Characterization of the lysogenic repressor (c) gene of the Pseudomonas aeruginosa transposable bacteriophage D3112
Characterization of the lysogenic repressor (c) gene of the Pseudomonas aeruginosa transposable bacteriophage D3112
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DOI：
10.1006/viro.2000.0341
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发表时间：
2000-06-20
期刊：
影响因子：
3.7
通讯作者：
DuBow, MS
中科院分区：
文献类型：
--
作者：
Salmon, KA;Freedman, O;DuBow, MS
噬菌体 D3112 是铜绿假单胞菌的 Mu 样温带转座噬菌体。遗传图谱和 DNA 序列分析已确定噬菌体基因组的左端编码转座酶 (A) 和溶原性 (c) 阻遏蛋白。 c开放阅读框（ORF）位于噬菌体基因组的最左端，从右向左转录，有四个可能的GTG起始密码子。使用定点诱变，四个 GTG 密码子中的每一个都被修改为 GTA，GTA 不能作为起始密码子。构建表达野生型阻遏物 ORF 或含有突变 GTA 密码子的 ORF 的质粒。当引入铜绿假单胞菌时，当第二个 GTG 发生突变时，没有观察到 D3112 对重复感染的免疫力。 Northern 印迹分析表明 D3112 c 阻遏蛋白被转录为 900 nt mRNA。通过转录 lacZ 融合和从噬菌体基因组左端到 bp 972-940 的引物延伸分析来定义启动子区域。当 D3112 c 阻遏蛋白过表达并纯化为具有 C 端六组氨酸延伸 (cts15-His6) 的融合蛋白时，它对位于 c 阻遏蛋白 ORF 直接上游的 261 bp PvuII 片段表现出高亲和力。我们的结果表明，虽然 D3112c 与 lambda 阻遏蛋白家族的氨基酸相似性高于与 Mu 和 D108 的氨基酸相似性，但其结构和功能似乎更准确地反映了与转座大肠杆菌噬菌体 Mu 和 D108 的进化祖先。 (C) 2000 年学术出版社。
Bacteriophage D3112 is a Mu-like temperate transposable phage of Pseudomonas aeruginosa. Genetic mapping and DNA sequence analysis have identified the left end of the phage genome as encoding the transposase enzyme (A) and the lysogenic (c) repressor. The c open reading frame (ORF), located at the leftmost end of the phage genome and transcribed from right to left, has four possible GTG initiation codons. Using site-directed mutagenesis, each of the four GTG codons was modified to GTA, which cannot serve as an initiation codon. Plasmids were constructed expressing either the wild-type repressor ORF or the ORFs containing the mutated GTA codons. When introduced into Pseudomonas aeruginosa, no immunity to superinfection by D3112 was observed when the second GTG had been mutated. Northern blotting analysis demonstrated that the D3112 c repressor is transcribed as a 900-nt mRNA. The promoter region was defined by transcriptional lacZ fusions and primer extension analyses to bp 972-940 from the left end of the phage genome. When the D3112 c repressor was overexpressed and purified as a fusion protein with a C-terminal six-histidine extension (cts15-His6), it showed high affinity for a 261-bp PvuII fragment localized directly upstream of the c repressor ORF. Our results indicate that although D3112 c shows higher amino acid similarity to the lambda family of repressors than it does to those of Mu and D108, it appears that its structure and function more accurately reflect an evolutionary ancestry with those from transposable coliphages Mu and D108. (C) 2000 Academic Press.