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. 1999 Sep;181(17):5234-41.
doi: 10.1128/JB.181.17.5234-5241.1999.

Analysis of BvgA activation of the pertactin gene promoter in Bordetella pertussis

S M Kinnear  1 , P E Boucher, S Stibitz, N H Carbonetti
Affiliations

Analysis of BvgA activation of the pertactin gene promoter in Bordetella pertussis

S M Kinnear et al. J Bacteriol. 1999 Sep.

Abstract

Bordetella pertussis, the causative agent of whooping cough, regulates expression of its virulence factors via a two-component signal transduction system encoded by the bvg regulatory locus. It has been shown by activation kinetics that several of the virulence factors are differentially regulated. fha is transcribed at 10 min following an inducing signal, while ptx is not transcribed until 2 to 4 h after the inducing signal. We present data indicating that prn is transcribed at 1 h, an intermediate time compared to those of fha and ptx. We have identified cis-acting sequences necessary for expression of prn in B. pertussis by using prn-lac fusions containing alterations in the sequence upstream of the prn open reading frame. In vitro transcription and DNase I footprinting analyses provided evidence to support our hypothesis that BvgA binds to this sequence upstream of prn to activate transcription from the promoter. Our genetic data indicate that the region critical for prn activation extends upstream to position -84. However, these data do not support the location of the prn transcription start site as previously published. We used a number of methods, including prn-lac fusions, reverse transcriptase PCR, and 5' rapid amplification of cDNA ends, to localize and identify the bvg-dependent 5' end of the prn transcript to the cytosine at -125 with respect to the published start site.

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Figures

FIG. 1
FIG. 1
RT-PCR analysis of promoter activation kinetics. RT-PCR was used to detect transcripts of sodB, fha, ptx, and prn after induction of the Bvg system. The time course of induction is shown on ethidium bromide-stained agarose gels at 0, 30, 60, 240, and 480 min for the bvg-dependent fha, ptx, and prn promoters and the bvg-independent standard, sodB. Results from a typical experiment are shown.
FIG. 2
FIG. 2
Effect of deletions and replacements upstream of the chromosomal prn-lac transcriptional fusion on prn promoter activity. A restriction map of the prn upstream region (orf, upstream open reading frame) is shown at the top (not drawn to scale). Constructs are shown on the left, and promoter activities (103 Lac units) with standard deviation bars are shown on the right. The gray bars represent β-galactosidase levels when the strains were grown under nonmodulating conditions, and the black bars represent β-galactosidase levels when the strains were grown in the presence of 50 mM MgSO4. (A) NMD616, wild type; (B) NMD618, strain containing additional EcoRI and ClaI cloning sites inserted at the BglI site; (C) NMD623, strain with deletion of sequence between engineered and wild-type EcoRI sites; (D) NMD625, strain with replacement of the EcoRI fragment deletion with unrelated sequence of same size; (E) NMD630, strain with replacement of EcoRI fragment deletion with a 16-bp wild-type sequence upstream from the EcoRI site; (F) NMD631, strain with replacement of EcoRI deletion with a 26-bp wild-type sequence upstream from the EcoRI site.
FIG. 3
FIG. 3
BvgA-mediated in vitro transcription analysis of the prn promoter. Transcription reaction mixtures contained 0.5 pmol of supercoiled pTE-PRN plasmid, 150 nM E. coli ς70-saturated RNAP or 1.4 μM purified B. bronchiseptica RNAP, and between 0 and 0.78 μM BvgA. Where indicated, Ac∼P was added at a final concentration of 15 mM. Lanes: 3 and 7, 0.20 μM BvgA; 4 and 8, 0.39 μM BvgA; 2, 5, 6, and 9, 0.78 μM BvgA. Reaction mixtures were electrophoresed on a 6% polyacrylamide sequencing gel and exposed for autoradiography in a PhosphorImager cassette. The arrow indicates the prn transcript.
FIG. 4
FIG. 4
DNase I footprinting analysis of the prn promoter. Protection reaction mixtures, where indicated, contained 150 nM E. coli ς70-saturated RNAP, 15 mM Ac∼P, and BvgA at 0.58 (lane 3) or 1.2 (lanes 2, 4, and 5) μM. Reaction mixtures were electrophoresed on a 6% acrylamide sequencing gel and exposed for autoradiography in a PhosphorImager cassette. The black rectangle represents the region of primary protection by BvgA from positions −94 to −52, and the open rectangle represents the region of secondary protection by BvgA from −51 to +22.
FIG. 5
FIG. 5
Effect of sequential deletion of the sequence upstream of the prn ORF on prn promoter activity. Alterations are shown on the left, and promoter activities (103 Lac units) with standard deviation bars are shown on the right. The gray bars represent β-galactosidase levels when the strains were grown under nonmodulating conditions, and the black bars represent β-galactosidase levels when the strains were grown in the presence of 50 mM MgSO4. (A) NMD616, wild type; (B) NMD637, strain with deletion from lac fusion junction to position +61; (C) NMD638, strain with deletion from lac fusion junction to position +22; (D) NMD643, strain with deletion from lac fusion junction to position −8; (E) NMD642, strain with deletion from lac fusion junction to position −23. The putative −10 sequence and +1 are indicated in bold, and the ClaI site is italicized.
FIG. 6
FIG. 6
Schematic of prn promoter sequence and oligonucleotides used in RT-PCR analysis. Sequential oligonucleotide primers were paired with an antisense primer in the prn ORF in PCRs with total B. pertussis cDNA used as a template to determine the 5′ extent of the prn transcript. PCR product results are shown to the right of the respective oligonucleotide primers. +, strong band; +/−, faint band; −, absence of PCR product. The putative −35 and −10 sequences and +1 are indicated in bold.
FIG. 7
FIG. 7
Schematic diagram of the sequence upstream of prn. The end of an ORF with codon usage typical of B. pertussis is followed by 335 bp of intergenic sequence and the start of the prn ORF. A putative transcription terminator for the upstream ORF is depicted by inverted arrows. Relevant restriction sites are indicated in italics, and the reported transcription start site (12) is indicated in bold and marked by #. The region of primary DNase I protection by BvgA (from positions −94 to −52) is outlined with a solid line, while the region of weak, secondary protection by BvgA (from positions −51 to +22) is outlined with a dashed line. DNase I-hypersensitive sites in the presence of BvgA are noted with an asterisk. The transcription start site identified in this paper is indicated in bold and marked as +1, and the numbering is in reference to this start site. Putative −10 and −35 promoter sequences are in boldface type and labeled. A putative primary BvgA binding site is indicated in lowercase letters.
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