JOURNAL OF BACTERIOLOGY, Mar. 2005, p. 1648–1658
Vol. 187, No. 5
Activation of the vrg6 Promoter of Bordetella pertussis by RisA
Tadhg O ´Cro ´inı ´n,† Vanessa K. Grippe, and Tod J. Merkel*
Laboratory of Respiratory and Special Pathogens, Division of Bacterial, Parasitic and Allergenic Products,
Center for Biologics Evaluation and Research, Food and Drug Administration, Bethesda, Maryland
Received 4 June 2004/Accepted 17 November 2004
The BvgAS two-component system positively regulates the expression of the virulence genes of Bordetella
pertussis and negatively regulates a second set of genes whose function is unknown. The BvgAS-mediated
regulation of the bvg-repressed genes is accomplished through the activation of expression of the negative
regulator, BvgR. A second two-component regulatory system, RisAS, is required for expression of the bvg-
repressed surface antigens VraA and VraB. We examined the roles of BvgR and RisA in the regulation of four
bvg-repressed genes in B. pertussis. Our analyses demonstrated that all four genes are repressed by the product
of the bvgR locus and are activated by the product of the risA locus. Deletion analysis of the vrg6 promoter
identified the upstream and downstream boundaries of the promoter and, in contrast to previously published
results, demonstrated that sequences downstream of the start of transcription are not required for the
regulation of expression of vrg6. Gel mobility-shift experiments demonstrated sequence-specific binding of
RisA to the vrg6 and vrg18 promoters, and led to the identification of two putative RisA binding sites. Finally,
transcriptional analysis and Western blot analysis demonstrated that BvgR regulates neither the expression
nor the stability of RisA.
Bordetella pertussis is the causative agent of the respiratory
infection of humans known as whooping cough (13). A wide
variety of virulence factors have been identified which contrib-
ute to the bacterium’s ability to colonize the host and cause
disease (10–12, 14, 18, 23, 24, 26–28, 40). These factors include
adhesins such as filamentous hemagglutinin, pertactin, and
fimbriae, as well as several virulence determinants that cause
damage to host tissues, including pertussis toxin, adenylate
cyclase, and dermonecrotic toxin. The expression of most of
these virulence factors is regulated in response to environmen-
tal signals by a two-component regulatory system encoded by
the bvg locus (1, 29, 33, 34, 37). BvgS is a membrane-spanning
protein that presumably acts as a sensor of the external envi-
ronment, and BvgA is a soluble transcriptional activator. When
B. pertussis is grown under normal laboratory conditions in rich
media at 37°C, the BvgS protein is autophosphorylated and
mediates the phosphorylation of BvgA through a series of
phospho-transfer reactions (36, 38, 39). Upon phosphoryla-
tion, BvgA binds to the promoters of the virulence genes,
inducing transcription of those genes (5–7, 9, 16, 29). Although
the signals sensed by BvgS in vivo are unknown, it has been
shown that when B. pertussis is grown at low temperatures, or
in the presence of MgSO4or nicotinic acid, the BvgS-mediated
phospho-transfer reactions are inhibited, and the expression of
the bvg-activated genes is down regulated (a condition referred
to as modulation) (36, 38, 39).
In addition to the set of genes that is activated by the bvg
locus, a second set of genes have been identified which are
repressed by the bvg locus (17). Initial studies identified five
bvg-repressed genes: vrg6, vrg18, vrg24, vrg53, and vrg73 (17). A
conserved 21-bp sequence, located within the coding region,
was identified in each of these genes, and mutations in the
conserved region in the vrg6 gene were reported to cause a loss
of repression resulting in constitutive expression of the gene
(4). Replacement of the vrg6 promoter sequence with that of
the nonregulated asd gene was reported to have no effect on
the bvg-mediated repression of the gene (2, 3). Southwestern
analysis demonstrated the binding of a bvg-activated, 34-kDa
protein to the consensus sequence of the vrg6 gene (3). Taken
together, these results suggested that the expression of the
bvg-repressed genes was repressed by the binding of a bvg-
activated repressor to the conserved element found at the 5?
end of each of the bvg-repressed genes.
