盐限制下仍然是可诱导的，表明磷酸盐趋化性不受phoB的调节控制。我们进一步尝试克隆铜绿假单胞菌参与其感觉转导的基因。用S.鼠伤寒焦油转换基因。该探针与铜绿假单胞菌菌株PAOI的EcoRI消化的基因组DNA良好杂交，并将与探针强烈杂交的5.4-kb EcoRI DNA片段克隆到高拷贝数质粒pUC 18中。亚克隆后，我们确定了克隆片段的1.98 kb区域的核苷酸序列。核苷酸序列分析表明，该片段含有一个开放阅读框架，与鼠伤寒沙门氏菌tar的开放阅读框架具有高度的同源性。少