Purification and characterization of VSH-1, a generalized transducing bacteriophage of Serpulina hyodysenteriae
Purification and characterization of VSH-1, a generalized transducing bacteriophage of Serpulina hyodysenteriae
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DOI：
10.1128/jb.179.2.323-329.1997
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发表时间：
1997-01-01
影响因子：
3.2
通讯作者：
Zuerner, RL
中科院分区：
文献类型：
--
作者：
Humphrey, SB;Stanton, TB;Zuerner, RL
用丝裂霉素（20 μ g丝裂霉素/ml培养液）处理的猪舌盘菌B204细胞裂解并释放噬菌体。用聚乙二醇沉淀并经CsCl密度梯度超离心纯化的噬菌体颗粒，其浮力密度为1.375 g/cm（3），（45 nm直径）和超微结构简单（非收缩）尾（64 × 9 nm），由至少13种分子量在13和101 kDa之间的蛋白质组成，纯化的噬菌体被命名为VSH-1（VSH用于猪链球菌的病毒），VSH-1不能在代表三种Serpulina物种的五种肠螺旋体菌株中的任何一种上裂解生长，VSH-1核酸的大小被确定为约7.5 kb，并且基于差异染色、吖啶橙子、DNA酶I敏感性、电泳迁移率和通过电子显微镜测量的轮廓长度，是线性双链DNA，用限制性内切酶SspI、AseI、EcoRV和AflII消化的噬菌体DNA的电泳带型与猪链球菌消化的染色体DNA的电泳带型几乎相同。此外，VSH-1 DNA片段与猪链球菌染色体基因nor和flaA 1互补的探针杂交。当从猪链球菌A203（Δ flaA 1 593-762：：cat）培养物中诱导的纯化噬菌体加入到菌株A216（Δ nox 438-760：：kan）的生长细胞中时，以每个噬菌体颗粒1.5 × 10（-6）的频率获得转导子（Cm-r Km（r））（通过电子显微镜计数）。这些发现表明诱导的VSH-1病毒粒子包装猪链球菌DNA，并且能够在该螺旋体的细胞之间转移宿主基因。据我们所知，这是第一个螺旋体遗传转导的报告。
Serpulina hyodysenteriae B204 cells treated with mitomycin (20 mu g of mitomycin/ml of culture broth) lysed and released bacteriophages. Bacteriophage particles, precipitated by using polyethylene glycol and purified by CsCl density gradient ultracentrifugation, had a buoyant density of 1.375 g/cm(3) and consisted of a head (45-nm diameter) and an ultrastructurally simple (noncontractile) tail (64 by 9 nm) composed of at least 13 proteins with molecular masses ranging between 13 and 101 kDa, The purified bacteriophage has been designated VSH-1 (VSH for virus of S, hyodysenteriae), VSH-1 was incapable of lytic growth on any of five intestinal spirochete strains, representing three Serpulina species, VSH-1 nucleic acid was determined to be approximately 7.5 kb in size and to be linear, double-stranded DNA based on differential staining,vith acridine orange, DNase I sensitivity, electrophoretic mobility, and contour length as measured by electron microscopy, Phage DNA digested by the restriction enzymes SspI, AseI, EcoRV, and AflII gave electrophoretic banding patterns nearly identical to those of digested chromosomal DNA from S, hyodysenteriae. Additionally, VSH-1 DNA fragments hybridized with probes complementary to S, hyodysenteriae chromosomal genes nor and flaA1. When purified bacteriophages induced from cultures of S, hyodysenteriae A203 (Delta flaA1 593-762::cat) were added to growing cells of strain A216 (Delta nox 438-760::kan), transductants (Cm-r Km(r)) were obtained at a frequency of 1.5 x 10(-6) per phage particle (enumerated by electron microscopy), These findings indicate that induced VSH-1 virions package DNA of S, hyodysenteriae and are capable of transferring host genes between cells of that spirochete. To our knowledge, this is the first report of genetic transduction of a spirochete.