Erwinia carotovora subsp. carotovora 低分子量細菌素選殖與表現

Abstract

The Erwinia carotovora subsp. carotovovra (E.c.c.) causes soft-rot disease of many plant species. Despite its economic importance, no efficient method has been found to control this disease. The bacterium produces the antibacterial substances called bacteriocins owning the ability to identify the similar bacteria and kills them. We want to find out the bacteriocin of E.c.c. being able to kill most of subspecies, and use the biological control this disease. By bacterial mating experiment a transposon Tn5 insertion mutant TF1-2 was obtained, which lost the low-molecular-weight bacteriocin(LMWB) producing ability. After the first TAIL-PCR experiment, the PCR products were isolated and the DNA sequences were determined from the Tn5 insertion junction site. The DNA probe was prepared from TAIL-PCR result and it was used in Southern hybridization experiments. A 5705bp fragment was isolated from 3F-3 DNA library, and it contains six open reading frames (ORFs). The ORF2 encodes a protein 38% identical to colicin D activity protein, which is ribonuclease activity. The ORF3 encodes a protein 32% identical to immunity protein of colicin D, which inhibition the bacteriocin activity to host cell. It was therefore proposed that ORF2 be designated as caroS2K, and ORF3 as caroS2I. The two genes were named carocin S2 gene. We cloned the carocin S2 gene into pBR322 or pMCL210 vector, and the expression plasmids pBR322-119 and pMCL210-119 were obtained. After pBR322-119 or pMCL210-119 into E. coli DH5α, Ea1068, or TF1-2 cells, the results showed that DH5α/pMCL210-119, Ea1068/pMCL210-119 and TF1-2/pMCL210-119 can produce the low-molecular-weight bacteriocin by bacteriocin assay experiments. This result suggests that the carocin S2 gene is a LMWB genes contain a killing protein and an immunity protein.