Defining a rob regulon in Escherichia coli by using transposon mutagenesis

The Rob protein of Escherichia,coli is a member of the AraC-XylS family of prokaryotic transcriptional regulators and is expressed constitutively. Del etion of the rob gene increases susceptibility to organic solvents, while o verexpression of Rob increases tolerance to organic solvents and resistance to a variety of antibiotics and to the superoxide-generating compound phen azine methosulfate. To determine whether constitutive levels of Rob regulat e basal gene expression, we performed a MudJ transposon screen in a rob del etion mutant containing a plasmid that allows for controlled rob gene expre ssion. We identified eight genes and confirmed that seven are transcription ally activated by normal expression of Rob from the chromosomal rob gene (i naA, 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 m utant. Rob binding to the promoter regions of most of these genes was subst antiated in electrophoretic mobility shift assays. However, Mn insertions i n individual Rob-regulated genes did not affect solvent sensitivity, This p henotype may depend on changes in the expression of several of these Rob-re gulated genes or on other genes that were not identified. Rob clearly affec ts the basal expression of genes with a broad range of functions, including antibiotic resistance, acid adaptation, carbon metabolism, cell wall synth esis, central intermediary metabolism, and transport. The magnitudes of Rob 's effects are modest, however, and the protein may thus play a role as a g eneral transcription cofactor.