A MarR-Type Regulator Directly Activates Transcription from the Brucella abortus virB Promoter by Sharing a Redundant Role with HutC

Abstract

Type IV secretion systems (T4SS) are multiprotein structures that direct the translocation of specific molecules across the bacterial cell envelope. As in other bacteria, pathogenicity of the genus Brucella essentially depends on the integrity of the T4SS-encoding virB operon, whose expression is regulated by multiple transcription factors belonging to different families. Previously, we identified IHF and HutC, two direct regulators of the virB genes that were isolated from total protein extracts of Brucella. Here, we report the identification of MdrA, a third regulatory element that was isolated using the same screening procedure. This transcription factor, which belongs to the MarR-family of transcriptional regulators, binds at two different sites of the virB promoter and regulates expression in a growth phase-dependent manner. Like other members of the MarR family, specific ligands were able to dissociate MdrA from DNA in vitro. Determination of the MdrA-binding sites by DNase I footprinting and analyses of protein-DNA complexes by electrophoresis mobility shift assays (EMSAs) showed that MdrA competes with IHF and HutC for the binding to the promoter because their target DNA sequences overlap. Unlike IHF, both MdrA and HutC bound to the promoter without inducing bending of DNA. Moreover, the two latter transcription factors activated virB expression to similar extents, and in doing so, they are functionally redundant. Taken together, our results show that MdrA is a regulatory element that directly modulates the activity of the virB promoter and is probably involved in coordinating gene expression in response to specific environmental signals.

Regulation of Virulence in Brucella : An Eclectic Repertoire of Transcription Factors Defines the Complex Architecture of the virB Promoter

“…According to these overlapping target DNA sequences, it was also observed that MdrA competes with IHF and HutC to bind to the promoter [43]. Interestingly, the superposition of the HutC and MdrAbinding sites probably accounts for the functional interplay between these two transcription factors.…”

“…In Gramneg ative bacteria, the inducer of histidine utilization is urocanic acid, the first intermediate of the Hut catabolic pathway. This small molecule inter acts with HutC and promotes its dissociation [43] BvrR OmpR ND -ND -Yes Yes [65,67] BabR/BlxR future science group Review Sieira from DNA, leading to derepression of the genes coding for the Hut catabolic enzymes and also hutC in the case of Klebsiella, Pseudomonas and Brucella [42,50,51].…”

“…The analysis of the protected regions indicated that the MdrA binding site II contains two partially conserved 11bp motifs arranged as inverted repeats with dyad symmetry (Table 1). On the other hand, the downstreamlocated MdrAbinding site I showed no obvious dyad symmetry, but contains a similar 11bp motif located at the center of the protected region [43].…”

“…It belongs to the MarR family of transcription factors involved in regulation of virulence and bacterial responses to antibi otic stress [59]. Using EMSA, it was initially observed that MdrA binds with high affinity to a DNA probe that contains sequences cor responding to the upstream region of the virB promoter (positions 430 to 202) [43]. In addi tion, incubation of MdrA with a DNA probe corresponding to the downstream region (posi tions 201 to +24) also resulted in a specific pro tein-DNA complex, indicating that this regu lator recognizes two different binding sites in the virB promoter, which is a trait that was also found in other promoters regulated by MarR type transcription factors.…”

“…Using EMSA, affinity chromatography and mass spectrometry, Ugalde's group implemented a strategy that allowed the identification of dif ferent DNAbinding proteins that specifically interact with sequences of the Brucella virB promoter [22,42,43]. One of them, integration host factor (IHF), belongs to the family of the socalled nucleoidassociated factors [44].…”