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

Lactococcus lactis is best known for its role in mesophilic dairy fermentations, including those used in production of cheddar cheese, buttermilk, and sour cream. Its long history of safe use in the food industry and generally recognized as safe (GRAS) status provide new opportunities for using L. lactis in important roles in food biotechnology, particularly in the presentation of vaccines, antimicrobial agents, or intracellular peptidases involved in cheese ripening, outside of the cell. Three major mechanisms have been exploited in these studies. First, signal sequences of the lactococcal secreted protein Usp45 (43), the lactococcal proteinase (46), and the S-layer protein (encoded by slpA) (45) of Lactobacillus brevis have been employed to secrete heterologous proteins from L. lactis via the secretory pathway. ATP-binding cassette–transporter export systems have also been used to export heterologous bacteriocins from L. lactis (for a review, see reference 1). Lastly, induction of cell autolysis can result in efficient release of homologous and heterologous proteins from the cell. Interest in naturally occurring autolytic strains has focused on the release of intracellular enzymes, namely peptidases, into the cheese medium to enhance flavor development and accelerate cheese ripening. Strains of L. lactis differ widely in their ability to undergo autolysis (25). Recent studies have implicated both the major lactococcal autolysin AcmA (5) and “leaky” low-level expression of a prophage-encoded lysin (21, 27) as causes of cell autolysis. Advances in the molecular techniques available for studying L. lactis have increased efforts to genetically engineer autolytic strains to control cell lysis. For instance, the lactococcal autolysin AcmA was cloned under the control of two regulated promoters, the chloride-inducible promoter (39) and the promoter-operator region of the temperate phage r1t (5, 32), generating strains which, upon induction of the promoter, lyse to release intracellular enzymes into the supernatant. Another major advance in this area has been the cloning and characterization of several lactococcal bacteriophage lysins and holins (reviewed in reference 15). In contrast to the lactococcal autolysin AcmA, the bacteriophage lysins do not possess a signal sequence (4). Externalization occurs via a small, transmembrane holin which oligomerizes to form nonspecific pores in the host membrane (for reviews, see references 3, 15, 52, and 53). The genes encoding several bacteriophage lysins and/or holins have been exploited to design strains which lyse under controlled conditions. For example, the holin and lysin genes of phages r1t and US3 were placed under the control of PnisA (6) and the chloride-inducible promoter (39), generating strains which released intracellular peptidases into the medium upon induction of the promoter. Overproduction of only the holin from phage φUS3, via PnisA, resulted in an immediate inhibition of cell growth and partial lysis of the host cell (6). The use of bacteriophage lytic cassettes is an exciting advance in the continued development of expression systems allowing the delivery of proteins and enzymes to the outside environment. However, these systems often cause rapid cessation of growth and, eventually, cell lysis. Moreover, since lysin works extracellularly, the growth of other lactococcal strains used in combination would also be affected. In this study, we describe a novel expression system which allows the release of significant levels of a Streptococcus thermophilus β-galactosidase (β-gal) (40) in L. lactis without the use of signal sequences and without causing severe losses of cell viability or integrity. The system utilizes the phage transcriptional activator Tac31A (formerly open reading frame 2 [ORF2] and tac [36, 47]) to activate a late promoter residing on a prophage integrated in the genome of L. lactis. Low-level activation of the late promoter, driving downstream expression of holin and lysin, resulted in a leaky behavior that efficiently externalized an enzyme and a vaccine, with minimal losses of other cytoplasmic enzymes.