Transcriptional activation of promoters of the superoxide and multiple antibiotic resistance regulons by Rob, a binding protein of the Escherichia coli origin of chromosomal replication

Abstract

The Rob protein, isolated on the basis of its ability to bind to the right arm of the Escherichia coli origin of chromosomal replication, is about 50% identical in amino acid sequence to SoxS and MarA, the direct regulators of the superoxide (soxRS) and multiple antibiotic resistance (mar) regulons, respectively. Having previously demonstrated that SoxS (as a MalE-SoxS fusion protein) and MarA are essentially identical in their abilities to activate in vitro transcription of genes of the sox-mar regulons, we investigated the properties of Rob as a transcriptional activator. We found that Rob (i) activates the transcription of zwf, fpr, fumC, micF, nfo, and sodA, (ii) requires a 21-bp soxbox-marbox-robbox sequence to activate zwf transcription, (iii) protects the soxbox/marbox/robbox from attack by DNase I, (iv) is ambidextrous, i.e., requires the C-terminal domain of the ␣ subunit of RNA polymerase for activation of zwf but not fumC or micF, (v) bends zwf and fumC DNA, and (vi) binds zwf and fumC DNA as a monomer. Since these transcription activation properties of Rob are virtually identical to those of MalE-SoxS and MarA, it appears as if the E. coli genome encodes three genes with the same functional capacity. However, in contrast to SoxS and MarA, whose syntheses are induced by specific environmental stimuli and elicit a clear defense response, Rob is expressed constitutively and its normal function is unknown.

Defining a rob Regulon in Escherichia coli by Using Transposon Mutagenesis

“…Similarly, the demonstrated dependence of inaA on Rob is consistent with previously reported transcriptional activation of inaA by Rob overexpression (5). Other genes reported to be activated by Rob in vitro (fpr, fumC, micF, nfo, sodA, and zwf) (15) or by Rob overexpression in vivo (fumC, inaA, micF, and sodA) (5) were not found in our study; thus, it appears that the mutagenesis and screening procedure was not saturating.…”

“…To establish specific binding of Rob protein to the DNA fragments, we used a double-stranded DNA fragment lacking known Rob binding sites (Randomdir plus Randomrev; see Table 2) as a nonspecific competitor DNA and the micF promoter (double-stranded 35-mer micF promoter site 1; see Materials and Methods) as a specific competitor DNA. In vitro binding of Rob to the micF promoter has been demonstrated previously (5,19), and Rob activates the transcription of micF in vitro (15). Here, the relevance of Rob-dependent expression of micF was further established by Northern blotting of micF mRNA (Fig.…”

“…These values were compared with the predicted masses of the proteins encoded by the Rob-regulated genes and operons identified in this study and with those of proteins encoded by genes known to be induced by Rob in vivo (5) or in vitro (15,32) 7 kDa); however, previous studies showed no in vivo induction of zwf by Rob (5). The band with an M r of ϳ60,000 could correspond with MdlAB (66.0 and 65.2 kDa).…”

“…Furthermore, in vivo studies (5) have shown that overexpression of Rob (or its N-terminal domain alone) activates transcription of sodA (encoding manganese-containing superoxide dismutase), fumC (encoding fumarase C), inaA (encoding a weak acid-inducible protein), and micF (gene for an antisense RNA repressing the outer membrane porin OmpF). In vitro studies (15) demonstrated Robactivated transcription of sodA, fumC, micF, zwf (encoding glucose-6-phosphate dehydrogenase), nfo (encoding DNA repair endonuclease IV), and fpr (encoding NADPH-ferredoxin oxidoreductase). However, it is not known whether normal expression of Rob contributes to the in vivo expression of these genes.…”

The Escherichia coli Transcriptional Regulator MarA Directly Represses Transcription of purA and hdeA

“…The transcriptional start sites of the purA gene (14) and the hdeAB operon (23) have been defined. We mixed the promoter-containing DNA of purA or hdeA with that of the control gnd (a promoter that is unresponsive to MarA) (21,22,24,25). The promoter for the MarA-activated gene nfnB (20), also mixed with gnd DNA, was tested in parallel as a positive control for MarA activity.…”