Skip to main content
NCBI home page
Journal of Bacteriology logoLink to Journal of Bacteriology
. 1997 Oct;179(20):6294–6301. doi: 10.1128/jb.179.20.6294-6301.1997

Increase of methicillin resistance in Staphylococcus aureus caused by deletion of a gene whose product is homologous to lytic enzymes.

T Fujimura 1, K Murakami 1
PMCID: PMC179542  PMID: 9335275

Abstract

A spontaneous high-level methicillin-resistant mutant, SRM1648, for which the MIC of methicillin is 1,600 microg/ml, was isolated on a plate containing 400 microg of the antibiotic/ml on which had been cultured the low-level methicillin-resistant Staphylococcus aureus SR17238, for which the MIC is 6.3 microg/ml. Analysis of the chromosomal DNAs of the mutant and the parental strains by the restriction landmark genomic scanning method with two-dimensional electrophoresis of restriction fragments revealed a 1.6-kb deletion in the chromosome of the mutant. The HindIII fragment of 2.5 kb containing this deleted region was cloned into a plasmid vector and introduced into the parental strain. A deletion mutant reconstructed in the presence of a low concentration of methicillin by integration and excision of the recombinant plasmid exhibited a high level of resistance (methicillin MIC, 1,600 microg/ml), confirming that the deletion had caused the elevation of the resistance level. Sequence analysis indicated that the deletion occurred in three consecutive open reading frames (ORFs). The predicted amino acid sequence of the first ORF showed high homology with both RelA and SpoT of Escherichia coli, which are involved in the synthesis and hydrolysis of guanosine 5',3'-polyphosphate, and that of the third ORF showed a relatively high homology to the lytic enzyme encoded by the lytC gene of Bacillus subtilis. We also isolated another high-level resistant mutant with a deletion within the third ORF, which suggested that inactivation of some lytic enzyme resulted in the increased resistance.

