| Research Abstract |
A major cause of antibiotic resistance of Pseudomonas aeruginosa has recently appeared to be the energy-dependent antibiotic efflux pump, MexA,B-OprM.Analysis of chimeric pump between MexA,B-OprM and its homologous counterpart, MexC,D-OprJ, suggests that MexA and MexB form a substrate recognition complex. In this study, membrane topology of inner membrane subunits, MexA and MexB, were analyzed by gene-fusion technique, and MexB mutants were obtained by random mutagenesis method. These experiments shows : (1) MexA has lipid modification at the 24th cystein residue and anchors inner membrane through the lipid. The region beyond the cystein residue localizes in the periplasm. (2) The hydrophilic periplasmic region of MexA is important, but lipid modification is not essential for function of the MexA.(3) MexB laas 12 trans membrane segments (TMS) and the large 1st and 4th loops with about 300 amino acid residues located in the periplasm. (4) MexB mutants obtained by random mutagenesis has mutations in the cytoplasmic loop, TMS, and periplasmic loops. (5) Suppressor mutants obtained from the MexB mutant having a mutation in the TMS10 have the same second mutation in TMS4. From these results, TMS4 and TMS10 are suggested to pack in the close vicinity in inner membrane and the periplasmic loops 1 and 4 might interact with MexA to form substrate recognition complex.
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