一株产1，3-丙二醇的克雷伯氏菌的噬菌体生物学特性

Characterization of a bacteriophage of Klebsiella pneumoniae producing 1，3-propanediol

摘要:

摘要:【目的】克雷伯氏菌发酵生产1，3-丙二醇过程中发生噬菌体感染会严重影响宿主菌的生长和目标产物的生成，因此分离克雷伯氏菌噬菌体并考察其生物学特性对预防和控制噬菌体感染具有重要意义。【方法】采用敏感指示菌法及Adams双层平板法从感染噬菌体的肺炎克雷伯氏杆菌发酵液中分离得到一株噬菌体;纯化后用磷钨酸负染法电镜观察;手工法提取噬菌体核酸，酶切后琼脂糖凝胶电泳分析; 同时考察了其最佳感染复数、一步生长曲线及对温度、pH、紫外线、乙醚和氯仿等理化因素的敏感性等生理特性;最后考察了噬菌体对肺炎克雷伯氏杆菌生长和发酵的影响。【结果】分离出一株肺炎克雷伯氏杆菌溶源性噬菌体，其噬菌斑为无晕环透明圆斑，直径约1.5mm;其头部为直径约60 nm－70nm的球体，有一长约160nm的丝状长尾;基因组核酸能被双链DNA内切酶EcoRⅠ及HindⅢ切开，大小约42 kb;对高温和紫外线敏感，耐碱性而受强酸抑制，对氯仿不敏感;最佳感染复数为1，潜伏期与裂解期均为50 min，裂解量为343个;感染噬菌体的肺炎克雷伯氏杆菌的细胞生长延滞约8 h，代谢流偏向乳酸途径。【结论】该噬菌体属于无包膜长尾噬菌体，能改变克雷伯氏菌发酵生产1，3-丙二醇的代谢规律，为1，3-丙二醇发酵生产过程中噬菌体感染的预防和控制研究奠定了基础。

Abstract:

Abstract:［Objective］Phage infection could seriously influence cell growth and metabolism in the fermentation of 1，3-propanediol from glycerol by Klebsiella pneumoniae．Isolation of the Klebsiella pneumoniae phage and research on its physiological characteristics would be of great significance.［Methods］ A K．pneumoniae phage was isolated by using Adams double plate method from the infected fermentation broth of 1，3-propanediol. After the electron microscope observation，the genome of the phage was extracted and its size was identified with restriction enzyme analysis. The physiological characteristics of the phage were also tested，such as the optimal multiplicity of infection，the one-step growth curves and the sensitivity to temperature，pH，UV light and chloroform. And the phage infected fermentation was carried out and compared with normal fermentation.［Results］ The phage had an isometric polyhedral head (about 60 nm－70 nm in diameter) and a long noncontractile tail (about 160nm long)．The nucleic acid could be cut off by dsDNA restriction enzyme EcoRⅠor HindⅢ and its complete size was about 42 kb．It was sensitive to high temperature and UV light，insensitive to chloroform．The optimal multiplicity of infection for the phage was 1，the latent phase and rise phase were both 50min，and the burst size was 343．Compared with the normal fed-batch fermentation of 1，3-propanediol， the phage infected fermentation indicated that cell growth was delayed about 8 h and metabolic flow was changed to organic acid (e. g. lactic acid) pathway．［Conclusion］The phage was a non-envelop long-tailed phage，and could change the metabolism of the 1，3-propanediol fermentation from glycerol by Klebsiella pneumoniae． This work would be helpful for prevention and controlling of phage infection during the 1，3-propanediol fermentation．