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. 1995 Oct;177(20):5872–5877. doi: 10.1128/jb.177.20.5872-5877.1995

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

T Shiba 1, K Ishiguro 1, N Takemoto 1, H Koibuchi 1, K Sugimoto 1
PMCID: PMC177412  PMID: 7592337

Abstract

The protein NfxB, involved in conferring resistance to quinolones in Pseudomonas aeruginosa, has a helix-turn-helix motif which is similar to that of other DNA-binding proteins. It appears to affect the membrane-associated 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 phi (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.

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Selected References

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  1. Bradford M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976 May 7;72:248–254. doi: 10.1016/0003-2697(76)90527-3. [DOI] [PubMed] [Google Scholar]
  2. Casadaban M. J., Chou J., Cohen S. N. In vitro gene fusions that join an enzymatically active beta-galactosidase segment to amino-terminal fragments of exogenous proteins: Escherichia coli plasmid vectors for the detection and cloning of translational initiation signals. J Bacteriol. 1980 Aug;143(2):971–980. doi: 10.1128/jb.143.2.971-980.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Chandler M. S. New shuttle vectors for Haemophilus influenzae and Escherichia coli: P15A-derived plasmids replicate in H. influenzae Rd. Plasmid. 1991 May;25(3):221–224. doi: 10.1016/0147-619x(91)90016-p. [DOI] [PubMed] [Google Scholar]
  4. Dodd I. B., Egan J. B. Improved detection of helix-turn-helix DNA-binding motifs in protein sequences. Nucleic Acids Res. 1990 Sep 11;18(17):5019–5026. doi: 10.1093/nar/18.17.5019. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Duchêne M., Schweizer A., Lottspeich F., Krauss G., Marget M., Vogel K., von Specht B. U., Domdey H. Sequence and transcriptional start site of the Pseudomonas aeruginosa outer membrane porin protein F gene. J Bacteriol. 1988 Jan;170(1):155–162. doi: 10.1128/jb.170.1.155-162.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Hirai K., Suzue S., Irikura T., Iyobe S., Mitsuhashi S. Mutations producing resistance to norfloxacin in Pseudomonas aeruginosa. Antimicrob Agents Chemother. 1987 Apr;31(4):582–586. doi: 10.1128/aac.31.4.582. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Johnson A. D., Meyer B. J., Ptashne M. Interactions between DNA-bound repressors govern regulation by the lambda phage repressor. Proc Natl Acad Sci U S A. 1979 Oct;76(10):5061–5065. doi: 10.1073/pnas.76.10.5061. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  9. Li X. Z., Livermore D. M., Nikaido H. Role of efflux pump(s) in intrinsic resistance of Pseudomonas aeruginosa: resistance to tetracycline, chloramphenicol, and norfloxacin. Antimicrob Agents Chemother. 1994 Aug;38(8):1732–1741. doi: 10.1128/aac.38.8.1732. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Masuda N., Sakagawa E., Ohya S. Outer membrane proteins responsible for multiple drug resistance in Pseudomonas aeruginosa. Antimicrob Agents Chemother. 1995 Mar;39(3):645–649. doi: 10.1128/AAC.39.3.645. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Matsumoto H., Ohta S., Kobayashi R., Terawaki Y. Chromosomal location of genes participating in the degradation of purines in Pseudomonas aeruginosa. Mol Gen Genet. 1978 Nov 29;167(2):165–176. doi: 10.1007/BF00266910. [DOI] [PubMed] [Google Scholar]
  12. Nikaido H., Nikaido K., Harayama S. Identification and characterization of porins in Pseudomonas aeruginosa. J Biol Chem. 1991 Jan 15;266(2):770–779. [PubMed] [Google Scholar]
  13. Nikaido H. Prevention of drug access to bacterial targets: permeability barriers and active efflux. Science. 1994 Apr 15;264(5157):382–388. doi: 10.1126/science.8153625. [DOI] [PubMed] [Google Scholar]
  14. Okazaki T., Hirai K. Cloning and nucleotide sequence of the Pseudomonas aeruginosa nfxB gene, conferring resistance to new quinolones. FEMS Microbiol Lett. 1992 Oct 1;76(1-2):197–202. doi: 10.1016/0378-1097(92)90386-3. [DOI] [PubMed] [Google Scholar]
  15. Okazaki T., Iyobe S., Hashimoto H., Hirai K. Cloning and characterization of a DNA fragment that complements the nfxB mutation in Pseudomonas aeruginosa PAO. FEMS Microbiol Lett. 1991 Mar 15;63(1):31–35. doi: 10.1016/0378-1097(91)90522-c. [DOI] [PubMed] [Google Scholar]
  16. Poole K., Krebes K., McNally C., Neshat S. Multiple antibiotic resistance in Pseudomonas aeruginosa: evidence for involvement of an efflux operon. J Bacteriol. 1993 Nov;175(22):7363–7372. doi: 10.1128/jb.175.22.7363-7372.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Schell M. A. Molecular biology of the LysR family of transcriptional regulators. Annu Rev Microbiol. 1993;47:597–626. doi: 10.1146/annurev.mi.47.100193.003121. [DOI] [PubMed] [Google Scholar]
  18. Shinagawa H., Iwasaki H., Kato T., Nakata A. RecA protein-dependent cleavage of UmuD protein and SOS mutagenesis. Proc Natl Acad Sci U S A. 1988 Mar;85(6):1806–1810. doi: 10.1073/pnas.85.6.1806. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Studier F. W., Rosenberg A. H., Dunn J. J., Dubendorff J. W. Use of T7 RNA polymerase to direct expression of cloned genes. Methods Enzymol. 1990;185:60–89. doi: 10.1016/0076-6879(90)85008-c. [DOI] [PubMed] [Google Scholar]
  20. Tabor S., Richardson C. C. A bacteriophage T7 RNA polymerase/promoter system for controlled exclusive expression of specific genes. Proc Natl Acad Sci U S A. 1985 Feb;82(4):1074–1078. doi: 10.1073/pnas.82.4.1074. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Wang L., Helmann J. D., Winans S. C. The A. tumefaciens transcriptional activator OccR causes a bend at a target promoter, which is partially relaxed by a plant tumor metabolite. Cell. 1992 May 15;69(4):659–667. doi: 10.1016/0092-8674(92)90229-6. [DOI] [PubMed] [Google Scholar]

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