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. 1997 Aug;41(8):1770–1774. doi: 10.1128/aac.41.8.1770

Cloning and sequencing of a novel gene (recG) that affects the quinolone susceptibility of Staphylococcus aureus.

T Niga 1, H Yoshida 1, H Hattori 1, S Nakamura 1, H Ito 1
PMCID: PMC164002  PMID: 9257758

Abstract

In a study of the quinolone resistance genes in Staphylococcus aureus, a recG homolog was cloned as a gene affecting quinolone susceptibility. Sequencing analysis revealed that the gene consists of 2,061 nucleotides and encodes a 686-amino-acid polypeptide, which shows 38, 39, and 50% amino acid identity with the RecGs of Escherichia coli, Haemophilus influenzae, and Streptococcus pneumoniae, respectively. Seven helicase motifs are well conserved in the gene product. A plasmid carrying the gene complemented a recG-deficient mutant of E. coli with respect to mitomycin hypersusceptibility, demonstrating that the gene product is functionally equivalent to E. coli RecG. These results indicate that the gene is the recG gene of S. aureus. S. aureus RCM101 (recG::Tn551), designated S. aureus 3f33, is four to eight times more susceptible to quinolones than the parent strain, RCM101. The transformation of strain 3f33 with a plasmid carrying the S. aureus recG gene made it as quinolone resistant as strain RCM101. These results suggest that the recG gene is involved in the repair of DNA damage resulting from quinolone treatment in S. aureus.

