title: Bordetella pertussis fim3 gene regulation by BvgA: phosphorylation controls the formation of inactive vs. active transcription complexes.
abstract: Two-component systems [sensor kinase/response regulator (RR)] are major tools used by microorganisms to adapt to environmental conditions. RR phosphorylation is typically required for gene activation, but few studies have addressed how and if phosphorylation affects specific steps during transcription initiation. We characterized transcription complexes made with RNA polymerase and the Bordetella pertussis RR, BvgA, in its nonphosphorylated or phosphorylated (BvgA∼P) state at P(fim3), the promoter for the virulence gene fim3 (fimbrial subunit), using gel retardation, potassium permanganate and DNase I footprinting, cleavage reactions with protein conjugated with iron bromoacetamidobenzyl-EDTA, and in vitro transcription. Previous work has shown that the level of nonphosphorylated BvgA remains high in vivo under conditions in which BvgA is phosphorylated. Our results here indicate that surprisingly both BvgA and BvgA∼P form open and initiating complexes with RNA polymerase at P(fim3). However, phosphorylation of BvgA is needed to generate the correct conformation that can transition to competent elongation. Footprints obtained with the complexes made with nonphosphorylated BvgA are atypical; while the initiating complex with BvgA synthesizes short RNA, it does not generate full-length transcripts. Extended incubation of the BvgA/RNA polymerase initiated complex in the presence of heparin generates a stable, but defective species that depends on the initial transcribed sequence of fim3. We suggest that the presence of nonphosphorylated BvgA down-regulates P(fim3) activity when phosphorylated BvgA is present and may allow the bacterium to quickly adapt to the loss of inducing conditions by rapidly eliminating P(fim3) activation once the signal for BvgA phosphorylation is removed.
authors.affiliation: Gene Expression and Regulation Section, Laboratory of Cell and Molecular Biology, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892;
authors.forename: Alice
authors.initials: A
authors.lastname: Boulanger
authors.affiliation: Gene Expression and Regulation Section, Laboratory of Cell and Molecular Biology, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892;
authors.forename: Kyung
authors.initials: K
authors.lastname: Moon
authors.affiliation: Gene Expression and Regulation Section, Laboratory of Cell and Molecular Biology, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892;
authors.forename: Kimberly B
authors.initials: KB
authors.lastname: Decker
authors.affiliation: Division of Bacterial, Parasitic, and Allergenic Products, Center For Biologics Evaluation and Research, Food and Drug Administration, Silver Spring, MD 20993.
authors.forename: Qing
authors.initials: Q
authors.lastname: Chen
authors.affiliation: Gene Expression and Regulation Section, Laboratory of Cell and Molecular Biology, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892;
authors.forename: Leslie
authors.initials: L
authors.lastname: Knipling
authors.affiliation: Division of Bacterial, Parasitic, and Allergenic Products, Center For Biologics Evaluation and Research, Food and Drug Administration, Silver Spring, MD 20993.
authors.forename: Scott
authors.initials: S
authors.lastname: Stibitz
authors.affiliation: Gene Expression and Regulation Section, Laboratory of Cell and Molecular Biology, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892; dhinton@helix.nih.gov.
authors.forename: Deborah M
authors.initials: DM
authors.lastname: Hinton
mesh_terms: D000222:Adaptation, Physiological / Q000235:genetics / Q000502:physiology*; D000942:Antigens, Bacterial / Q000235:genetics / Q000378:metabolism*; D001426:Bacterial Proteins / Q000378:metabolism*; D001886:Bordetella pertussis / Q000235:genetics* / Q000472:pathogenicity; D018983:DNA Footprinting; D017931:DNA Primers / Q000235:genetics; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism; D004338:Drug Combinations; D024202:Electrophoretic Mobility Shift Assay; D036781:Fimbriae Proteins / Q000235:genetics / Q000378:metabolism*; D015964:Gene Expression Regulation, Bacterial / Q000235:genetics / Q000502:physiology*; D046912:Multiprotein Complexes / Q000235:genetics / Q000378:metabolism*; D009821:Oils; D010636:Phenols; D010766:Phosphorylation; D014157:Transcription Factors / Q000378:metabolism*; D014158:Transcription, Genetic / Q000235:genetics / Q000502:physiology*; D014774:Virulence; D010566:Virulence Factors, Bordetella / Q000235:genetics / Q000378:metabolism*
chemical_list: D000942:Antigens, Bacterial; D001426:Bacterial Proteins; C065601:BvgA protein, Bacteria; D017931:DNA Primers; D004338:Drug Combinations; D046912:Multiprotein Complexes; D009821:Oils; C027222:P & S Liquid; D010636:Phenols; D014157:Transcription Factors; D010566:Virulence Factors, Bordetella; C101542:fim3 protein, Bordetella; D036781:Fimbriae Proteins; D012321:DNA-Directed RNA Polymerases
keywords: Bordetella pertussis; BvgA; RNA polymerase; fim3; transcription