Identification of the quorum-sensing target DNA sequence and N-Acyl homoserine lactone responsiveness of the Brucella abortus virB promoter.
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.
Arocena
Gastón M
GM
Fundación Instituto Leloir (IIBBA-CONICET), Av. Patricias Argentinas 435, Buenos Aires C1405BWE, Argentina.
Sieira
Rodrigo
R
Comerci
Diego J
DJ
Ugalde
Rodolfo A
RA
D054742:Acyl-Butyrolactones / Q000494:pharmacology*; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites / Q000235:genetics; D002003:Brucella abortus / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D018983:DNA Footprinting; D004269:DNA, Bacterial / Q000235:genetics; D024202:Electrophoretic Mobility Shift Assay; D009876:Operon / Q000235:genetics; D011401:Promoter Regions, Genetic / Q000235:genetics / Q000502:physiology; D011485:Protein Binding / Q000235:genetics; D053038:Quorum Sensing / Q000187:drug effects*; D011994:Recombinant Proteins
D054742:Acyl-Butyrolactones; D001426:Bacterial Proteins; D004269:DNA, Bacterial; D011994:Recombinant Proteins
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