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Selected References

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  1. Fasching C. E., Tenover F. C., Slama T. G., Fisher L. M., Sreedharan S., Oram M., Willard K., Sinn L. M., Gerding D. N., Peterson L. R. gyrA mutations in ciprofloxacin-resistant, methicillin-resistant Staphylococcus aureus from Indiana, Minnesota, and Tennessee. J Infect Dis. 1991 Nov;164(5):976–979. doi: 10.1093/infdis/164.5.976. [DOI] [PubMed] [Google Scholar]
  2. Ferrero L., Cameron B., Crouzet J. Analysis of gyrA and grlA mutations in stepwise-selected ciprofloxacin-resistant mutants of Staphylococcus aureus. Antimicrob Agents Chemother. 1995 Jul;39(7):1554–1558. doi: 10.1128/aac.39.7.1554. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Ferrero L., Cameron B., Manse B., Lagneaux D., Crouzet J., Famechon A., Blanche F. Cloning and primary structure of Staphylococcus aureus DNA topoisomerase IV: a primary target of fluoroquinolones. Mol Microbiol. 1994 Aug;13(4):641–653. doi: 10.1111/j.1365-2958.1994.tb00458.x. [DOI] [PubMed] [Google Scholar]
  4. Fleischmann R. D., Adams M. D., White O., Clayton R. A., Kirkness E. F., Kerlavage A. R., Bult C. J., Tomb J. F., Dougherty B. A., Merrick J. M. Whole-genome random sequencing and assembly of Haemophilus influenzae Rd. Science. 1995 Jul 28;269(5223):496–512. doi: 10.1126/science.7542800. [DOI] [PubMed] [Google Scholar]
  5. Gorbalenya A. E., Koonin E. V., Donchenko A. P., Blinov V. M. Two related superfamilies of putative helicases involved in replication, recombination, repair and expression of DNA and RNA genomes. Nucleic Acids Res. 1989 Jun 26;17(12):4713–4730. doi: 10.1093/nar/17.12.4713. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Goswitz J. J., Willard K. E., Fasching C. E., Peterson L. R. Detection of gyrA gene mutations associated with ciprofloxacin resistance in methicillin-resistant Staphylococcus aureus: analysis by polymerase chain reaction and automated direct DNA sequencing. Antimicrob Agents Chemother. 1992 May;36(5):1166–1169. doi: 10.1128/aac.36.5.1166. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Hiasa H., Yousef D. O., Marians K. J. DNA strand cleavage is required for replication fork arrest by a frozen topoisomerase-quinolone-DNA ternary complex. J Biol Chem. 1996 Oct 18;271(42):26424–26429. doi: 10.1074/jbc.271.42.26424. [DOI] [PubMed] [Google Scholar]
  8. Hori S., Ohshita Y., Utsui Y., Hiramatsu K. Sequential acquisition of norfloxacin and ofloxacin resistance by methicillin-resistant and -susceptible Staphylococcus aureus. Antimicrob Agents Chemother. 1993 Nov;37(11):2278–2284. doi: 10.1128/aac.37.11.2278. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Howard B. M., Pinney R. J., Smith J. T. Function of the SOS process in repair of DNA damage induced by modern 4-quinolones. J Pharm Pharmacol. 1993 Jul;45(7):658–662. doi: 10.1111/j.2042-7158.1993.tb05673.x. [DOI] [PubMed] [Google Scholar]
  10. Isegawa Y., Sheng J., Sokawa Y., Yamanishi K., Nakagomi O., Ueda S. Selective amplification of cDNA sequence from total RNA by cassette-ligation mediated polymerase chain reaction (PCR): application to sequencing 6.5 kb genome segment of hantavirus strain B-1. Mol Cell Probes. 1992 Dec;6(6):467–475. doi: 10.1016/0890-8508(92)90043-w. [DOI] [PubMed] [Google Scholar]
  11. Ito H., Yoshida H., Bogaki-Shonai M., Niga T., Hattori H., Nakamura S. Quinolone resistance mutations in the DNA gyrase gyrA and gyrB genes of Staphylococcus aureus. Antimicrob Agents Chemother. 1994 Sep;38(9):2014–2023. doi: 10.1128/aac.38.9.2014. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Kaatz G. W., Seo S. M., Ruble C. A. Efflux-mediated fluoroquinolone resistance in Staphylococcus aureus. Antimicrob Agents Chemother. 1993 May;37(5):1086–1094. doi: 10.1128/aac.37.5.1086. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Kornblum J., Hartman B. J., Novick R. P., Tomasz A. Conversion of a homogeneously methicillin-resistant strain of Staphylococcus aureus to heterogeneous resistance by Tn551-mediated insertional inactivation. Eur J Clin Microbiol. 1986 Dec;5(6):714–718. doi: 10.1007/BF02013311. [DOI] [PubMed] [Google Scholar]
  14. Kreiswirth B. N., Löfdahl S., Betley M. J., O'Reilly M., Schlievert P. M., Bergdoll M. S., Novick R. P. The toxic shock syndrome exotoxin structural gene is not detectably transmitted by a prophage. Nature. 1983 Oct 20;305(5936):709–712. doi: 10.1038/305709a0. [DOI] [PubMed] [Google Scholar]
  15. Lewin C. S., Howard B. M., Ratcliffe N. T., Smith J. T. 4-quinolones and the SOS response. J Med Microbiol. 1989 Jun;29(2):139–144. doi: 10.1099/00222615-29-2-139. [DOI] [PubMed] [Google Scholar]