Transposon mutagenesis studies have identified bvgR, a bvg-
activated gene located immediately downstream of bvgS, as the
repressor of the bvg-repressed genes (19, 21). In-frame dele-
tions of bvgR result in constitutive expression of the bvg-re-
pressed genes without affecting the regulated expression of the
bvg-activated genes (19, 21). A more recent study demon-
strated that expression of BvgR is activated by the binding of
phosphorylated BvgA to the bvgR promoter (20). Taken to-
gether, these studies indicate that BvgA represses the expres-
sion of the bvg-repressed genes through the activation of the
In addition to the bvgAS regulatory system that represses
expression of the bvg-repressed genes, a second two-compo-
nent regulatory system that is required for the expression of
the bvg-repressed genes was identified. This locus was desig-
nated as the risAS locus due to its association with reduced
intracellular survival by B. bronchiseptica (15). Two groups
independently identified the risAS locus: Jungnitz et al. iden-
tified the risAS locus as a region that is required for intracel-
lular survival of B. bronchiseptica, while Stenson et al. identi-
fied the same locus as a region that is required for the
* Corresponding author. Mailing address: Laboratory of Respira-
tory and Special Pathogens, DBPAP/CBER/FDA, Building 29, Rm.
418, 8800 Rockville Pike, Bethesda, MD 20892. Phone: (301) 496-5564.
Fax: (301) 402-2776. E-mail: email@example.com.
† Present address: Department of Microbiology, Moyne Institute of
Preventative Medicine, Trinity College Dublin, Dublin 2, Ireland.
expression of two bvg-repressed surface antigens, VraA and
VraB, in B. pertussis (15, 31, 32).
In this study, we investigated the regulation of the bvg-
repressed gene, vrg6, by the bvgASR and risAS regulatory sys-
tems. We found that risA is essential for expression of the
bvg-repressed genes and that the RisA protein binds to the
promoter region of both the vrg6 and vrg18 genes. Although
our data clearly demonstrate that BvgR is required for the
repression of the bvg-repressed genes, we show that the puta-
tive repressor-binding site, conserved in each of the five bvg-
repressed genes, is not required for the Bvg- or Ris-mediated
regulation of the bvg-repressed genes in B. pertussis.
MATERIALS AND METHODS
Bacterial strains, plasmids, and media. The bacterial strains and plasmids
used in this study are presented in Table 1. Escherichia coli strains were grown on
L agar or in L broth supplemented with antibiotics when appropriate. B. pertussis
strains were grown on Bordet-Gengou (BG) agar (Difco, Detroit, Mich.) con-
taining 1% proteose peptone (Difco) and 15% defibrinated sheep blood. Unless
otherwise noted, the concentrations of antibiotics included in the medium were
10-?g/ml gentamicin sulfate, 10-?g/ml kanamycin sulfate, 100-?g/ml streptomy-
cin sulfate, and 10-?g/ml nalidixic acid.
Bacterial conjugations. Prior to mating, B. pertussis strains were grown for 3
days and E. coli strains were grown overnight at 37°C. Matings between E. coli
and B. pertussis strains were performed by swabbing bacteria from fresh plate
cultures of each strain onto a BG agar plate supplemented with 10 mM MgCl2.
After 3 h of incubation at 37°C, bacteria were swabbed onto BG agar plates
containing the appropriate antibiotics for the selection of exconjugants, and the
plates were incubated at 37°C.
Construction of isogenic bvgR and risA knockout mutants. Strain TM1627,
which bears an internal, in-frame deletion in risA, was constructed as follows:
Oligonucleotide risA-F (5?-GCAGCGGGAAGACGAAGTTTCGA-3?) was
used in combination with oligonucleotide risA-R (5?-CCGTATGCGAATAGA
CCAGGGCCGT-3?) in a PCR using Tohama I chromosomal DNA as template.
