一株希瓦氏菌低温噬菌体的分离及特征研究

Isolation and Characterization of a Shewanellacold-active Bacteriophage

采用双层平板法从新疆东帕米尔高原喀拉库勒湖中分离纯化低温噬菌体,并对其特征进行研究。结果分离得到1株烈性低温噬菌体,命名为KLP17,其宿主菌KLB17经16SrRNA基因序列分析鉴定为Shewanella菌株。KLP17裂解性好,可在双层平板上感染宿主菌KLB17形成直径2-4mm的圆形、透明噬菌斑。电镜下其颗粒形态呈头尾型,头部为正多面体对称,直径约88nm,收缩性尾长约160nm,属肌尾噬菌体科(Myoviridae)。噬菌体KLP17对高温敏感,耐碱不耐酸,对氯仿和异丙醇均不敏感,但对SDS和蛋白酶K十分敏感,推测其衣壳组分为蛋白质而无脂质包被。KLP17最佳感染复数为0.01,感染宿主菌KLB17的潜伏期、裂解期分别为55min和30min,裂解量为27。酶切结果显示其基因组为dsDNA,大小约67~69kb,研究结果不仅为进一步探究微生物适冷机制积累重要素材,而且为深入解析低温噬菌体与宿主菌及环境的互作关系提供数据参考。

This study focused on the isolation and purification of cold-active bacteriophages from Kara-kul Lake, East Pamir by double-layer plate method, and their biological characteristics were observed. A lytic cold-active bacteriophages against Shewanella KLB17, which was named KLP17, was isolated from the water samples of Karakul Lake. The plaques of KLP17 phages, which were round and trans- parent with a diameter of about 2-4 mm, had a good lytic ability in low temperature. Transmission elec- tron microscope showed that KLP17 had an isometric head about 88nm in diameter and a long contrac- tile tail about 160nm in length. KLP17 belongs to the Myoviridaefamily. Physiological analysis results showed that KLP17 had poor thermalstability, alkaline resisting but sensitive to acid. The phage was insensitive to chloroform and isopropyl alcohol but sensitive to SDS and Proteinase K, which indicated that KLP17 was a lytic phage with capsid protein but not a lipid envelope. The optimal multiplicity of infection for KLP17 was 0.01, the latent phase and rise phage were 55 min and 30 min, and the burst size was 27. Its genome was made of linear double-stranded DNA, 67~69 kb. The results not only ac- cumulate important material for exploring the cold-adapted mechanism of microorganisms, but provide a reference for the interaction analysis between low temperature phage, host bacteria and their environ- ment.