Sequence analysis of a bacteriocinogenic plasmid of Clostridium butyricum and expression of the bacteriocin gene in Escherichia coli.
A small cryptic plasmid, namely, pCBM588, was obtained from Clostridium butyricum MIYAIRI 588 (CBM588)--a bacterium used in probiotics. The complete sequence of pCBM588 was determined. The size of pCBM588 was 8060 bp and the G + C content was 24.3%. Nine open reading frames (ORFs) were predicted, and ORF3 showed significant homologies with a structural bacteriocin gene of Clostridium tyrobutyricum. The putative bacteriocin gene was inserted into the pET21d expression vector in frame; it was expressed as a His-tagged recombinant protein in Escherichia coli BL21 (DE3). A total of 10240AU of the recombinant bacteriocin were purified from 100 ml of E. coli culture. The bacteriocin was cleaved into 2 portions, and the small C-terminal polypeptide consisting of 83 amino acids possessed bactericidal activity. These results demonstrated that the ORF3 of pCBM588 encoded a bacteriocin, which is identical or very similar to the previously reported butyricin 7423.
Nakanishi
Shusuke
S
Miyarisan Pharmaceutical Co. Research Laboratory, 102-15, Sakaki, Hanishina, Nagano 389-0682, Japan. s.nakanishi@miyarisan.com
Tanaka
Mamoru
M
D000595:Amino Acid Sequence; D001430:Bacteriocins / Q000096:biosynthesis* / Q000235:genetics; D046972:Clostridium butyricum / Q000235:genetics*; D004926:Escherichia coli / Q000235:genetics; D005798:Genes, Bacterial / Q000235:genetics; D008969:Molecular Sequence Data; D016366:Open Reading Frames; D010957:Plasmids / Q000235:genetics*; D015202:Protein Engineering; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000235:genetics; D017421:Sequence Analysis; D020539:Sequence Analysis, Protein
D001430:Bacteriocins; D011994:Recombinant Proteins