Detection of an ORF-less group II intron in the alpha toxin gene of Clostridium perfringens isolated from a broiler chicken

Department of Veterinary Public Health, Faculty of Agriculture, University of Miyazaki, 1-1 Gakuenkibanadai-nishi, Miyazaki, 889-2192, Japan; Department of Microbiology, Graduate School of Medical Science, Kanazawa University, 13-1, Takara-Machi, Kanazawa, Ishikawa 920-8640, Japan

^* To whom correspondence should be addressed. Email: a0d901u{at}cc.miyazaki-u.ac.jp.

	Abstract

A DNA insertion of 834 bp, designated as CPF-G2Im, was identified within the alpha toxin gene (cpa) of Clostridium perfringens strain CPBC16ML isolated from a broiler chicken. Sequence analysis of the CPF-G2Im indicated that it was integrated 340 nt downstream of the start codon of cpa. However, the insertion did not abolish the phospholipase C (PLC) and hemolytic activities of CPBC16ML. To investigate the expression of its alpha toxin, the intact copy of cpa was cloned into an expression vector, and transformed into Escherichia coli M15 cells. Immunoblotting analysis showed that the protein expressed from the transformant as well as culture supernatant of C. perfringens strain CPBC16ML was the expected molecular weight as detected in reference strains of C. perfringens. Northern hybridization and RT-PCR analysis revealed that the entire CPF-G2Im was completely spliced from the cpa precursor mRNA transcripts. The sequence of the insertion fragment has 95% and 97% identity to two non-coding regions corresponding to sequences that flank a predicted group II reverse transcriptase (RT) present in pCPF4969 plasmid of C. perfringens. However, a RT was not encoded by the CPF-G2Im fragment. Based on the secondary structure prediction analysis, CPF-G2Im revealed typical features of group II introns. The present study showed that CPF-G2Im was capable of splicing in both C. perfringens and E. coli. To our knowledge, this is the first report that an ORF-less group II intron is located in the cpa ORF of C. perfringens.