Characterization of a bacteriocinogenic plasmid from Clostridium perfringens and molecular genetic analysis of the bacteriocin-encoding gene

Abstract

The bacteriocinogenic plasmid pIP404 from Clostridium perfringens was isolated and cloned in Eschenichia coli, and its physical map was deduced. Expression of the bcn gene, encoding bacteriocin BCN5, is inducible by UV irradiation of C. perfiringens and thus resembles the SOS-regulated bacteriocin genes of enteric bacteria.The location of bcn on pIP4W4 was established by a dot-blot procedure, using specific hybridization probes to analyze mRNA samples from induced and uninduced cultures. From the nucleotide sequence of its gene, the molecular weight of BCN5 was deduced to be 96,591, and a protein of this size was secreted by bacteriocinproducing cultures of C. perfiringens. The primary structure of the protein suggests that it may function as an ionophore, since a hydrophobic domain, resembling those of the ionophoric colicins, is present at the COOH terminus. No bacteriocin activity could be detected in E. coli harboring plasmids bearing the bcn gene, even when the transcriptional and translational signals were replaced by those of lacZ. A possible explanation may be found in the unusual codon usage of the adenine-thymine-rich bcn gene, as this shows a preference for codons with a high adenine-plus-thymine content, especially in the wobble position. Many of the frequently used codons correspond to those recognized by minor tRNA species in E. coli. Consequently, bcn expression might be limnited by tRNA availability in this bacterium.

Nucleotide sequence of the Clostridium acidiurici ("Clostridium acidi-urici") gene for 10-formyltetrahydrofolate synthetase shows extensive amino acid homology with the trifunctional enzyme C1-tetrahydrofolate synthase from Saccharomyces cerevisiae

“…None of the three other genes demonstrate the preferred C in the third position as does the synthase gene. It has been suggested that codon bias hinders the expression of the C. perfringens bacteriocin gene in E. coli (13). Although the C. acidiurici 10-formyl-THF synthetase gene also shows strong codon bias, it is well expressed in E. coli (41).…”

“…It is of interest to compare the codon usage in the synthetase gene with the usage in the nifHJ and nifH2 genes of Clostridium pasteurianum (7) and the plasmid-encoded bacteriocin gene from Clostridium perfringens (13), the only other genes reported for large proteins from a clostridial species with a G+C content of less than 30%. None of the three other genes demonstrate the preferred C in the third position as does the synthase gene.…”

Cloning, phenotypic expression, and DNA sequence of the gene for lactacin F, an antimicrobial peptide produced by Lactobacillus spp

“…5A and B). The laf gene contained all 25 N-terminal amino acids of lactacin F which were identified previously through protein sequencing (34 (17,25,29). Amino acid composition analyses of purified lactacin F did not show the presence of lanthionine or other unusual residues as found in bacteriocins now classified as lantibiotics (34).…”

Toxin Plasmids of Clostridium perfringens

“…While early studies of C. perfringens plasmids focused primarily on antibiotic resistance and bacteriocin plasmids (224)(225)(226)(227)(228)(229)(230)(231)(232), the first linkage of C. perfringens toxin production with plasmids occurred over 30 years ago, when loss of CPB production was shown to correlate with the disappearance of a plasmid from a type C strain (233). Later studies then definitively localized several toxin genes to extrachromosomal DNA in a few C. perfringens strains (234).…”