title: Assembly of the MexAB-OprM multidrug efflux system of Pseudomonas aeruginosa: identification and characterization of mutations in mexA compromising MexA multimerization and interaction with MexB.
abstract: The membrane fusion protein (MFP) component, MexA, of the MexAB-OprM multidrug efflux system of P. aeruginosa is proposed to link the inner (MexB) and outer (OprM) membrane components of this pump as a probable oligomer. A cross-linking approach confirmed the in vivo interaction of MexA and MexB, while a LexA-based assay for assessing protein-protein interaction similarly confirmed MexA multimerization. Mutations compromising the MexA contribution to antibiotic resistance but yielding wild-type levels of MexA were recovered and shown to map to two distinct regions within the N- and C-terminal halves of the protein. Most of the N-terminal mutations occurred at residues that are highly conserved in the MFP family (P68, G72, L91, A108, L110, and V129), consistent with these playing roles in a common feature of these proteins (e.g., oligomerization). In contrast, the majority of the C-terminal mutations occurred at residues poorly conserved in the MFP family (V264, N270, H279, V286, and G297), with many mapping to a region of MexA that corresponds to a region in the related MFP of Escherichia coli, AcrA, that is implicated in binding to its RND component, AcrB (C. A. Elkins and H. Nikaido, J. Bacteriol. 185:5349-5356, 2003). Given the noted specificity of MFP-RND interaction in this family of pumps, residues unique to MexA may well be important for and define the MexA interaction with its RND component, MexB. Still, all but one of the MexA mutations studied compromised MexA-MexB association, suggesting that native structure and/or proper assembly of the protein may be necessary for this.
authors.affiliation: Department of Microbiology and Immunology, Queen's University, Kingston, Ontario K7L 3N6, Canada.
authors.forename: Dominic
authors.initials: D
authors.lastname: Nehme
authors.forename: Xian-Zhi
authors.initials: XZ
authors.lastname: Li
authors.forename: Rachel
authors.initials: R
authors.lastname: Elliot
authors.forename: Keith
authors.initials: K
authors.lastname: Poole
mesh_terms: D000595:Amino Acid Sequence; D001425:Bacterial Outer Membrane Proteins / Q000737:chemistry*; D002352:Carrier Proteins / Q000737:chemistry*; D017124:Conserved Sequence; D019281:Dimerization; D024881:Drug Resistance, Bacterial; D026901:Membrane Transport Proteins / Q000737:chemistry*; D008969:Molecular Sequence Data; D009154:Mutation; D011550:Pseudomonas aeruginosa / Q000737:chemistry* / Q000235:genetics
chemical_list: D001425:Bacterial Outer Membrane Proteins; D002352:Carrier Proteins; D026901:Membrane Transport Proteins; C118598:MexA protein, Pseudomonas aeruginosa; C118597:MexB protein, Pseudomonas aeruginosa; C402263:OprM protein, Pseudomonas aeruginosa