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

Abstract

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 ␤-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 ␤-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 ␤-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-C1) 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. Bordetella pertussis, the causative agent of whooping cough, synthesizes toxins and adhesins which appear to aid the bacterium in colonization and multiplication (15,23,28,38,56,59). Expression of toxins (pertussis toxin, adenylate cyclasehemolysin, and dermonecrotic toxin) and adhesins (filamentous hemagglutinin, fimbriae, and pertactin) is positively regulated by the products of the bvg operon (11,57,59). In contrast to the multiple loci activated by bvg, vrg genes of B. pertussis (5,6,25), siderophore production in some Bordetella bronchiseptica strains (14), and motility genes of B. bronchiseptica (1-3) are repressed by bvg.Sequence analysis and in vitro studies have indicated that BvgA and BvgS are members of the two-component family of signal transduction proteins. BvgS is a 135-kDa sensor protein localized to the cytoplasmic membrane, and BvgA is a 23-kDa cytoplasmic response regulator (4, 7, 37, 49, 50-53). The signals sensed by BvgAS in vivo are not known, but BvgAS can be inactivated in vitro by growth at a low temperature or by addition of sulfate anion or nicotinic acid to the culture medium. This reversible inactivation of BvgAS is known as phenot...

A new gene locus of Bordetella pertussis defines a novel family of prokaryotic transcriptional accessory proteins

“…By the introduction of the respective loci into these mutants, several of these suppressor mutations could be mapped to either the bvg or the rpoA locus. Recent results obtained demonstrated that the BvgA transcription factor is able to interact directly with all virulence promoters analyzed so far, the ptx, cyaA, fha, and bvg loci (5,31,38,42). The localization of suppressor mutations within the bvg and rpoA loci is in agreement with these results and further strengthens the hypothesis that BvgA and the RNA polymerase ␣ subunit may interact physically during the transcription initiation process at the toxin promoters (7).…”

“…Therefore, these mutations in the bvg locus lead to avirulent bacteria, the so-called phase variants (23). Despite the coordinate regulation mediated by this two-component system, differential regulatory phenomena such as different induction kinetics of transcription of the various factors or different expression patterns in the heterologous host Escherichia coli were noted, which led to the classification of the virulence factors into two groups, (i) the two toxins PTX and CyaA and (ii) the other factors (33,34,42). These differences in their expression are also reflected by differences in the promoter structures of the two groups.…”

Autoregulation Is Essential for Precise Temporal and Steady-State Regulation by theBordetellaBvgAS Phosphorelay

“…The fact that RBH was capable of expressing all of the same phenotypic phases as RB50, however, indicates that BvgAϳP levels can be adjusted precisely and maintained indefinitely at specific levels in response to specific environmental cues, even when BvgA is maintained at a high level. Since there is considerable evidence that phosphorylation and dephosphorylation of BvgA are controlled only by BvgS (41,(45)(46)(47)(48), these data provide strong evidence that the kinase and/or phosphatase activity of BvgS can be adjusted to and maintained at intermediate levels. Our data also suggest that it is the absolute amount of BvgAϳP, rather than the ratio of BvgAϳP to BvgA, that is important in controlling gene expression, because this ratio must be well below one in RBH cells grown under Bvg iphase conditions but one or nearly one in RBL cells grown under Bvg ϩ -phase conditions and in RB50 cells immediately after a shift from Bvg Ϫ -phase conditions to Bvg ϩ -phase conditions, yet in all of these cases, the Bvg i phase is expressed.…”

Synergistic binding of RNA polymerase and BvgA phosphate to the pertussis toxin promoter of Bordetella pertussis

“…Mechanistically, this could be due to a lower binding affinity of BvgA at ptx or to the need for higher-order multimers of BvgA. A recent study demonstrates that under certain growth conditions, BvgAS is in fact sufficient for modulation-responsive activation of the ptx locus in E. coli cells (39). Uhl and Miller suggest that the relevant difference between the growth conditions promoting ptx expression and conditions previously used is a slower rate of growth with a concomitant increase in BvgA levels.…”