Mutations in the bvgA gene of Bordetella pertussis that differentially affect regulation of virulence determinants

Stibitz, Scott

doi:10.1128/jb.176.18.5615-5621.1994

Cited by 41 publications

(37 citation statements)

References 35 publications

(30 reference statements)

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“…Furthermore, initial reports indicated that bvgAS was not sufficient for activation of ptxA and cya transcriptional fusions in E. coli (17,33). These results suggested that an intermediate factor was required for expression of pertussis toxin and adenylate cyclase; however, such a factor has not yet been described (9,31,48). …”

mentioning

confidence: 43%

“…This has led to speculation that the bvg virulence control system may have a downstream activator that participates in ptxA expression, much like the ToxRS-ToxT regulatory system of Vibrio cholerae (13). Several mutations conferring an Fha ϩ Cya Ϫ Ptx Ϫ phenotype have been isolated and mapped (9,48). These mutations are not located in a putative downstream transcriptional activator but are instead found upstream of the translational start site of rpoA or in the C terminus of BvgA, invoking a model in which BvgA interacts with RpoA at the ptxA promoter.…”

Section: Discussionmentioning

confidence: 99%

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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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mentioning

confidence: 43%

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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“…Both mutations mapped to the C terminus of BvgA. Stibitz suggested that ptx and cya expression may require the interaction of an accessory factor with the C terminus of BvgA (42). This proposed factor may be Baf.…”

Section: Discussionmentioning

confidence: 99%

“…It is interesting that no such mutants have been identified in studies utilizing transposon mutagenesis of the B. pertussis chromosome (7,19,49,50). Stibitz recently used chemical mutagenesis in an effort to identify a putative Bvg accessory factor (42). While no accessory factor mutants were identified, two mutants were found that exhibited wildtype fhaB expression but decreased ptx and cya expression.…”

Section: Discussionmentioning

confidence: 99%

Identification of a Bordetella pertussis regulatory factor required for transcription of the pertussis toxin operon in Escherichia coli

1995

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Transcription of the pertussis toxin operon (ptx) is positively regulated in Bordetella pertussis by the bvgAS locus. However, a ptx-lacZ transcriptional fusion in Escherichia coli cannot be activated by bvgAS in trans. This suggests that an additional factor(s) is required for transcription of ptx. A gene encoding a Bvg accessory factor (Baf) was identified by its ability to activate an E. coli ptx-lacZ fusion in the presence of bvgAS. The expression of ptx-lacZ was decreased by the addition of 40 mM MgSO 4 , a compound that also modulates ptx expression in B. pertussis. Baf alone did not activate expression of an E. coli fhaB-lacZ fusion, nor did it increase expression of fhaB-lacZ in trans with bvgAS. The gene encoding Baf was localized, sequenced, and found to produce a novel 28-kDa protein. Sequences homologous to B. pertussis baf were identified in Bordetella bronchiseptica and Bordetella parapertussis but not in Bordetella avium. When an additional copy of baf was integrated into the chromosome of BC75, a B. pertussis mutant that produces a low level of pertussis toxin, pertussis toxin production was partially complemented in the cointegrate strain.Bordetella pertussis, a gram-negative coccobacillus, causes the upper respiratory tract disease pertussis (whooping cough) in humans. B. pertussis synthesizes multiple attachment factors and toxins that act in concert to produce disease (8,39,48). Filamentous hemagglutinin, pertactin, pertussis toxin (PT), and fimbriae are important in mediating attachment to a variety of cell types. The organism elaborates several factors that are toxic to host cells, including tracheal cytotoxin, adenylate cyclase toxin, and PT.All of the virulence factors listed above, with the exception of tracheal cytotoxin, are coordinately regulated by the products of the bvgAS locus, BvgA and BvgS (6,22,34,37,51). These molecules are members of a family of transcriptional regulators that utilize two components, a sensor and a regulator, to activate or repress the transcription of genes in response to a variety of environmental stimuli (15,30). BvgS is a 135-kDa transmembrane sensor protein that regulates the transcription of virulence genes in response to environmental stimuli such as MgSO 4 , nicotinic acid, or growth at low temperatures (25ЊC) (26,27,29,46). In the presence of such stimuli, there is no transcription of B. pertussis virulence-activated genes (vag), a phenomenon termed ''phenotypic modulation' ' (21, 26). In the absence of these signals (e.g., at 37ЊC), BvgS activates BvgA, a 23-kDa cytoplasmic protein (46). BvgA binds to regulatory sequences upstream of some virulence genes and positively influences their transcription (36). Uhl and Miller (47) have recently shown that the cytoplasmic domain of BvgS phosphorylates BvgA in vitro. The phosphorylation of BvgA correlates with the activation of an fhaB-lacZ transcriptional fusion in Escherichia coli (47), suggesting that phosphorylated BvgA mediates the transcriptional activation of virulence-associated genes. Similar res...

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“…These observations gave rise to the hypothesis that a secondary bvg-dependent activator is involved in ptx promoter activation. However, attempts to identify a secondary activator have been unsuccessful, and mutations causing specific loss of ptx (and cya ), but not fha, promoter activity were found to map to either the rpoA gene (expressing the ␣ subunit of RNA polymerase (RNAP)) Stibitz and Carbonetti, 1994) or the bvgA gene itself (Stibitz, 1994). Upon closer examination, Uhl and Miller (1995b) demonstrated Bvgdependent activation of a ptx-lacZYA fusion in E. coli that was affected by the growth rate of the strain, and proposed that bvg is the only locus required for ptx activation.…”

Section: Introductionmentioning

confidence: 99%

Genetic analysis of pertussis toxin promoter activation in Bordetella pertussis

Marques

Carbonetti

1997

Molecular Microbiology

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SummaryBordetella pertussis regulates expression of its virulence factors such as pertussis toxin (Ptx) via the bvg locus, which encodes a two-component system composed of a sensor protein, BvgS, and a transcription activator, BvgA. We used a ptx -lac fusion on the B. pertussis chromosome to analyse promoter activation by alteration of specific sequences upstream of and within the promoter. Our data demonstrate that a pair of heptanucleotide inverted repeats separated by a turn of the DNA helix within the upstream repeat region (centred around nucleotide ¹136.5) are crucial cis-activating elements, and probably represent the initial BvgA-binding site. In addition, we demonstrate that the sequence between these repeats and the promoter plays a role in activation. Our data are most consistent with a model of co-operative binding of BvgA dimers to this intervening region and interaction with RNA polymerase at the promoter to activate ptx transcription. In the core promoter region both the non-consensus 21 bp spacing and the specific sequence between the ¹35 and ¹10 elements are crucial for promoter activity.

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Mutations in the bvgA gene of Bordetella pertussis that differentially affect regulation of virulence determinants

Cited by 41 publications

References 35 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

Identification of a Bordetella pertussis regulatory factor required for transcription of the pertussis toxin operon in Escherichia coli

Genetic analysis of pertussis toxin promoter activation in Bordetella pertussis

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