The PCR product generated by the reaction was cloned into pCR2.1-TOPO
(Invitrogen, Carlsbad, Calif.), generating pTM255. Plasmid pTM255 was di-
gested with SacII and was religated, generating pTM266, which has a deletion of
a 204-bp fragment from the risA gene. Plasmid pTM266 was digested with
EcoRI, and the fragment bearing the truncated risA gene was inserted into the
EcoRI site of pSS1577 (35), generating pTM268. E. coli strain S17 bearing
pTM268 was mated with B. pertussis strain BP536, and exconjugates in which the
plasmid sequences had integrated into the chromosome were isolated by selec-
tion with gentamicin. An isolate in which plasmid sequences were lost from the
chromosome, but in which the in-frame truncation of risA was retained, was
isolated by selection for streptomycin resistance on BG plates and by screening
with PCR. This strain was designated TM1627.
Strain TM1793, which bears an internal in-frame deletion in bvgR, was con-
structed as follows: The SalI restriction fragment that contains the 3? end of the
bvgR gene, and extends 304 bp downstream of bvgR, was excised from pBBR:BgB
(19) and inserted into the SalI site in pTM025 (21) to generate pTM119. Plasmid
pTM119 was digested with ApaI, followed by treatment with mung bean nuclease
to generate blunt ends, digestion with StuI, and finally religation to circularize
the plasmid. These manipulations resulted in the loss of an internal 606-bp
fragment from bvgR. The plasmid bearing the in-frame internal truncation of
bvgR was designated pTM120. E. coli strain S17 bearing pTM120 was mated with
B. pertussis strain BP536, and exconjugates in which the plasmid sequences had
integrated into the chromosome were isolated by selection with gentamicin. An
isolate in which plasmid sequences were lost from the chromosome, but in which
the in-frame truncation in bvgR was retained, was isolated by selection for
streptomycin resistance on BG plates and by screening with PCR. This strain was
Construction of vrg promoter fusions. B. pertussis strains bearing fusions of the
vrg6, vrg18, vrg24, and vrg73 promoters to lacZ were constructed as follows.
Oligonucleotide pairs vrg6-F1/vrg6-B1, vrg18-F1/vrg18-B1, vrg24-F1/vrg24-B1,
and vrg73-F1/vrg73-B1 were used in PCRs using Tohama I chromosomal DNA as
a template (Table 2). The PCR products generated by the aforementioned PCRs
were cloned into pCR2.1-TOPO (Invitrogen, Carlsbad, Calif.). The full-length
vrg6, vrg18, vrg24, and vrg73 promoter fragments were excised from pCR2.1-
TOPO using the restriction enzymes, XbaI and SalI, and the excised fragments
were cloned into the previously described reporter plasmid, pSS2809 (8). The
pSS2809 derivatives were subsequently transferred by conjugation from E. coli
TABLE 1. Bacterial strains and plasmids
Strain or plasmidRelevant featuresSource
E. coli K-12
Tra functions of IncP plasmids integrated into chromosome
lacl, PlacUV5-T7 RNA polymerase, ?ompT, ?lon
Invitrogen, Carlsbad, Calif.
Novagen, San Diego, Calif.
Tohama I, NalrStrr
Tohama I, NalrStrr?risA
Tohama I, Nalr, Strr, ?bvgR
CBER Bordetella Collectiona
CBER Bordetella Collection
CBER Bordetella Collection
CBER Bordetella Collection
AprKanrrpsL oriT cos
AprGmroriT; B. pertussis DNA, E. coli lac operon
TA cloning vector
Lacl-repressed T7 promoter, 6His tag
AprKmrrpsL oriT cos bvgR
AprKmrrpsL oriT cos ?bvgR
AprGmrrpsL oriT cos ?risA
aCBER, Center for Biologics Evaluation and Research.
VOL. 187, 2005RisA ACTIVATION OF vrg61649
strain S17 into B. pertussis strains BP536, TM1627, and TM1793. Selection for
exconjugates was performed by plating onto BG plates containing gentamicin
and nalidixic acid. The identities of the resulting strains were confirmed by PCR.
