Weng Shu-Fen, Fu Yung-Chieh, Lin Juey-Wen, Tseng Tsai-Tien
Institute of Molecular Biology, National Chung Hsing University, Taichung, Taiwan.
Institute of Biochemistry, National Chung Hsing University, Taichung, Taiwan.
J Mol Microbiol Biotechnol. 2018;28(2):78-86. doi: 10.1159/000488678. Epub 2018 Jul 3.
Virion-associated peptidoglycan hydrolases (VAPGH) in bacteriophages are potential antimicrobials. Xop411 is a syphophage infecting the Gram-negative Xanthomonas oryzae pv. oryzae that causes bacterial leaf blight in rice plants. The Xop411 gp21 protein was identified here as a peptidoglycan glycohydrolase by Western blotting and zymogram assay, and localized to the phage tail by immunogold-labelling electron microscopy. This protein showed an apparent molecular mass of 17 kDa in SDS-polyacrylamide gels, larger than that calculated from the amino acid sequence, 15 kDa with 130 residues. The recombinant gp21 expressed in Escherichia coli formed inclusion bodies, which gained enzyme activity after in-gel renaturation. In contrast, the secreted recombinant protein (s-gp21His) expressed in Pichia pastoris was soluble and enzymatically active. Plate assays showed that s-gp21His was capable of killing 3 species of Xanthomonas, a genus containing 27 closely related plant pathogenic species, as well as the opportunistic Pseudomonas aeruginosa and Stenotrophomonas maltophilia causing nosocomial infections. These results indicate that the Xop411 gp21 has possible wide applications as an antimicrobial against xanthomonads and at least 2 opportunistic bacteria. Several other VAPGH from Xanthomonas phages were also identified by bioinformatic analysis, with 1 being confirmed by Western blotting.
噬菌体中的病毒体相关肽聚糖水解酶（VAPGH）是潜在的抗菌剂。Xop411是一种感染革兰氏阴性水稻白叶枯病菌（Xanthomonas oryzae pv. oryzae）的梅毒噬菌体，该病菌会导致水稻植株发生细菌性叶枯病。通过蛋白质免疫印迹和酶谱分析，Xop411的gp21蛋白在此被鉴定为一种肽聚糖糖水解酶，并通过免疫金标记电子显微镜定位到噬菌体尾部。该蛋白在SDS-聚丙烯酰胺凝胶中显示出17 kDa的表观分子量，大于根据氨基酸序列计算得出的15 kDa（含130个残基）。在大肠杆菌中表达的重组gp21形成包涵体，经凝胶内复性后获得酶活性。相比之下，在毕赤酵母中表达的分泌型重组蛋白（s-gp21His）是可溶且具有酶活性的。平板试验表明，s-gp21His能够杀死3种黄单胞菌，黄单胞菌属包含27种密切相关的植物病原菌，以及导致医院感染的机会致病菌铜绿假单胞菌和嗜麦芽窄食单胞菌。这些结果表明，Xop411 gp21作为一种抗黄单胞菌和至少两种机会致病菌的抗菌剂可能具有广泛的应用。通过生物信息学分析还鉴定了几种来自黄单胞菌噬菌体的其他VAPGH，其中1种经蛋白质免疫印迹确认。
