PhoP-PhoQ homologues in Pseudomonas aeruginosa regulate expression of the outer-membrane protein OprH and polymyxin B resistance

Rapid adaptation to environmental challenge is essential for the survival of many bacterial species, end is often effectively mediated by two-component regulatory systems. Part of the adaptive response of Pseudomonas aeruginosa to Mg2+ starvation is overexpression of the outer-membrane protein OprH and increased resistance to the polycationic antibiotic polymyxin B. Two overlapping open reading frames that encoded proteins with high similarities to the PhoP-Phoa two-component regulatory system of Salmonella typhimurium were identified downstream of the oprH gene. A P. aeruginosa PhoP-null mutant, H851, was constructed by means of a phoP::xylE-Gm(R) transcriptional fusion, and shown to be deficient in OprH expression. In contrast, an analogous PhoQ-null mutant, H854 (phoQ::xylE-GmR), exhibited constitutive overexpression of OprH. Normal Mg2+-regulated OprH expression could be restored in both mutants by complementation with a plasmid carrying the phoP and phoQ genes. Measurement of the catechol-2,3-dioxygenase activity, expressed from the xylE transcriptional fusion in strains H851 and H854, indicated that PhoP-PhoQ is involved in the regulation of phoP-phoQ as well as oprH, Reverse transcription polymerase chain reaction experiments and Northern blot analysis revealed linkage of oprH, phoP and phoQ into an operon that was demonstrated to be under the joint control of PhoP-PhoQ and Mg2+ ion concentration. In addition, studies of the polymyxin B resistance of the two mutant strains, H851 and H854, indicated that PhoP-PhoQ is involved in regulating P, aeruginosa polymyxin resistance in response to external Mg2+ concentrations.