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. 1997 Jun;179(11):3625–3631. doi: 10.1128/jb.179.11.3625-3631.1997

Molecular cloning, sequencing, and expression of lytM, a unique autolytic gene of Staphylococcus aureus.

L Ramadurai 1, R K Jayaswal 1
PMCID: PMC179157  PMID: 9171409

Abstract

A gene encoding an autolytic activity was identified in an autolysis-deficient mutant (Lyt-) of Staphylococcus aureus which produces only a single band in autolytic-activity gels (N. Mani, P. Tobin, and R. K. Jayaswal, J. Bacteriol. 175:1493-1499, 1993). An open reading frame, designated lytM, of 948 bp that could encode a polypeptide of 316 amino acid residues was identified. The calculated molecular mass of the lytM gene product (34.4 kDa) corresponded to that of the autolytic activity detected (approximately 36 kDa) in the Lyt- mutant. Results deduced from amino acid sequence analysis and N-terminal amino acid sequencing data suggest that LytM is a secreted protein. The C-terminal region of the putative protein encoded by lytM showed 51% identity with the N-terminal region of the mature lysostaphin from Staphylococcus simulans and 50% identity with the N-terminal region of ALE-1 from Staphylococcus capitis EPK1. Northern blot analysis showed that lytM expresses a transcript of approximately 955 bp, as predicted from the DNA sequence. Escherichia coli clones carrying the lytM gene exhibited autolytic-activity bands of approximately 36 kDa as well as of 19 and 22 kDa in activity gels. The lytM gene was mapped to the SmaI-D fragment on the S. aureus chromosome. Mapping data and results of hybridization experiments with primers generated from gene sequences of known autolytic genes of S. aureus clearly indicate that the lytM gene is distinct from other staphylococcal autolytic genes reported to date.

