Expression and Functional Characterization of orf151 and orf106 of Xanthomonas fragariae Bacteriocin

• Main Authors: Hsuan-Wu Hou, 侯萱吾
• Other Authors: 楊明德
• Format: Others
• Language: zh-TW
• Published: 2014
• Online Access: http://ndltd.ncl.edu.tw/handle/87214634391203392156

碩士 === 國立中興大學 === 分子生物學研究所 === 102 === A high-molecular weight bacteriocin produced by plant pathogen Xanthomonas fragarae (Xf) was identified in this laboratory and classify to be a phage tail-like bacteriocin. The complete nucleotide sequences of the cloned Xf bacteriocin gene cluster were determined to include 22,921 bp and predicted to have 32 putative orfs. The lysis cassette, orf74 (orf79), orf169, and the downstream orf151 and orf106 of Xf bacteriocin has been suggested to play the same role as holin (H), lysozyme (LSer), Rz and Rz1 as λ phage. Biochemical and functional characterization of the putative Rz (ORF151) and the putative Rz1 (ORF106) were investigated in this study. Polyclonal antibodies raised against ORF151 and ORF106 were prepared from immunized mice with purified recombinant proteins overexpressed in E. coli. Sucrose density gradient fractionation and Western blot analyses were applied to determine the subcellular localizations of the ORF151 and ORF106. Result showed that the overexpressed ORF151 and ORF106 could be identified in the inner and outer membrane fractions, respectively. The truncated ORF151 lacking the trans-membrane domain and fused with the CBD-Ssp intein sequence (designated as intein-ORF151ΔTMD), and the truncated ORF106 lacking the lipobox (designated as ORF106ΔLpp), were used for soluble proteins production in E. coli. Co-immunoprecipitation and gel-filtration chromatography results indicated that these two proteins forming a complex through direct interaction with each other. To elucidate the effect of lysis cassette genes on the growth of their host cells, each of the lysis cassette genes (orf74, orf79, orf169 , orf151 and orf106) and genes encoded by two (orf74 and orf169, orf151 and orf106) or four components of the cassette, were separately cloned into pBBad22k vector and individually introduced into E. coli DH5α. In addition, the 15 residues of (Gly4 Ser)3 was used as linker to fuse H and LSer protein, the constructs designated as HlL74 (or HlL79), were also used in this study. Changes of the culture turbidity were monitored after induction the expression of the recombinant lysis cassette proteins with arabinose. Results demonstrated that expression of the all four lysis genes (HLRR) or orf74 and orf169 (HL and HlL79) displayed significant lysis of E. coli hosts than the other constructs. The viable cell counts and the release of the cytoplasmic enzyme β-galactosidase correlated well with the rate turbidity reduction. The same lysis cassette genes were then introduced into M13 phage. However, after infection with the modified M13 phage, the E. coli JM103 hosts showed no significant difference in viable cell count.