Zymogram and preliminary characterization of Lactobacillus helveticus autolysins

• Published in: Applied and Environmental Microbiology 1995-09, Vol.61 (9), p.3391-3399
• Main Authors: VALENCE, F, LORTAL, S
• Format: Article
• Language: English
• Subjects: actividad enzimatica; activite enzymatique; Bacteriology; Biological and medical sciences; biological differences; Biology of microorganisms of confirmed or potential industrial interest; Biotechnology; Cellular biology; diferencias biologicas; difference biologique; Ecology, environment; electroforesis; electrophorese; electrophoresis; enzymic activity; Fundamental and applied biological sciences. Psychology; hidrolasas; hydrolase; hydrolases; Lactobacillus helveticus; Life Sciences; Mission oriented research; Physiology and metabolism
• ISSN: 0099-2240; 1098-5336
• DOI: 10.1128/aem.61.9.3391-3399.1995

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.