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Selected References

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  1. Berry A. M., Lock R. A., Hansman D., Paton J. C. Contribution of autolysin to virulence of Streptococcus pneumoniae. Infect Immun. 1989 Aug;57(8):2324–2330. doi: 10.1128/iai.57.8.2324-2330.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Brunskill E. W., Bayles K. W. Identification and molecular characterization of a putative regulatory locus that affects autolysis in Staphylococcus aureus. J Bacteriol. 1996 Feb;178(3):611–618. doi: 10.1128/jb.178.3.611-618.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Brunskill E. W., Bayles K. W. Identification of LytSR-regulated genes from Staphylococcus aureus. J Bacteriol. 1996 Oct;178(19):5810–5812. doi: 10.1128/jb.178.19.5810-5812.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Darzins A., Chakrabarty A. M. Cloning of genes controlling alginate biosynthesis from a mucoid cystic fibrosis isolate of Pseudomonas aeruginosa. J Bacteriol. 1984 Jul;159(1):9–18. doi: 10.1128/jb.159.1.9-18.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Foster S. J. Molecular characterization and functional analysis of the major autolysin of Staphylococcus aureus 8325/4. J Bacteriol. 1995 Oct;177(19):5723–5725. doi: 10.1128/jb.177.19.5723-5725.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Heinrich P., Rosenstein R., Böhmer M., Sonner P., Götz F. The molecular organization of the lysostaphin gene and its sequences repeated in tandem. Mol Gen Genet. 1987 Oct;209(3):563–569. doi: 10.1007/BF00331163. [DOI] [PubMed] [Google Scholar]
  7. Huff E., Silverman C. S., Adams N. J., Awkard W. S. Extracellular cell wall lytic enzyme from Staphylococcus aureus: purification and partial characterization. J Bacteriol. 1970 Sep;103(3):761–769. doi: 10.1128/jb.103.3.761-769.1970. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Höltje J. V., Tuomanen E. I. The murein hydrolases of Escherichia coli: properties, functions and impact on the course of infections in vivo. J Gen Microbiol. 1991 Mar;137(3):441–454. doi: 10.1099/00221287-137-3-441. [DOI] [PubMed] [Google Scholar]
  9. 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]
  10. Mani N., Baddour L. M., Offutt D. Q., Vijaranakul U., Nadakavukaren M. J., Jayaswal R. K. Autolysis-defective mutant of Staphylococcus aureus: pathological considerations, genetic mapping, and electron microscopic studies. Infect Immun. 1994 Apr;62(4):1406–1409. doi: 10.1128/iai.62.4.1406-1409.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Mani N., Tobin P., Jayaswal R. K. Isolation and characterization of autolysis-defective mutants of Staphylococcus aureus created by Tn917-lacZ mutagenesis. J Bacteriol. 1993 Mar;175(5):1493–1499. doi: 10.1128/jb.175.5.1493-1499.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Mead D. A., Szczesna-Skorupa E., Kemper B. Single-stranded DNA 'blue' T7 promoter plasmids: a versatile tandem promoter system for cloning and protein engineering. Protein Eng. 1986 Oct-Nov;1(1):67–74. doi: 10.1093/protein/1.1.67. [DOI] [PubMed] [Google Scholar]
  13. Novick R. Properties of a cryptic high-frequency transducing phage in Staphylococcus aureus. Virology. 1967 Sep;33(1):155–166. doi: 10.1016/0042-6822(67)90105-5. [DOI] [PubMed] [Google Scholar]
  14. 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]
  15. Oshida T., Tomasz A. Isolation and characterization of a Tn551-autolysis mutant of Staphylococcus aureus. J Bacteriol. 1992 Aug;174(15):4952–4959. doi: 10.1128/jb.174.15.4952-4959.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Potvin C., Leclerc D., Tremblay G., Asselin A., Bellemare G. Cloning, sequencing and expression of a Bacillus bacteriolytic enzyme in Escherichia coli. Mol Gen Genet. 1988 Oct;214(2):241–248. doi: 10.1007/BF00337717. [DOI] [PubMed] [Google Scholar]
  17. Recsei P. A., Gruss A. D., Novick R. P. Cloning, sequence, and expression of the lysostaphin gene from Staphylococcus simulans. Proc Natl Acad Sci U S A. 1987 Mar;84(5):1127–1131. doi: 10.1073/pnas.84.5.1127. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. 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]
  19. Shockman G. D., Barrett J. F. Structure, function, and assembly of cell walls of gram-positive bacteria. Annu Rev Microbiol. 1983;37:501–527. doi: 10.1146/annurev.mi.37.100183.002441. [DOI] [PubMed] [Google Scholar]
  20. Sugai M., Fujiwara T., Akiyama T., Ohara M., Komatsuzawa H., Inoue S., Suginaka H. Purification and molecular characterization of glycylglycine endopeptidase produced by Staphylococcus capitis EPK1. J Bacteriol. 1997 Feb;179(4):1193–1202. doi: 10.1128/jb.179.4.1193-1202.1997. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Wang X., Mani N., Pattee P. A., Wilkinson B. J., Jayaswal R. K. Analysis of a peptidoglycan hydrolase gene from Staphylococcus aureus NCTC 8325. J Bacteriol. 1992 Oct;174(19):6303–6306. doi: 10.1128/jb.174.19.6303-6306.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Wang X., Wilkinson B. J., Jayaswal R. K. Sequence analysis of a Staphylococcus aureus gene encoding a peptidoglycan hydrolase activity. Gene. 1991 Jun 15;102(1):105–109. doi: 10.1016/0378-1119(91)90547-o. [DOI] [PubMed] [Google Scholar]
  23. 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]
  24. Yanisch-Perron C., Vieira J., Messing J. Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mp18 and pUC19 vectors. Gene. 1985;33(1):103–119. doi: 10.1016/0378-1119(85)90120-9. [DOI] [PubMed] [Google Scholar]
  25. von Heijne G. A new method for predicting signal sequence cleavage sites. Nucleic Acids Res. 1986 Jun 11;14(11):4683–4690. doi: 10.1093/nar/14.11.4683. [DOI] [PMC free article] [PubMed] [Google Scholar]

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