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

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Abstract
When grown in divalent cation-limited medium,Pseudomonas aeruginosabecomes resistant to cationic antimicrobial peptides and polymyxin B. This resistance is regulated by the PhoP-PhoQ and PmrA-PmrB two-component regulatory systems. To further characterize Mg2+regulation inP. aeruginosa, microarray transcriptional profiling was conducted to compare wild-typeP. aeruginosagrown under Mg2+-limited and Mg2+-replete conditions to isogenicphoPandpmrAmutants grown under Mg2+-limited conditions. Under Mg2+-limited conditions (0.02 mM Mg2+), approximately 3% of theP. aeruginosagenes were differentially expressed compared to the expression in bacteria grown under Mg2+-replete conditions (2 mM Mg2+). Only a modest subset of the Mg2+-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 knownoprH-phoP-phoQoperon and thepmrHFIJKLM-ugdoperon, the PA0921 and PA1343 genes, encoding small basic proteins, were regulated by Mg2+in a PhoP-dependent manner. The number of known PmrA-regulated genes was expanded to include the PA1559-PA1560, PA4782-PA4781, andfeoABoperons, in addition to the previously known PA4773-PA4775-pmrABandpmrHFIJKLM-ugdoperons.