The resident TP712 prophage of Lactococcus lactis MG1363 provides extra holin functions to the new P335 phage CAP for effective host lysis

Prophages are widely present in Lactococcus lactis , a lactic acid bacterium (LAB) that plays a key role in dairy fermentations. L. lactis MG1363 is a laboratory strain used worldwide as a model LAB. Initially regarded as plasmid- and prophage-free, MG1363 carries two complete prophages TP712 and MG-3. Only TP712 seems to be inducible but unable to lyse the host. Several so-called TP712 lysogens able to lyse upon prophage induction were reported in the past, but the reason for their lytic phenotype remained unknown. In this work, we describe CAP, a new P335 prophage detected in the “lytic TP712 lysogens”, which had remained unnoticed. CAP is able to excise after mitomycin C treatment, along with TP712, and able to infect L. lactis MG1363-like strains but not the lytic TP712 lysogens. Both phages cooperate for efficient host lysis. While the expression i n trans of the CAP lytic genes was sufficient to trigger cell lysis, this process was boosted when the resident TP712 prophage was concomitantly induced. Introduction of mutations into the TP712 lytic genes revealed that its holin but not its endolysin plays a major role. Accordingly, it is shown that the lytic activity of the recombinant CAP endolysin relies on membrane depolarization. Revisiting the seminal work to generate the extensively used L. lactis MG1363 strain led us to conclude that the CAP phage was originally present in its ancestor L. lactis NCDO712 and our results solved long-standing mysteries around the MG1363 resident prophage TP712 reported in the “pre-sequencing” era. Importance Prophages are bacterial viruses that integrate in the chromosome of bacteria until an environmental trigger induces their lytic cycle ending with lysis of the host. Prophages present in dairy starters can compromise milk fermentation and represent a serious threat in dairy plants. In this work, we have discovered that two temperate phages TP712 and CAP infecting the laboratory strain Lactococcus lactis MG1363 join forces to lyse the host. Based on the in vitro lytic activity of the LysCAP endolysin, in combination with mutated versions of TP712 lacking either its holin or endolysin, we conclude that this cooperation relies on the combined activity of the holins of both phages that boost the activity of LysCAP. The presence of an additional prophage explains the lytic phenotype of the formerly thought to be single TP712 lysogens that had remained a mystery for many years.