Contribution of the PhoP-PhoQ and PmrA-PmrB Two-Component Regulatory Systems to Mg²⁺-Induced Gene Regulation inPseudomonas aeruginosa

Abstract

. 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...

The sensor kinase PhoQ mediates virulence in Pseudomonas aeruginosa

“…Microarray analysis also indicated many interesting genes that were strongly affected by mutation of the phoQ gene, but that were not previously shown to be Mg 2+ regulated or within the PhoP regulon (McPhee et al, 2006). These included two type II secretion genes, hxcQ (PA0685; probable type II secretion system protein) and xqhA (PA1868; type II secretion protein), which were downregulated.…”

“…Data analysis of DNA microarrays was carried out as previously described (McPhee et al, 2006). Slide images from the five biologically independent experiments were quantified using ImaGene 6.0 Standard Edition software (BioDiscovery).…”

“…RNA quality was assessed spectrophotometrically and by agarose gel electrophoresis. RNA was converted to cDNA and hybridized as previously described (McPhee et al, 2006).…”

“…The phoQ mutation dysregulated many genes that we had previously shown to be regulated by the PhoP response regulator and/or the PmrA-PmrB two-component system in an Mg 2+ -dependent manner (McPhee et al, 2006). These genes are listed in Supplementary Table S2.…”

“…Although P. aeruginosa PhoQ responds normally to limiting concentrations of Mg 2+ (causing resistance), unlike in Salmonella, phoQ mutants display increased resistance to peptides and polymyxin B when grown in high concentrations of Mg 2+ (Macfarlane et al, 2000;McPhee et al, 2006). In contrast to Salmonella (Bader et al, 2005;, P. aeruginosa PhoQ is not activated by cationic antimicrobial peptides and only very slightly activated under acidic conditions (McPhee et al, 2006). Furthermore, while Salmonella PhoQ is able to both phosphorylate (activate) and dephosphorylate (repress) PhoP, the Pseudomonas PhoQ sensor appears to regulate PhoP only by dephosphorylation (Macfarlane et al, 1999).…”