Analysis of the bacteriolytic enzymes of the autolytic lactococcus lactis subsp. cremoris strain AM2 by renaturing polyacrylamide gel electrophoresis: identification of a prophage-encoded enzyme

Abstract

Lactococcus lactis subsp. cremoris AM2 was previously shown to lyse early and extensively during cheese ripening (M.-P. Chapot-Chartier, C. Deniel, M. Rousseau, L. Vassal, and J.-C. Gripon, Int. Dairy J. 4:251-269, 1994). We analyzed the bacteriolytic activities of autolytic strain AM2 by using renaturing sodium dodecyl sulfate-polyacrylamide gel electrophoresis performed with two different substrates in the gel, Micrococcus lysodeikticus and L. lactis autoclaved cells. Several lytic activities were detected in L. lactis AM2; a major lytic activity, designated A2 (46 kDa), was found only with the L. lactis cell substrate. This activity appears to be different from major peptidoglycan hydrolase AcmA characterized previously (G. Buist, J. Kok, K. J. Leenhouts, M. Dabrowska, G. Venema, and A. J. Haandrickman, J. Bacteriol. 177:1554-1563, 1995), which has a similar molecular mass. The two enzymes differ in substrate specificity as well as in sensitivity to pH and different chemical compounds. L. lactis AM2 is lysogenic and mitomycin C inducible. Enzyme A2 was shown to be inducible by mitomycin C and to be prophage encoded. It was identified as an enzyme similar to the lysin encoded by lactococcal small isometric temperate bacteriophages. A prophage-cured derivative of L. lactis AM2 was obtained, and this isolate exhibited different autolytic properties than AM2. After prolonged incubation in the stationary phase after growth on M17 medium, the extent of lysis of an AM2 culture was 60%, whereas over the same period there was almost no lysis in a prophage-cured derivative strain culture. These results suggest that the prophage lytic system is involved in the strain AM2 lysis observed in liquid medium and that it could also be involved in the lysis observed during cheese ripening.

FIG. 1

Detection of bacteriolytic activities of L. lactis MG1363, NCDO763, and AM2 by renaturing SDS-PAGE. (A) Gel containing autoclaved M. lysodeikticus cells as the substrate. (B) Gel containing autoclaved L. lactis cells as the substrate. Lane 1, MG1363 SDS cell extract; lane 2, NCDO763 SDS cell extract; lane 3, AM2 SDS cell extract. The positions of the lytic bands are indicated by arrowheads. The numbers on the left are molecular masses (in kilodaltons).

FIG. 2

Detection of bacteriolytic activities of L. lactis NCDO763 and AM2 by renaturing SDS-PAGE in a gel containing autoclaved M. lysodeikticus cells as the substrate. Lane 1, native cell wall preparation from strain AM2; lane 2, NCDO763 SDS cell extract. The positions of the lytic bands are indicated by arrowheads.

FIG. 3

Effect of mitomycin C on the growth of L. lactis subsp. cremoris AM2 (A) and on its bacteriolytic activity profile (B and C). (A) Symbols: ▴, untreated culture; •, mitomycin C induction. The arrow indicates the time of mitomycin C (1 μg/ml) addition. (B and C) Samples were removed at 1-h intervals from the untreated culture (B) or from the mitomycin C-induced culture (C), and SDS cell extracts containing the same amount of proteins were analyzed by renaturing SDS-PAGE. Lanes 1 through 6, samples taken at zero time and at 1, 2, 3, 4, and 5 h after mitomycin C addition. The position of lytic band A2 is indicated by an arrowhead.

FIG. 4

Electron micrograph of bacteriophage particles observed in an L. lactis AM2 lysate after mitomycin C induction. Bar = 50 nm.

FIG. 5

Bacteriolytic activity profile of prophage-cured derivative AM2-C of L. lactis AM2 as determined by renaturing SDS-PAGE performed with a gel containing autoclaved L. lactis cells as the substrate. Lane 1, AM2 SDS cell extract; lane 2, AM2-C SDS cell extract. The same amount of proteins was loaded into each well.

FIG. 6

Southern blot hybridization analysis of total DNA from different strains and bacteriophage φAM2. Lane 1, L. lactis subsp. cremoris AM2; lane 2, L. lactis subsp. cremoris AM2-C; lane 3, bacteriophage φAM2. DNA was cut with ClaI and hybridized with the 1.2-kb lys PCR product as the probe. The numbers on the left indicate molecular sizes (in kilobases).

FIG. 7

Autolysis after prolonged incubation in the stationary phase after growth in M17-lac broth at 30°C of L. lactis subsp. cremoris AM2 (A) and L. lactis subsp. cremoris AM2-C (B). Bacterial lysis was monitored by determining the changes in broth turbidity (•) (expressed as percentages of the maximum OD₆₅₀), in cell viability (○) (expressed as percentages of the maximum number of CFU per milliliter), and in the PepX activity released into the culture supernatant (▴) (expressed in nanokatals per milliliter of culture supernatant). PepX activity was measured with Ala-Pro-paranitroanilide as the substrate. Similar results were obtained in three independent experiments.