Table_3_The Helicobacter pylori Heat-Shock Repressor HspR: Definition of Its Direct Regulon and Characterization of the Cooperative DNA-Binding Mechanism on Its Own Promoter.XLSX
The ability of pathogens to perceive environmental conditions and modulate gene expression accordingly is a crucial feature for bacterial survival. In this respect, the heat-shock response, a universal cellular response, allows cells to adapt to hostile environmental conditions and to survive during stress. In the major human pathogen Helicobacter pylori the expression of chaperone-encoding operons is under control of two auto-regulated transcriptional repressors, HrcA and HspR, with the latter acting as the master regulator of the regulatory circuit. To further characterize the HspR regulon in H. pylori, we used global transcriptome analysis (RNA-sequencing) in combination with Chromatin Immunoprecipitation coupled with deep sequencing (ChIP-sequencing) of HspR genomic binding sites. Intriguingly, these analyses showed that HspR is involved in the regulation of different crucial cellular functions through a limited number of genomic binding sites. Moreover, we further characterized HspR-DNA interactions through hydroxyl-radical footprinting assays. This analysis in combination with a nucleotide sequence alignment of HspR binding sites, revealed a peculiar pattern of DNA protection and highlighted sequence conservation with the HAIR motif (an HspR-associated inverted repeat of Streptomyces spp.). Site-directed mutagenesis demonstrated that the HAIR motif is fundamental for HspR binding and that additional nucleotide determinants flanking the HAIR motif are required for complete binding of HspR to its operator sequence spanning over 70 bp of DNA. This finding is compatible with a model in which possibly a dimer of HspR recognizes the HAIR motif overlapping its promoter for binding and in turn cooperatively recruits two additional dimers on both sides of the HAIR motif.
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