|Budget Amount *help
¥1,900,000 (Direct Cost : ¥1,900,000)
Fiscal Year 1992 : ¥400,000 (Direct Cost : ¥400,000)
Fiscal Year 1991 : ¥1,500,000 (Direct Cost : ¥1,500,000)
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
.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