Characterization of the highly autolytic Lactococcus lactis subsp. cremoris strains CO and 2250

Two highly autolytic Lactococcus lactis subsp. cremoris strains (CO and 2250) were selected and analyzed for their autolytic properties. Both strains showed maximum lysis when grown in M17 broth containing a limiting concentration of glucose (0.4 to 0.5%) as the carbohydrate source. Lysis did not vary greatly with pH or temperature but was reduced when strains were grown on lactose or galactose. Growth in M17 containing excess glucose (1%) prevented autolysis, although rapid lysis of L. lactis subsp. cremoris CO did occur in the presence of 1% glucose if sodium fluoride (an inhibitor of glycolysis) was added to the medium. Maximum cell lysis in a buffer system was observed early in the stationary phase, and for CO, two pH optima were observed for log-phase and stationary-phase cells (6.5 and 8.5, respectively). Autolysins were extracted from the cell wall fraction of each strain by using either 4% sodium dodecyl sulfate (SDS), 6 M guanidine hydrochloride, or 4 M lithium chloride, and their activities were analyzed by renaturing SDS-polyacrylamide gel electrophoresis on gels containing Micrococcus luteus or L. lactis subsp. cremoris CO cells as the substrate. More than one lytic band was observed on each substrate, with the major band having an apparent molecular mass of 48 kDa for CO. Each lytic band was present throughout growth and lysis. These results suggest that at least two different autolytic enzymes are present in the autolytic L. lactis subsp. cremoris strains. The presence of the lactococcal cell wall hydrolase gene, acmA (G. Buist, J. Kok, K.J. Leenhouts, M. Dabrowska, G. Venema, and A.J. Haandrikman, J. Bacteriol. 177:1554-1563, 1995), in strains 2250 and CO was confirmed by Southern hybridization. Analysis of an acmA deletion mutant of 2250 confirmed that the gene was involved in cell separation and had a role in cell lysis.