MarA-mediated Transcriptional Repression of the rob Promoter

Abstract

The Escherichia coli transcriptional regulator MarA affects functions that include antibiotic resistance, persistence, and survival. MarA functions as an activator or repressor of transcription utilizing similar degenerate DNA sequences (marboxes) with three different binding site configurations with respect to the RNA polymerase-binding sites. We demonstrate that MarA down-regulates rob transcripts both in vivo and in vitro via a MarA-binding site within the rob promoter that is positioned between the ؊10 and ؊35 hexamers. As for the hdeA and purA promoters, which are repressed by MarA, the rob marbox is also in the "backward" orientation. Protein-DNA interactions show that SoxS and Rob, like MarA, bind the same marbox in the rob promoter. Electrophoretic mobility shift analyses with a MarA-specific antibody demonstrate that MarA and RNA polymerase form a ternary complex with the rob promoter DNA. Transcription experiments in vitro and potassium permanganate footprinting analysis show that MarA affects the RNA polymerase-mediated closed to open complex formation at the rob promoter.The Rob protein is an abundant nucleoid protein that was initially discovered bound to the right origin of replication of the Escherichia coli chromosome (1). Despite its association with the origin of replication, no experimental evidence supports the role of Rob in chromosome replication, chromatin structure, and superhelicity (2).Rob is, however, a member of a subgroup within the AraC/XylS family of transcriptional regulators, which includes the MarA and SoxS proteins that are involved in a wide range of regulatory functions (3). The N-terminal domain of Rob shares similarity with MarA and SoxS, which is in contrast to the other members of the subgroup that share homology within the C-terminal domain (3). When induced by bile salts and dipyridyl, the C-terminal domain undergoes post-transcriptional modification that results in the conversion of Rob from a low to a high activity state in the cell (4). However, in the absence of these compounds, overexpression of either the full or C-terminal domain deleted protein is sufficient for activity and promoter binding in vitro (5, 6) and in vivo (5, 7). This observation is consistent with structural data derived from the Rob-micF complex, where only the N-terminal domain makes DNA-specific contacts (8). Overexpression of rob confers multidrug, organic solvent and heavy metal resistance (6, 7); in accord, some experimental data indicate that rob null mutants are hypersensitive to antibiotics, organic solvents, and heavy metals (5, 7, 9).Transposon mutagenesis experiments aimed at defining the Rob regulon revealed eight namely inaA, marRAB, aslB, ybaO, mdlA, yfhD, ybiS, and galT (10), some of which are also known members of the mar and sox regulons (11). In addition, studies demonstrate that Rob induced by decanoate or bile salts results in the increased expression of acrAB in the absence of both the mar and sox loci (12).The expression levels of rob do not vary dramatically during differ...

AraC/XylS Family Stress Response Regulators Rob, SoxS, PliA, and OpiA in the Fire Blight Pathogen Erwinia amylovora

“…The regulation of the acrAB genes has been studied extensively in human-pathogenic enterobacteria. Several members of the AraC/XylS family, including MarA, RamA, Rob, and SoxS, have been identified as positive transcriptional regulators of this efflux system (10,13,14,21,54,58). In this study, we identified four regulators from the plant pathogen E. amylovora that are closely related to the MarA/Rob/ SoxS group of AraC/XylS transcriptional activators from E. coli (see Fig.…”

Multiple Regulatory Pathways Associated with High-Level Ciprofloxacin and Multidrug Resistance in Salmonella enterica Serovar Enteritidis: Involvement of ramA and Other Global Regulators

“…These data suggest that soxS plays a greater role than marA in MDR in 104-cip. Expression of marA decreased following deletion of soxS and vice versa, highlighting the cross-regulation that exists between these transcriptional factors (12,43). Recently, ramA has been reported to contribute to fluoroquinolone resistance and MDR in serovar Typhimurium through activation of acrB (1).…”

“…As overexpression of rob has been shown to confer MDR in E. coli through activation of acrB (46), it is reasonable to assume a lack of involvement of this regulator in the development of MDR in this study. The decreased expression of rob in 104-cip is likely due to downregulation by soxS and marA (28,43). The nature and extent of the cross talk between ramA and other global regulators are currently unknown.…”

MarR-Like Transcriptional Regulator Involved in Detoxification of Aromatic Compounds in Sulfolobus solfataricus

“…As an example, the MexR protein isolated from Pseudomonas aeruginosa is a repressor of the MexAB-OprM operon, which encodes a tripartite multidrug efflux system (1). In general, the mar loci mediate a global stress response involving a large network of genes (3,39,51), and the same operon contributes to ethanol tolerance in engineered ethanologenic E. coli cells (28).…”