| Budget Amount *help |
¥3,500,000 (Direct Cost: ¥3,500,000)
Fiscal Year 1999: ¥1,000,000 (Direct Cost: ¥1,000,000)
Fiscal Year 1998: ¥2,500,000 (Direct Cost: ¥2,500,000)
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| Research Abstract |
Two chemotactic transducers for inorganic phosphate (PィイD2iィエD2) designated CtpH and CtpL, have been identified in Pseudomonas aeruginosa. The corresponding genes (ctpH and ctpL) were inactivated by inserting kanamycin and tetracycline resistance gene cassettes into the wild-type genes in the P. aeruginosa PAO1 genome. The computer-assisted capillary assays showed that the ctpH single mutant failed to exhibit PィイD2iィエD2 taxis when the concentration of PィイD2iィエD2 in the capillary was higher than 5 mM. Conversely, the ctpL single mutant could not respond to PィイD2iィエD2 at the concentration of 0.01 mM. Like PAO1, these single mutants were induced by PィイD2iィエD2 limitation for PィイD2iィエD2 taxis. The ctpH ctpL double mutant was defective in PィイD2iィエD2 taxis at any concentration ranging from 0.01 to 10 mM. To investigate regulation of PィイD2iィエD2 taxis, the ctpH and ctpL genes were also disrupted individually in the P aeruginosa phoU and phoB single mutants. The ctpH phoU and crpH phoB double mu
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tants were defective in PィイD2iィエD2 taxis, regardless of whether the cells were starved for PィイD2iィエD2' The crpL phoU and ctpL phoB double mutants exhibited PィイD2iィエD2 taxis at PィイD2iィエD2 concentrations higher than 5 mM. In addition, the ctpL phoU double mutant was constitutive for PィイD2iィエD2 taxis, whereas the ctpL phoB double mutant was induced by PィイD2iィエD2 limitation for PィイD2iィエD2 taxis. The region upstream of ctpL, but not ctpH, contained a putative pho box sequence. Expression of ctpL: :lacZ was induced by PィイD2iィエD2 limitation in PAO1, while it was constitutive in the phoU mutant. In contrast, the phoB mutant showed only background levels of atpL: : lacZ expression. These results showed that ctpL is involved in the pho regulon genes in P aeruginosa and, therefore, could explain that the ctpH phoB mutant was defective in PィイD2iィエD2 taxis. Furthermore, the fact that the ctpH phoU mutant failed to exhibit PィイD2iィエD2 taxis despite inducible expression of ctpL suggested that the PィイD2iィエD2 detection by CtpL requires PhoU. On the other hand, like PAO1 , the phoB and phoU single mutants were constitutive for expression of ctpH: :lacZ. Thus, the evidence that the ctpL pho U mutant but not the ctpL phoB mutant and PAO1 , was constitutive for PィイD2iィエD2 taxis raised the possibility that PhoU exerts a negative control on the PィイD2iィエD2 detection by CtpH at posttranscriptional level.
A chemotaxis-defective mutants of Enterobacter cloacae IFO3320, designated EC1, was isolated after N-methyl-N'-nitro-N-nitrosoguanidine (NTG). Computer-assisted capillary assays revealed that they failed to show chemotactic responses to peptone and PィイD2iィエD2 Cloning and sequence analysis showed that both of EC1 are a cheR mutant. These results suggest that PィイD2iィエD2 taxis by E. cloacae is dependent on methyl-accepting chemotaxis proteins (MCPs). EC1 was further mutagenized with NTG to isolate cheR pstS and cheR pstA double mutants, designated ECAP1 and ECAP2, respectively. pECT01.1 containing the E. cloacae cheR gene restored the ability of both double mutants to exhibit chemotactic responses toward peptone but not toward PィイD2iィエD2' These results confirm that both of CheR and Pst are simultaneously required for PィイD2iィエD2 taxis in E. cloacae. Less
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