Xanthomonas fragariae 穿孔素基因 (orf74) 及溶菌酶基因 (orf169) 表現及功能分析

Abstract

Xanthomonas fragarae (Xf) is a plant pathogenic bacterium that mainly infected strawberry and secreted a high-molecular-weight bacteriocin that classify to phage tail-like bacteriocin. In this study, the complete nucleotide sequences of Xf bacteriocin has been sequenced. The Xf bacteriocin gene cluster is composed of 22,921 bp and predicted to have 32 putative orfs which reveal high similarity to the Pantoea ananatis LMG 20103 prophage except some putative hypothetical proteins. The biological functions of the putative holin (orf74) and the putative lysozyme (orf169) were investigated in this study. The gene organization shows that orf74 is located upstream of orf169 and the translated protein has a highly charge C-terminal region. ORF74 has two predicted transmembrane domains and is classified as Class II holin. According to result of RT-PCR analyses, orf74, orf169 and the downstream orf 151 and orf 106 were transcribed as a single mRNA. This indicates that orf74, orf169, orf 151 and orf 106 of Xf bacteriocin may play the same role as holin, endolysin, Rz and Rz1 as lambda phage. When orf74 and orf169 were co-expressed in the same plasmid, cell growth or death of the E. coli host were observed. Co-expression of pLysS, which encoded T7 lysozyme, and orf74 showed the same effect but no significant effect was found when expressed with orf74 or orf169 alone. The beta-galactosidase activity in the supernatant of the pLysS-orf74-co-expressed transformant, suggesting that ORF74 formed non-specific holes in the cell membrane and the size is sufficient for beta-galactosidase to pass the membrane. Furthermore, the same morphology of lytic phage can be observed in the modified M13 phage when orf74 and orf169 were cloned into M13mp18 plasmid. Overexpression of ORF74 and ORF169 recombinant proteins in E. coli result in death or stop growing of the host. The location of the expressed ORF74 was detected in the inner membrane fraction. The oligomerization of ORF74 in cell membrane was also analyzed by native PAGE. All of the described characteristics of ORF74 is similar to lambda phage holin. ORF169 was found to locate at the host outer membrane fraction. ORF169 was predicted to have a transmembrane domain at N-terminal region and this region may also function as signal peptide. The amino acid sequence of ORF169 is similar to T4 lysozyme family which contain SAR (signal-arrest-release) domain at N-terminal region. From this point of view, ORF169 differ from lysozyme that located in the typical lysis cassette. In order to determine the biological function of Xf lysozyme, ORF146 which elimination of ORF169 N-terminal region but still retain muramidase activity region was used. ORF146 showed bactericidal activity to chloroform-treated Xanthomonas species such as X. fragarae.