Differential impact of MexB mutations on substrate selectivity of the MexAB-OprM multidrug efflux pump of Pseudomonas aeruginosa

J Bacteriol. 2004 Mar;186(5):1258-69. doi: 10.1128/JB.186.5.1258-1269.2004.

Abstract

The integral inner membrane resistance-nodulation-division (RND) components of three-component RND-membrane fusion protein-outer membrane factor multidrug efflux systems define the substrate selectivity of these efflux systems. To gain a better understanding of what regions of these proteins are important for substrate recognition, a plasmid-borne mexB gene encoding the RND component of the MexAB-OprM multidrug efflux system of Pseudomonas aeruginosa was mutagenized in vitro by using hydroxylamine and mutations compromising the MexB contribution to antibiotic resistance identified in a DeltamexB strain. Of 100 mutants that expressed wild-type levels of MexB and showed increased susceptibility to one or more of carbenicillin, chloramphenicol, nalidixic acid, and novobiocin, the mexB genes of a representative 46 were sequenced, and 19 unique single mutations were identified. While the majority of mutations occurred within the large periplasmic loops between transmembrane segment 1 (TMS-1) and TMS-2 and between TMS-7 and TMS-8 of MexB, mutations were seen in the TMSs and in other periplasmic as well as cytoplasmic loops. By threading the MexB amino acid sequence through the crystal structure of the homologous RND transporter from Escherichia coli, AcrB, a three-dimensional model of a MexB trimer was obtained and the mutations were mapped to it. Unexpectedly, most mutations mapped to regions of MexB predicted to be involved in trimerization or interaction with MexA rather than to regions expected to contribute to substrate recognition. Intragenic second-site suppressor mutations that restored the activity of the G220S mutant version of MexB, which was compromised for resistance to all tested MexAB-OprM antimicrobial substrates, were recovered and mapped to the apparently distal portion of MexB that is implicated in OprM interaction. As the G220S mutation likely impacted trimerization, it appears that either proper assembly of the MexB trimer is necessary for OprM interaction or OprM association with an unstable MexB trimer might stabilize it, thereby restoring activity.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Anti-Bacterial Agents / metabolism*
  • Anti-Bacterial Agents / pharmacology
  • Bacterial Outer Membrane Proteins / chemistry
  • Bacterial Outer Membrane Proteins / genetics*
  • Bacterial Outer Membrane Proteins / metabolism*
  • Carrier Proteins / chemistry
  • Carrier Proteins / genetics*
  • Carrier Proteins / metabolism*
  • Drug Resistance, Bacterial
  • Membrane Transport Proteins / metabolism*
  • Microbial Sensitivity Tests
  • Models, Molecular
  • Mutation*
  • Pseudomonas aeruginosa / drug effects*
  • Pseudomonas aeruginosa / genetics
  • Pseudomonas aeruginosa / growth & development
  • Solvents / pharmacology
  • Substrate Specificity

Substances

  • Anti-Bacterial Agents
  • Bacterial Outer Membrane Proteins
  • Carrier Proteins
  • Membrane Transport Proteins
  • MexA protein, Pseudomonas aeruginosa
  • MexB protein, Pseudomonas aeruginosa
  • OprM protein, Pseudomonas aeruginosa
  • Solvents