Walker M J, Birch R G, Pemberton J M
Department of Microbiology, University of Queensland, St. Lucia, Australia.
Mol Microbiol. 1988 Jul;2(4):443-54. doi: 10.1111/j.1365-2958.1988.tb00050.x.
A DNA fragment containing a gene for resistance to the antibiotic albicidin was isolated from Klebsiella oxytoca and shown to be expressed in Escherichia coli, where it also protected bacteriophage T7 replication from inhibition by albicidin. In vivo translation analysis demonstrated that the cloned 2.2kb DNA fragment coded for a 36 kiloDalton (kD) protein and a 25kD protein. The DNA sequence was determined for a 654-base-pair open reading frame contained within a 1.2kb subcloned DNA fragment encoding albicidin resistance. The predicted molecular weight of the polypeptide translated from the open reading frame was 25.8kD. A putative Shine-Dalgarno sequence precedes the open reading frame but a potential promoter sequence was not detected. A possible rho-independent transcription termination signal was found directly following the stop codon. The functional protein for albicidin resistance was isolated and purified. Both the molecular weight and NH2-terminal amino acid sequence of this protein correspond with that predicted from the DNA sequence of the open reading frame. The cloned albicidin resistance gene had no effect on the tsx (nupA) nucleoside uptake gene associated with spontaneous albicidin resistance in E. coli; also, it did not complement any of a range of E. coli DNAts mutants at restrictive temperatures. The cloned resistance gene product remained intracellular in exponential cultures of K. oxytoca and E. coli. Cell-free extracts from E. coli containing the resistance gene protected a sensitive strain of E. coli from inhibition by albicidin, as did the purified albicidin resistance protein. The mechanism of this albicidin resistance protein involved binding to albicidin to form a complex without antibiotic activity, but without catalysing further chemical modification of the antibiotic.
从产酸克雷伯菌中分离出一个含有抗杀稻瘟菌素抗生素基因的DNA片段，并证明其在大肠杆菌中表达，在该菌中它还能保护噬菌体T7复制免受杀稻瘟菌素的抑制。体内翻译分析表明，克隆的2.2kb DNA片段编码一种36千道尔顿（kD）的蛋白质和一种25kD的蛋白质。对包含在一个1.2kb亚克隆DNA片段中的654个碱基对的开放阅读框进行了DNA序列测定，该片段编码杀稻瘟菌素抗性。从开放阅读框翻译的多肽的预测分子量为25.8kD。在开放阅读框之前有一个假定的Shine-Dalgarno序列，但未检测到潜在的启动子序列。在终止密码子之后直接发现了一个可能的不依赖于ρ因子的转录终止信号。分离并纯化了具有杀稻瘟菌素抗性的功能蛋白。该蛋白的分子量和NH2末端氨基酸序列均与从开放阅读框的DNA序列预测的一致。克隆的杀稻瘟菌素抗性基因对与大肠杆菌中自发的杀稻瘟菌素抗性相关的tsx（nupA）核苷摄取基因没有影响；此外，它在限制温度下不能互补一系列大肠杆菌DNAts突变体中的任何一个。克隆的抗性基因产物在产酸克雷伯菌和大肠杆菌的指数培养物中保持在细胞内。含有抗性基因的大肠杆菌无细胞提取物保护敏感的大肠杆菌菌株免受杀稻瘟菌素的抑制，纯化的杀稻瘟菌素抗性蛋白也有同样的作用。这种杀稻瘟菌素抗性蛋白的作用机制涉及与杀稻瘟菌素结合形成一种没有抗生素活性的复合物，但不催化抗生素的进一步化学修饰。
