DNA-Binding Activities of the HilC and HilD Virulence Regulatory Proteins ofSalmonella entericaSerovar Typhimurium

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Abstract
The HilC and HilD proteins of Salmonella enterica serovar Typhimurium are members of the AraC/XylS family of transcription regulators. They are encoded on Salmonella pathogenicity island 1 (SPI1) and control expression of the hilA gene, which encodes the major transcriptional activator for many genes encoded on SPI1 and elsewhere that contribute to invasion of host cells. Gel electrophoretic shift and DNase footprinting assays revealed that purified HilC and HilD proteins can bind to multiple regions in the hilA and hilC promoters and to a single region in the hilD promoter. Although both HilC and -D proteins can bind to the same DNA regions, they showed different dependencies on the sequence and lengths of their DNA targets. To identify the binding-sequence specificity of HilC and HilD, a series of single base substitutions changing each position in a DNA fragment corresponding to positions −92 to −52 of the hilC promoter was tested for binding to HilC and HilD in a gel shift DNA-binding assay. This mutational analysis in combination with sequence alignments allowed deduction of consensus sequences for binding of both proteins. The consensus sequences overlap but differ so that HilC can bind to both types of sites but HilD only to one. The hilA and hilC promoters contain multiple binding sites of each type, whereas the hilD promoter contains a site that binds HilC but not HilD without additional binding elements. The HilC and HilD proteins had no major effect on transcription from the hilA or hilD promoters using purified proteins in vitro but changed the choice of promoter at hilC. These results are consistent with a model derived from analysis of lacZ fusions stating that HilC and HilD enhance hilA expression by counteracting a repressing activity.