Positive transcriptional feedback at the bvg locus controls expression of virulence factors in Bordetella pertussis.

Scarlato, Vincenzo; Prugnola, Anna; Aricò, Beatrice; Rappuoli, R

doi:10.1073/pnas.87.17.6753

Cited by 109 publications

(150 citation statements)

References 19 publications

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“…Plasmids are as follows: pJM660 contains BvgA under control of the IPTG-inducible trc promoter, pMU112 is a derivative of pJM660 with the bvgA-C1 mutation, and pMU352 is the autoregulated bvg locus with an in-frame BvgA mutation. pDM20 contains the entire positively autoregulated wild-type bvg locus (41,46), and pTrc99A is the vector used to create pJM660 and pMU112 and contains neither bvgA nor bvgS. Values are means Ϯ SD of assays done in triplicate.…”

Section: Discussionmentioning

confidence: 99%

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

Uhl

Miller

1995

J Bacteriol

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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...

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Section: Discussionmentioning

confidence: 99%

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

Uhl

Miller

1995

J Bacteriol

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“…The sensor component, usually membrane associated, senses a signal from the outside of the cell and transmits this signal to the response-regulator component, usually via phosphorylation, which then results either in repression or activation of one or more genes. A. tumefaciens, S. typhimurium, and B. pertussis are a few pathogens that utilize two-component regulatory systems to regulate virulence-associated genes (3,35,41,42). PilA and PilB compose a two-component system that was iden- Competition assay for PilA binding to P pilE with excess unlabelled DNAs.…”

Section: Discussionmentioning

confidence: 99%

“…This complex control is often mediated by regulatory systems that activate and/or repress the expression of a battery of virulence genes that compose a regulon. For example, the bvg, phoP phoQ, and virA virG loci regulate the expression of several virulence genes in Bordetella pertussis (35,42), Salmonella typhimurium (3, 26), and Agrobacterium tumefaciens (41), respectively.Many bacteria employ a system of two-protein components to regulate the expression of genes in response to environmental changes (30). These systems consist of a sensor component, often found in the cytoplasmic membrane, and a responseregulator, which is usually cytoplasmic.…”

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Interaction of the Neisseria gonorrhoeae PilA protein with the pilE promoter involves multiple sites on the DNA

Arvidson

1995

J Bacteriol

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PilA is the putative DNA-binding component of a two-component system that regulates transcription of the pilin expression locus (pilE) of Neisseria gonorrhoeae. Here we report the purification of the PilA protein and characterization of its DNA-binding activity. PilA was overproduced in Escherichia coli with an isopropyl-␤-D-thiogalactopyranoside (IPTG)-inducible expression vector. Cell extracts were prepared by sonication and fractionated by anion-exchange chromotography, followed by dye affinity chromatography with Cibacron Blue. Proteins were eluted by using a gradient of KCl, and PilA-containing fractions were identified by immunoblot analysis with a polyclonal anti-PilA antiserum. Purified PilA was judged to be >90% pure, as determined by Coomassie blue staining and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. PilA purified in this manner was used to develop a gel retardation assay with a 301-bp fragment containing the pilE promoter (P pilE ) and upstream sequences as a probe. A fragment of similar size containing the E. coli aroH promoter was used as a negative control. Competition experiments using a 100-to 1,000-fold excess of unlabelled DNA fragments confirmed the specificity of PilA binding to the pilE promoter. To localize the PilA binding site within the 301-bp P pilE fragment, stepwise deletions were generated by PCR and the fragments were examined in the gel shift assay. The results of these experiments show that there are two regions upstream of P pilE that are required for binding by PilA. Taken together, these data indicate that while PilA binds specifically to the upstream region of the pilE gene, this interaction is complex and likely involves multiple regions of this DNA sequence.Neisseria gonorrhoeae is a major sexually transmitted pathogen that infects only humans. The gram-negative diplococcus normally infects cells of the urogenital tract, although it can also infect other human tissues such as the conjunctiva and pharynx. Experimental infection of Fallopian tube organ cultures shows that the bacteria attach to nonciliated epithelial cells of the mucosa, invade these cells, and transcytose to the subepithelial matrix (21). Attachment of the bacteria to epithelial cells occurs via adhesins including pili and the opacity protein (Opa or PII;17,44). Pili are considered a major virulence factor, because only piliated variants produce infection in healthy volunteers (14,45).The gonococcal chromosome harbors numerous variant pil genes that participate in phase and antigenic variation of these proteins. In vitro, pilin antigenic variation can occur via DNA transformation and recombination or gene conversion involving silent pil loci (pilS) and the pilin expression site (pilE) (7, 36). Phase variation can also occur by this mechanism, i.e., if a recombination event results in an internal frameshift, P ϩ (piliated)-to-P Ϫ (nonpiliated) and P Ϫ -to-P ϩ transitions can occur (1a). Recombination can also result in deletion of all or part of the transcriptional or translational signals, ...

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“…Like other bvg-activated loci, this bvg-dependent increase in expression can be inhibited by environmental stimuli such as growth in MgSO4. Two differentially regulated promoters have been identified upstream of the bvgAS operon (16,18). The bvgP1 promoter is positively autoregulated and therefore requires the bvgAS gene products for activity.…”

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confidence: 99%

Identification of Bordetella pertussis regulatory sequences required for transcriptional activation of the fhaB gene and autoregulation of the bvgAS operon

1991

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Transcription of numerous virulence genes in Bordetella pertussis is positively regulated by the products of the bvgAS genes. In this study a series of lacZYA fusions containing deletions in either the fhaB or bvgA promoter regions was used to identify cis-acting regulatory regions required for bvg activation of these two genes. Gel retardation and DNase I protection analyses have shown that specific protein-DNA interactions occur at these regulatory regions and that these interactions require the transcriptional activator protein BvgA. The regulatory regions found upstream of fhaB and bvgA which are involved in protein binding both contain the sequence TTTCCTA. This sequence is part of an inverted repeat upstream of fhaB and a direct repeat upstream of bvgA. Homologous repeats are not apparent upstream of other bvg-activated genes, such as ptx and cyaA. These data suggest that the mechanism for transcriptional regulation of the other bvg-activated genes is complex and may require regulatory factors in addition to the bvgAS gene products.

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Positive transcriptional feedback at the bvg locus controls expression of virulence factors in Bordetella pertussis.

Cited by 109 publications

References 19 publications

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

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

Interaction of the Neisseria gonorrhoeae PilA protein with the pilE promoter involves multiple sites on the DNA

Identification of Bordetella pertussis regulatory sequences required for transcriptional activation of the fhaB gene and autoregulation of the bvgAS operon

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