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中的感觉传递。一种用于测量细菌趋化反应的广泛使用的方法是毛细血管测定技术。尽管该测定技术提供了有关趋化性的定量信息，但它很耗时，并且从试验到试验都显示出很大的差异。因此，我们开发了毛细管测定技术的计算机辅助版本，以便可以在几分钟内评估对吸引剂的细菌反应。在这种快速技术下，我们发现在磷酸盐限制下生长时，Paoi Paoi Paoi暴露的磷酸盐出租车暴露了磷酸盐。与酒精磷酸酶活性并行，观察到在磷酸盐限制下生长过程中磷酸盐出租车的强度会增加。我们分离了用于酒精磷酸酶的组成型突变体。所有突变体均显示出对磷酸盐的反应，而不管细胞是否饿死了磷酸盐。该结果表明，P…More .Aeruginosa在与Phoa相同的调节基因的控制之下。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，在磷酸盐限制下，Phob突变体的磷酸出租车仍然可以诱导，显示磷酸出租车不受PHOB的调节控制。我们进一步试图克隆与其感觉转导有关的p.aeruginosa基因。使用鼠伤寒链球菌tar透射蛋白基因制备杂交探针。探针与P.铜绿假单胞菌菌株PAOI的ECORI消化基因组DNA充分杂交，并将探测器强烈杂交的5.4-kb Ecori DNA片段融合到高拷贝质量质体PUC18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，该片段包含一个开放式阅读框，该片段与S. typhimurium tar具有高度的同源性。较少的