Construction of promoter deletion fusions. A nested set of 5? deletions of the
vrg6 promoter fused to the lacZ gene were constructed as follows. Oligonucle-
otide vrg6-B1 (Table 2) was used in combination with oligonucleotides vrg6-F1,
-F2, -F3, -F4, -F5, -F6, -F7, and -F8 in PCRs using Tohama I chromosomal DNA
as a template. A nested set of 3? deletions of the vrg6 promoter fused to the lacZ
gene were constructed as follows. Oligonucleotide vrg6-F1 (Table 2) was used in
combination with oligonucleotides vrg6-B2, -B3, -B4, -B5, and -B6 in PCRs using
Tohama I chromosomal DNA as a template. The PCR products generated by the
aforementioned PCRs were cloned into pCR2.1-TOPO. The full-length pro-
moter fragment and each of the 5? and 3? deletions were excised by digesting with
XbaI and SalI, and the resulting fragments were cloned into the previously
described reporter plasmid, pSS2809 (8). These plasmids were then transferred
by conjugation from E. coli strain S17 into B. pertussis strains BP536, TM1627,
and TM1793. Selection for exconjugates was performed by plating onto BG
plates containing gentamicin and nalidixic acid. The identities of the resulting
strains were confirmed by PCR.
Quantitative ?-galactosidase assays. ?-Galactosidase assays were performed
as described by Miller (22) with minor modifications. Bacteria were recovered
from the plates with a sterile swab and were resuspended in 3.5 ml of 1 M
Tris-HCl, pH 8.0. The A600was measured. For measurement of ?-galactosidase
activity, 50 ?l of cell suspension was added to 1 ml of Z-buffer (0.1 M sodium
phosphate [pH 7.0], 10 mM KCl, 1 mM MgSO4, 50 mM mercaptoethanol). Cells
were permeabilized by adding 30 ?l of 0.1% sodium dodecyl sulfate and 30 ?l of
chloroform, followed by vortexing. The remainder of the assay was performed as
described by Miller (22). For quantification of ?-galactosidase activity, units were
defined by the following equation: Units ? 1,000 ? [A420? (1.75 ? A550)]/(T ?
V ? A600), where T is the incubation time in minutes and V is the volume (in
milliliters) of permeabilized cells added to the assay.
Sequence analysis of the risAS locus. Oligonucleotide risAS-F (5?-GCCGGC
GCGTGCCAGCAATTCCCGT-3?) was used in combination with oligonucleo-
tide risAS-B (5?-GGCCTCAAGCCCTAAATTCTACGCT-3?) in PCRs using
chromosomal DNA from four randomly selected B. pertussis clinical isolates
(Bp106, Bp188, Bp509, and Bp10536) as the template. The resulting PCR prod-
ucts containing the risAS locus were sequenced with a BigDye Terminator v1.1
sequencing kit (Applied Biosystems), and the reactions were analyzed on ABI
PRISM 3730xl DNA analyzers using Applied Biosystems sequence analysis soft-
ware. The sequences were edited with Sequencher version 4.1.2, and the data
obtained were assembled into contiguous sequences. The sequence of both
strands of each of the four amplified DNA fragments was determined and
compared to published sequences (15, 25).
Preparation of recombinant protein. An E. coli strain expressing recombinant
RisA protein was constructed as follows. Oligonucleotide risA-F1 (5?-CTCGA
GATGAACACGCAAAACACCACTCCT-3?) was used in combination with oli-
gonucleotide risA-B1 (5?-CTCGAGACTGCCGCCATCCGGAACGAAAAC-
3?) in a PCR using Tohama I chromosomal DNA as a template. The resulting
PCR product containing the risA open reading frame was cloned into pCR2.1-
TOPO, generating pTM275. The risA open reading frame was excised from
pTM275 as an XhoI fragment and was inserted into the XhoI site in the expres-
sion vector pET22b (Novagen/EMD Biosciences, Inc., San Diego, Calif.), gen-
erating pTM276. Plasmid pTM276 bears a C-terminal fusion of a sequence
encoding six histidine residues to the risA open reading frame under the tran-
scriptional regulation of a recombinant T7 promoter engineered to be regulated
by the E. coli lac repressor (LacI). Plasmid pTM276 was transformed into E. coli
strain BL21(DE3)pLysS (Novagen/EMD Biosciences, Inc., San Diego, Calif.). E.
