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.

FIG. 1.

Bacteriocin production by C. beijerinckii ATCC 25752 and C. tyrobutyricum ADRIAT 932. Bacterial growth, followed by measurement of the optical density of the cultures at 600 nm, is indicated by circles and squares, respectively, while bacteriocin activity is shown by diamonds and triangles, respectively. The optical densities at 600 nm (OD600) are plotted on the left y axes, while the right y axes show the bacteriocin activities (in AU per milliliter) in both culture supernatants.

FIG. 2.

Circularin A and closticin 574 activity in a Tricine-SDS-PAGE gel overlay assay. Lanes: 1, 0.125 μl of C. beijerinckii ATCC 25752 supernatant; 2, 7.5 μl of C. tyrobutyricum ADRIAT 932 supernatant; M, 4 μl of Rainbow molecular weight marker RPN 755. The band of inhibition in lane M corresponds to lysozyme activity. The indicator strain used was L. saké ATCC 15521.

FIG. 3.

Nucleotide sequence of cirA and circularization of circularin A. The fragments A (underlined), B (boldface), and B extended (boldface and italic) are indicated throughout the figure. (A) Amino acid sequences of fragments A and B of CnBr-cleaved circularin A. The sequence of fragment A was obtained by Edman degradation on a purified fragment of CnBr-cleaved circularin A. The sequence of fragment B was obtained by deduction as described in the text. The extended sequence of fragment B was used to determine the circularization point. The degenerate nucleotide sequences of the primers (514CB1a1 and 514CB2a2) used to obtain an internal cirA DNA fragment by PCR (dotted line) are indicated. (B) Nucleotide and deduced amino acid sequences of the structural gene of circularin A (cirA). The vertical arrow indicates the circularization point. Putative Shine-Dalgarno (S.D.) and promoter sequences are underlined. Translation starts with a formyl-methionine (fMet). Two large arrows indicate an inverted repeat downstream of cirA. (C) Putative maturation process of circularin A. The peptide bond between Leu³ and Val⁴ is cleaved, and a new bond is formed between Tyr⁷² and Val⁴. The reaction yields the peptide MFL and the circular bacteriocin. The thin line in the pre-CirA sequence is a schematic representation of the newly forming peptide bond.

FIG. 4.

Nucleotide and derived amino acid sequences of closticin 574. (A) N-terminal amino acid sequence of closticin 574 as determined by Edman degradation on the purified protein. Amino acids within parentheses were determined with a confidence level of >90% certainty. Parenthetic amino acids with a question mark obtained a confidence level of 50%. At position 16, signals of both Ala and Gly were derived, probably as a consequence of high background levels of glycine. (B) Nucleotide and deduced amino acid sequences of the structural gene of closticin 574 (cloA). The deduced amino acid sequence of mature closticin 574 is indicated in boldface. The predicted N-terminal signal sequence is underlined. Putative Shine Dalgarno (S.D) and promoter sequences are underlined. The putative UUG start codon is indicated in boldface.