Pseudomonas aeruginosa LysR PA4203 regulator acts as a repressor of the PA4202 gene encoding a nitronate monooxygenase. Pseudomonas aeruginosa PAO1

More than 7% of the Pseudomonas aeruginosa genes are encoding transcriptional regulators, many of which with unknown functions. Among them, those belonging to the LysR family are the most represented. The PA4203 gene lies upstream of the previously characterized ppgL gene (PA4204), which encodes a periplasmic gluconolactonase, which detoxifies gluconolactone by converting it to gluconate. Upstream of PA4203 and in the opposite orientation are the PA4202 gene coding for a nitronate monooxygenase and ddlA (PA4201) encoding a D-alanine alanine ligase. This genetic organization is conserved in all P. aeruginosa genomes, but not in other pseudomonads. The intergenic regions between PA4203 and ppgL, and PA4202 are very short (79 and 107 nucleotides, respectively). PA4203 is a repressor of PA4202 and of its own transcription. A chromatin immunoprecipation analysis confirmed the presence of a single PA4203 binding site between PA4202 and PA4203. Electrophoretic mobility shift assays (EMSAs) with the purified PA4203 protein and in41 gel footprinting with the 1, 10-phenanthroline-copper ion, combined with primer extension analysis to determine transcriptional startpoints allowed the identification of a LysR binding motive in the PA4202 and PA4203 intergenic region. Despite this, a transcriptome analysis revealed more genes to be affected in a PA4203 mutant, likely due to the overexpression of the nitronate monooxygenase (PA4202). Deletion of the PA4202 gene resulted in an increased sensitivity of the cells to 3- nitropropionic acid (3-NPA). Overall design: [expression] Two samples of a Delta PA4203 mutant compared to two samples of PAO1 wild type. [ChIP] Two samples of PAO1-pUCP4203 compared to one sample of PAO1-wt.

铜绿假单胞菌（Pseudomonas aeruginosa）中超过7%的基因编码转录调控因子（transcriptional regulators），其中多数功能尚未明确。在这类调控因子中，隶属于LysR家族（LysR family）的成员占比最高。PA4203基因位于已表征的ppgL基因（PA4204）的上游，该基因编码一种周质葡糖酸内酯酶（periplasmic gluconolactonase），可通过将葡糖酸内酯转化为葡糖酸实现解毒功能。PA4203的上游且反向排布的是PA4202基因与ddlA（PA4201）：前者编码硝基单加氧酶（nitronate monooxygenase），后者编码D-丙氨酸-丙氨酸连接酶（D-alanine alanine ligase）。该基因组织结构在所有铜绿假单胞菌基因组中均保守，但在其他假单胞菌中并不保守。PA4203与ppgL、PA4202之间的基因间区长度极短，分别仅为79 nt与107 nt（核苷酸）。PA4203可对PA4202基因及其自身的转录发挥阻遏作用。染色质免疫沉淀（chromatin immunoprecipitation, ChIP）分析证实，在PA4202与PA4203的基因间区存在唯一的PA4203结合位点。研究人员利用纯化的PA4203蛋白开展电泳迁移率变动分析（electrophoretic mobility shift assay, EMSA），结合1,10-邻菲啰啉-铜离子（1,10-phenanthroline-copper ion）的凝胶足迹实验与引物延伸分析（primer extension analysis）以确定转录起始位点（transcriptional startpoints），最终在PA4202与PA4203的基因间区中鉴定得到一个LysR结合基序（LysR binding motif）。尽管如此，转录组分析（transcriptome analysis）结果显示，PA4203突变体中受影响的基因数量更多，这可能是由于PA4202编码的硝基单加氧酶过表达所导致的。敲除PA4202基因会使菌体对3-硝基丙酸（3-nitropropionic acid, 3-NPA）的敏感性升高。整体实验设计：[转录组测序] 以PAO1野生型菌株的2个样本作为对照，与2个ΔPA4203突变体样本进行比对；[染色质免疫沉淀测序] 以PAO1野生型菌株的1个样本作为对照，与2个携带pUCP4203质粒的PAO1菌株样本进行比对。