title: Structural insights into the regulation of foreign genes in Salmonella by the Hha/H-NS complex.
abstract: BACKGROUND
  Hha facilitates H-NS-mediated silencing of foreign genes in bacteria.
  
  
  RESULTS
  Two Hha monomers bind opposing faces of the H-NS N-terminal dimerization domain.
  
  
  CONCLUSIONS
  Hha binds the dimerization domain of H-NS and may contact DNA via positively charged surface residues.
  
  
  CONCLUSIONS
  The structure of Hha and H-NS in complex provides a mechanistic model of how Hha may affect gene regulation. The bacterial nucleoid-associated proteins Hha and H-NS jointly repress horizontally acquired genes in Salmonella, including essential virulence loci encoded within Salmonella pathogenicity islands. Hha is known to interact with the N-terminal dimerization domain of H-NS; however, the manner in which this interaction enhances transcriptional silencing is not understood. To further understand this process, we solved the x-ray crystal structure of Hha in complex with the N-terminal dimerization domain of H-NS (H-NS(1-46)) to 3.2 Å resolution. Two monomers of Hha bind to symmetrical sites on either side of the H-NS(1-46) dimer. Disruption of the Hha/H-NS interaction by the H-NS site-specific mutation I11A results in increased expression of the Hha/H-NS co-regulated gene hilA without affecting the expression levels of proV, a target gene repressed by H-NS in an Hha-independent fashion. Examination of the structure revealed a cluster of conserved basic amino acids that protrude from the surface of Hha on the opposite side of the Hha/H-NS(1-46) interface. Hha mutants with a diminished positively charged surface maintain the ability to interact with H-NS but can no longer regulate hilA. Increased expression of the hilA locus did not correspond to significant depletion of H-NS at the promoter region in chromatin immunoprecipitation assays. However, in vitro, we find Hha improves H-NS binding to target DNA fragments. Taken together, our results show for the first time how Hha and H-NS interact to direct transcriptional repression and reveal that a positively charged surface of Hha enhances the silencing activity of H-NS nucleoprotein filaments.
authors.affiliation: Departments of Molecular Genetics, University of Toronto, Toronto, Ontario M5S 1A8, Canada.
authors.forename: Sabrina S
authors.initials: SS
authors.lastname: Ali
authors.forename: John C
authors.initials: JC
authors.lastname: Whitney
authors.forename: James
authors.initials: J
authors.lastname: Stevenson
authors.forename: Howard
authors.initials: H
authors.lastname: Robinson
authors.forename: P Lynne
authors.initials: PL
authors.lastname: Howell
authors.forename: William Wiley
authors.initials: WW
authors.lastname: Navarre
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D017124:Conserved Sequence; D018360:Crystallography, X-Ray; D004269:DNA, Bacterial / Q000737:chemistry / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D015964:Gene Expression Regulation, Bacterial; D020868:Gene Silencing; D022761:Gene Transfer, Horizontal; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011485:Protein Binding; D054730:Protein Interaction Domains and Motifs; D020836:Protein Structure, Quaternary; D017433:Protein Structure, Secondary; D012486:Salmonella typhimurium / Q000235:genetics* / Q000378:metabolism; D013499:Surface Properties; D059467:Transcriptome
chemical_list: D001426:Bacterial Proteins; D004269:DNA, Bacterial; D004268:DNA-Binding Proteins; C031908:H-NS protein, bacteria
keywords: Bacterial Genetics; DNA-binding Protein; Gene Regulation; Gene Transfer; Microbial Pathogenesis; Microbiology