  16. Lloyd R. G., Buckman C. Genetic analysis of the recG locus of Escherichia coli K-12 and of its role in recombination and DNA repair. J Bacteriol. 1991 Feb;173(3):1004–1011. doi: 10.1128/jb.173.3.1004-1011.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Lloyd R. G., Sharples G. J. Molecular organization and nucleotide sequence of the recG locus of Escherichia coli K-12. J Bacteriol. 1991 Nov;173(21):6837–6843. doi: 10.1128/jb.173.21.6837-6843.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Lloyd R. G., Sharples G. J. Processing of recombination intermediates by the RecG and RuvAB proteins of Escherichia coli. Nucleic Acids Res. 1993 Apr 25;21(8):1719–1725. doi: 10.1093/nar/21.8.1719. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Low B. Formation of merodiploids in matings with a class of Rec- recipient strains of Escherichia coli K12. Proc Natl Acad Sci U S A. 1968 May;60(1):160–167. doi: 10.1073/pnas.60.1.160. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Martin B., Sharples G. J., Humbert O., Lloyd R. G., Claverys J. P. The mmsA locus of Streptococcus pneumoniae encodes a RecG-like protein involved in DNA repair and in three-strand recombination. Mol Microbiol. 1996 Mar;19(5):1035–1045. doi: 10.1046/j.1365-2958.1996.445975.x. [DOI] [PubMed] [Google Scholar]
  21. Miller V. L., Mekalanos J. J. A novel suicide vector and its use in construction of insertion mutations: osmoregulation of outer membrane proteins and virulence determinants in Vibrio cholerae requires toxR. J Bacteriol. 1988 Jun;170(6):2575–2583. doi: 10.1128/jb.170.6.2575-2583.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Ng E. Y., Trucksis M., Hooper D. C. Quinolone resistance mediated by norA: physiologic characterization and relationship to flqB, a quinolone resistance locus on the Staphylococcus aureus chromosome. Antimicrob Agents Chemother. 1994 Jun;38(6):1345–1355. doi: 10.1128/aac.38.6.1345. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Nishibuchi M., Kumagai K., Kaper J. B. Contribution of the tdh1 gene of Kanagawa phenomenon-positive Vibrio parahaemolyticus to production of extracellular thermostable direct hemolysin. Microb Pathog. 1991 Dec;11(6):453–460. doi: 10.1016/0882-4010(91)90042-9. [DOI] [PubMed] [Google Scholar]
  24. Novick R. P. Genetic systems in staphylococci. Methods Enzymol. 1991;204:587–636. doi: 10.1016/0076-6879(91)04029-n. [DOI] [PubMed] [Google Scholar]
  25. Shaw J. H., Clewell D. B. Complete nucleotide sequence of macrolide-lincosamide-streptogramin B-resistance transposon Tn917 in Streptococcus faecalis. J Bacteriol. 1985 Nov;164(2):782–796. doi: 10.1128/jb.164.2.782-796.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Shen L. L., Kohlbrenner W. E., Weigl D., Baranowski J. Mechanism of quinolone inhibition of DNA gyrase. Appearance of unique norfloxacin binding sites in enzyme-DNA complexes. J Biol Chem. 1989 Feb 15;264(5):2973–2978. [PubMed] [Google Scholar]
  27. Sreedharan S., Oram M., Jensen B., Peterson L. R., Fisher L. M. DNA gyrase gyrA mutations in ciprofloxacin-resistant strains of Staphylococcus aureus: close similarity with quinolone resistance mutations in Escherichia coli. J Bacteriol. 1990 Dec;172(12):7260–7262. doi: 10.1128/jb.172.12.7260-7262.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Sreedharan S., Peterson L. R., Fisher L. M. Ciprofloxacin resistance in coagulase-positive and -negative staphylococci: role of mutations at serine 84 in the DNA gyrase A protein of Staphylococcus aureus and Staphylococcus epidermidis. Antimicrob Agents Chemother. 1991 Oct;35(10):2151–2154. doi: 10.1128/aac.35.10.2151. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Whitby M. C., Vincent S. D., Lloyd R. G. Branch migration of Holliday junctions: identification of RecG protein as a junction specific DNA helicase. EMBO J. 1994 Nov 1;13(21):5220–5228. doi: 10.1002/j.1460-2075.1994.tb06853.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Willmott C. J., Critchlow S. E., Eperon I. C., Maxwell A. The complex of DNA gyrase and quinolone drugs with DNA forms a barrier to transcription by RNA polymerase. J Mol Biol. 1994 Sep 30;242(4):351–363. doi: 10.1006/jmbi.1994.1586. [DOI] [PubMed] [Google Scholar]
  31. Yamagishi J., Kojima T., Oyamada Y., Fujimoto K., Hattori H., Nakamura S., Inoue M. Alterations in the DNA topoisomerase IV grlA gene responsible for quinolone resistance in Staphylococcus aureus. Antimicrob Agents Chemother. 1996 May;40(5):1157–1163. doi: 10.1128/aac.40.5.1157. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Yoshida H., Bogaki M., Nakamura S., Ubukata K., Konno M. Nucleotide sequence and characterization of the Staphylococcus aureus norA gene, which confers resistance to quinolones. J Bacteriol. 1990 Dec;172(12):6942–6949. doi: 10.1128/jb.172.12.6942-6949.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Yoshida H., Nakamura M., Bogaki M., Ito H., Kojima T., Hattori H., Nakamura S. Mechanism of action of quinolones against Escherichia coli DNA gyrase. Antimicrob Agents Chemother. 1993 Apr;37(4):839–845. doi: 10.1128/aac.37.4.839. [DOI] [PMC free article] [PubMed] [Google Scholar]

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