Defining a rob regulon in Escherichia coli by using transposon mutagenesis

Abstract

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, Mu 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.

FIG. 1

Regulated rob expression. (A) Northern blot of rob mRNA. RNA was extracted from cultures of E. coli GC4468 (wt), MB4468 (Δrob), or MB4468/pMB101 after exposure to different concentrations of IPTG for 30 min at 37°C. Autoradiograms were exposed for ∼8 h. (B) To confirm that equivalent amounts of RNA were loaded, rRNA bands were visualized by ethidium bromide staining. (C) Expression of the rob-regulated gene inaA. The β-galactosidase activity of E. coli MB9701 (Δrob inaA1::lacZ) containing plasmid pMB101 was quantified as a function of IPTG concentration in the growth medium.

FIG. 2

Locations of insertions of transcriptional fusions of MudJ containing promoterless lacZ in Rob-regulated genes identified in this study. Arrows indicate the direction of transcription of genes and of lacZ.

FIG. 3

Expression of micF depends on rob. Total RNA was extracted from cultures of E. coli GC4468 (wt) and MB4468 (Δrob) that were grown in LB medium to an OD₆₀₀ of 0.6 at 37°C. RNA was hybridized with probes specific for micF (A) and rob (B). Autoradiograms were exposed for ∼20 h. To confirm that equivalent amounts of RNA were loaded, rRNA bands were visualized by ethidium bromide staining (C).

FIG. 4

Binding of Rob-His₆ to the promoter regions of Rob-regulated genes. F, free probe; C1 through C5, DNA-Rob-His₆ complexes; X, contaminating labeled DNA strand. Lanes 1, probe alone (no protein); lanes 2 through 6, Rob-His₆ (see below for amounts) with no competitor (lanes 2), a 10-fold excess of nonspecific competitor (lanes 3), a 100-fold excess of nonspecific competitor (lanes 4), a 10-fold excess of specific competitor (lanes 5), or a 100-fold excess of specific competitor (lanes 6). (A) Rob-His₆ (3 nM) binding to mar promoter DNA; (B) Rob-His₆ (9 nM) binding to gal promoter DNA; (C) Rob-His₆ (13.5 nM) binding to aslB promoter DNA; (D) Rob-His₆ (3 nM) binding to ybaO upstream DNA region (free probe of lane 1 ran off gel); (E) Rob-His₆ (11 nM) binding to mdlA upstream DNA region.