Genetic characterization and heterologous expression of brochocin-C, an antibotulinal, two-peptide bacteriocin produced by Brochothrix campestris ATCC 43754

Brochocin-C, produced by Brochothrix campestris ATCC 43754, is active again st many strains of the closely related meat spoilage organism Brochothrix t hermosphacta and a wide range of other gram-positive bacteria, including sp ores of Clostridium botulinum. Purification of the active compound and gene tic characterization of brochocin-C revealed that it is a chromosomally enc oded, two-peptide nonlantibiotic bacteriocin. Both peptides of brochocin-C are ribosomally synthesized as prepeptides that are typical of class II bac teriocins, They are cleaved following Gly-Gly cleavage sites to yield the m ature peptides, BrcA and BrcB, containing 59 and 43 amino acids, respective ly. Fusion of the nucleotides encoding the signal peptide of the bacterioci n divergicin A in front of the structural genes for either BrcA or BrcB all owed independent expression of each component by the general protein secret ion pathway. This revealed the two-component nature of brochocin-C and the necessity for both peptides for activity. A 53-amino-acid peptide encoded d ownstream of brcB functions as the immunity protein (BrcI) for brochocin-C. In addition, the cloned chromosomal fragment revealed open reading frames downstream of brcI designated brcT and brcD, that encode proteins with homo logy to ATP-binding cassette translocator and accessory proteins, respectiv ely, involved in the secretion of Gly-Gly-type bacteriocins.