Hertwig Stefan, Klein Iris, Lurz Rudi, Lanka Erich, Appel Bernd
Department of Biological Safety, Robert Koch-Institut, D-13353 Berlin, Germany.
Mol Microbiol. 2003 May;48(4):989-1003. doi: 10.1046/j.1365-2958.2003.03458.x.
PY54 is a temperate phage isolated from Yersinia enterocolitica. Lysogenic Yersinia strains harbour the PY54 prophage as a plasmid (pY54). The plasmid has the same size (46 kb) as the PY54 genome isolated from phage particles. By electron microscopy, restriction analysis and DNA sequencing, it was demonstrated that the phage and the plasmid DNAs are linear, circularly permuted molecules. Unusually for phages of Gram-negative bacteria, the phage genome has 3'-protruding ends. The linear plasmid pY54 has covalently closed ends forming telomere-like hairpins. The equivalent DNA sequence of the phage genome is a 42 bp perfect palindrome. Downstream from the palindrome, an open reading frame (ORF) was identified that revealed strong DNA homology to the telN gene of Escherichia coli phage N15 encoding a protelomerase. Similar to PY54, the N15 prophage is a linear plasmid with telomeres. The N15 protelomerase has cleaving/joining activity generating the telomeres by processing a 56 bp palindrome (telomere resolution site tel RL). To study the activity of the PY54 protein, the telN-like gene was cloned and expressed in E. coli. A 77 kDa protein was obtained and partially purified. The protein was found to process recombinant plasmids containing the 42 bp palindrome. Telomere resolution of plasmids under in vivo conditions was also investigated in Yersinia infected with PY54. Processing required a plasmid containing the palindrome as well as adjacent DNA sequences from the phage including an additional inverted repeat. Regions on the phage genome important for plasmid maintenance were defined by the construction of linear and circular miniplasmid derivatives of pY54, of which the smallest miniplasmid comprises a 4.5 kb DNA fragment of the plasmid prophage.
PY54是一种从小肠结肠炎耶尔森氏菌中分离出的温和噬菌体。溶原性耶尔森氏菌菌株将PY54原噬菌体作为质粒（pY54）携带。该质粒的大小（46 kb）与从噬菌体颗粒中分离出的PY54基因组相同。通过电子显微镜、限制性分析和DNA测序表明，噬菌体和质粒DNA是线性的、环状排列的分子。与革兰氏阴性菌的噬菌体不同，该噬菌体基因组具有3'突出末端。线性质粒pY54具有共价闭合末端，形成类似端粒的发夹结构。噬菌体基因组的等效DNA序列是一个42 bp的完美回文序列。在回文序列下游，鉴定出一个开放阅读框（ORF），该阅读框与编码原端粒酶的大肠杆菌噬菌体N15的telN基因具有很强的DNA同源性。与PY54相似，N15原噬菌体是一种带有端粒的线性质粒。N15原端粒酶具有切割/连接活性，通过加工一个56 bp的回文序列（端粒切割位点tel RL）产生端粒。为了研究PY54蛋白的活性，将telN样基因克隆并在大肠杆菌中表达。获得了一种77 kDa的蛋白并进行了部分纯化。发现该蛋白可加工含有4 bp回文序列的重组质粒。还在感染了PY54的耶尔森氏菌中研究了体内条件下质粒的端粒切割。加工需要一个含有回文序列以及来自噬菌体的相邻DNA序列（包括一个额外的反向重复序列）的质粒。通过构建pY54的线性和环状小质粒衍生物，确定了噬菌体基因组上对质粒维持重要的区域，其中最小的小质粒包含质粒原噬菌体的一个4.5 kb DNA片段。
