Dasgupta Shreya, Basu Pallabi, Pal Ritesh Ranjan, Bag Satyabrata, Bhadra Rupak K
Infectious Diseases and Immunology Division, CSIR-Indian Institute of Chemical Biology, Kolkata-700 032, India.
Microbiology (Reading). 2014 Sep;160(Pt 9):1855-1866. doi: 10.1099/mic.0.079319-0. Epub 2014 Jul 1.
In Vibrio cholerae, the causative agent of cholera, products of three genes, relA, spoT and relV, govern nutritional stress related stringent response (SR). SR in bacteria is critically regulated by two intracellular small molecules, guanosine 3'-diphosphate 5'-triphosphate (pppGpp) and guanosine 3',5'-bis(diphosphate) (ppGpp), collectively called (p)ppGpp or alarmone. Evolution of relV is unique in V. cholerae because other Gram-negative bacteria carry only relA and spoT genes. Recent reports suggest that RelV is needed for pathogenesis. RelV carries a single (p)ppGpp synthetase or RelA-SpoT domain (SYNTH/RSD) and belongs to the small alarmone synthetase (SAS) family of proteins. Here, we report extensive functional characterizations of the relV gene by constructing several deletion and site-directed mutants followed by their controlled expression in (p)ppGpp(0) cells of Escherichia coli or V. cholerae. Substitution analysis indicated that the amino acid residues K107, D129, R132, L150 and E188 of the RSD region of RelV are essential for its activity. While K107, D129 and E188 are highly conserved in RelA and SAS proteins, L150 appears to be conserved in the latter group of enzymes, and the R132 residue was found to be unique in RelV. Extensive progressive deletion analysis indicated that the amino acid residues at positions 59 and 248 of the RelV protein are the functional N- and C-terminal boundaries, respectively. Since the minimal functional length of RelV was found to be 189 aa, which includes the 94 aa long RSD region, it seems that the flanking residues of the RSD are also important for maintaining the (p)ppGpp synthetase activity.
在霍乱弧菌（霍乱的病原体）中，relA、spoT和relV这三个基因的产物控制着与营养应激相关的严紧反应（SR）。细菌中的SR由两种细胞内小分子鸟苷3'-二磷酸5'-三磷酸（pppGpp）和鸟苷3',5'-双（二磷酸）（ppGpp）严格调控，这两种分子统称为（p）ppGpp或警报素。relV在霍乱弧菌中的进化是独特的，因为其他革兰氏阴性菌仅携带relA和spoT基因。最近的报告表明，RelV是致病所必需的。RelV携带一个单一的（p）ppGpp合成酶或RelA-SpoT结构域（SYNTH/RSD），属于小警报素合成酶（SAS）蛋白家族。在此，我们通过构建几个缺失突变体和定点突变体，随后在大肠杆菌或霍乱弧菌的（p）ppGpp(0)细胞中对其进行可控表达，报告了relV基因广泛的功能特征。取代分析表明，RelV的RSD区域的氨基酸残基K107、D129、R132、L150和E188对其活性至关重要。虽然K107、D129和E188在RelA和SAS蛋白中高度保守，但L150似乎在后者这一组酶中保守，并且发现R132残基在RelV中是独特的。广泛的渐进缺失分析表明，RelV蛋白第59位和第248位的氨基酸残基分别是功能性的N端和C端边界。由于发现RelV的最小功能长度为189个氨基酸，其中包括94个氨基酸长的RSD区域，因此似乎RSD的侧翼残基对于维持（p）ppGpp合成酶活性也很重要。
