Transcription of virulence genes of Bordetella pertussis is co-ordinately regulated by the BvgA and BvgS proteins, which are members of the two-component family of bacterial signal-transduction proteins. BvgS is the transmembrane sensor and BvgA the transcriptional regulator. By gel mobility shift assays we demonstrate that phosphorylated BvgA (BvgA approximately P) forms distinct complexes with the filamentous haemagglutinin (PFHA) promoter DNA at different BvgA approximately P: DNA ratios. DNase I protection analyses show that phosphorylation of BvgA not only enhances affinity of the protein for the binding sites of the PFHA and bvgP1 promoters, but it extends significantly the bound region towards position -35 of these promoters. Conversely, a 10-fold higher amount of BvgA approximately P is required for binding to a large DNA region, from -168 to -60, of the pertussis toxin (Ptox) promoter sequence. These findings suggest that the molecular interaction of BvgA approximately P with the Ptox promoter is different from its interaction with the PFHA and bvgP1 promoters. The sigma 70 Escherichia coli RNA polymerase (RNP) does not bind to the bvg-regulated promoters. However, following the formation of a BvgA approximately P-promoter complex, the E. coli RNP specifically recognizes and binds to the bvg-regulated promoters. Thus, BvgA approximately P exerts its action at the level of promoter recognition by directing promoter selectivity by RNP.

Intracellular signaling by mitogen-activated protein (MAP) kinase cascades plays an essential role in the cellular response to environmental stress. In the yeast Saccharomyces cerevisiae, the PKC1-regulated, stress-activated MAP kinase pathway, the MPK1 cascade, is activated by heat and by a decrease in osmolarity. The genes WSC1, WSC2 and WSC3 encode putative receptors that maintain cell wall integrity under conditions of heat stress. Genetic studies place the function of the WSC genes upstream of the MPK1 kinase cascade. To further define the role of the WSC family in the stress response we determined whether: (1) the wscdelta mutants are sensitive to other environmental stress conditions, in addition to heat shock; (2) expression from four transcriptional control elements, known to be activated by stress, is impaired in wscdelta mutants; and (3) Wsc4, a Wsc homolog, has functions that overlap with those of the other Wsc family members. We report here that deletion of WSC and PKC1 causes hypersensitivity to ethanol, hydrogen peroxide and DNA-damaging drugs. In wscdelta mutants expression of beta-galactosidase from the AP-1 response element (ARE), the heat shock response element (HSE) or the stress response element (STRE) is not reduced. In contrast, expression of a reporter gene placed under the control of the Rlm1 (transcription factor)-dependent response element is significantly reduced in wscdelta mutants. This suggests that the lysis defect of wscdelta mutants is at least in part caused by a defect in transcriptional regulation by Rlm1. Phenotypic analysis of the effect of deleting WSC4 in a wsc1delta mutant show that, unlike WSC2 or WSC3, deletion of WSC4 does not exacerbate the lysis defect of a wsc1delta strain. In contrast, deletion of WSC4 enhances the sensitivity of the wsc1delta mutant to heat shock, ethanol, and a DNA-damaging drug, suggesting that WSC4 plays a role in the response to environmental stress but that its function may differ from those of the other WSC family members.

BpH1, the Bordetella pertussis H1 homolog, interacts with chromosomal DNA. With DNase I protection assays, we demonstrate in this study that BpH1 binds DNA in a nonspecific manner and that it may cover DNA fragments from end to end. Although the binding was shown to be nonspecific, preferential binding sites and sites resistant to BpH1 binding were identified within and upstream of the pertussis toxin promoter sequence. In the presence of DNA ligase, BpH1 favored the formation of multimeric DNA fragments of various sizes and prevented ring closures, suggesting a diminished flexibility of the DNA fragments and thus indicating that BpH1 acts as a macromolecular crowding agent.Many bacteria have genes that code for small basic and relatively abundant proteins, called histone-like proteins, with no significant amino acid sequence similarity to eukaryotic histone proteins. These proteins are associated with either the bacterial chromosome or bacteriophage DNA. The histonelike proteins possess the capability to bind DNA in a nonspecific manner and are able to compact chromosomal DNA many thousand fold (7,14,21). In addition, these proteins can regulate transcription of a specific gene by influencing the local promoter DNA supercoiling (6,12,13,16,24).We have recently shown that Bordetella pertussis directs the synthesis of a small basic and abundant protein, BpH1, that has a high level of amino acid similarity (Ϸ54% identity) to eukaryotic histone H1 (18). Another similar protein, Hc1, has been described for Chlamydia trachomatis (9). In both cases, it has been demonstrated that the proteins bind DNA nonspecifically and that they constrain supercoiled structures (2, 18). Furthermore, overexpression of Hc1 in Escherichia coli induces condensation of chromosomal DNA (3).Here, we show that BpH1 binds DNA nonspecifically and that it is able to stimulate intermolecular ligation of linear DNA fragments, very likely by altering DNA flexibility.Footprinting studies of BpH1 with Bordetella DNA. To investigate whether BpH1 has preferential DNA binding sites, we carried out DNase I footprinting experiments with B. pertussis promoter and coding regions. DNA probes were from the bvg regulatory locus and from the pertussis toxin operon. In B. pertussis, the bvg locus coordinately regulates expression of virulence factors, while the pertussis toxin is its major virulence factor (1, 4, 8, 10).The results of a DNase I protection analysis of BpH1 bound to bvg promoter fragments are shown in Fig. 1. As expected, at high concentrations of BpH1, the probes were completely insensitive to DNase I digestion (Fig. 1A and B, lanes 3). With addition of competitor DNA, the two probes were not bound by BpH1, giving rise to a pattern of DNase I digestion indistinguishable from that of the naked DNAs (Fig. 1A and B, lanes 4 to 6). DNase I protection experiments with BpH1 bound to fragments from the coding regions of the filamentous hemagglutinin (5) and pertussis toxin (8) genes gave similar results (data not shown). We conclude that binding of BpH...