The Clostridium perfringens TetA(P) efflux protein contains a functional variant of the Motif A region found in major facilitator superfamily transport proteins
The Clostridium perfringens TetA(P) efflux protein contains a functional variant of the Motif A region found in major facilitator superfamily transport proteins
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DOI：
10.1099/mic.0.26614-0
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发表时间：
2004-01-01
期刊：
影响因子：
2.8
通讯作者：
Rood, JI
中科院分区：
文献类型：
--
作者：
Bannam, TL;Johanesen, PA;Rood, JI
产气荚膜梭菌四环素抗性蛋白 TetA(P) 是一种内膜蛋白，介导四环素从细菌细胞主动流出。该蛋白质包含 420 个氨基酸，预计有 12 个跨膜结构域 (TMD)。将 TetA(P) 氨基酸序列与主要促进子超家族 (MFS) 的几个成员的氨基酸序列进行比较，鉴定出保守基序 A 的变异副本。该区域由序列 E(59)xPxxxxxDxxxRK(72) 组成，位于 TetA(P) 蛋白预测结构模型中连接 TMD 2 和 3 的推定环内。为了研究保守残基的功能重要性，使用定点诱变构建了 17 个点突变，然后分析它们对四环素耐药性的影响及其产生免疫反应性 TetA(P) 蛋白的能力。保守的 Phe-58 残基的变化是可以耐受的，而 Pro-61 的三个独立取代消除了四环素耐药性。对碱性残基的检查表明，Arg-71 是功能所必需的，而当 Lys-72 被精氨酸取代时，四环素抗性得以保留。这些结果证实，编码该基序的区域对于四环素抗性很重要，并且代表了在其他外排蛋白和 MFS 家族成员中发现的基序 A 区域的远距离版本。此外，研究表明，TetA(P) 蛋白的 Glu-117（预计位于 TMD4 中）对于耐药性很重要，尽管在该位置具有天冬氨酸残基的衍生物也具有功能。
The Clostridium perfringens tetracycline resistance protein, TetA(P), is an inner-membrane protein that mediates the active efflux of tetracycline from the bacterial cell. This protein comprises 420 aa and is predicted to have 12 transmembrane domains (TMDs). Comparison of the TetA(P) amino acid sequence to that of several members of the major facilitator superfamily (MFS) identified a variant copy of the conserved Motif A. This region consists of the sequence E(59)xPxxxxxDxxxRK(72) and is located within the putative loop joining TMDs 2 and 3 in the predicted structural model of the TetA(P) protein. To study the functional importance of the conserved residues, site-directed mutagenesis was used to construct 17 point mutations that were then analysed for their effect on tetracycline resistance and their ability to produce an immunoreactive TetA(P) protein. Changes to the conserved Phe-58 residue were tolerated, whereas three independent substitutions of Pro-61 abolished tetracycline resistance. Examination of the basic residues showed that Arg-71 is required for function, whereas tetracycline resistance was retained when Lys-72 was substituted with arginine. These results confirm that the region encoding this motif is important for tetracycline resistance and represents a distant version of the Motif A region found in other efflux proteins and members of the MFS family. In addition, it was shown that Glu-117 of the TetA(P) protein, which is predicted to be located in TMD4, is important for resistance although a derivative with an aspartate residue at this position is also functional.