Development of bacteriocin expression systems in lactic acid bacteria

Lipophilic antimicrobial peptides or proteins produced by bacteria are defined as bacteriocins. Class II bacteriocins from gram-positive bacteria are small, ribosomally synthesized, heat stable peptides that undergo minimal posttranslational modification that is generally limited to the cleavage of a leader peptide. Export of class II bacteriocins requires this leader peptide and is also dependent on dedicated ATP-binding cassette secretion proteins. Divergicin A is an atypical bacteriocin produced from Carnobacterium divergens LV13. Predivergicin A contains a signal peptide and utilizes the general secretory pathway (Sec-) for export. Carnobacteriocin B2 is a well characterized bacteriocin from Carnobacterium piscicola LV17. Secretion of active carnobacteriocin B2 was achieved by replacing the natural leader peptide with the signal peptide of divergicin A. N-terminal sequencing of the purified compound revealed that proper processing of the signal peptide occurred. When these expression constructs were transformed into the wildtype divergicin A producer, multiple bacteriocin expression via the Sec-pathway was achieved. Colicin V is a small, proteinaceous bacterial toxin produced by many strains of Escherichia coli that fits the definition of class II bacteriocins from gram-positive bacteria. Colicin V was expressed in lactic acid bacteria using the divergicin A signal peptide, and in this case, the immunity protein was not required. Brochocin-C is a two-component bacteriocin produced by Brochothrix campestris that is active against many strains of the closely related meat spoilage organism Brochothrix thermosphacta and a wide range of other gram-positive bacteria, including spores of Clostridium botulinum. Expression of individual peptides from the two-component bacteriocin brochocin-C revealed that both peptides are required for bacteriocin activity. A plasmid was constructed that expressed both brochocin-C peptides from the same host. This novel bacteriocin expression system functions in a variety of heterologous hosts and it may prove useful in the development of multiple bacteriocin expression cassettes. As well, this system may serve as a model for the heterologous expression of other small bacteriocins from gram-negative bacteria in lactic acid bacteria.