Regulation of Membrane Permeability by a Two-Component Regulatory System in Pseudomonas aeruginosa (original) (raw)
2003, Antimicrobial Agents and Chemotherapy
Membrane impermeability is the major contributing factor to multidrug resistance in clinical isolates of Pseudomonas aeruginosa. By using laboratory strain PAK, a spontaneous P. aeruginosa mutant (mutant PAK1-3) whose membrane had reduced permeability and which displayed increased levels of resistance to various antibiotics, especially aminoglycosides, was isolated. By complementation of the mutant with a genomic clone library derived from wild-type strain PAK, a novel two-component regulatory system (PprA and PprB) was identified and was found to be able to increase the permeability of the bacterial membrane and render PAK1-3 sensitive to antibiotics. Furthermore, specific phosphorylation of the response regulator (PprB) by histidine kinase (PprA) was observed in vitro, demonstrating that they are cognate two-component regulatory genes. Introduction of a plasmid expressing the pprB gene into randomly chosen clinical isolates (n ؍ 17) resulted in increased sensitivity to aminoglycosides in the majority of isolates (n ؍ 13) tested. This is the first demonstration that P. aeruginosa membrane permeability can be regulated, providing an important clue in the understanding of the mechanism of membrane impermeability-mediated multidrug resistance in P. aeruginosa. on June 11, 2015 by guest http://aac.asm.org/ Downloaded from FIG. 2. (A) Purification of PprA and PprB proteins from E. coli overexpressing the pprA and pprB genes, respectively. Lane 1, total cellular protein of M15/pREP4/pYW021; lane 2, purified six-His-tagged PprA fusion protein; lane 3, total cellular protein of M15/pREP4/pYW024; lane 4, purified six-His-tagged PprB fusion protein; lane M, molecular size marker. (B) Coomassie blue stain of an SDS-polyacrylamide gel showing the PprA and PprB proteins used in the phosphorylation study. (C) X-ray film after exposure to the SDS-polyacrylamide gel shown in panel B.
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