Identification of the quorum-sensing target DNA sequence and N-Acyl homoserine lactone responsiveness of the Brucella abortus virB promoter

Abstract

VjbR is a LuxR-type quorum-sensing (QS) regulator that plays an essential role in the virulence of the intracellular facultative pathogen Brucella, the causative agent of brucellosis. It was previously described that VjbR regulates a diverse group of genes, including the virB operon. The latter codes for a type IV secretion system (T4SS) that is central for the pathogenesis of Brucella. Although the regulatory role of VjbR on the virB promoter (P(virB)) was extensively studied by different groups, the VjbR-binding site had not been identified so far. Here, we identified the target DNA sequence of VjbR in P(virB) by DNase I footprinting analyses. Surprisingly, we observed that VjbR specifically recognizes a sequence that is identical to a half-binding site of the QS-related regulator MrtR of Mesorhizobium tianshanense. As shown by DNase I footprinting and electrophoretic mobility shift assays, generation of a palindromic MrtR-like-binding site in P(virB) increased both the affinity and the stability of the VjbR-DNA complex, which confirmed that the QS regulator of Brucella is highly related to that of M. tianshanense. The addition of N-dodecanoyl homoserine lactone dissociated VjbR from the promoter, which confirmed previous reports that indicated a negative effect of this signal on the VjbR-mediated activation of P(virB). Our results provide new molecular evidence for the structure of the virB promoter and reveal unusual features of the QS target DNA sequence of the main regulator of virulence in Brucella.

FIG. 1.

Identification of the VjbR-binding site in P_virB. (A) DNase I footprinting analysis performed with a probe corresponding to 224 bp of P_virB and increasing concentrations of VjbR, as indicated. The contents of lanes A and G are from DNA sequencing reactions performed by the Sanger method. The VjbR-protected region is indicated by an open rectangle. Arrowheads indicate DNase I hypersensitivity sites. (B) Schematic representation of the VjbR-protected sequence. The protected region and DNase I hypersensitivity sites are as indicated in panel A. Nucleotides that match the MrtR-binding site of Mesorhizobium tianshansense are highlighted in gray. The orientation of the half-dyad symmetric sequence corresponding to the MrtR-binding site is indicated by an arrow. The position relative to the transcription start site is indicated. (C) DNase I footprinting experiment performed with probe P_virB or control probe P_{virB hM⁻} in the absence or in the presence of 400 nM VjbR.

FIG. 2.

Generation of a dyad symmetric MrtR-binding site in P_virB increased the affinity and stability of the VjbR-DNA complex. (A) Schematic representation of sequences corresponding to wild-type probe P_virB, probe P_{virB-MrtR bs}, or the mrtI promoter (P_mrtI) of M. tianshanense. Sequences that match the MrtR-binding site of P_mrtI are highlighted in gray. The dyad symmetry of the MrtR-binding site is indicated by arrows. (B) DNase I footprinting analysis performed with probe P_{virB-MrtR bs} and increasing concentrations of VjbR, as indicated. The VjbR-protected region is indicated by an open rectangle. Arrowheads indicate DNase I hypersensitivity sites. (C) EMSA performed with a control probe, probe P_virB, or probe P_{virB-MrtR bs} and increasing concentrations of VjbR.

FIG. 3.

The addition of C₁₂-HSL dissociates VjbR from P_virB. DNase I footprinting analysis was performed with P_virB, VjbR, and different concentrations of C₁₂-HSL. Concentration of VjbR: lane 1, no protein; lanes 2 to 5, 300 nM. The concentrations of C₁₂-HSL were 0 (lanes 1, 2, and 5), 10 μM (lane 3), and 20 μM (lane 4). Lane 5, acetonitrile added to a final concentration equivalent to that in lanes 3 and 4. The VjbR-protected region is indicated by an open rectangle. DNase I hypersensitivity sites are indicated by arrowheads.

FIG. 4.

Schematic representation of the structure of P_virB and different regulatory inputs that control virB expression. Solid line, sequences corresponding to the virB promoter. The 5′ region of the first gene of the virB operon (virB1) is indicated. Open, black, and gray rectangles, binding sites for HutC, IHF, and VjbR, respectively. The position relative to the transcription start site (+1) is indicated in each case. HutC-mediated activation on P_virB and repression on the hut operon is indicated. Dashed arrow, unidentified pathways that activate virB expression in response to different stimuli.