The Bordetella pertussis model of exquisite gene control by the global transcription factor BvgA.
ABSTRACT Bordetella pertussis causes whooping cough, an infectious disease that is reemerging despite widespread vaccination. A more complete understanding of B. pertussis pathogenic mechanisms will involve unravelling the regulation of its impressive arsenal of virulence factors. Here we review the action of the B. pertussis response regulator BvgA in the context of what is known about bacterial RNA polymerase and various modes of transcription activation. At most virulence gene promoters, multiple dimers of phosphorylated BvgA (BvgA~P) bind upstream of the core promoter sequence, using a combination of high- and low-affinity sites that fill through cooperativity. Activation by BvgA~P is typically mediated by a novel form of class I/II mechanisms, but two virulence genes, fim2 and fim3, which encode serologically distinct fimbrial subunits, are regulated using a previously unrecognized RNA polymerase/activator architecture. In addition, the fim genes undergo phase variation because of an extended cytosine (C) tract within the promoter sequences that is subject to slipped-strand mispairing during replication. These sophisticated systems of regulation demonstrate one aspect whereby B. pertussis, which is highly clonal and lacks the extensive genetic diversity observed in many other bacterial pathogens, has been highly successful as an obligate human pathogen.
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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.Molecular Microbiology 03/2013; DOI:10.1111/mmi.12177 · 5.03 Impact Factor
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ABSTRACT: Response regulator proteins within two-component signal transduction systems are activated by phosphorylation and can catalyze their own covalent phosphorylation using small molecule phosphodonors. To date, comprehensive kinetic characterization of response regulator autophosphorylation is limited to CheY, which follows a simple model of phosphodonor binding followed by phosphorylation. We characterized autophosphorylation of the response regulator PhoB, known to dimerize upon phosphorylation. In contrast to CheY, PhoB time traces exhibited an initial lag phase and gave apparent pseudo-first order rate constants that increased with protein concentration. Furthermore, plots of the apparent autophosphorylation rate constant versus phosphodonor concentration were sigmoidal, as were PhoB binding isotherms for the phosphoryl group analog BeF3-. Successful mathematical modeling of the kinetic data necessitated inclusion of the formation of a PhoB heterodimer (one phosphorylated and one unphosphorylated monomer) with an enhanced rate of phosphorylation. Specifically, dimerization constants for the PhoB heterodimer and homodimer (two phosphorylated monomers) were similar, but the rate constant for heterodimer phosphorylation was ~10-fold higher than for the monomer. In a test of the model, disruption of the known PhoBN dimerization interface by mutation led to markedly slower and non-cooperative autophosphorylation kinetics. Furthermore, phosphotransfer from the sensor kinase PhoR was enhanced by dimer formation. Phosphorylation-mediated dimerization allows many response regulators to bind to tandem DNA binding sites and regulate transcription. Our data challenge the notion that response regulator dimers primarily form between two phosphorylated monomers, and raise the possibility that response regulator heterodimers containing one phosphoryl group may participate in gene regulation.Journal of Biological Chemistry 06/2013; DOI:10.1074/jbc.M113.471763 · 4.60 Impact Factor
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ABSTRACT: Multisubunit RNA polymerases are complex protein machines that require a specificity factor for the recognition of a specific transcription start site. Although bacterial σ factors are thought to be quite different from the specificity factors employed in higher organisms, a comparison of the σ/RNA polymerase structures with recent structures of eukaryotic Pol II together with TFIIB highlights significant functional similarities. Other work reveals that both bacterial and eukaryotic promoters are composed of modular elements that are used in different combinations. Bacteria, archaea, and eukaryotes also utilize similar strategies to alter core promoter specificity, from specificity factor exchange to the employment of activators that bind close to or overlap core promoter sequences, directing the transcriptional machinery to a new start site. Here we examine the details of core promoter recognition in bacteria that reveal the transcriptional similarities throughout biology. Expected final online publication date for the Annual Review of Microbiology Volume 67 is September 08, 2013. Please see http://www.annualreviews.org/catalog/pubdates.aspx for revised estimates.Annual review of microbiology 06/2013; 67. DOI:10.1146/annurev-micro-092412-155756 · 13.02 Impact Factor