Identification of PhoP-PhoQ homologues in Pseudomonas aeruginosa responsible for regulation of the outer membrane protein OprH

Expression of the Pseudomonas aeruginosa outer membrane protein OprH is induced in low magnesium growth conditions (Nicas and Hancock, 1980; 1983). This protein has been proposed to play a role in stabilizing the outer membrane in the absence of Mg²⁺ by interacting with LPS at sites where these cations would bind. Adaptation to magnesium limitation in Salmonella typhimurium has been shown to occur through activation of the two-component regulatory system, PhoP-PhoQ (Soncini et al., 1996). Putative PhoP and PhoQ proteins were identified in the P. aeruginosa genome through homology searches using the corresponding S. typhimurium protein sequences. The genes encoding these proteins were located directly downstream of the gene encoding OprH. Transcriptional linkage of oprH, phoP and phoQ was demonstrated and the hypothesis that this system regulates expression of OprH in P. aeruginosa was tested in the following study. Through construction of aphoP null mutants and transformation of this mutant with PhoP encoding plasmids, it was shown that PhoP is required for expression of OprH. Furthermore, PhoP was demonstrated to be an activator of oprH, phoP and phoQ transcription from a promoter upstream of oprH. In contrast, a phoQ null mutant showed high-level, unregulated activation of oprH and phoP transcription and OprH expression. Complementation of this mutant demonstrated a requirement for PhoQ in down regulation of transcription and response to magnesium. Analysis of the oprH promoter enabled identification of the start of transcription and delineation of the sequences required for regulated OprH expression to within 90 basepairs of the ATG.Science, Faculty ofMicrobiology and Immunology, Department ofGraduat