Rapid adaptation to environmental challenge is essential for the survival of many bacterial species, and 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–PhoQ 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‐GmR 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.

. Only a modest subset of the Mg 2؉ -regulated genes were regulated through either PhoP or PmrA. To determine which genes were directly regulated, a bioinformatic search for conserved binding motifs was combined with confirmatory reverse transcriptase PCR and gel shift promoter binding assays, and the results indicated that very few genes were directly regulated by these response regulators. It was found that in addition to the previously known oprH-phoP-phoQ operon and the pmrHFIJKLM-ugd operon, the PA0921 and PA1343 genes, encoding small basic proteins, were regulated by Mg 2؉ in a PhoP-dependent manner. The number of known PmrAregulated genes was expanded to include the PA1559-PA1560, PA4782-PA4781, and feoAB operons, in addition to the previously known PA4773-PA4775-pmrAB and pmrHFIJKLM-ugd operons.Pseudomonas aeruginosa is an important opportunistic pathogen that is capable of infecting a large number of hosts, including nematodes, insects, plants, animals, and especially humans. It is the third-leading cause of nosocomial infections and is also the leading cause of morbidity and mortality in cystic fibrosis (CF) patients (33). P. aeruginosa is also noted for its metabolic diversity, which allows it to colonize a large number of environmental habitats. The versatility of this organism is believed to be related to the large number of regulatory proteins found in its genome (469 of 5,570 open reading frames) (43).The two-component response regulators constitute one of the larger families of regulatory proteins in P. aeruginosa (43). These systems typically contain a sensor protein that responds to some chemical or physical stimulus, which leads to phosphorylation of the sensor protein at a conserved histidine residue, thus altering the conformation of the sensor and promoting phosphotransfer to a cognate response regulator protein (9). The phosphorylated response regulator then recognizes and binds to a specific DNA sequence, leading to modulation of transcription from that promoter. Although this is often the mechanism, regulation may also occur through phosphatase activity of the sensor kinase with the response regulator (37) or through integration into the signaling cascade of multiple signals from other proteins (32). In P. aeruginosa, there are 64 response regulators and 63 histidine kinases, as well as 16 atypical kinases (36). The functions of the majority of these regulatory proteins have not been established yet.In P. aeruginosa, two separate two-component regulatory systems, PmrA-PmrB (26) and PhoP-PhoQ (21), are known to respond to the presence of limiting concentrations of Mg 2ϩ and to separately regulate certain operons. The PhoPQ system autoregulates the oprH-phoP-phoQ operon (21) under Mg 2ϩ -limiting growth conditions and is also involved in resistance to cationic antimicrobial peptides and polymyxin B and in virulence, as phoQ mutants exhibit increased resistance to cationic antimicrobial peptides and polymyxin B and have reduced virulence (20). Similarly, the PmrAB system regulates resistanc...

Resistance to the polycationic antibiotic polymyxin B and expression of the outer-membrane protein OprH in the opportunistic pathogen Pseudomonas aeruginosa both involve the PhoP-PhoQ two-component regulatory system. The genes for this system form an operon with oprH, oprH-phoP-phoQ, that responds to Mg 2M starvation and PhoP levels. In this study, the Mg 2M -regulated promoter for this operon was mapped upstream of oprH by primer-extension experiments. An oprH ::xylE-Gm R mutant H855 was constructed and measurement of the catechol 2,3-dioxygenase activity expressed from this transcriptional fusion provided evidence for a second, weak promoter for phoP-phoQ. Wild-type P. aeruginosa PAO1 strain H103 was found to exhibit Mg 2M -regulated resistance to the α-helical antimicrobial cationic peptide CP28 in addition to its previously characterized resistance to polymyxin B. Resistance to this peptide was unchanged in the OprH-null mutant H855 and a PhoP-null mutant H851. In contrast, PhoQ-null mutant H854 demonstrated constitutive CP28 resistance. Northern blot analysis revealed constitutive expression of phoP in this strain, implicating PhoP-PhoQ in the resistance of P. aeruginosa to cationic peptides. Furthermore, all three null-mutant strains demonstrated increased resistance to the aminoglycoside antibiotics streptomycin, kanamycin and amikacin. Two additional mutant strains, H895 and H896, were constructed that carried unmarked deletions in oprH and were found to exhibit aminoglycoside susceptibility equivalent to that of the wildtype. This result provided definitive evidence that OprH is not involved in P. aeruginosa aminoglycoside resistance and that the changes in resistance in strain H855 and a previously reported oprH mutant were due to polar effects on phoP-phoQ rather than loss of OprH expression. A role for PhoP-PhoQ in resistance to aminoglycosides is envisaged that is distinct from that in resistance to cationic peptides and polymyxin B.

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