Detection and characterization of the S. typhimurium HilA protein

In order to detect and localize the HilA protein in S. typhimurium, we raised polyclonal antiserum against purified His-tagged HilA. This allowed us to study the effect of environmental conditions on the production of HilA. We also used the antiserum to examine the fractionation properties and SDS-PAGE mobility of native HilA. Our results indicate that S. typhimurium initiates translation of HilA from the first AUG codon in the hilA open-reading frame (ORF), producing a soluble 553 amino acid (63 kDa) protein product.Materials and methods are now available to study the environmental regulation of the HilA protein in S. typhimurium. Our results also indicate that future in vitro studies of the interaction between HilA and DNA should utilize soluble preparations of HilA. Previous analyses used preparations of HilA in which the protein fractionated with the membrane, greatly limiting the types of experiments that could be conducted.Salmonella pathogenicity island 1 (SPI1), a 40 kb region of the Salmonella typhimurium chromosome, encodes a type III secretion system that is important for virulence [1,2]. SPI1 genes that encode components of the type III secretion apparatus are directly activated by HilA [3,4]. HilA is a member of the OmpR/ToxR family of transcription regulators based on homology of its N-terminal domain to the conserved OmpR/ToxR DNA binding and transcription activation domain [5]. Members of this family typically bind degenerate direct repeats as a dimer [6,7]. Although HilA also appears to activate gene expression by binding to direct repeats, other features of HilA deviate from those of well-characterized family members [5,7,8]. Whereas OmpR and PhoB are regulated by phosphorylation of an N-terminal phosphoryl-acceptor domain, HilA lacks homology to such a domain. Although ToxR and CadC also lack a phosphoryl-acceptor domain, HilA lacks a predicted membrane spanning domain which is characteristic of these two family members.HilA is thought to coordina