THE ROLE OF MEX-GENE PRODUCTS IN ANTIBIOTIC EXTRUSION IN PSEUDOMONAS-AERUGINOSA

The antibiotic extrusion machinery in Pseudomonas aeruginosa is assemb led from the mex-operon encoded proteins, OprM and MexA-MexB, connecti ng the outer and inner membranes. To envisage the role of these protei ns in antibiotic extrusion and resistance, we employed the gene replac ement technique to construct mutants deficient in mexA, mexB, or oprM, and all possible combinations of these genes. Using the Southern and the Western blotting methods, we confirmed that only the target genes were disrupted. All the mutants deficient in OprM exhibited a 4 to 16 times higher susceptibility against quinolone antibiotics, chloramphen icol, and gentamicin than the parent strain, The mutants deficient in MexA or MexB or both MexA and MexB were only 2 to 4 times more suscept ible to these antibiotics than the parent strain. All the mutants lack ing MexA, MexB, or OprM showed stereospecific hypersusceptibility to p -lactam antibiotics than the parent strain, However, the extent of sus ceptibility to each p-lactam was comparable among the mutants. Strains lacking OprM accumulated the highest level of ciprofloxacin among all these isogenic strains. The strains lacking either MexA or MexB accum ulated lower levels of ciprofloxacin than the mutant lacking OprM, but the levels were still higher than in the parent strain. The results a re consistent with the antibiotic susceptibility of these strains. The se results suggest that the extrusion of antibiotics occurs most effic iently with a whole assembly of MexA/B-OprM, but it remains a possibil ity that OprM interacts with a putative inner membrane pump(s). (C) 19 97 Academic Press.