Function of the membrane fusion protein, MexA, of the MexA, B-OprM efflux pump in Pseudomonas aeruginosa without an anchoring membrane

Resistance of Pseudomonas aeruginosa to multiple species of antibiotics is largely attributable to expression of the MexA, B-OprM efflux pump. The Mex A protein is thought to be located at the inner membrane and has been assum ed to link the xenobiotics-exporting subunit, MexB, and the outer membrane channel protein, OprM, To verify this assumption, we analyzed membrane anch oring and localization of the MexA protein. n-[9,10-H-3]Palmitic acid incor poration experiments revealed that MexA was radiolabeled with palmitic acid , suggesting that the MexA anchors the inner membrane via the fatty acid mo iety, To evaluate the role of lipid modification and inner membrane anchori ng, we substituted cysteine 24 with phenylalanine or tyrosine and tested wh ether or not these mutant MexAs function properly. When the mutant mexAs we re expressed in the strain lacking chromosomal mexA in the presence of n-[9 ,10-H-3]palmitic acid, we found undetectable radiolabeling at the MexA band , These transformants restored antibiotic resistance to the level of the wi ld-type strain, indicating that lipid modification is not essential for Mex A function. These mutant strains contained both processed and unprocessed f orms of the MexA proteins. Cellular fractionation experiments revealed that an unprocessed form of MexA anchored the inner membrane probably via an un cleaved signal sequence, whereas the processed form was undetectable in the membrane fraction. To assure that the lipid-free MexA polypeptide could be unbound to the membrane, we analyzed the two-dimensional membrane topology by the gene fusion technique. A total of 78 mexA-blaM fusions covering the entire MexA polypeptide were constructed, and all fusion sites were shown to be located at the periplasm, To answer the question of whether or not me mbrane anchoring is essential for the MexA function, we replaced the signal sequence of the MexA protein with that of the azurin protein, which contai ns a cleavable signal sequence but no lipid modification site. The signal s equence of the azurin-MexA hybrid protein was properly processed and bore t he mature MexA which was fully recovered in the soluble fraction. The trans formant, which expressed azurin-MexA hybrid protein restored the antibiotic resistance to a level indistinguishable from that of the wild-type strain. We concluded from these results that the MexA protein is fully functional as expressed in the periplasmic space without anchoring the inner membrane. This finding questioned the assumption that the membrane fusion proteins c onnect the inner and outer membranes.