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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.MM

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.

Authors+Show Affiliations

Department of Microbiology and Immunology, Queen's University, Kingston, Ontario, Canada. poolek@post.queensu.caNo affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info available

Pub Type(s)

Journal Article
Research Support, Non-U.S. Gov't

Language

eng

PubMed ID

8878035

Citation

Poole, K, et al. "Overexpression of the mexC-mexD-oprJ Efflux Operon in nfxB-type Multidrug-resistant Strains of Pseudomonas Aeruginosa." Molecular Microbiology, vol. 21, no. 4, 1996, pp. 713-24.
Poole K, Gotoh N, Tsujimoto H, et al. Overexpression of the mexC-mexD-oprJ efflux operon in nfxB-type multidrug-resistant strains of Pseudomonas aeruginosa. Mol Microbiol. 1996;21(4):713-24.
Poole, K., Gotoh, N., Tsujimoto, H., Zhao, Q., Wada, A., Yamasaki, T., Neshat, S., Yamagishi, J., Li, X. Z., & Nishino, T. (1996). Overexpression of the mexC-mexD-oprJ efflux operon in nfxB-type multidrug-resistant strains of Pseudomonas aeruginosa. Molecular Microbiology, 21(4), 713-24.
Poole K, et al. Overexpression of the mexC-mexD-oprJ Efflux Operon in nfxB-type Multidrug-resistant Strains of Pseudomonas Aeruginosa. Mol Microbiol. 1996;21(4):713-24. PubMed PMID: 8878035.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Overexpression of the mexC-mexD-oprJ efflux operon in nfxB-type multidrug-resistant strains of Pseudomonas aeruginosa. AU - Poole,K, AU - Gotoh,N, AU - Tsujimoto,H, AU - Zhao,Q, AU - Wada,A, AU - Yamasaki,T, AU - Neshat,S, AU - Yamagishi,J, AU - Li,X Z, AU - Nishino,T, PY - 1996/8/1/pubmed PY - 1996/8/1/medline PY - 1996/8/1/entrez SP - 713 EP - 24 JF - Molecular microbiology JO - Mol. Microbiol. VL - 21 IS - 4 N2 - 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. SN - 0950-382X UR - https://www.unboundmedicine.com/medline/citation/8878035/Overexpression_of_the_mexC_mexD_oprJ_efflux_operon_in_nfxB_type_multidrug_resistant_strains_of_Pseudomonas_aeruginosa_ L2 - https://onlinelibrary.wiley.com/resolve/openurl?genre=article&sid=nlm:pubmed&issn=0950-382X&date=1996&volume=21&issue=4&spage=713 DB - PRIME DP - Unbound Medicine ER -