Construction and analysis of F plasmid traR, trbJ, and trbH mutants.
Construction and analysis of F plasmid traR, trbJ, and trbH mutants.
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F 质粒 traR、trbJ 和 trbH 突变体的构建和分析。
DOI：
10.1128/jb.175.5.1528-1531.1993
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发表时间：
1993
影响因子：
3.2
通讯作者：
Ippen-Ihler,K
中科院分区：
文献类型：
--
作者：
Maneewannakul,K;Ippen-Ihler,K
构建了携带转移区基因traR、trbJ和trbH插入突变的F质粒衍生物。标准试验表明，这些基因座对于大肠杆菌K-12宿主之间F菌的产生或F菌转移并不是必需的。在traR和trbH突变体中，kan盒的取向对突变体的表型没有影响。在每种情况下，对F- pili的外观，转移频率或F-pilus特异性噬菌体的电镀效率都没有显著影响。携带与转录方向相反的kan基因的trbJ插入对噬菌体的敏感性影响很小，但明显降低了质粒的转移效率。然而，在同一位点的kan插入突变，在定向转移中，似乎没有影响这两种性质。对携带trbJ序列的克隆进行分析，发现trbJ表达了一个约11 kda的蛋白产物。TrbJ蛋白在TrbJ序列中携带kan插入或终止密码子连接子插入的克隆中不表达。然而，它是从不包含该区域113密码子开放阅读框开头序列的克隆中表达的。我们的数据表明，trbJ的翻译必须从更远端的GUG密码子开始。这将导致表达一个93个氨基酸的多肽。
F plasmid derivatives carrying kan insertion mutations in the transfer region genes traR, trbJ, and trbH were constructed. Standard tests indicated that these loci are not essential for F pilus production or F transfer among Escherichia coli K-12 hosts. Among the traR and trbH mutants tested, the orientation of the kan cassette had no effect on the mutant phenotype. In each case, there was no significant effect on the appearance of F pili, the transfer frequency, or the plating efficiency of F-pilus-specific phages. The trbJ insertion carrying a kan gene oriented in the direction opposite to tra transcription had very little effect on phage sensitivity but markedly reduced the plasmid transfer efficiency. However, the kan insertion mutation at the same site, in the tra orientation, did not seem to affect either property. Analysis of clones carrying trbJ sequences regulated by a phage T7 promoter showed that trbJ expresses an approximately 11-kDa protein product. The TrbJ protein was not expressed from clones carrying a kan insertion or stop codon linker insertion in the trbJ sequence. However, it was expressed from clones that did not include sequences at the beginning of the 113-codon open reading frame in this region. Our data indicated that translation of trbJ must be initiated at the more distal GUG codon in this frame. This would result in expression of a 93-amino-acid polypeptide.