DESIGN AND CONSTRUCTION OF TOOLS FOR CHIP-SEQ ANALYSIS OF THE vjbR REGULON IN Brucella

ROMINA M. RODRÍGUEZ; ANGELES ZORREGUIETA; RODRIGO SIEIRA

Mar del Plata

Congreso; X Congreso de la Sociedad Argentina de Microbiología General; 2014

The genus Brucella comprises several species of facultative intracellular bacteria that are the causative agent of brucellosis, a widespread zoonotic disease that affects animals and humans. VjbR is a LuxR-familiy regulator that plays a central role in the virulence of Brucella and affects transcription of a vast number of genes including virulence determinants. Despite extensive efforts to identify the VjbR regulon by microarray and proteomic analyses, the only VjbR-binding site characterized so far corresponds to the virB promoter. The aim of this work is to identify all VjbR binding sites in the genome of Brucella. To achieve this, we took the challenge to apply an innovative technique called ChIP-seq. This technique comprises several steps including crosslinking of bacteria, chromatin fragmentation, and immunoprecipitation of specific protein-DNA complexes. In order to mimic the intraphagosomal environment found by Brucella within the host, bacteria were incubated under a restricted set of conditions that trigger the expression of the VjbR protein, thus allowing the regulator to bind to its target sequences. Following incubation under low nutrients and low pH (5.5), bacteria were crosslinked and disrupted. Sonication of chromatin was optimized and a fragment size of 250 bp suitable for further analysis was obtained. Proper folding, expression, and integrity of the protein after sonication and decrosslinking was analyzed by Western blot. In order to prepare a VjbR derivative that can be used for chromatin immunoprecipitation through the use of a high affinity and high specificity monoclonal antibody, we constructed a mutant strain containing a 3xFLAG epitope fused to the N-terminus of the protein VjbR. To check that the 3xFLAG-VjbR protein retains its biological activity, we constructed transcriptional fusions between a target promoter of VjbR and the lacZ reporter gene, in order to perform b-Galactosidase assays. The developed tool will allow to carry out the subsequent steps, that consist of immunoprecipitation of VjbR-DNA complexes, confirmation by real-time PCR by measuring the amount of immunoprecipitated DNA corresponding to the known binding site for VjbR, and de-crosslinking of DNA. Finally, co-immunoprecipitated DNA fragments will be analyzed by deep sequencing on a platform Illumina.