Identification of residues critical for the function of the Vibrio cholerae virulence regulator ToxT by scanning alanine mutagenesis
Identification of residues critical for the function of the Vibrio cholerae virulence regulator ToxT by scanning alanine mutagenesis
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DOI：
10.1016/j.jmb.2007.01.061
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发表时间：
2007-04-13
影响因子：
5.6
通讯作者：
Klose, Karl E.
中科院分区：
文献类型：
--
作者：
Childers, Brandon M.;Weber, Gregor G.;Klose, Karl E.
霍乱弧菌毒力因子的表达受转录调控蛋白ToxT的调控。ToxT可激活霍乱毒素(CTX)和毒素共调节菌毛(TCP)基因的转录，以及辅助定植因子(ACF)基因的转录。ToxT是AraC家族中的一员，以往的研究表明它由两个结构域组成，一个是N端二聚化和环境敏感结构域，另一个是C端DNA结合域。在这项研究中，利用全面的扫描丙氨酸突变来鉴定对ToxT功能至关重要的氨基酸。在霍乱弧菌ACFA-PhoA报告菌株和鼠伤寒沙门氏菌ctxAp-LacZ报告菌株中，有48个Ala替换的蛋白质(总共267个)显示出ToxT依赖的激活缺陷(>；90%减少)。在体外毒力因子诱导条件下，这些突变蛋白中的大多数还导致霍乱毒素(CT)和毒素共调节菌毛(TCP)在A毒素T V霍乱菌株中的表达减少。用基于LexA的报告系统进一步分析表明，N末端(F151a)的20个Ala取代之一减少了二聚化，并且该残基位于预测的a-螺旋结构区域，从而识别了可能的二聚体界面。导致差异启动子激活的两个可能的螺旋-转角螺旋(HTH)识别螺旋(K203A和S249A)上的ALA替换似乎没有改变特异的DNA结合，表明这些残基参与了转录激活的其他方面。还发现了一些Ala替换导致ToxT转录活性升高，这些突变几乎只在N端发现，这与参与ToxT活性调控的这个结构域是一致的。这项研究阐明了特定氨基酸对ToxT的二聚化、DNA结合和转录活性的贡献。(C)2007爱思唯尔有限公司。保留所有权利。
Virulence factor expression in Vibrio cholerae is controlled by the transcriptional regulatory protein ToxT. ToxT activates transcription of the genes encoding cholera toxin (ctx) and the toxin co-regulated pilus (tcp), as well as accessory colonization factor (acf) genes. Previous studies of ToxT, a member of the AraC family of proteins, have revealed that it consists of two domains, an N-terminal dimerization and environmental sensing domain, and a C-terminal DNA binding domain. In this study, comprehensive scanning alanine mutagenesis was utilized to identify amino acids critical for the function of ToxT. Forty-eight proteins with Ala substitutions (of 267 total) exhibited defects in ToxT-dependent activation (> 90% reduction) in both a V cholerae acfA-phoA reporter strain and a Salmonella typhimurium ctxAp-lacZ reporter strain. Most of these mutant proteins also caused reductions in cholera toxin (CT) and toxin coregulated pilus (TCP) expression in a A toxT V cholerae strain under in vitro virulence factor inducing conditions. Further analysis with a LexA-based reporter system revealed that one of the 20 Ala substitutions in the N terminus (F151A) diminishes dimerization, and this residue is located in a region of predicted a-helical structure, thus identifying a putative dimer interface. Ala substitutions in two putative helix-turn-helix (HTH) recognition helices that caused differential promoter activation (K203A and S249A) did not appear to alter specific DNA binding, suggesting these residues contribute to other aspects of transcriptional activation. A number of Ala substitutions were also found that result in a higher level of ToxT transcriptional activity, and these mutations were almost exclusively found within the N terminus, consistent with this domain being involved in modulation of ToxT activity. This study illuminates the contribution of specific amino acids to the dimerization, DNA binding, and transcriptional activity of ToxT. (c) 2007 Elsevier Ltd. All rights reserved.