Study of pchP regulation in Pseudomonas aeruginosa

MASSIMELI J; MANSILLA, MC; CHOI, KH; LISA, AT; SCHWEIZER, HP

Laramie, Wyoming, USA

Congreso; Rocky Mountain Branch-American Society for Microbiology; 2006

American Society for Microbiology

Our research group previously documented the presence of an inducible phosphorylcholine phosphatase, PChP, in P. aeruginosa. This protein plus the hemolytic phospholipase C (PlcH), are synthesized when bacteria are grown in culture media containing high or low phosphate concentration with choline or derivatives as the sole nutritional source. It was postulated that through the coordinated action of PlcH and PChP, bacteria may break down the choline-containing compounds of the host cells, and in this way, colonize host tissues under varied environmental conditions. The PChP enzyme has been extensively studied in our group and recently the gene responsible for its transcription was identified and named pchP. Taking advantage of this development, we proposed to elucidate the molecular mechanisms governing its expression in P. aeruginosa, as model of choline regulated genes. Preliminary data showed that this gene is principally transcribed as a monocistronic mRNA of 1Kb. Then, a 250 bp intergenic fragment upstream of pchP was PCR amplified and fused to lacZ in a mini-Tn7 derivative plasmid allowing the integration of the region to the PAO1 chromosome in single copy.  Consistently with preliminary data, a functional promoter was found in the region immediately upstream of pchP. PA5293, a putative LysR-type regulator-encoding gene, is located usptream of pchP. A deletion mutant of this gene, which will henceforth be called pchT, was constructed. By overlap extension PCR a mutant fragment was generated. This fragment was cloned in the Gateway-compatible replacement vector pEX18ApGW by BP and LR clonase reactions. The suicide plasmid pEX18ApGW-gene::Gm was transferred into P. aeruginosa by electroporation. Colony PCR utilizing the gene-specific up and down primers showed that double crossing-over events occurred replacing the wild type gene by the mutant fragment. PChP measurements in pchT deletion mutant showed a reduction of 60% in PChP activity compared to the WT strain in the periplasmic space. This is consistent with previous observations seen with transpositional mutants. Mariner transposon mutagenesis was performed to genetically identify repressor genes involved in regulation of pchP transcription. Identification of mutant with a derepressed pchP expression was performed by selection of blue colonies on LB-gentamycin Xgal plates, and bgalactosidase activity assay. Several up-regulated mutants were generated using this approach with an increased promoter activity in cells grown in the presence of preferred carbon and nitrogen source when compared to the wild type strain.