Overexpression of the mexC-mexD-oprJ Efflux Operon in nfxB-type Multidrug-Resistant Strains of Pseudomonas Aeruginosa

Mol Microbiol. 1996 Aug;21(4):713-24. doi: 10.1046/j.1365-2958.1996.281397.x.

Abstract

OprJ, overproduced in nfxB multidrug-resistant strains of Pseudomonas aeruginosa, and OprK, overproduced in the multidrug-resistant strain K385, were demonstrated to be immunologically cross-reactive using an OprJ-specific monoclonal antibody. Treatment of the purified proteins with trypsin or chymotrypsin yielded virtually indistinguishable digestion patterns, and the N-terminal sequence of two trypsin fragments was identical for both proteins, indicating that OprJ and OprK share identity. The N-terminal amino acid sequences were used to facilitate cloning of the oprJ gene on a 5kbp Kpnl fragment and a 10 kbp BamHl fragment. Nucleotide sequencing of portions of these fragments revealed that oprJ was the terminal gene in a putative three-gene operon, mexC-mexD-oprJ. The predicted mexC-mexD-oprJ gene products exhibit homology to the MexA-MexB-OprM components of the multidrug-resistance efflux pump of P. aeruginosa (43-46% identity). Consistent with an implied role for mexC-mexD-oprJ in drug efflux, the mexC-mexD-oprJ-hyperexpressing strain K385 showed reduced accumulation of a variety of antibiotics as compared with its parent strain, and this drug 'exclusion' was abrogated by energy inhibitors. The mexC and oprJ products are putative lipoproteins of a molecular mass of 40,707 and 51,742 Da, respectively, while mexD was predicted to encode a protein of 111 936 Da. Sequencing upstream of mexC revealed the presence of the nfxB gene transcribed divergently from the efflux genes. Overproduction of OprJ and the attendant multiple-antibiotic resistance of strain K385 was shown to result from a point mutation in nfxB, resulting in a H87-->R change in the predicted NfxB polypeptide. OprJ overproduction and multidrug resistance in K385 was reversed by the cloned nfxB gene, suggesting that nfxB encodes a repressor of mexC-mexD-oprJ expression. Consistent with this, the cloned nfxB gene repressed synthesis of a mexC-lacZ fusion in Escherichia coli. nfxB also repressed expression of a nfxB-lacZ fusion, indicating that NfxB negatively regulates its own expression. These data indicate that the multidrug resistance of nfxB strains is due to overexpression of an efflux operon, mexC-mexD-oprJ, encoding components of a second efflux pump in P. aeruginosa.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Anti-Bacterial Agents / pharmacology
  • Bacterial Outer Membrane Proteins / analysis
  • Bacterial Outer Membrane Proteins / genetics
  • Bacterial Proteins / genetics*
  • Carbonyl Cyanide m-Chlorophenyl Hydrazone / pharmacology
  • Chloramphenicol / pharmacology
  • Cloning, Molecular
  • DNA-Binding Proteins / genetics*
  • Drug Resistance, Microbial / genetics
  • Drug Resistance, Multiple / genetics*
  • Gene Expression Regulation, Bacterial / physiology*
  • Genes, Bacterial / genetics
  • Ionophores / pharmacology
  • Molecular Sequence Data
  • Norfloxacin / pharmacology
  • Operon / genetics*
  • Point Mutation
  • Pseudomonas aeruginosa / drug effects
  • Pseudomonas aeruginosa / genetics*
  • Transcription Factors*

Substances

  • Anti-Bacterial Agents
  • Bacterial Outer Membrane Proteins
  • Bacterial Proteins
  • DNA-Binding Proteins
  • Ionophores
  • NfxB protein, Pseudomonas aeruginosa
  • Transcription Factors
  • Carbonyl Cyanide m-Chlorophenyl Hydrazone
  • Chloramphenicol
  • Norfloxacin

Associated data

  • GENBANK/U57969