Skip to main content
NCBI home page
Journal of Bacteriology logoLink to Journal of Bacteriology
. 1993 Mar;175(5):1493–1499. doi: 10.1128/jb.175.5.1493-1499.1993

Isolation and characterization of autolysis-defective mutants of Staphylococcus aureus created by Tn917-lacZ mutagenesis.

N Mani 1, P Tobin 1, R K Jayaswal 1
PMCID: PMC193237  PMID: 8095258

Abstract

Two autolysis-defective mutants (Lyt-1 and Lyt-2) of Staphylococcus aureus have been isolated by transposon Tn917-lacZ mutagenesis. The mutants exhibited normal growth rate, cell division, cell size, and adaptive responses to environmental changes. No autolytic activities were detected in a crude autolytic enzyme preparation from the Lyt- mutants. The rate of autolysis of whole cells and cell walls in the mutants were negligible, but mutant cell wall preparations were degraded by crude enzyme preparations from the wild-type strain. Zymographic analyses of enzyme extracts from the mutants showed a single autolytic enzyme band, compared with more than 10 autolytic enzyme bands from the parent strain. Analyses of intracellular and exoprotein fractions gave results similar to those in experiments with total-cell extracts. Southern blot analysis indicated the insertion of a single copy of the transposon into the chromosome of Lyt mutants. Isogenic Lyt mutants constructed by phage phi 11 transduction showed similar phenotypes. Because both Lyt- mutants had Tn917-lacZ inserted in the appropriate orientation, it was possible to determine gene activity under various conditions by measuring beta-galactosidase activity. The gene activity was found to be induced by low pH, low temperature, and high sucrose and high sodium chloride concentrations. From these data, we propose that the mutation lies in either a master regulatory gene or a structural gene which is responsible for the synthesis or processing of a majority of the autolytic enzyme bands.

