Michan C, Zhou L, Gallegos M T, Timmis K N, Ramos J L
Consejo Superior de Investigaciones Cientificas, Estación Experimental del Zaidin, Departamento de Bioquímica Vegetal, Granada, Spain.
J Biol Chem. 1992 Nov 15;267(32):22897-901.
The XylS protein is the positive regulator of the promoter controlling the meta-cleavage pathway (Pm) for catabolism of certain alkylbenzoates on the TOL plasmid of Pseudomonas. Transcription from Pm is mediated by XylS either in the presence of benzoate effectors or through XylS hyperproduction. Two regions of the NH2 terminus of XylS (residues 37-45) had been predicted to be involved in effector control of XylS transcriptional activation. Different methods were used to induce mutations in this region, including genetic selections (where Pm controlled a tetracycline resistance gene), bisulfite mutagenesis at a unique restriction site, and extensive oligonucleotide mutagenesis at residues 41 and 45. The mutants fell into four classes based on their phenotypes with respect to effector-mediated activation of a Pm-lacZ fusion: (a) effector profiles similar to wild type, (b) no Pm stimulation with benzoates, (c) altered effector specificity, and (d) higher basal Pm activities, in some cases including changes in effector specificity. In some mutants, higher basal Pm activity was apparently due to mutations that increased XylS stability. Substitutions at Arg-41 resulted in all four mutant phenotypes, indicating that this is a critical residue in XylS for effector stimulation of transcription activity.
XylS蛋白是一种正向调节因子，可调控启动子，该启动子控制着假单胞菌TOL质粒上某些烷基苯甲酸分解代谢的间位裂解途径（Pm）。无论在苯甲酸效应物存在的情况下，还是通过XylS的过量表达，Pm的转录均由XylS介导。XylS氨基末端的两个区域（第37 - 45位氨基酸残基）被预测参与XylS转录激活的效应物控制。使用了不同方法在该区域诱导突变，包括遗传筛选（其中Pm控制一个四环素抗性基因）、在一个独特限制位点进行亚硫酸氢盐诱变，以及对第41和45位氨基酸残基进行广泛的寡核苷酸诱变。根据它们对于Pm - lacZ融合体效应物介导激活的表型，这些突变体分为四类：（a）效应物谱与野生型相似，（b）苯甲酸不存在对Pm的刺激，（c）效应物特异性改变，以及（d）基础Pm活性更高，在某些情况下包括效应物特异性的改变。在一些突变体中，基础Pm活性较高显然是由于增加XylS稳定性的突变。第41位精氨酸的取代导致了所有四种突变体表型，表明这是XylS中对于效应物刺激转录活性至关重要的一个残基。
