Overexpression of the mexC-mexD-oprJ efflux operon in nfxB-type multidrug-resistant strains of Pseudomonas aeruginosa.
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.
Poole
K
K
Department of Microbiology and Immunology, Queen's University, Kingston, Ontario, Canada. poolek@post.queensu.ca
Gotoh
N
N
Tsujimoto
H
H
Zhao
Q
Q
Wada
A
A
Yamasaki
T
T
Neshat
S
S
Yamagishi
J
J
Li
X Z
XZ
Nishino
T
T
D000595:Amino Acid Sequence; D000900:Anti-Bacterial Agents / Q000494:pharmacology; D001425:Bacterial Outer Membrane Proteins / Q000032:analysis / Q000235:genetics; D001426:Bacterial Proteins / Q000235:genetics*; D002258:Carbonyl Cyanide m-Chlorophenyl Hydrazone / Q000494:pharmacology; D002701:Chloramphenicol / Q000494:pharmacology; D003001:Cloning, Molecular; D004268:DNA-Binding Proteins / Q000235:genetics*; D004352:Drug Resistance, Microbial / Q000235:genetics; D018432:Drug Resistance, Multiple / Q000235:genetics*; D015964:Gene Expression Regulation, Bacterial / Q000502:physiology*; D005798:Genes, Bacterial / Q000235:genetics; D007476:Ionophores / Q000494:pharmacology; D008969:Molecular Sequence Data; D009643:Norfloxacin / Q000494:pharmacology; D009876:Operon / Q000235:genetics*; D017354:Point Mutation; D011550:Pseudomonas aeruginosa / Q000187:drug effects / Q000235:genetics*; D014157:Transcription Factors
D000900:Anti-Bacterial Agents; D001425:Bacterial Outer Membrane Proteins; D001426:Bacterial Proteins; D004268:DNA-Binding Proteins; D007476:Ionophores; C077901:NfxB protein, Pseudomonas aeruginosa; D014157:Transcription Factors; D002258:Carbonyl Cyanide m-Chlorophenyl Hydrazone; D002701:Chloramphenicol; D009643:Norfloxacin