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Applied and Environmental Microbiology, December 1998, p. 4757-4766, Vol. 64, No. 12
0099-2240/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.

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

John K. McCormick,1,dagger Alison Poon,2 Miloslav Sailer,3,Dagger Yan Gao,2 Ken L. Roy,1 Lynn M. McMullen,2 John C. Vederas,3 Michael E. Stiles,2,* and Marco J. Van Belkum2

Departments of Biological Sciences,1 Agricultural, Food and Nutritional Science,2 and Chemistry,3 University of Alberta, Edmonton, Alberta, Canada T6G 2P5

Received 20 March 1998/Accepted 18 September 1998

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 of Clostridium 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 of brcI, 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.

* Corresponding author. Mailing address: Department of Agricultural, Food and Nutritional Science, 4-10 Ag/For Centre, University of Alberta, Edmonton, Alberta, Canada T6G 2P5. Phone: (403) 492 2386. Fax: (403) 492 8914. E-mail: mstiles{at}afns.ualberta.ca.

dagger Present address: Department of Microbiology, University of Minnesota Medical School, Minneapolis, MN 55455.

Dagger Present address: Apotex Fermentations, Winnipeg, Manitoba, Canada R3Y 1G4.

Applied and Environmental Microbiology, December 1998, p. 4757-4766, Vol. 64, No. 12
0099-2240/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.


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