Full text

PDF
1493

Images in this article

1496Image
on p.1496
1496Image
on p.1496
1496Image
on p.1496

Selected References

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

  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. Bierbaum G., Sahl H. G. Autolytic system of Staphylococcus simulans 22: influence of cationic peptides on activity of N-acetylmuramoyl-L-alanine amidase. J Bacteriol. 1987 Dec;169(12):5452–5458. doi: 10.1128/jb.169.12.5452-5458.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. 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]
  4. Béliveau C., Potvin C., Trudel J., Asselin A., Bellemare G. Cloning, sequencing, and expression in Escherichia coli of a Streptococcus faecalis autolysin. J Bacteriol. 1991 Sep;173(18):5619–5623. doi: 10.1128/jb.173.18.5619-5623.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Casadaban M. J., Cohen S. N. Lactose genes fused to exogenous promoters in one step using a Mu-lac bacteriophage: in vivo probe for transcriptional control sequences. Proc Natl Acad Sci U S A. 1979 Sep;76(9):4530–4533. doi: 10.1073/pnas.76.9.4530. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Chatterjee A. N., Wong W., Young F. E., Gilpin R. W. Isolation and characterization of a mutant of Staphylococcus aureus deficient in autolytic activity. J Bacteriol. 1976 Mar;125(3):961–967. doi: 10.1128/jb.125.3.961-967.1976. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Cheung H. Y., Freese E. Monovalent cations enable cell wall turnover of the turnover-deficient lyt-15 mutant of Bacillus subtilis. J Bacteriol. 1985 Mar;161(3):1222–1225. doi: 10.1128/jb.161.3.1222-1225.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Cleveland R. F., Holtje J. V., Wicken A. J., Tomasz A., Daneo-Moore L., Shockman G. D. Inhibition of bacterial wall lysins by lipoteichoic acids and related compounds. Biochem Biophys Res Commun. 1975 Dec 1;67(3):1128–1135. doi: 10.1016/0006-291x(75)90791-3. [DOI] [PubMed] [Google Scholar]
  9. Dolinger D. L., Schramm V. L., Shockman G. D. Covalent modification of the beta-1,4-N-acetylmuramoylhydrolase of Streptococcus faecium with 5-mercaptouridine monophosphate. Proc Natl Acad Sci U S A. 1988 Sep;85(18):6667–6671. doi: 10.1073/pnas.85.18.6667. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Fein J. E., Rogers H. J. Autolytic enzyme-deficient mutants of Bacillus subtilis 168. J Bacteriol. 1976 Sep;127(3):1427–1442. doi: 10.1128/jb.127.3.1427-1442.1976. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Foster S. J. Analysis of the autolysins of Bacillus subtilis 168 during vegetative growth and differentiation by using renaturing polyacrylamide gel electrophoresis. J Bacteriol. 1992 Jan;174(2):464–470. doi: 10.1128/jb.174.2.464-470.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Foster S. J., Johnstone K. Purification and properties of a germination-specific cortex-lytic enzyme from spores of Bacillus megaterium KM. Biochem J. 1987 Mar 1;242(2):573–579. doi: 10.1042/bj2420573. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. García E., García J. L., Ronda C., García P., López R. Cloning and expression of the pneumococcal autolysin gene in Escherichia coli. Mol Gen Genet. 1985;201(2):225–230. doi: 10.1007/BF00425663. [DOI] [PubMed] [Google Scholar]
  14. Gilpin R. W., Chatterjee A. N., Young F. E. Autolysis of microbial cells: salt activation of autolytic enzymes in a mutant of Staphylococcus aureus. J Bacteriol. 1972 Jul;111(1):272–283. doi: 10.1128/jb.111.1.272-283.1972. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. 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]
  16. 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]
  17. 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]
  18. Kuroda A., Sekiguchi J. Cloning, sequencing and genetic mapping of a Bacillus subtilis cell wall hydrolase gene. J Gen Microbiol. 1990 Nov;136(11):2209–2216. doi: 10.1099/00221287-136-11-2209. [DOI] [PubMed] [Google Scholar]
  19. Leclerc D., Asselin A. Detection of bacterial cell wall hydrolases after denaturing polyacrylamide gel electrophoresis. Can J Microbiol. 1989 Aug;35(8):749–753. doi: 10.1139/m89-125. [DOI] [PubMed] [Google Scholar]
  20. Madiraju M. V., Brunner D. P., Wilkinson B. J. Effects of temperature, NaCl, and methicillin on penicillin-binding proteins, growth, peptidoglycan synthesis, and autolysis in methicillin-resistant Staphylococcus aureus. Antimicrob Agents Chemother. 1987 Nov;31(11):1727–1733. doi: 10.1128/aac.31.11.1727. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. 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]
  22. 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]
  23. 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]
  24. 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]
  25. Qoronfleh M. W., Wilkinson B. J. Effects of growth of methicillin-resistant and -susceptible Staphylococcus aureus in the presence of beta-lactams on peptidoglycan structure and susceptibility to lytic enzymes. Antimicrob Agents Chemother. 1986 Feb;29(2):250–257. doi: 10.1128/aac.29.2.250. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Raychaudhuri D., Chatterjee A. N. Use of resistant mutants to study the interaction of triton X-100 with Staphylococcus aureus. J Bacteriol. 1985 Dec;164(3):1337–1349. doi: 10.1128/jb.164.3.1337-1349.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Recsei P., Kreiswirth B., O'Reilly M., Schlievert P., Gruss A., Novick R. P. Regulation of exoprotein gene expression in Staphylococcus aureus by agar. Mol Gen Genet. 1986 Jan;202(1):58–61. doi: 10.1007/BF00330517. [DOI] [PubMed] [Google Scholar]
  28. Sanchez-Puelles J. M., Ronda C., Garcia J. L., Garcia P., Lopez R., Garcia E. Searching for autolysin functions. Characterization of a pneumococcal mutant deleted in the lytA gene. Eur J Biochem. 1986 Jul 15;158(2):289–293. doi: 10.1111/j.1432-1033.1986.tb09749.x. [DOI] [PubMed] [Google Scholar]
  29. Satta G., Varaldo P. E., Grazi G., Fontana R. Bacteriolytic activity in staphylococci. Infect Immun. 1977 Apr;16(1):37–42. doi: 10.1128/iai.16.1.37-42.1977. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Singer H. J., Wise E. M., Jr, Park J. T. Properties and purification of N-acetylmuramyl-L-alanine amidase from Staphylococcus aureus H. J Bacteriol. 1972 Nov;112(2):932–939. doi: 10.1128/jb.112.2.932-939.1972. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. 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]
  32. Tomasz A., Moreillon P., Pozzi G. Insertional inactivation of the major autolysin gene of Streptococcus pneumoniae. J Bacteriol. 1988 Dec;170(12):5931–5934. doi: 10.1128/jb.170.12.5931-5934.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Townsend D. E., Wilkinson B. J. Proline transport in Staphylococcus aureus: a high-affinity system and a low-affinity system involved in osmoregulation. J Bacteriol. 1992 Apr;174(8):2702–2710. doi: 10.1128/jb.174.8.2702-2710.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Valisena S., Varaldo P. E., Satta G. Purification and characterization of three separate bacteriolytic enzymes excreted by Staphylococcus aureus, Staphylococcus simulans, and Staphylococcus saprophyticus. J Bacteriol. 1982 Aug;151(2):636–647. doi: 10.1128/jb.151.2.636-647.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Ved H. S., Gustow E., Mahadevan V., Pieringer R. A. Dodecylglycerol. A new type of antibacterial agent which stimulates autolysin activity in Streptococcus faecium ATCC 9790. J Biol Chem. 1984 Jul 10;259(13):8115–8121. [PubMed] [Google Scholar]
  36. Ved H. S., Gustow E., Pieringer R. A. The involvement of the proteinase of Streptococcus faecium ATCC 9790 in the stimulation of its autolysin activity by dodecylglycerol. J Biol Chem. 1984 Jul 10;259(13):8122–8124. [PubMed] [Google Scholar]
  37. Vitković L. Bacillus subtilis Lyt+ and Lyt- strains secrete peptidoglycan hydrolases. Can J Microbiol. 1987 Jun;33(6):563–565. doi: 10.1139/m87-095. [DOI] [PubMed] [Google Scholar]
  38. WELSCH M., SALMON J. Quelques aspects de la staphylolyse. Ann Inst Pasteur (Paris) 1950 Nov;79(5):802–813. [PubMed] [Google Scholar]
  39. 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]
  40. 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]
  41. Wecke J., Lahav M., Ginsburg I., Giesbrecht P. Cell wall degradation of Staphylococcus aureus by lysozyme. Arch Microbiol. 1982 Mar;131(2):116–123. doi: 10.1007/BF01053992. [DOI] [PubMed] [Google Scholar]
  42. Wecke J., Lahav M., Ginsburg I., Kwa E., Giesbrecht P. Inhibition of wall autolysis of staphylococci by sodium polyanethole sulfonate "liquoid". Arch Microbiol. 1986 Mar;144(2):110–115. doi: 10.1007/BF00414719. [DOI] [PubMed] [Google Scholar]

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

RESOURCES