Full Text

The Full Text of this article is available as a PDF (735.0 KB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Berger-Bächi B., Barberis-Maino L., Strässle A., Kayser F. H. FemA, a host-mediated factor essential for methicillin resistance in Staphylococcus aureus: molecular cloning and characterization. Mol Gen Genet. 1989 Oct;219(1-2):263–269. doi: 10.1007/BF00261186. [DOI] [PubMed] [Google Scholar]
  2. Brenner D. G., Shaw W. V. The use of synthetic oligonucleotides with universal templates for rapid DNA sequencing: results with staphylococcal replicon pC221. EMBO J. 1985 Feb;4(2):561–568. doi: 10.1002/j.1460-2075.1985.tb03665.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Freedman R., Gibson B., Donovan D., Biemann K., Eisenbeis S., Parker J., Schimmel P. Primary structure of histidine-tRNA synthetase and characterization of hisS transcripts. J Biol Chem. 1985 Aug 25;260(18):10063–10068. [PubMed] [Google Scholar]
  4. Gustafson J. E., Berger-Bächi B., Strässle A., Wilkinson B. J. Autolysis of methicillin-resistant and -susceptible Staphylococcus aureus. Antimicrob Agents Chemother. 1992 Mar;36(3):566–572. doi: 10.1128/aac.36.3.566. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Gustafson J. E., Wilkinson B. J. Lower autolytic activity in a homogeneous methicillin-resistant Staphylococcus aureus strain compared to derived heterogeneous-resistant and susceptible strains. FEMS Microbiol Lett. 1989 May;50(1-2):107–111. doi: 10.1016/0378-1097(89)90468-0. [DOI] [PubMed] [Google Scholar]
  6. Hartman B. J., Tomasz A. Expression of methicillin resistance in heterogeneous strains of Staphylococcus aureus. Antimicrob Agents Chemother. 1986 Jan;29(1):85–92. doi: 10.1128/aac.29.1.85. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Hartman B. J., Tomasz A. Low-affinity penicillin-binding protein associated with beta-lactam resistance in Staphylococcus aureus. J Bacteriol. 1984 May;158(2):513–516. doi: 10.1128/jb.158.2.513-516.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Hatada I., Hayashizaki Y., Hirotsune S., Komatsubara H., Mukai T. A genomic scanning method for higher organisms using restriction sites as landmarks. Proc Natl Acad Sci U S A. 1991 Nov 1;88(21):9523–9527. doi: 10.1073/pnas.88.21.9523. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Henze U., Sidow T., Wecke J., Labischinski H., Berger-Bächi B. Influence of femB on methicillin resistance and peptidoglycan metabolism in Staphylococcus aureus. J Bacteriol. 1993 Mar;175(6):1612–1620. doi: 10.1128/jb.175.6.1612-1620.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Hershey H. V., Taylor M. W. Nucleotide sequence and deduced amino acid sequence of Escherichia coli adenine phosphoribosyltransferase and comparison with other analogous enzymes. Gene. 1986;43(3):287–293. doi: 10.1016/0378-1119(86)90218-0. [DOI] [PubMed] [Google Scholar]
  11. Hirotsune S., Shibata H., Okazaki Y., Sugino H., Imoto H., Sasaki N., Hirose K., Okuizumi H., Muramatsu M., Plass C. Molecular cloning of polymorphic markers on RLGS gel using the spot target cloning method. Biochem Biophys Res Commun. 1993 Aug 16;194(3):1406–1412. doi: 10.1006/bbrc.1993.1981. [DOI] [PubMed] [Google Scholar]
  12. Jayaswal R. K., Lee Y. I., Wilkinson B. J. Cloning and expression of a Staphylococcus aureus gene encoding a peptidoglycan hydrolase activity. J Bacteriol. 1990 Oct;172(10):5783–5788. doi: 10.1128/jb.172.10.5783-5788.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. KNOX R., SMITH J. T. The nature of penicillin resistance in staphylococci. Lancet. 1961 Sep 2;2(7201):520–522. doi: 10.1016/s0140-6736(61)92958-0. [DOI] [PubMed] [Google Scholar]
  14. Kuroda A., Asami Y., Sekiguchi J. Molecular cloning of a sporulation-specific cell wall hydrolase gene of Bacillus subtilis. J Bacteriol. 1993 Oct;175(19):6260–6268. doi: 10.1128/jb.175.19.6260-6268.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Kyte J., Doolittle R. F. A simple method for displaying the hydropathic character of a protein. J Mol Biol. 1982 May 5;157(1):105–132. doi: 10.1016/0022-2836(82)90515-0. [DOI] [PubMed] [Google Scholar]
  16. Lazarevic V., Margot P., Soldo B., Karamata D. Sequencing and analysis of the Bacillus subtilis lytRABC divergon: a regulatory unit encompassing the structural genes of the N-acetylmuramoyl-L-alanine amidase and its modifier. J Gen Microbiol. 1992 Sep;138(9):1949–1961. doi: 10.1099/00221287-138-9-1949. [DOI] [PubMed] [Google Scholar]
  17. Maidhof H., Reinicke B., Blümel P., Berger-Bächi B., Labischinski H. femA, which encodes a factor essential for expression of methicillin resistance, affects glycine content of peptidoglycan in methicillin-resistant and methicillin-susceptible Staphylococcus aureus strains. J Bacteriol. 1991 Jun;173(11):3507–3513. doi: 10.1128/jb.173.11.3507-3513.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Maki H., Yamaguchi T., Murakami K. Cloning and characterization of a gene affecting the methicillin resistance level and the autolysis rate in Staphylococcus aureus. J Bacteriol. 1994 Aug;176(16):4993–5000. doi: 10.1128/jb.176.16.4993-5000.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Mechold U., Cashel M., Steiner K., Gentry D., Malke H. Functional analysis of a relA/spoT gene homolog from Streptococcus equisimilis. J Bacteriol. 1996 Mar;178(5):1401–1411. doi: 10.1128/jb.178.5.1401-1411.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Mechold U., Steiner K., Vettermann S., Malke H. Genetic organization of the streptokinase region of the Streptococcus equisimilis H46A chromosome. Mol Gen Genet. 1993 Oct;241(1-2):129–140. doi: 10.1007/BF00280210. [DOI] [PubMed] [Google Scholar]
