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. 1995 Sep;61(9):3391–3399. doi: 10.1128/aem.61.9.3391-3399.1995

Zymogram and Preliminary Characterization of Lactobacillus helveticus Autolysins

F Valence, S Lortal
PMCID: PMC1388579  PMID: 16535125

Abstract

The autolysins of Lactobacillus helveticus ISLC5 were detected and partially characterized by renaturing sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis with substrate-containing gels (zymogram). By using lyophilized Micrococcus luteus cells or heated whole cells of L. helveticus ISLC5 (0.2% [wt/vol]) as a substrate, several lytic activities were detected in the whole-cell SDS extract of strain ISLC5 (i) one activity at 42.4 kDa, which was named autolysin A, and (ii) six other activities having very similar molecular weights (29.1, 29.6, 30, 30.8, 31.7, and 32.8 kDa), which were named autolysins B (B1 through B6, respectively). As regards the temporal distribution of the enzymes, autolysins A and B were detected in the cells harvested from the beginning of the exponential growth phase. Autolysin A appeared to be associated only with viable cells, whereas the autolysins B remained associated with the cell envelope several days after the complete loss of culture viability. When SDS-treated walls of L. helveticus ISLC5 were used as a substrate, a supplementary lytic activity appeared at 37.5 kDa; it was considered a peptidoglycan hydrolase, since it was not able to induce lysis of whole-cell substrate. The autolysins of 30 other strains of L. helveticus from various geographical origins were also analyzed by zymogram; all the activity profiles obtained were similar to that of strain ISLC5 in terms of the number of lytic bands and their apparent molecular weights. Only the relative intensities of the lytic bands corresponding to autolysins A and B were variable depending on the strains. This observation suggested that autolysins are highly conserved enzymes. A concentrated crude lysate of the virulent bacteriophage 832-B1 infecting L. helveticus was also analyzed by zymogram; one lytic activity with an apparent molecular weight of 31.7 kDa, very close to the weights of the autolysins B, was observed. Finally, the autolysins of L. helveticus ISLC5 were successfully extracted from whole cells by using a 1 M lithium chloride solution; they were partially purified by precipitation, selective resolubilization, and gel filtration chromatography, which led to a 20-fold increase in specific activity.

