Screening and Characterization of the Xanthomonas fragariae Bacteriocin Mutants with Different Host Specificity

• Main Authors: Si-Ru Lin, 林思汝
• Other Authors: Ming-Te Yang
• Format: Others
• Language: zh-TW
• Published: 2012
• Online Access: http://ndltd.ncl.edu.tw/handle/zv384h

碩士 === 國立中興大學 === 分子生物學研究所 === 100 === Abstract Bacteriocins are proteins secreted by bacteria with bactericidal or bacteriostatic activities toward related species of the producer strain. Previous studies from this laboratory found that bacteriocin produced by Xanthomonas fragariae (Xf), a plant pathogenic bacterium that mainly infects strawberry, has a narrow bactericidal spectrum. Moreover, Xf bacteriocin is a high-molecular-weight protein complex and a phage tail-like particle was observed in the purified fraction of Xf bacteriocin under electron microscope. It has been suggested that tail fiber proteins are the primary determinants of host range of the phage tail-like bacteriocins. The aim of this study was to alter the host range specificity of Xf bacteriocin by engineering tail fiber protein OFR612. Although several attempts have been made, construction of the orf612 deleted strain by site-specific homologous recombination was unsuccessful. Meanwhile, random mutagenesis of orf612 was carried out by error prone PCR and the mutated orf612 were then cloned into pBBad22k vector. After transformation of the constructed plasmids into Xf and induced by arabinose, mutated bacteriocins produced by the complementary strains that altering host range specificity were selected. Three mutant strains of orf612, and designated N3C7, N3C7 and TM#15 with altered host range specificities were identified. DNA sequence analysis revealed that the mutant strains have three amino acid changes in the C-terminus of ORF612 (E43A、E48V、K305R). The identification of the exact site of amino acid responsible for the host range specificity requires more specific mutations in orf612 by site-directed mutagenesis in the future studies.