Purification and characterization of brochocin A and brochocin B(10-43), a functional fragment generated by heterologous expression in Carnobacterium piscicola.
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.
Garneau
Sylvie
S
Department of Chemistry, University of Alberta, Edmonton, Alberta T6G 2G2, Canada.
Ference
Claire A
CA
van Belkum
Marco J
MJ
Stiles
Michael E
ME
Vederas
John C
JC
D000595:Amino Acid Sequence; D001430:Bacteriocins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism / Q000494:pharmacology; D001483:Base Sequence; D002942:Circular Dichroism; D003470:Culture Media; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005520:Food Preservatives; D007777:Lactobacillaceae / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D013058:Mass Spectrometry / Q000379:methods; D008826:Microbial Sensitivity Tests; D008969:Molecular Sequence Data; D017422:Sequence Analysis, DNA
D001430:Bacteriocins; D003470:Culture Media; D005520:Food Preservatives; C116792:brochocin C