coli strain BL21(DE3)pLysS, bearing pTM276, was grown in Luria broth at 37°C
to an optical density of 0.6, and subsequently the expression of the risA gene was
induced by the addition of IPTG (isopropyl-?-D-thiogalactopyranoside) to a final
concentration of 1 mM. Four hours after induction, cells were harvested by
centrifugation for 10 min at 5,000 ? g and were lysed with 8 M urea. Denatured
protein was purified with the QIAGEN Ni-nitrilotriacetic acid protein purifica-
tion kit (QIAGEN, Valencia, Calif.) according to the manufacturer’s instruc-
tions. Refolding of denatured protein was carried out by slow gradient dialysis
against dialysis buffer (10 mM HEPES-NaOH, 1 mM EDTA, 0.1 mM dithio-
threitol [DTT], pH 7.4). After removal of insoluble protein by centrifugation, the
supernatant was concentrated in a Centricon-10 centrifugal concentrator (Mil-
lipore, Billerica, Mass.). Glycerol was added to a final concentration of 40%, and
the samples were stored at ?20°C.
Gel shift assays. The vrg6 promoter fragment generated by PCR using oligo-
nucleotides F4 and B1 as described above, was excised from pCR2.1-TOPO by
digestion with XbaI and SalI. Following gel purification, the fragments were end
labeled with32P by T4-polynucleotide kinase reaction (Lofstrand, Gaithersburg,
Md.). Gel-shift reaction mixtures contained 10 ng of probe (2 ? 104dpm/ng), 100
ng of poly(dI:dC), and10 ?g of purified protein in binding buffer (10 mM
Tris-HCl [pH 7.8], 2 mM MgCl2, 50 mM KCl, 0.2 mM DTT). Reactions were
incubated for 20 min at 30°C. Prior to loading samples, the 6% polyacrylamide–
Tris-borate-EDTA (TBE) gels (Invitrogen, Carlsbad, Calif.) were pre-electro-
phoresed for 30 min. Electrophoresis was performed at 15 V/cm, after which gels
were exposed to a PhosphorImager screen (Molecular Dynamics, Sunnyvale,
Calif.), and the images were visualized with Imagequant software (Molecular
Generating mouse antiserum to recombinant RisA. Recombinant RisA was
mixed 1:1 (vol/vol) with a 0.65% solution of Alhydrogel (aluminum hydroxide;
Superfos a/s, Vedbaek, Denmark) to give a final protein concentration of 500
?g/ml. Each of five female BALB/c mice was injected intraperitoneally with 0.1
ml of the protein-adjuvant solution (50 ?g of protein). Booster doses were given
2 and 4 weeks after the initial injection. After the second booster dose, blood was
collected from the periorbital artery of each mouse. The serum was collected by
centrifugation and was stored at ?20°C.
Immunoblotting. After growth on BG plates in the presence or absence of 50
mM MgSO4, bacteria were resuspended in Laemmli buffer (62.5 mM Tris-HCl
[pH 6.8], 2.35% SDS, 100 mM DTT, 10% glycerol, 1 mM EDTA, 0.001%
bromphenol blue), and were lysed by boiling. Samples were separated by SDS-
polyacrylamide gel electrophoresis (SDS-PAGE) (10% polyacrylamide), and
proteins were transferred to nitrocellulose membranes using a wet tank immu-
noblotter (Bio-Rad, Hercules, Calif.). Nonspecific binding sites on the mem-
branes were blocked using 5% (wt/vol) dehydrated milk (Marvel) in phosphate-
buffered saline (PBS; blocking solution), and were probed with anti-RisA mouse
polyclonal antiserum diluted 1:1,000 in blocking solution. Membranes were
washed in PBS, and antigen-antibody complexes were detected with rabbit anti-
mouse immunoglobulin G (IgG) antibodies conjugated to horseradish peroxi-
dase diluted 1:500 in blocking solution. Cross-reacting proteins were visualized
with the TMB membrane peroxidase substrate system (KPL, Gaithersburg, Md.).
TABLE 2. Sequences of oligonucleotides used to generate
promoter deletion mutants
1650O ´ CRO ´INI´N ET AL.J. BACTERIOL.
