Many Lactococcus strains used as dairy starters carry prophages, which may eventually be activated and lyse their host. Bacteriophage infection in cheese manufacture is a major threat as milk acidification relies on the metabolic activity of the dairy starter. On the other hand, cheese ripening and flavor development profit from starter (auto-)lysis, a process in which prophage-encoded endolysins play a role. In this work, we have studied two isogenic strains derived from the model lactic acid bacteria L. lactis MG1363 that differ in their prophage content and their lytic response after mitomycin C induction. L. lactis TP+CAP- carries the prophage TP712 which is unable to cause substantial lysis after induction with Mitomycin C. The other strain, L. lactis TP+CAP+, harbors an additional novel prophage, named CAP, and bulk lysis is observed upon prophage activation. Previous insights into the lytic genes of TP712 showed that they were not very proficient in lysing host cells when expressed in trans. Thus, we asked the question whether the lytic phenotype of the double lysogen was solely due to the CAP lytic functions or not. Based on sequence comparisons, the CAP lysis module encodes a predicted pinholin with two transmembrane domains and a modular endolysin (LysCAP) which possesses a N-terminal catalytic domain with a putative Amidase_5 enzymatic activity, and a C-terminal region with a PG_binding_1 domain, likely involved in substrate binding. Recombinant LysCAP production was approached in E. coli by designing a synthetic gene. Protein was produced with a 6xHis C-terminal tag, purified by Ni-affinity chromatography and the activity of the recombinant endolysin was tested in zymograms and quantified by standard turbidity assays. When added exogenously, lysis was observed only when target cells were sensitized through the addition of nisin, which is an effective ionophore that binds to the cell wall precursor lipid II and forms pores in the cytoplasmic membrane causing membrane depolarization. Lysis from within in L. lactis was also examined. Only expression of the CAP holin-endolysin genes together triggered cell lysis, in line with the dependence of LysCAP on membrane depolarization. Interestingly, concomitant induction of the TP712 prophage speeded lysis. Further experiments with mutated TP712 prophages confirmed that the TP712 holins but not its endolysin accounted for shortening the onset of host lysis, proving the cooperation beween the lytic functions of both prophages.

Trabajo presentado en el World Microbe Forum. ASM-FEMS Congress, celebrado online del 20 al 24 de junio de 2021