title: Identification of amino acid residues of Salmonella SlyA that are critical for transcriptional regulation.
abstract: The type III secretion system encoded by Salmonella pathogenicity island 2 (SPI-2) is essential for the intracellular survival and replication of Salmonella enterica. The expression of SPI-2 genes is dependent on a two-component regulatory system, SsrA (SpiR)/SsrB, encoded in the SPI-2 region. This paper shows that SlyA regulates transcription of the sensor kinase SsrA by binding to the ssrA promoter, indicating that SlyA is directly involved in the regulation of SPI-2 gene expression. A structure model of the SlyA dimer in complex with DNA was constructed. The model of SlyA indicated that its structure is very similar to that of other MarR family proteins. Based on this model, site-directed mutagenesis of residues located in the winged-helix region required for DNA binding and in the alpha-helices of the N-terminal and C-terminal regions required for dimerization of the SlyA protein was performed to identify the residues that are critical for SlyA function. Nine mutants of SlyA with single substitutions were unable to activate ssrA transcription in vivo. These mutant SlyA proteins revealed that the residues Leu-63, Val-64, Arg-65, Leu-67, Leu-70, Arg-86 and Lys-88 within the winged-helix region are required for DNA binding, and residues Leu-12 and Leu-126 within the alpha-helices of the N-terminal and C-terminal regions are required for efficient dimer formation. A Salmonella slyA mutant strain carrying a plasmid expressing SlyA derivatives containing mutations at these amino acid positions did not exhibit restored SlyA function in infected HeLa cells, thereby confirming the structural and functional relationships of the SlyA protein.
authors.affiliation: Department of Microbiology, School of Pharmaceutical Sciences, Kitasato University, Tokyo 108-8641, Japan. okadan@pharm.kitasato-u.ac.jp
authors.forename: Nobuhiko
authors.initials: N
authors.lastname: Okada
authors.forename: Yorie
authors.initials: Y
authors.lastname: Oi
authors.forename: Mayuko
authors.initials: M
authors.lastname: Takeda-Shitaka
authors.forename: Kazuhiko
authors.initials: K
authors.lastname: Kanou
authors.forename: Hideaki
authors.initials: H
authors.lastname: Umeyama
authors.forename: Takeshi
authors.initials: T
authors.lastname: Haneda
authors.forename: Tsuyoshi
authors.initials: T
authors.lastname: Miki
authors.forename: Sachiko
authors.initials: S
authors.lastname: Hosoya
authors.forename: Hirofumi
authors.initials: H
authors.lastname: Danbara
mesh_terms: D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D001483:Base Sequence; D019281:Dimerization; D015964:Gene Expression Regulation, Bacterial; D006367:HeLa Cells; D006801:Humans; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D012486:Salmonella typhimurium / Q000235:genetics / Q000378:metabolism / Q000472:pathogenicity*; D013329:Structure-Activity Relationship; D014157:Transcription Factors / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D014158:Transcription, Genetic
chemical_list: D001426:Bacterial Proteins; D014157:Transcription Factors