  21. Menguito C. A., Keherly M. J., Tang C., Papaconstantinou J., Weigel P. H. Molecular cloning, sequence, structural analysis and expression of the histidyl-tRNA synthetase gene from Streptococcus equisimilis. Nucleic Acids Res. 1993 Feb 11;21(3):615–620. doi: 10.1093/nar/21.3.615. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Metzger S., Dror I. B., Aizenman E., Schreiber G., Toone M., Friesen J. D., Cashel M., Glaser G. The nucleotide sequence and characterization of the relA gene of Escherichia coli. J Biol Chem. 1988 Oct 25;263(30):15699–15704. [PubMed] [Google Scholar]
  23. Murakami K., Nomura K., Doi M., Yoshida T. Production of low-affinity penicillin-binding protein by low- and high-resistance groups of methicillin-resistant Staphylococcus aureus. Antimicrob Agents Chemother. 1987 Sep;31(9):1307–1311. doi: 10.1128/aac.31.9.1307. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Murakami K., Tomasz A. Involvement of multiple genetic determinants in high-level methicillin resistance in Staphylococcus aureus. J Bacteriol. 1989 Feb;171(2):874–879. doi: 10.1128/jb.171.2.874-879.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Oshida T., Sugai M., Komatsuzawa H., Hong Y. M., Suginaka H., Tomasz A. A Staphylococcus aureus autolysin that has an N-acetylmuramoyl-L-alanine amidase domain and an endo-beta-N-acetylglucosaminidase domain: cloning, sequence analysis, and characterization. Proc Natl Acad Sci U S A. 1995 Jan 3;92(1):285–289. doi: 10.1073/pnas.92.1.285. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Pearson W. R., Lipman D. J. Improved tools for biological sequence comparison. Proc Natl Acad Sci U S A. 1988 Apr;85(8):2444–2448. doi: 10.1073/pnas.85.8.2444. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Rodionov D. G., Ishiguro E. E. Direct correlation between overproduction of guanosine 3',5'-bispyrophosphate (ppGpp) and penicillin tolerance in Escherichia coli. J Bacteriol. 1995 Aug;177(15):4224–4229. doi: 10.1128/jb.177.15.4224-4229.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Ryffel C., Strässle A., Kayser F. H., Berger-Bächi B. Mechanisms of heteroresistance in methicillin-resistant Staphylococcus aureus. Antimicrob Agents Chemother. 1994 Apr;38(4):724–728. doi: 10.1128/aac.38.4.724. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Sanger F., Nicklen S., Coulson A. R. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. doi: 10.1073/pnas.74.12.5463. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Sarubbi E., Rudd K. E., Xiao H., Ikehara K., Kalman M., Cashel M. Characterization of the spoT gene of Escherichia coli. J Biol Chem. 1989 Sep 5;264(25):15074–15082. [PubMed] [Google Scholar]
  31. Schenk S., Laddaga R. A. Improved method for electroporation of Staphylococcus aureus. FEMS Microbiol Lett. 1992 Jul 1;73(1-2):133–138. doi: 10.1016/0378-1097(92)90596-g. [DOI] [PubMed] [Google Scholar]
  32. Song M. D., Wachi M., Doi M., Ishino F., Matsuhashi M. Evolution of an inducible penicillin-target protein in methicillin-resistant Staphylococcus aureus by gene fusion. FEBS Lett. 1987 Aug 31;221(1):167–171. doi: 10.1016/0014-5793(87)80373-3. [DOI] [PubMed] [Google Scholar]
  33. Stahl M. L., Pattee P. A. Confirmation of protoplast fusion-derived linkages in Staphylococcus aureus by transformation with protoplast DNA. J Bacteriol. 1983 Apr;154(1):406–412. doi: 10.1128/jb.154.1.406-412.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Sugai M., Akiyama T., Komatsuzawa H., Miyake Y., Suginaka H. Characterization of sodium dodecyl sulfate-stable Staphylococcus aureus bacteriolytic enzymes by polyacrylamide gel electrophoresis. J Bacteriol. 1990 Nov;172(11):6494–6498. doi: 10.1128/jb.172.11.6494-6498.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Utsui Y., Yokota T. Role of an altered penicillin-binding protein in methicillin- and cephem-resistant Staphylococcus aureus. Antimicrob Agents Chemother. 1985 Sep;28(3):397–403. doi: 10.1128/aac.28.3.397. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Yamada S., Sugai M., Komatsuzawa H., Nakashima S., Oshida T., Matsumoto A., Suginaka H. An autolysin ring associated with cell separation of Staphylococcus aureus. J Bacteriol. 1996 Mar;178(6):1565–1571. doi: 10.1128/jb.178.6.1565-1571.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. de Jonge B. L., de Lencastre H., Tomasz A. Suppression of autolysis and cell wall turnover in heterogeneous Tn551 mutants of a methicillin-resistant Staphylococcus aureus strain. J Bacteriol. 1991 Feb;173(3):1105–1110. doi: 10.1128/jb.173.3.1105-1110.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. de Lencastre H., Tomasz A. Reassessment of the number of auxiliary genes essential for expression of high-level methicillin resistance in Staphylococcus aureus. Antimicrob Agents Chemother. 1994 Nov;38(11):2590–2598. doi: 10.1128/aac.38.11.2590. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Journal of Bacteriology are provided here courtesy of American Society for Microbiology (ASM)

RESOURCES