地中海拟无枝菌酸菌专一的噬菌体φMMR1的研究

CHARACTERIZATION OF φMMR1, A NEWLY ISOLATED NARROW HOST-RANGE LYTIC BACTERIOPHAGE OF AMYCOLATOPSIS MEDITERRANEI U119

从生产力复霉素ＳＶ的地中海拟无枝菌酸菌（Ａｍｙｃｏｌａｔｏｐｓｉｓｍｅｄｉｔｅｒｒａｎｅｉ）Ｕ１１９的工业发酵罐裂解液中，分离到一株新的噬菌体φＭＭＲ。该噬菌体经多次单斑分离、纯化，得到的噬斑多数为清亮的（约占９０％），其它噬斑为浑浊斑，但未能从中分离到溶源菌。在被测试的可能的宿主菌中，φＭＭＲ１不能感染除地中海拟无枝菌酸菌以外的菌株，说明寄主范围很窄。对φＭＭＲ１的增殖和储存条件，诸如ｐＨ值、温度、二价金属离子、有机溶剂等对φＭＭＲ１的影响进行了较详细的研究。电子显微镜观察揭示，φＭＭＲ１为长尾无收缩尾鞘，头为正多面体，属长尾噬菌体科Ｂ１亚群。制备了φＭＭＲ１的兔抗血清，并测定了φＭＭＲ１以地中海拟无枝菌酸菌Ｕ－３２为宿主菌的一步生长曲线。使用２４种限制性内切酶对φＭＭＲ１ＤＮＡ进行了单酶切分析。结果表明，φＭＭＲ１基因组ＤＮＡ为线状双链，未找到粘性末端，大小约为５９６ｋｂ。φＭＭＲ１ＤＮＡ的Ｇ＋Ｃ含量为６７％。利用ＳＤＳ－ＰＡＧＥ分析了噬菌体的外壳蛋白多肽的组成。纯化的裸露噬菌体ＤＮＡ可利用电穿孔技术成功地转染地中海拟无枝菌酸菌Ｕ－３２。

Bacteriophage φMMR1 was isolated from a lysed industrial fermentation culture of Amycolatopsis mediterranei U119. φMMR1 is likely to be lytic phage, because more than 90% of the plaques were clear and no lysogens, immune to phage infection were obtained. φ MMR1 could form plaques only on laboratory strains of A. mediterranei among 11 tested strains. The host range of the phage is so narrow that it failed to infect the very closely related species, A. orientalis, A. rugosus, A. sulphreaus and A. methanolica . The conditions of propagation and storage of φMMR1, such as pH, temperature, divalent cations and organic solvents, have been studied in detail. The virion structure observed under electron microscopy is similar to that of most known Streptomycetes' phage. It belongs to the B1 group of Styloviridae. Rabbit antiserum directed against φMMR1 was prepared and a onestep growth curve was obtained.Restriction enzyme analysis of the genomic DNA indicated a unique pattern different from those of other known Actinophages. The phage genome is linear, double-stranded and no cohesive ends could be tested. The genome is ca. 59.6 kb in size. The G+ C content of φMMR1 DNA was determined as 67% by HPLC. The proteins of the phage capsid were analyzed by SDS-PAGE. Purified phage DNA was able to transfect the standard laboratory strain A. mediterranei U-32 through electroporation.