Expression of bvgAS of Bordetella pertussis represses flagellar biosynthesis of Escherichia coli.

BvgAS is a two-component system of Bordetella pertussis involved in the reciprocal regulation of the virulence genes and the flagellar biosynthesis. In this study, we found that expression of bvgAS in Escherichia coli also results in reduced motility. The repression was relieved by the addition of known chemical modulators of BvgAS such as MgSO4 and nicotinic acid, indicating that functional BvgAS proteins are required for the negative control of E. coli motility. In addition, BvgAS repressed the transcription of the flhDC master operon of E. coli, which consequently caused non-flagellation on the cell surface. However, expression of BvgAS had no effect on stress-resistant motile mutants of E. coli. These data suggest that E. coli may have BvgA-like protein(s) involved in the regulatory interactions between the stress response and the flagellar biosynthesis.

Central role of the BvgS receiver as a phosphorylated intermediate in a complex two-component phosphorelay.

Two-component systems use phosphorylation reactions to regulate stimulus/response pathways. In Bordetella pertussis, a human respiratory pathogen, the infectious cycle of the organism is controlled by the BvgAS two-component system. BvgS has similarities to sensor and response regulator components and is an autophosphorylating kinase that phosphorylates BvgA. BvgA, a response regulator, is a DNA-binding protein that activates virulence gene transcription. Three phosphorylated BvgS domains, the transmitter, receiver, and C terminus, are essential for signal transduction. We now demonstrate that the BvgS transmitter is sufficient for autophosphorylation but is unable to phosphorylate the C terminus or BvgA. The BvgS receiver regulates several phenotypes: dephosphorylation of both the BvgS transmitter and C terminus as well as transfer of a phosphoryl group from the transmitter to the C terminus. Our results indicate that BvgAS signal transduction initiates with autophosphorylation of the transmitter followed by phosphotransfer to the receiver. The phosphorylated receiver can donate to the C terminus or to water. The phosphorylated C terminus is then able to transfer the phosphoryl group to BvgA.

Integration of multiple domains in a two-component sensor protein: the Bordetella pertussis BvgAS phosphorelay.

BvgS and BvgA, a two-component system, regulate virulence gene expression in Bordetella pertussis. BvgS is a transmembrane sensor protein that can autophosphorylate and phosphorylate BvgA. Phosphorylated BvgA activates transcription of virulence genes. The cytoplasmic region of BvgS contains three domains separated by alanine/proline-rich sequences--the transmitter, receiver and C-terminus. We report that the C-terminal domain, like the transmitter and receiver, is an essential part of the phosphorelay from BvgS to BvgA. The BvgS C-terminal domain is phosphorylated in trans via a phosphotransfer mechanism by the cytoplasmic portion of BvgS, and trans-phosphorylation of the C-terminal domain requires both the transmitter and receiver. We also demonstrate that phosphorylated, purified C-terminal domain alone is sufficient for phosphotransfer to BvgA. A point mutation in the C-terminal domain (His1172-->Gln) abolishes BvgS activity in vivo and eliminates detectable phosphorylation of BvgA in vitro. Activity of BvgS His 1172-->Gln could be restored by providing the wild-type C-terminal domain in trans. Our results indicate an obligatory role for an alternate phosphodonor module in the BvgAS phosphorelay.

BvgAS is sufficient for activation of the Bordetella pertussis ptx locus in Escherichia coli.

BvgA and BvgS, which regulate virulence gene expression in Bordetella pertussis, are members of the two-component signal transduction family. The effects of growth conditions on the ability of BvgAS to activate transcription of fhaB (encoding filamentous hemagglutinin) and ptxA (encoding the S1 subunit of pertussis toxin) were assessed in Escherichia coli by using chromosomal fhaB-lacZYA and ptxA-lacZYA fusions. Although it had previously been reported that a ptxA-lacZYA transcriptional fusion was not activated by bvgAS in E. coli (J. F. Miller, C. R. Roy, and S. Falkow, J. Bacteriol. 171:6345-6348, 1989), we now present evidence that ptxA is activated by bvgAS in E. coli in a manner that is highly dependent on the growth conditions. Higher levels of beta-galactosidase were produced by ptxA-lacZYA in the presence of bvgAS during growth in Stainer-Scholte medium or M9 minimal salts medium with glucose than in Luria-Bertani medium. In contrast, the level of fhaB-lacZYA expression was high during growth in all media. Addition of modulating stimuli which inhibit BvgAS function eliminated expression of ptxA-lacZYA. Levels of beta-galactosidase expressed from the ptx-lacZYA fusion correlated with growth rate and with the final optical density at 600 nm, suggesting that the lower growth rate in M9-glucose and Stainer-Scholte media was responsible for greater accumulation of beta-galactosidase than was seen in Luria-Bertani medium. Overproduction of BvgA was not sufficient for activation of ptxA expression but was sufficient for fhaB expression. However, overproduction of a constitutive BvgA allele (bvgA-Cl) or overproduction of BvgA in the presence of BvgS was able to activate ptxA. Our results demonstrate Bvg-dependent activation of a ptxA-lacZYA fusion in E. coli and indicate that bvg is the only Bordetella locus required for ptxA activation in this heterologous system.

Autophosphorylation and phosphotransfer in the Bordetella pertussis BvgAS signal transduction cascade.

Expression of adhesins, toxins, and other virulence factors of Bordetella pertussis is under control of the BvgA and BvgS proteins, members of a bacterial two-component signal transduction family. BvgA bears sequence similarity to regulator components, whereas BvgS shows similarity to both sensor and regulator components. BvgA and the cytoplasmic portion of BvgS ('BvgS) were overexpressed and purified. 'BvgS autophosphorylated with the gamma-phosphate from [gamma-32P]ATP and phosphorylated BvgA. Kinetic analysis indicated that BvgA receives its phosphate from 'BvgS. Mutations in the transmitter, receiver, and C-terminal domains of BvgS were tested for activation of a BvgAS-dependent fhaB::lacZ reporter fusion in vivo and for autophosphorylation and phosphotransfer to BvgA in vitro. All mutations abolished activation of the fhaB::lacZ fusion. A point mutation in the transmitter (H729Q) prevented autophosphorylation of 'BvgS. In contrast to other characterized sensor proteins, autophosphorylation also required sequences in the 'BvgS receiver and C-terminal domains. A 'BvgS receiver point mutation (D1023N) had the novel phenotype of being able to autophosphorylate but unable to transfer the phosphate to BvgA. Autophosphorylation activity of the D1023N mutant protein was kinetically and chemically indistinguishable from wild-type 'BvgS despite an uncoupling of phosphotransfer from autophosphorylation. 'BvgS was shown to contain primarily amidyl phosphate and BvgA an acyl phosphate linkage. We present a model for a phosphorelay controlling virulence gene expression in B. pertussis.