Leaky Lactococcus cultures that externalize enzymes and antigens independently of culture lysis and secretion and export pathways

A novel system that leaks beta-galactosidase (beta-gal) without a requirement for secretion or export signals was developed in Lactococcus lactis by controlled expression of integrated phage holin and lysin cassettes. The late promoter of the lytic lactococcal bacteriophage phi31 is an 888-bp fragment (P(15A10)) encoding the transcriptional activator. When a high-copy-number P(15A10)::lacZ.st fusion was introduced into L. lactis strains C10, ML8, NCK203, and R1/r1t, high levels of the resultant beta-gal activity were detected in the supernatant (approximately 85% of the total beta-gal activity for C10, ML8, and NCK203 and 45% for R1/r1t). Studies showed that the phenotype resulted from expression of Tac31A from the P(15A10) fragment, which activated a homologous late promoter in prophages harbored by the lactococcal strains. Despite the high levels of beta-gal obtained in the supernatant, the growth of the strains was not significantly affected, nor was there any evidence of severe membrane damage as determined by using propidium iodide or transmission electron microscopy. Integration of the holin-lysin cassette of phage r1t, under the control of the phage phi31 late promoter, into the host genome of MG1363 yielded a similar "leaky" phenotype, indicating that holin and lysin might play a critical role in the release of beta-gal into the medium. In addition to beta-gal, tetanus toxin fragment C was successfully delivered into the growth medium by this system. Interestingly, the X-prolyl dipeptidyl aminopeptidase PepXP (a dimer with a molecular mass of 176 kDa) was not delivered at significant levels outside the cell. These findings point toward the development of bacterial strains able to efficiently release relevant proteins and enzymes outside the cell in the absence of known secretion and export signals.