Identification and Characterization of Two Novel Clostridial Bacteriocins, Circularin A and Closticin 574

Abstract

Two novel antibacterial peptides of clostridial species were purified, N-terminally sequenced, and characterized. Moreover, their structural genes were identified. Closticin 574 is an 82-amino-acid bacteriocin produced by Clostridium tyrobutyricum ADRIAT 932. The supernatant of the producing strain showed a high level of activity against the indicator strain C. tyrobutyricum. The protein is synthesized as a preproprotein that is possibly secreted via the general secretion pathway, after which it is hydrolyzed at an Asp-Pro site. Circularin A is produced by Clostridium beijerinckii ATCC 25752 as a prepeptide of 72 amino acids. Cleavage of the prepeptide between the third leucine and fourth valine residues followed by a head-to-tail ligation between the N and C termini creates a circular antimicrobial peptide of 69 amino acids. The unusually small circularin A leader peptide of three amino acids is cleaved off in this process. The supernatant of C. beijerinckii ATCC 25752 showed a broad antibacterial activity range.Clostridia have a negative image as many species are known as pathogens (50), toxin producers (50), and food-spoilage bacteria (36). Various clostridia produce bacteriocins, but despite their long history as a means of typing clostridia (33, 39), only little sequence, functional, or structural information is available on these antibacterial peptides. The few clostridial bacteriocins that have been further characterized are BCN5 and boticin B (12, 18). BCN5 is a large (97-kDa) UV-inducible protein produced by Clostridium perfringens (18). Boticin B is produced by Clostridium botulinum as a small peptide with a predicted size of 50 amino acid residues (12). Little is known about the regulation of production or secretion of these bacteriocins. Generally, bacteriocins are small ribosomally synthesized antimicrobial peptides (27,35). They are mostly membrane permeabilizing and cationic, and they typically comprise fewer than 50 amino acid residues (35). Bacteriocins can be used as an additive to food products to prevent the growth of spoilage bacteria (10). Klaenhammer divided bacteriocins into four classes (35). Class I bacteriocins, known as lantibiotics, contain posttranslationally modified residues such as lanthionine, ␤-methyl lanthionine, and dehydrated residues. Class II bacteriocins lack these modifications and are linear peptides (35,44). Class III contains the large, heat-labile bacteriocins. Class IV bacteriocins are complex molecules composed of protein and chemical moieties. Both class I and class II bacteriocins have been intensively studied, and more than 20 and 100 representatives are known, respectively.The subclass IIc (class II bacteriocins other than the pediocin-like bacteriocins or the two-peptide bacteriocins) contains a few examples of bacteriocins that are produced as circular molecules. This circularization is the result of a head-to-tail peptide bond formation of a prepeptide. Bacteriocins with this typical structure are microcin J25, gassericin A, and AS-48/ Bac21 (6,31,40,59). Mi...

The Circular Bacteriocins Gassericin A and Circularin A

“…Circularin A was purified to homogeneity from the supernatant of a culture grown in an AC broth dialysate by reverse-phase HPLC and was shown to have an apparent molecular weight of 2,200 by SDS-PAGE. Circularin A showed antibacterial activity against C. tyrobutyricum, E. faecalis, L. alimentarius, L. sake and Lactococcus lactis [5].…”

“…From 12 clostridial strains tested, circularin A produced by C. beijerinckii ATCC25752 was the most effective bacteriocin against C. tyrobutyricum NIZOB570, a known cheese-spoilage bacterium [5]. Circularin A was purified to homogeneity from the supernatant of a culture grown in an AC broth dialysate by reverse-phase HPLC and was shown to have an apparent molecular weight of 2,200 by SDS-PAGE.…”

“…After cleavage of purified circularin A using cyanogen bromide, which cleaves methionyl bonds, and subsequent determination of the N-terminal amino acid sequence of the cleavage products, the structural gene of circularin A was cloned by reverse genetics [5]. The gene, cirA (GenBank databases, accession number AY164463), was shown to encode a protein of 72 amino acid residues.…”

“…Recently, a new class, class V, has been proposed: that of the "cyclic bacteriocins" [5]. These peptides are characterized by the fact that their N-and C-termini are covalently linked.…”

Lactobacillus gasseri LF221 and K7 — from isolation to application

“…Recently, a new class (V) of the cyclic bacteriocins has been proposed. These peptides have covalently linked their N-and C-termini (Kemperman et al, 2003, Kawai et al, 2004.…”