Zymogram and Preliminary Characterization of Lactobacillus helveticus Autolysins

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.

Characterization of the Highly Autolytic Lactococcus lactis subsp. cremoris Strains CO and 2250

“…For strain CO, zymogram analysis showed that while each lytic band was present throughout the growth, maximum clearance activities occurred for early-stationary-phase cells. These results differ from those obtained with Lactobacillus helveticus (33), where the major autolysin was essentially absent once stationary phase was reached.…”

Effects of a muramidase on a mixed bacterial community

“…lactis IL1403 [17]. Interestingly, phylogenetically more remote L. helveticus strains CNRZ10940 and CNRZ1102, described as autolysin positive [7] were unable to disrupt chains of MG1363acmAv1 or CNRZ7 under conditions described above. This can be explained by differences in peptidoglycan composition and speci¢city of peptidoglycan hydrolases of phylogenetically di¡erent bacteria [18,19].…”

Impact of broken cells of lactococci or propionibacteria on the ripening of Saint-Paulin UF-cheeses: extent of proteolysis and GC-MS profiles

“…Proteins in the aqueous cheese extracts, as well as CBS and CFE, were analyzed by denaturing polyacrylamide gel electrophoresis (SDS-PAGE), as described by Valence and Lortal [44]. Samples were mixed (v/v) in Laemmli buffer and loaded on the gel (14% separating gel).…”