Cloning and characterization of a DNA fragment that complements the nfxB mutation in Pseudomonas aeruginosa PAO

Okazaki, Takashi; Iyobe, Shizuko; Hashimoto, Hajime; Hirai, Keiji

doi:10.1016/0378-1097(91)90522-c

Cited by 15 publications

(24 citation statements)

References 17 publications

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“…P. aeruginosa PAO2142 (ilv-9001 lys-12 met-9011 tyr-9009) was provided by H. Matsumoto (12), and PK1013E (nfx13E transconjugant of PAO2142) [nfx13E (nfxB) met-9011 tyr-9009] was obtained from K. Hirai (17). E. coli BL21(DE3) (22) was used as the host strain for the plasmid that overproduced NfxB protein, and CSH26 was used (13) for the ␤-galactosidase assay.…”

Section: Methodsmentioning

confidence: 99%

“…Some nfxB mutants overproduce the 54-kDa outer membrane protein, OprJ (11), little of which is produced in the wild-type strain (7,17). Antibiotic resistance in the nfxB mutants is probably not due to altered outer membrane permeability but to multidrug efflux pump effects (14, 18).The nfxB gene cloned from the wild type as well as a mutant (the nfx13E mutation) has been sequenced (16,17). The amino acid sequence of NfxB revealed that it has a helix-turn-helix motif that might be responsible for its ability to bind in a sequence-specific manner to DNA, and it has no significant hydrophobic membrane-spanning regions (16).…”

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confidence: 99%

“…The mutants also show hypersusceptibility to ␤-lactam and aminoglycoside antibiotics (17). Some nfxB mutants overproduce the 54-kDa outer membrane protein, OprJ (11), little of which is produced in the wild-type strain (7,17). Antibiotic resistance in the nfxB mutants is probably not due to altered outer membrane permeability but to multidrug efflux pump effects (14, 18).…”

mentioning

confidence: 99%

“…The nfxB gene cloned from the wild type as well as a mutant (the nfx13E mutation) has been sequenced (16,17). The amino acid sequence of NfxB revealed that it has a helix-turn-helix motif that might be responsible for its ability to bind in a sequence-specific manner to DNA, and it has no significant hydrophobic membrane-spanning regions (16).…”

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Purification and characterization of the Pseudomonas aeruginosa NfxB protein, the negative regulator of the nfxB gene

et al. 1995

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The protein NfxB, involved in conferring resistance to quinolones in Pseudomonas aeruginosa, has a helixturn-helix motif which is similar to that of other DNA-binding proteins. It appears to affect the membraneassociated energy-driven efflux of some antibiotics (H. Nikaido, Science 264:382-388, 1994). We constructed a plasmid that overproduced NfxB in Escherichia coli and purified the protein. Two species of NfxB (23 and 21 kDa), which are probably translated from different initiation codons, were isolated. Both proteins are also expressed in vivo in P. aeruginosa, with the 23-kDa NfxB being the major species. NfxB specifically binds upstream of the nfxB coding region as demonstrated by gel retardation and DNase I footprinting. Expression of the ⌽(nfxB-lacZ ؉ )(Hyb) gene was repressed in the presence of the nfxB gene product provided by a second compatible plasmid in E. coli. In the P. aeruginosa wild-type strain (PAO2142), NfxB was undetectable by immunoblotting; however, it was detected in the nfxB missense mutant (PK1013E). These results suggested that NfxB negatively autoregulates the expression of nfxB itself. Since the 54-kDa outer membrane protein (OprJ) (N. Masuda, E. Sakagawa, and S. Ohya, Antimicrob. Agents Chemother. 39:645-649, 1995) was overproduced in nfxB mutants, NfxB may also regulate the expression of membrane proteins that are involved in the drug efflux machinery of P. aeruginosa.The intrinsic resistance of Pseudomonas aeruginosa to a large variety of antimicrobial agents was shown to be due mainly to efflux system effects and partly to the low-permeability outer membrane (10, 14).The P. aeruginosa nfxB mutants, which show a 16-fold increase in resistance to norfloxacin, were isolated spontaneously (7). The mutants also show hypersusceptibility to ␤-lactam and aminoglycoside antibiotics (17). Some nfxB mutants overproduce the 54-kDa outer membrane protein, OprJ (11), little of which is produced in the wild-type strain (7,17). Antibiotic resistance in the nfxB mutants is probably not due to altered outer membrane permeability but to multidrug efflux pump effects (14, 18).The nfxB gene cloned from the wild type as well as a mutant (the nfx13E mutation) has been sequenced (16,17). The amino acid sequence of NfxB revealed that it has a helix-turn-helix motif that might be responsible for its ability to bind in a sequence-specific manner to DNA, and it has no significant hydrophobic membrane-spanning regions (16). nfx13E is a missense mutation which replaces an arginine residue in the putative helix-turn-helix domain with a glycine residue in the mutant (16). The wild-type nfxB gene can restore susceptibility to norfloxacin in P. aeruginosa with the nfx13E mutation. This evidence suggests that NfxB binds to DNA and regulates the expression of genes that encode the outer membrane protein(s). It is possible that Nfx13E mutant protein is unable to bind to DNA and thus loses its regulatory function. In addition, nfxB expression might be autoregulated by NfxB protein itself. Like the LysR-type transcri...

