title: In vivo phosphorylation dynamics of the Bordetella pertussis virulence-controlling response regulator BvgA.
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.
authors.affiliation: Gene Expression and Regulation Section, Laboratory of Cell and Molecular Biology, NIDDK, National Institutes of Health, Bethesda, MD 20892, USA.
authors.forename: Alice
authors.initials: A
authors.lastname: Boulanger
authors.forename: Qing
authors.initials: Q
authors.lastname: Chen
authors.forename: Deborah M
authors.initials: DM
authors.lastname: Hinton
authors.forename: Scott
authors.initials: S
authors.lastname: Stibitz
mesh_terms: D000595:Amino Acid Sequence; D001224:Aspartic Acid / Q000235:genetics; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001886:Bordetella pertussis / Q000187:drug effects / Q000235:genetics / Q000378:metabolism* / Q000472:pathogenicity*; D004269:DNA, Bacterial / Q000378:metabolism; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000378:metabolism; D015964:Gene Expression Regulation, Bacterial / Q000187:drug effects; D007700:Kinetics; D008278:Magnesium Sulfate / Q000494:pharmacology; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics; D010766:Phosphorylation / Q000187:drug effects; D010769:Phosphothreonine / Q000378:metabolism; D014157:Transcription Factors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D014774:Virulence / Q000187:drug effects / Q000235:genetics
chemical_list: D001426:Bacterial Proteins; C065601:BvgA protein, Bacteria; D004269:DNA, Bacterial; D014157:Transcription Factors; C080662:bvgS protein, Bordetella pertussis; D010769:Phosphothreonine; D001224:Aspartic Acid; D008278:Magnesium Sulfate; D012321:DNA-Directed RNA Polymerases