Regulation of bvg-repressed genes by the risAS and bvgR
loci. Stenson et al. demonstrated that the expression of two
distinct bvg-repressed surface antigens was dependent upon an
intact risAS locus (31, 32). Based on this finding, we sought to
determine if other bvg-repressed genes in B. pertussis were
dependent upon the risAS locus for full expression. We cloned
the promoter regions of four bvg-repressed genes (vrg6, vrg18,
vrg24, and vrg73) into the promoter assay vector, pSS2809 (8),
as described in Materials and Methods. This vector has a
multiple cloning site for cloning promoter fragments upstream
of a promoterless lac operon. It also has multiple tandem
transcription terminators upstream of the cloning sites in order
to minimize transcriptional read-through in this region. A 2-kb
fragment of B. pertussis genomic DNA in the plasmid enables
insertion of the plasmid via homologous recombination into
the B. pertussis chromosome at a site distant from the vrg6,
vrg18, vrg24, or vrg73 loci. The activity of each promoter fusion
was assayed after growth of the bacteria in the absence or
presence of MgSO4in the wild-type, ?bvgR, and ?risA genetic
backgrounds (Fig. 1). As expected, all four bvg-repressed genes
were expressed in the wild-type strain when bacteria were
grown on BG plates in the presence of 50 mM MgSO4but were
not expressed when grown on BG plates in the absence of
MgSO4(Fig. 1). The expression of the four bvg-repressed
genes was increased between 6- and 20-fold when the bacteria
were grown in the presence of 50 mM MgSO4. When these
same promoter fusions were tested in a strain bearing an in-
frame deletion of the locus containing bvgR (?bvgR), all four
loci demonstrated the same high level of expression in the
presence of 50 mM MgSO4(Fig. 1). Although all four loci
demonstrated reduced levels of expression upon growth in the
absence of 50 mM MgSO4,the level of expression of each of
these genes was significantly higher than that observed in the
wild-type background in the absence of 50 mM MgSO4. This
result indicates that the expression of these four genes is de-
repressed in the absence of BvgR. When the transcriptional
activity of the four bvg-repressed promoters was tested in a
strain bearing an in-frame deletion of the risA gene (?risA), the
activity of each promoter was reduced to basal levels under all
conditions tested (Fig. 1). These results confirm that expres-
sion of the four bvg-repressed genes, examined herein, is re-
pressed by the product of the bvgR locus. These data also
demonstrate that expression of these four genes is dependent
on an intact risA locus.
RisS sequence analysis. The B. pertussis genome sequence
was determined by Parkhill and colleagues at The Wellcome
Trust Sanger Institute (25). According to the Sanger Institute
sequence, the B. pertussis risS coding sequence has a frameshift
mutation within a tract of three cytosine residues following
codon 233. This frameshift was not present in the B. pertussis
risS sequence reported by Jungnitz and colleagues (15). In
order to determine if risS is a pseudogene in B. pertussis, we
amplified and sequenced the DNA region containing the risAS
locus from four independent clinical isolates of B. pertussis.
The four isolates were selected randomly from the Center for
Biologics Evaluation and Research B. pertussis strain collec-
tion. Isolates Bp106 and Bp108 were isolated in the 1930s.
Isolate Bp509 was isolated in 1982. The isolation date of
Bp10536 is unknown. The sequence of the risAS locus in all
four clinical isolates was the same as that reported for the B.
pertussis risAS locus by Parkhill et al.
Deletion analysis of the vrg6 promoter. We generated dele-
tion mutants in an effort to define the upstream and down-
stream boundaries of the vrg6 promoter. A nested set of 5? and
3? deletions of the vrg6 promoter was constructed by PCR as
described in Materials and Methods (Fig. 2A). In order to
determine the activities of these promoter derivatives in vivo,
FIG. 1. Expression of bvg-repressed genes in bvgR and risA knock-
out strains. Fusions of the vrg6, vrg18, vrg24, and vrg73 promoters to the
E. coli gene encoding ?-galactosidase were constructed and crossed
onto the chromosome of wild-type strain BP536, strain BP536::?bvgR,
and strain BP536::?risA as described in Materials and Methods. The
?-galactosidase activity expressed by each of the resulting reporter
strains was determined after growth on BG plates in the presence
(black bars) or absence (gray bars) of 50 mM MgSO4. The statistical
significance for selected comparisons was determined by Student’s t
test analysis. The mean activity from each promoter in the ?bvgR
background when grown in the presence or absence of MgSO4was
compared to those levels measured in wild-type bacteria under the
same environmental conditions. Each result reported is the mean of at
least four independent assays. Error bars represent the standard de-
viation from the mean. Statistically significant differences (P ? 0.01)
are indicated with two asterisks.
