Sequence analysis of a bacteriocinogenic plasmid of Clostridium butyricum and expression of the bacteriocin gene in Escherichia coli |
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| Authors: | Shusuke Nakanishi Mamoru Tanaka |
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| Institution: | 1. Instituto Superior de Investigaciones Biológicas (INSIBIO), CONICET-UNT and Instituto de Química Biológica “Dr. Bernabé Bloj”, Facultad de Bioquímica, Química y Farmacia, UNT, Chacabuco 461, T4000ILI San Miguel de Tucumán, Argentina;2. Departamento de Bioquímica e Tecnologia Química, Instituto de Química, UNESP-Univ Estadual Paulista, Araraquara, SP, Brazil;3. Centro de Referencia para Lactobacilos, Chacabuco 145, San Miguel de Tucumán, Argentina;1. Key Laboratory for Food Microbial Technology of Zhejiang Province, School of Food Science and Biotechnology, Zhejiang Gongshang University, Hangzhou 310018, PR China;2. College of Life Sciences, Zhejiang University of Traditional Chinese Medicine, 548, Binwen Road, Binjiang District, Hangzhou 310053, PR China |
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| Abstract: | 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 10240 AU 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. |
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