Identification of HutC-mediated regulatory network: The histidine Utilization Represor recognizes more than one promoter.

GASTÓN M. AROCENA; RODRIGO SIEIRA

Villa Carlos Paz, Córdoba, Argentina.

Congreso; VI Reunión Anual de la Sociedad Argentina de Microbiología General (SAMIGE); 2009

HutC is the transcriptional regulator of the hut genes (for histidine-utilization), which confer the ability to use histidine as a 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 abortus. 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, which were identified by DNase I Footprinting experiments. 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 present in the genome of B. abortus. To test this possibility, we searched for additional HutC-binding sites using PATSER software. Neither the dyad symmetric sequence of Phut nor the HutC-recognized sequence of PvirB were found twice in the B. abortus genome. However, when we performed a search using a degenerate consensus sequence that matches both motifs, two putative additional HutC-binding sites were found. EMSA experiments showed that HutC binds specifically to one of the two identified sequences, which is located upstream of a gene that codes for a type-V autotransporter that shares homology with bacterial adhesins.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. These observations suggest that histidine catabolism triggers an adaptive response mediated by HutC through modulation of expression of virulence determinants and putative surface-exposed proteins.