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 beta-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.
Highlights
Bacteriocins are peptides secreted by lactic acid bacteria that often display antimicrobial activity against other related bacteria [10]
Our attention has been focused on brochocin C (BrcC) from Brochothrix campestris ATCC 43754, a heat-stable two-peptide unmodified bacteriocin system originally discovered by Siragusa and Cutter [19] and characterized by McCormick et al [12]
As a prelude to nuclear magnetic resonance (NMR) examination of two-peptide bacteriocin interactions, we characterized the peptides produced by separate Carnobacterium piscicola LV17C brcA and brcB clones and Escherichia coli brcA and brcB clones
Summary
Bacteriocins are peptides secreted by lactic acid bacteria that often display antimicrobial activity against other related bacteria [10]. A number of both modified and unmodified bacteriocins have been reported to occur as two-peptide systems in which both components are necessary for full antibacterial activity [4]. BrcC has a broad activity spectrum comparable to that of nisin [3]. As a prelude to NMR examination of two-peptide bacteriocin interactions, we characterized the peptides produced by separate Carnobacterium piscicola LV17C brcA and brcB clones and Escherichia coli brcA and brcB clones. The results show that the C. piscicola clones produce mature BrcA and a fully functional truncated fragment of BrcB missing the first nine amino acids, whereas the E. coli clones generate multiple fragments of BrcA or BrcB that are weakly active or inactive. The results presented in this paper provide insight into understanding how BrcC can be a potential meat preservative
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