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Selected References

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  1. Ayusawa D., Yoneda Y., Yamane K., Maruo B. Pleiotropic phenomena in autolytic enzyme(s) content, flagellation, and simultaneous hyperproduction of extracellular alpha-amylase and protease in a Bacillus subtilis mutant. J Bacteriol. 1975 Oct;124(1):459–469. doi: 10.1128/jb.124.1.459-469.1975. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. BADDILEY J., DAVISON A. L. The occurrence and location of teichoic acids in lactobacilli. J Gen Microbiol. 1961 Feb;24:295–299. doi: 10.1099/00221287-24-2-295. [DOI] [PubMed] [Google Scholar]
  3. Bernadsky G., Beveridge T. J., Clarke A. J. Analysis of the sodium dodecyl sulfate-stable peptidoglycan autolysins of select gram-negative pathogens by using renaturing polyacrylamide gel electrophoresis. J Bacteriol. 1994 Sep;176(17):5225–5232. doi: 10.1128/jb.176.17.5225-5232.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. 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: 10.1006/abio.1976.9999. [DOI] [PubMed] [Google Scholar]
  5. Brown W. C. Rapid methods for extracting autolysins from Bacillus subtilis. Appl Microbiol. 1973 Feb;25(2):295–300. doi: 10.1128/am.25.2.295-300.1973. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Cornett J. B., Johnson C. A., Shockman G. D. Release of autolytic enzyme from Streptococcus, faecium cell walls by treatment with dilute alkali. J Bacteriol. 1979 Jun;138(3):699–704. doi: 10.1128/jb.138.3.699-704.1979. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Forsberg C. W., Rogers H. J. Characterization of Bacillus licheniformis 6346 mutants which have altered lytic enzyme activities. J Bacteriol. 1974 May;118(2):358–368. doi: 10.1128/jb.118.2.358-368.1974. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Forsberg C., Rogers H. J. Autolytic enzymes in growth of bacteria. Nature. 1971 Jan 22;229(5282):272–273. doi: 10.1038/229272a0. [DOI] [PubMed] [Google Scholar]
  9. 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]
  10. García J. L., García E., Arrarás A., García P., Ronda C., López R. Cloning, purification, and biochemical characterization of the pneumococcal bacteriophage Cp-1 lysin. J Virol. 1987 Aug;61(8):2573–2580. doi: 10.1128/jvi.61.8.2573-2580.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Higgins M. L., Coyette J., Shockman G. D. Sites of cellular autolysis in Lactobacillus acidophilus. J Bacteriol. 1973 Dec;116(3):1375–1382. doi: 10.1128/jb.116.3.1375-1382.1973. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Hinks R. P., Daneo-Moore L., Shockman G. D. Cellular autolytic activity in synchronized populations of Streptococcus faecium. J Bacteriol. 1978 Feb;133(2):822–829. doi: 10.1128/jb.133.2.822-829.1978. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. IKAWA M., SNELL E. E. Cell wall composition of lactic acid bacteria. J Biol Chem. 1960 May;235:1376–1382. [PubMed] [Google Scholar]
  14. 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]
  15. 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]
  16. 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]
  17. Messner P., Sleytr U. B. Crystalline bacterial cell-surface layers. Adv Microb Physiol. 1992;33:213–275. doi: 10.1016/s0065-2911(08)60218-0. [DOI] [PubMed] [Google Scholar]
  18. Oterholm A., Ordal Z. J., Witter L. D. Glycerol ester hydrolase activity of lactic acid bacteria. Appl Microbiol. 1968 Mar;16(3):524–527. doi: 10.1128/am.16.3.524-527.1968. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Pooley H. M., Porres-Juan J. M., Shockman G. D. Dissociation of an autolytic enzyme-cell wall complex by treatment with unusually high concentrations of salt. Biochem Biophys Res Commun. 1970 Mar 27;38(6):1134–1140. doi: 10.1016/0006-291x(70)90357-8. [DOI] [PubMed] [Google Scholar]
  20. Pooley H. M., Shockman G. D. Relationship between the latent form and the active form of the autolytic enzyme of Streptococcus faecalis. J Bacteriol. 1969 Nov;100(2):617–624. doi: 10.1128/jb.100.2.617-624.1969. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. 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]
  22. Rogers H. J., Taylor C., Rayter S., Ward J. B. Purification and properties of autolytic endo-beta-N-acetylglucosaminidase and the N-acetylmuramyl-L-alanine amidase from Bacillus subtilis strain 168. J Gen Microbiol. 1984 Sep;130(9):2395–2402. doi: 10.1099/00221287-130-9-2395. [DOI] [PubMed] [Google Scholar]
  23. Romero A., Lopez R., Garcia P. Sequence of the Streptococcus pneumoniae bacteriophage HB-3 amidase reveals high homology with the major host autolysin. J Bacteriol. 1990 Sep;172(9):5064–5070. doi: 10.1128/jb.172.9.5064-5070.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Schleifer K. H., Kandler O. Peptidoglycan types of bacterial cell walls and their taxonomic implications. Bacteriol Rev. 1972 Dec;36(4):407–477. doi: 10.1128/br.36.4.407-477.1972. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. 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]
  26. Tuomanen E., Cozens R., Tosch W., Zak O., Tomasz A. The rate of killing of Escherichia coli by beta-lactam antibiotics is strictly proportional to the rate of bacterial growth. J Gen Microbiol. 1986 May;132(5):1297–1304. doi: 10.1099/00221287-132-5-1297. [DOI] [PubMed] [Google Scholar]
  27. Wadström T. Bacteriolytic enzymes from Staphylococcus aureus. Properties of the endo-beta-N-acetylglucosaminidase. Biochem J. 1970 Dec;120(4):745–752. doi: 10.1042/bj1200745. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Watt S. R., Clarke A. J. Initial characterization of two extracellular autolysins from Pseudomonas aeruginosa PAO1. J Bacteriol. 1994 Aug;176(15):4784–4789. doi: 10.1128/jb.176.15.4784-4789.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Williamson R., Ward J. B. Characterization of the autolytic enzymes of Clostridium perfringens. J Gen Microbiol. 1979 Oct;114(2):349–354. doi: 10.1099/00221287-114-2-349. [DOI] [PubMed] [Google Scholar]
  30. Yamamoto N., Akino A., Takano T. Purification and specificity of a cell-wall-associated proteinase from Lactobacillus helveticus CP790. J Biochem. 1993 Nov;114(5):740–745. doi: 10.1093/oxfordjournals.jbchem.a124247. [DOI] [PubMed] [Google Scholar]

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