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Applied and Environmental Microbiology, March 2003, p. 1352-1358, Vol. 69, No. 3
0099-2240/03/$08.00+0     DOI: 10.1128/AEM.69.3.1352-1358.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

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

Sylvie Garneau,¹ Claire A. Ference,¹ Marco J. van Belkum,² Michael E. Stiles,² and John C. Vederas^1*

Department of Chemistry,¹ Department of Agricultural, Food and Nutritional Science, University of Alberta, Edmonton, Alberta T6G 2G2, Canada²

Received 13 August 2002/ Accepted 10 December 2002

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 ß-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.

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* Corresponding author. Mailing address: Department of Chemistry, University of Alberta, Edmonton, Alberta T6G 2G2, Canada. Phone: (780) 492-5475. Fax: (780) 492-8231. E-mail: john.vederas{at}ualberta.ca.

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Applied and Environmental Microbiology, March 2003, p. 1352-1358, Vol. 69, No. 3
0099-2240/03/$08.00+0     DOI: 10.1128/AEM.69.3.1352-1358.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

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