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INVESTIGADORES

SIEIRA Rodrigo

congresos y reuniones científicas

Título:

HutC controls transcription of more than a single operon: the histidine utiliztion pathway of Brucella is integrated within a regultaory network that involves virulence genes and Type-V autotransporters

Autor/es:

GASTÓN M. AROCENA; RODRIGO SIEIRA

Lugar:

Baeza, Provincia de Jaén, España.

Reunión:

Workshop; Current trends in biomedicine: Bacterial Regulatory Networks; 2009

Resumen:

HutC is the transcriptional regulator of the hut genes (for histidine utilization), which confer the ability to use histidine asa sole carbon source to many bacterial species. Until now, the known target of HutC was restricted only to the operator sites present in the hut promoters. However, we recently found that in addition to the hut promoter, HutC also binds specifically to regulatory sequences of the virB genes of the animal and human pathogen Brucella obortus. Such genes code for the type-IV Secretion System VirB, a multicomponent secretion apparatus that, as in other pathogenic bacteria, is essential for the virulence of Brucella. Analyses of activity of the virB and hut promoters (PvirB and Phut, respectively) revealed that HutC modulates expression of both systems. Using electrophoresis mobility shift assays (EMSA), we determined dissociation constants for the binding of HutC to both PvirB and Phut, and observed that this regulator binds to each promoter with different affinities. Such differences can be explained by the different architectures of both HutC-binding sites, with were indentified by DNase I Footpriniting experiments. The 12-bp dyad symmetric sequence of the HutC-binding site of Phut is present in all hut promoters of other alpha-proteobacteria closely related to Brucella, which indicates that the ancestral sequence recongnized by HutC is fully conserved among this taxonomic group. Instead, the sequence recongnized in PvirB resembled more the HutC-binding site of Klebsiella or Pseudomonas rather than that of alpha-proteobacterial hut promoters, which could evidence an evolutionary trace of the acquisition of the virB operon during a horizontal transfer event in the common ancestor of Brucella. Following these observations, we hypothesized that in addition to the HutC-binding sites of PvirB and Phut, this transcriptional regulator could recognize other operator sequences presenting in the genome of B. abortus. To test this possibility, we searched for additional HutC-binding sites by means of Patser software. Using a degenerate consensus sequence that matches both PvirB and Phut Hutc-binding motifs, two putative additional HutC-binding sites were found. To determine whether these putative binding sites are functional, we performed EMSA experiments which showed that HutC binds specifically to one of the two identified sequences. This new functional HutC-binding site is located upstream of a gene that codes for a type-V autotransporter that shares homology with bacterial adhesins. In addition to HutC, we observed by EMSA that two other proteins that regulate PvirB (i.e.: IHF) also bind to this putative regulatory region, strongly suggesting that this Type-V autotransporter is co-regulated with the virB genes. In summary, our results show that, in contrast to the initial notion that HutC regulates transcription of a single locus, it interacts with at least three different promoters which may be part of a regulatory network involved in the survival of Brucella within its host. On the other hand, these observations highlight the need for genome scale ChIP-chip studies on different transcription factors involved in the virulence of this intracellular pathogen.

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