VOL. 187, 2005RisA ACTIVATION OF vrg61651
FIG. 2. Deletion analysis of the vrg6 promoter. 5? and 3? deletions of the vrg6 promoter, fused to the E. coli gene encoding ?-galactosidase, were
constructed and crossed onto the chromosome of wild-type strain BP536, strain BP536::?bvgR, and strain BP536::?risA as described in Materials
and Methods. (A) Schematic diagram of the vrg6 promoter deletions. The endpoints of each deletion, relative to the transcription start site, are
shown in parentheses. The vrg6 coding region is indicated by a black box, and the putative repressor-binding site is indicated by a gray box. (B) The
1652 O ´ CRO ´INI´N ET AL.J. BACTERIOL.
the deletion mutants were inserted into promoter assay vector
pSS2809. The promoter deletion constructs were crossed into
the wild-type, ?bvgR, and ?risA genetic backgrounds, and the
?-galactosidase activity of each reporter strain was determined
after growth of the bacteria in the presence or absence of 50
mM MgSO4. In the ?risA background, all of the deletion
fragments exhibited only a very low level of activity (Fig. 2B).
This observation was in agreement with the previous finding
that an intact risA locus is required for expression of the vrg6
promoter (Fig. 1). In the wild-type background, promoter de-
letions ?1 to ?4 showed normal expression when the cells were
grown in the presence of 50 mM MgSO4, and approximately
sixfold repression when the cells were grown in the absence of
MgSO4(Fig. 2B; constructs ?1 to ?4). These levels are similar
to that seen with the full-length promoter. In the ?bvgR back-
ground, promoter deletions ?1 to ?4 had the same high level
of expression as the full-length vrg6 promoter when the bacte-
ria were grown in the presence of 50 mM MgSO4. In the
absence of 50 mM MgSO4, the level of expression observed in
the ?bvgR background was lower than that observed in the
presence of MgSO4but was significantly higher than the ex-
pression observed in the wild-type background in the absence
of MgSO4. This analysis demonstrated that deletion of all of
the sequences upstream of position ?271, relative to the tran-
scription start site, does not affect the regulated expression
from the vrg6 promoter. Deletion of the sequences up to po-
sition ?156 resulted in a complete loss of vrg6 promoter ac-
tivity under all conditions tested, indicating that sequences
between ?271 and ?156 are essential for vrg6 promoter activ-
ity (Fig. 2B; constructs ?5 to ?8).
Examination of the 3? promoter deletions revealed that de-
letion of all of the sequences downstream of position ?24,
relative to the transcription start site, does not affect the acti-
vation or repression of the vrg6 promoter (Fig. 2B; constructs
?C and ?E). In the wild-type background, promoter deletions
?C and ?E showed normal levels of expression when the cells
were grown in the presence of 50 mM MgSO4and approxi-
mately sixfold repression when the cells were grown in the
absence of MgSO4. In the ?bvgR background, promoter dele-
tions ?C and ?E demonstrated levels of expression similar to
that of the full-length vrg6 promoter when cells were grown in
the presence of 50 mM MgSO4. In the absence of 50 mM
MgSO4, the level of expression observed in the ?bvgR back-
ground was lower than that observed in the presence of MgSO4
but was significantly higher than the expression observed in the
wild-type background in the absence of MgSO4. In both the
wild-type and ?bvgR backgrounds, promoter deletion ?D,
which has a 3? endpoint between deletions ?C and ?E, dem-
onstrated a higher level of expression than the full-length pro-
moter and promoter deletions ?C and ?E. The expression of
promoter deletion ?D was repressed upon growth in the ab-
sence of MgSO4in the wild-type background, and that degree
of repression was significantly reduced in the ?bvgR back-
ground. Deletion of sequences from the 3? end up to position
?63 and beyond resulted in a complete loss of vrg6 promoter
activity under all conditions tested (Fig. 2B; constructs ?A and
?B). Taken together, these results indicate that no sequences
upstream of position ?271 or downstream of position ?24 are
required for the RisA-mediated activation or the BvgR-medi-
ated repression of the vrg6 gene. The results also indicate that
the sequences downstream of ?1, previously identified as the
BvgR-binding site, are not required for repression of vrg6 ex-
In vitro binding of RisA to the vrg6 promoter. The 5? and 3?
