In Vivo Phosphorylation Dynamics of the Bordetella Pertussis Virulence-Controlling Response Regulator BvgA

Mol Microbiol. 2013 Apr;88(1):156-72. doi: 10.1111/mmi.12177. Epub 2013 Mar 14.

Abstract

We have used protein electrophoresis through polyacrylamide gels derivatized with the proprietary ligand Phos-tag™ to separate the response regulator BvgA from its phosphorylated counterpart BvgA∼P. This approach has allowed us to readily ascertain the degree of phosphorylation of BvgA in in vitro reactions, or in crude lysates of Bordetella pertussis grown under varying laboratory conditions. We have used this technique to examine the kinetics of BvgA phosphorylation after shift of B. pertussis cultures from non-permissive to permissive conditions, or of its dephosphorylation following a shift from permissive to non-permissive conditions. Our results provide the first direct evidence that levels of BvgA∼P in vivo correspond temporally to the expression of early and late BvgA-regulated virulence genes. We have also examined a number of other aspects of BvgA function predicted from previous studies and by analogy with other two-component response regulators. These include the site of BvgA phosphorylation, the exclusive role of the cognate BvgS sensor kinase in its phosphorylation in Bordetella pertussis, and the effect of the T194M mutation on phosphorylation. We also detected the phosphorylation of a small but consistent fraction of BvgA purified after expression in Escherichia coli.

Publication types

  • Research Support, N.I.H., Intramural

MeSH terms

  • Amino Acid Sequence
  • Aspartic Acid / genetics
  • Bacterial Proteins / chemistry
  • Bacterial Proteins / genetics
  • Bacterial Proteins / metabolism*
  • Bordetella pertussis / drug effects
  • Bordetella pertussis / genetics
  • Bordetella pertussis / metabolism*
  • Bordetella pertussis / pathogenicity*
  • DNA, Bacterial / metabolism
  • DNA-Directed RNA Polymerases / metabolism
  • Electrophoresis, Polyacrylamide Gel
  • Escherichia coli / metabolism
  • Gene Expression Regulation, Bacterial / drug effects
  • Kinetics
  • Magnesium Sulfate / pharmacology
  • Molecular Sequence Data
  • Mutation / genetics
  • Phosphorylation / drug effects
  • Phosphothreonine / metabolism
  • Transcription Factors / chemistry
  • Transcription Factors / genetics
  • Transcription Factors / metabolism*
  • Virulence / drug effects
  • Virulence / genetics

Substances

  • Bacterial Proteins
  • BvgA protein, Bacteria
  • DNA, Bacterial
  • Transcription Factors
  • bvgS protein, Bordetella pertussis
  • Phosphothreonine
  • Aspartic Acid
  • Magnesium Sulfate
  • DNA-Directed RNA Polymerases