Purification and Characterization of Brochocin A and Brochocin B(10-43), a Functional Fragment Generated by Heterologous Expression in Carnobacterium piscicola

ABSTRACT Brochothrix campestris ATCC 43754 produces a heat-stable, two-component, nonlantibiotic, class IIb bacteriocin, brochocin C (BrcC), that is active against a broad range of gram-positive bacteria, including spores of Clostridium botulinum. An improved purification method was developed for BrcC, in which n-butanol and chloroform extraction are used. Mass spectral characterization of the two components, brochocin A (BrcA) and brochocin B (BrcB), showed that both components are excreted into the medium by B. campestris as mature peptides consisting of 59 and 43 amino acids, respectively. Separate expression clones of BrcA and BrcB were constructed previously in Carnobacterium piscicola LV17C, but the products were not chemically characterized. Purification by the new protocol showed that BrcA is expressed as the mature 59-amino-acid peptide but that BrcB is produced by C. piscicola as a fragment, BrcB(10-43), which is cleaved at an internal Gly-Gly site. This fragment is not antimicrobial by itself, but in combination with BrcA it displays the full activity of the BrcC complex. Circular dichroism measurements revealed a high β-sheet content in the secondary structure of both BrcA and BrcB(10-43), as well as in a 1:1 BrcA-BrcB(10-43) mixture. Separate expression clones of brcA and brcB were also constructed in Escherichia coli, but these clones only produced multiple fragments of the desired peptides with little or no activity.

The Outer Membrane of Gram-Negative Bacteria Inhibits Antibacterial Activity of Brochocin-C

ABSTRACT Brochocin-C is a two-peptide bacteriocin produced byBrochothrix campestris ATCC 43754 that has a broad activity spectrum comparable to that of nisin. Brochocin-C has an inhibitory effect on EDTA-treated gram-negative bacteria, Salmonella enterica serovar Typhimurium lipopolysaccharide mutants, and spheroplasts of Typhimurium strains LT2 and SL3600. Brochocin-C treatment of cells and spheroplasts of strains of LT2 and SL3600 resulted in hydrolysis of ATP. The outer membrane of gram-negative bacteria protects the cytoplasmic membrane from the action of brochocin-C. It appears that brochocin-C is similar to nisin and possibly does not require a membrane receptor for its function; however, the difference in effect of the two bacteriocins on intracellular ATP indicates that they cause different pore sizes in the cytoplasmic membrane.

Genetic Characterization and Heterologous Expression of Brochocin-C, an Antibotulinal, Two-Peptide Bacteriocin Produced by Brochothrix campestris ATCC 43754

ABSTRACT Brochocin-C, produced by Brochothrix campestris ATCC 43754, 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 ofClostridium botulinum. Purification of the active compound and genetic characterization of brochocin-C revealed that it is a chromosomally encoded, two-peptide nonlantibiotic bacteriocin. Both peptides of brochocin-C are ribosomally synthesized as prepeptides that are typical of class II bacteriocins. They are cleaved following Gly-Gly cleavage sites to yield the mature peptides, BrcA and BrcB, containing 59 and 43 amino acids, respectively. Fusion of the nucleotides encoding the signal peptide of the bacteriocin divergicin A in front of the structural genes for either BrcA or BrcB allowed independent expression of each component by the general protein secretion pathway. This revealed the two-component nature of brochocin-C and the necessity for both peptides for activity. A 53-amino-acid peptide encoded downstream of brcB functions as the immunity protein (BrcI) for brochocin-C. In addition, the cloned chromosomal fragment revealed open reading frames downstream ofbrcI, designated brcT and brcD, that encode proteins with homology to ATP-binding cassette translocator and accessory proteins, respectively, involved in the secretion of Gly-Gly-type bacteriocins.