Structural and functional analysis of the C-terminal DNA binding domain of the Salmonella typhimurium SPI-2 response regulator SsrB

Ronan K. Carroll, Xiubei Liao, Leslie K. Morgan, Elisha M. Cicirelli, Yuanhe Li, Wanyun Sheng, Xiuhong Feng, Linda Kenney

Research output: Contribution to journalArticle

25 Citations (Scopus)

Abstract

In bacterial pathogenesis, virulence gene regulation is controlled by two-component regulatory systems. In Escherichia coli, the EnvZ/OmpR two-component system is best understood as regulating expression of outer membrane proteins, but in Salmonella enterica, OmpR activates transcription of the SsrA/B two-component system located on Salmonella pathogenicity island 2 (SPI-2). The response regulator SsrB controls expression of a type III secretory system in which effectors modify the vacuolar membrane and prevent its degradation via the endocytic pathway. Vacuolar modification enables Salmonella to survive and replicate in the macrophage phagosome and disseminate to the liver and spleen to cause systemic infection. The signals that activate EnvZ and SsrA are unknown but are related to the acidic pH encountered in the vacuole. Our previous work established that SsrB binds to regions of DNA that are AT-rich, with poor sequence conservation. Although SsrB is a major virulence regulator in Salmonella, very little is known regarding how it binds DNA and activates transcription. In the present work, we solved the structure of the C-terminal DNA binding domain of SsrB (SsrBC) by NMR and analyzed the effect of amino acid substitutions on function. We identified residues in the DNA recognition helix (Lys179, Met186) and the dimerization interface (Val197, Leu201) that are important for SsrB transcriptional activation and DNA binding. An essential cysteine residue in the N-terminal receiver domain was also identified (Cys45), and the effect of Cys203 on dimerization was evaluated. Our results suggest that although disulfide bond formation is not required for dimerization, dimerization occurs upon DNA binding and is required for subsequent activation of transcription. Disruption of the dimer interface by a C203E substitution reduces SsrB activity. Modification of Cys203 or Cys45 may be an important mode of SsrB inactivation inside the host.

Original languageEnglish (US)
Pages (from-to)12008-12019
Number of pages12
JournalJournal of Biological Chemistry
Volume284
Issue number18
DOIs
StatePublished - May 1 2009
Externally publishedYes

Fingerprint

Genomic Islands
Functional analysis
Salmonella
Salmonella typhimurium
Structural analysis
Dimerization
DNA
Transcription
Transcriptional Activation
Virulence
Substitution reactions
Chemical activation
AT Rich Sequence
Phagosomes
Salmonella enterica
Macrophages
Amino Acid Substitution
Vacuoles
Gene expression
Disulfides

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

Cite this

Structural and functional analysis of the C-terminal DNA binding domain of the Salmonella typhimurium SPI-2 response regulator SsrB. / Carroll, Ronan K.; Liao, Xiubei; Morgan, Leslie K.; Cicirelli, Elisha M.; Li, Yuanhe; Sheng, Wanyun; Feng, Xiuhong; Kenney, Linda.

In: Journal of Biological Chemistry, Vol. 284, No. 18, 01.05.2009, p. 12008-12019.

Research output: Contribution to journalArticle

Carroll, Ronan K. ; Liao, Xiubei ; Morgan, Leslie K. ; Cicirelli, Elisha M. ; Li, Yuanhe ; Sheng, Wanyun ; Feng, Xiuhong ; Kenney, Linda. / Structural and functional analysis of the C-terminal DNA binding domain of the Salmonella typhimurium SPI-2 response regulator SsrB. In: Journal of Biological Chemistry. 2009 ; Vol. 284, No. 18. pp. 12008-12019.
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T1 - Structural and functional analysis of the C-terminal DNA binding domain of the Salmonella typhimurium SPI-2 response regulator SsrB

AU - Carroll, Ronan K.

AU - Liao, Xiubei

AU - Morgan, Leslie K.

AU - Cicirelli, Elisha M.

AU - Li, Yuanhe

AU - Sheng, Wanyun

AU - Feng, Xiuhong

AU - Kenney, Linda

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AB - In bacterial pathogenesis, virulence gene regulation is controlled by two-component regulatory systems. In Escherichia coli, the EnvZ/OmpR two-component system is best understood as regulating expression of outer membrane proteins, but in Salmonella enterica, OmpR activates transcription of the SsrA/B two-component system located on Salmonella pathogenicity island 2 (SPI-2). The response regulator SsrB controls expression of a type III secretory system in which effectors modify the vacuolar membrane and prevent its degradation via the endocytic pathway. Vacuolar modification enables Salmonella to survive and replicate in the macrophage phagosome and disseminate to the liver and spleen to cause systemic infection. The signals that activate EnvZ and SsrA are unknown but are related to the acidic pH encountered in the vacuole. Our previous work established that SsrB binds to regions of DNA that are AT-rich, with poor sequence conservation. Although SsrB is a major virulence regulator in Salmonella, very little is known regarding how it binds DNA and activates transcription. In the present work, we solved the structure of the C-terminal DNA binding domain of SsrB (SsrBC) by NMR and analyzed the effect of amino acid substitutions on function. We identified residues in the DNA recognition helix (Lys179, Met186) and the dimerization interface (Val197, Leu201) that are important for SsrB transcriptional activation and DNA binding. An essential cysteine residue in the N-terminal receiver domain was also identified (Cys45), and the effect of Cys203 on dimerization was evaluated. Our results suggest that although disulfide bond formation is not required for dimerization, dimerization occurs upon DNA binding and is required for subsequent activation of transcription. Disruption of the dimer interface by a C203E substitution reduces SsrB activity. Modification of Cys203 or Cys45 may be an important mode of SsrB inactivation inside the host.

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