Defining a rob regulon in Escherichia coli by using transposon mutagenesis
Defining a rob regulon in Escherichia coli by using transposon mutagenesis
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DOI：
10.1128/jb.182.13.3794-3801.2000
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发表时间：
2000-07-01
影响因子：
3.2
通讯作者：
Demple, B
中科院分区：
文献类型：
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作者：
Bennik, MHJ;Pomposiello, PJ;Demple, B
大肠杆菌中的Rob蛋白是原核转录调控因子AraC-XylS家族的一员，是结构性表达的。Rob基因的缺失增加了对有机溶剂的易感性，而过表达的rob则增加了对有机溶剂的耐受性，并增加了对各种抗生素和产生超氧化合物吩嗪甲硫酸盐的耐药性。为了确定ROB的构成水平是否调节基础基因的表达，我们在一个ROB缺失突变体中进行了MudJ转座子筛选，该突变体包含一个允许控制ROB基因表达的质粒。我们鉴定了8个基因，其中7个基因(INAA、MARR、ASLB、ybaO、mdlA、yfhD和Ybis)被正常表达的rob基因激活，一个基因GalT被Rob抑制。我们还通过Northern分析证明，MICF在野生型大肠杆菌中的基础表达显著高于ROB缺失突变体。在电泳迁移率改变分析中证实了ROB与大多数这些基因的启动子区域的结合。然而，单个Rob调控基因中的Mn插入并不影响溶剂敏感性，这种表型可能取决于其中几个Rob调控基因的表达变化，或者取决于其他未被鉴定的基因。ROB明显影响具有广泛功能的基因的基础表达，包括抗生素耐药性、酸适应、碳代谢、细胞壁合成、中央中间代谢和运输。然而，Rob的影响程度不大，因此该蛋白可能起到一般转录辅助因子的作用。
The Rob protein of Escherichia,coli is a member of the AraC-XylS family of prokaryotic transcriptional regulators and is expressed constitutively. Deletion of the rob gene increases susceptibility to organic solvents, while overexpression of Rob increases tolerance to organic solvents and resistance to a variety of antibiotics and to the superoxide-generating compound phenazine methosulfate. To determine whether constitutive levels of Rob regulate basal gene expression, we performed a MudJ transposon screen in a rob deletion mutant containing a plasmid that allows for controlled rob gene expression. We identified eight genes and confirmed that seven are transcriptionally activated by normal expression of Rob from the chromosomal rob gene (inaA, marR, aslB, ybaO, mdlA, yfhD, and ybiS), One gene, galT, was repressed by Rob. We also demonstrated by Northern analysis that basal expression of micF is significantly higher in wild-type E. coli than in a rob deletion mutant. Rob binding to the promoter regions of most of these genes was substantiated in electrophoretic mobility shift assays. However, Mn insertions in individual Rob-regulated genes did not affect solvent sensitivity, This phenotype may depend on changes in the expression of several of these Rob-regulated genes or on other genes that were not identified. Rob clearly affects the basal expression of genes with a broad range of functions, including antibiotic resistance, acid adaptation, carbon metabolism, cell wall synthesis, central intermediary metabolism, and transport. The magnitudes of Rob's effects are modest, however, and the protein may thus play a role as a general transcription cofactor.