Two transcriptional regulators compete with IHF for the binding to the Brucella abortus virB promoter

RODRIGO SIEIRA; DIEGO J. COMERCI; RODOLFO A. UGALDE

Rosario, Pcia. de Santa Fe, Argentina

Congreso; XLII Reunión Anual de la Sociedad Argentina de Investigación en Bioquímica y Biología Molecular (SAIB); 2006

Sociedad Argentina de Investigación en Bioquímica y Biología Molecular (SAIB)

Type-IV Secretio Systems (T4SS) are multicomponent machineries that are present in many bacterial intracellular pathogens. They are involved in the translocation of effector proteins from the bacteriunm to the host cell cytoplasm. In Brucella abortusm, the ethiological agent of brucellosis, the virB operon encondes a T4SS that is essential for virulence and intracllular mitlipoication The nucleoid protein IHF plays an essential role in transcriptional regulationr of the virB operon during intracellular infection of J774 macrophages. Recently, we identified two proteins that bind specifically to different regions of the virB promoter. These proteins belong to the GntR and MarR families of negative transcriptional regulators. In this work, both transcriptional regulators were cloned and expressed as recombinant proteins. Using Electrophoresis Mobility Shift Assays we determined specificity of binding, identification of binding regions and apparent dissociation constants for each protein. It was observed that incubation of the transcriptional regulators with recombinant IHF and the virB promoter did not produce a ternary complex, indicating that the three factors compete for the same binding site. These results suggest that both transcriptional regulators could be acting as a double check point for the control of transcription of the B. abortus virB operon.