Phenotype Enhancement Screen of a Regulatory spx Mutant Unveils a Role for the ytpQ Gene in the Control of Iron Homeostasis.. Bacillus subtilis

Spx is a global regulator of genes that are induced by disulfide stress in Bacillus subtilis. Most of the Spx-regulated genes (SRGs) are of unknown function, but many encode products conserved in low %GC Gram positive bacteria. Using a gene-disruption library of B. subtilis genomic mutations, the SRGs were screened for phenotypes related to Spx-controlled activities, such as growth in minimal medium and sensitivity to methylglyoxal, but nearly all of the SRG mutations showed little if any phenotype. To uncover SRG function, the mutations were rescreened in an spx mutant background to determine which mutant SRG allele would enhance the spx mutant phenotype. One of the SRGs, ytpQ, was the site of a mutation that, when combined with an spx null mutation, elevated the severity of the Spx mutant phenotype, as shown by reduced growth in a minimal medium and by hypersensitivity to methylglyoxal. Proteomic and transcriptomic data indicated that the ytpQ mutation caused the derepression of the Fur and PerR regulons, as well as heightened LexA-controlled gene expression. Our study suggests that the ytpQ gene, encoding a conserved DUF1444 protein, functions directly or indirectly in repairing or stabilizing Fe-bearing proteins, which are sensitive to thiol reactive agents and, therefore, likely influenced by Spx-controlled genes. Overall design: The wild type parent, ytpQ and spx mutants were grown in a glucose minimal medium, with and without 2.8 mM methylglyoxal, to mid-log phase. Cells were harvested and RNA was extracted for microarray hybridization analysis to determine if any changes in the transcriptome could be detected that were attributable to the ytpQ mutation. Microarray hybridizations were performed with RNA from three biological replicates.

Spx是枯草芽孢杆菌（Bacillus subtilis）中由二硫胁迫诱导的基因的全局调控因子。大多数受Spx调控的基因（Spx-regulated genes, SRGs）功能未知，但其中许多编码的产物在低GC%革兰氏阳性菌中保守存在。研究人员利用枯草芽孢杆菌基因组突变的基因敲除文库，针对与Spx调控活性相关的表型（如基本培养基中的生长能力、对甲基乙二醛的敏感性）对SRGs进行了筛选，但几乎所有SRG突变体均未表现出显著或可检测的表型。为揭示SRGs的功能，研究人员在spx突变体背景下重新筛选这些突变，以确定哪些突变的SRG等位基因会增强spx突变体的表型。其中一个SRG——ytpQ，其突变位点与spx敲除突变（spx null mutation）结合后，会加剧Spx突变体的表型：表现为基本培养基中生长能力下降，以及对甲基乙二醛的超敏感性。蛋白质组学（proteomics）和转录组学（transcriptomics）数据显示，ytpQ突变会导致Fur调节子（Fur regulon）与PerR调节子（PerR regulon）的去阻遏，同时增强LexA调控的基因表达。本研究表明，编码保守DUF1444结构域蛋白的ytpQ基因，可直接或间接参与修复或稳定对巯基反应试剂敏感的含铁蛋白质——这类蛋白质易受Spx调控基因的影响。总体实验设计：将野生型亲本菌株、ytpQ突变体与spx突变体置于添加或不添加2.8 mM甲基乙二醛的葡萄糖基本培养基中培养至对数中期（mid-log phase）。收集细胞并提取RNA，进行微阵列杂交（microarray hybridization）分析，以检测是否存在由ytpQ突变导致的转录组变化。本次微阵列杂交实验采用三份生物学重复（biological replicates）的RNA样本完成。