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Purification and characterization of the Pseudomonas aeruginosa NfxB protein, the negative regulator of the nfxB gene

et al. 1995

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Cloning and nucleotide sequence of the Pseudomonas aeruginosa nfxB gene, conferring resistance to new quinolones

Okazaki¹,

Hirai²

1992

FEMS Microbiology Letters

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The gene nfxB is one of the genes which affect the cell membrane permeability of quinolones in Pseudomonas aeruginosa PAO. Both wild-type nfxB and a mutant nfxB (nfx13E) were cloned and the DNA sequences were determined. The wild-type gene was dominant in PAO strains. The nfxB mutation was a point mutation (cytosine----guanine) which generates an amino acid exchange (arginine----glycine) in the putative nfxB product. The amino acid sequence of the wild-type NfxB protein revealed that it has a helix-turn-helix motif which may be responsible for the ability to bind in a sequence-specific manner to DNA. This finding indicated that the NfxB protein may regulate the expression of genes that are associated with cell permeability of drugs in P. aeruginosa. The position of the amino acid substitution between the NfxB protein and the Nfx13E protein was located within a possible DNA-binding domain, suggesting that the mutant protein (Nfx13E) may have lost DNA binding ability and regulator activity.

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Antibiotic combination therapy can select for broad-spectrum multidrug resistance in Pseudomonas aeruginosa

Vestergaard

Paulander

Marvig

et al. 2016

International Journal of Antimicrobial Agents

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Combination therapy with several antibiotics is one strategy that has been applied in order to limit the spread of antimicrobial resistance. We compared the de novo evolution of resistance during combination therapy with the β-lactam ceftazidime and the fluoroquinolone ciprofloxacin with the resistance evolved after single-drug exposure. Combination therapy selected for mutants that displayed broad-spectrum resistance, and a major resistance mechanism was mutational inactivation of the repressor gene mexR that regulates the multidrug efflux operon mexAB-oprM. Deregulation of this operon led to a broad-spectrum resistance phenotype that decreased susceptibility to the combination of drugs applied during selection as well as to unrelated antibiotic classes. Mutants isolated after single-drug exposure displayed narrow-spectrum resistance and carried mutations in the MexCD-OprJ efflux pump regulator gene nfxB conferring ciprofloxacin resistance, or in the gene encoding the non-essential penicillin-binding protein DacB conferring ceftazidime resistance. Reconstruction of resistance mutations by allelic replacement and in vitro fitness assays revealed that in contrast to single antibiotic use, combination therapy consistently selected for mutants with enhanced fitness expressing broad-spectrum resistance mechanisms.

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Cloning and characterization of a DNA fragment that complements the nfxB mutation in Pseudomonas aeruginosa PAO

Cited by 15 publications

References 17 publications

Purification and characterization of the Pseudomonas aeruginosa NfxB protein, the negative regulator of the nfxB gene

Purification and characterization of the Pseudomonas aeruginosa NfxB protein, the negative regulator of the nfxB gene

Cloning and nucleotide sequence of the Pseudomonas aeruginosa nfxB gene, conferring resistance to new quinolones

Antibiotic combination therapy can select for broad-spectrum multidrug resistance in Pseudomonas aeruginosa

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