Structural insights into the regulation of foreign genes in Salmonella by the Hha/H-NS complex

J Biol Chem. 2013 May 10;288(19):13356-69. doi: 10.1074/jbc.M113.455378. Epub 2013 Mar 20.


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.

Conclusion: Hha binds the dimerization domain of H-NS and may contact DNA via positively charged surface residues.

Significance: 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.

Keywords: Bacterial Genetics; DNA-binding Protein; Gene Regulation; Gene Transfer; Microbial Pathogenesis; Microbiology.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Amino Acid Sequence
  • Amino Acid Substitution
  • Bacterial Proteins / chemistry*
  • Bacterial Proteins / genetics
  • Bacterial Proteins / metabolism
  • Conserved Sequence
  • Crystallography, X-Ray
  • DNA, Bacterial / chemistry
  • DNA, Bacterial / metabolism
  • DNA-Binding Proteins / chemistry*
  • DNA-Binding Proteins / genetics
  • DNA-Binding Proteins / metabolism
  • Gene Expression Regulation, Bacterial*
  • Gene Silencing
  • Gene Transfer, Horizontal
  • Models, Molecular
  • Molecular Sequence Data
  • Mutagenesis, Site-Directed
  • Protein Binding
  • Protein Interaction Domains and Motifs
  • Protein Structure, Quaternary
  • Protein Structure, Secondary
  • Salmonella typhimurium / genetics*
  • Salmonella typhimurium / metabolism
  • Surface Properties
  • Transcriptome


  • Bacterial Proteins
  • DNA, Bacterial
  • DNA-Binding Proteins
  • H-NS protein, bacteria