deletion analyses identified the upstream and downstream
boundaries of the vrg6 promoter, defining a 295-bp region
(?271 to ?24) that was required for the regulated activity of
the vrg6 promoter. Since both RisA and BvgR have been
shown to affect expression of vrg6, we hypothesized that bind-
ing sites for both proteins may be found within this 295-bp
region. In order to directly evaluate the interaction between
RisA and BvgR with the bvg-repressed promoters, we con-
ducted gel-shift assays. The coding sequences for both BvgR
and RisA were cloned into expression vector pET22b, and the
expressed proteins were purified as described in the Materials
and Methods. Both the BvgR and RisA proteins were ex-
pressed in large quantities upon induction and formed inclu-
sion bodies in E. coli. RisA refolded in soluble form upon serial
dialysis. However, BvgR remained an insoluble aggregate even
after dialysis. Therefore, we conducted the gel-shift assays us-
ing only RisA. The addition of purified RisA protein to a
reaction mixture containing
ment resulted in a mobility shift of the labeled promoter frag-
ment (Fig. 3A). Increasing the amount of protein added to a
constant amount of32P-labeled promoter fragment resulted in
an increase in the amount of probe that was shifted upward
(Fig. 3A). The binding interaction was specific as demon-
strated by competition for binding of RisA to the32P-labeled
fragment by unlabeled vrg promoter fragments (Fig. 3B). The
RisA protein bound to the32P-labeled vrg6 promoter fragment
in the absence of unlabeled DNA competitor. This binding was
completely blocked by the addition of unlabeled vrg6 and vrg18
promoter DNA but was not blocked by the addition of unla-
beled asd or sodB promoter DNA (Fig. 3B).
We utilized 45-bp, double-stranded oligonucleotides bearing
sequences derived from the vrg6 promoter region, to identify
RisA binding regions on the vrg6 promoter. We evaluated the
ability of the double-stranded oligonucleotides to compete
with the32P-labeled vrg6 probe for binding of RisA. A set of 15
double-stranded oligonucleotides that spanned the vrg6 pro-
32P-labeled vrg6 promoter frag-
?-galactosidase activity expressed by each of the reporter strains was determined after growth on BG plates in the presence (black bars) or absence
(gray bars) of 50 mM MgSO4. The statistical significance for selected comparisons was determined by Student’s t test analysis. The mean of the
activity of each promoter deletion in the presence of MgSO4, in the wild-type and ?bvgR backgrounds, was compared to the mean of the activity
of the full-length promoter in the presence of MgSO4, in the wild-type background. *, P ? 0.05; **, P ? 0.01. For each promoter, no statistically
significant difference was observed between the mean of the activity in the presence of MgSO4, in the ?bvgR background, and the mean of the
activity of the same construct in the presence of MgSO4, in the wild-type background. For each promoter, the mean of the activity in the absence
of MgSO4, in the ?bvgR background, was compared to the mean of the activity of the same construct in the absence of MgSO4, in the wild-type
background. †, P ? 0.01. Each result reported is the mean of at least four independent assays. Error bars represent the standard deviation from
VOL. 187, 2005RisA ACTIVATION OF vrg61653
moter was constructed (Table 3). The first eight oligonucleo-
tides (no. 1 to 8) spanned the 295-bp region known to be
required for the RisA-mediated activation of the promoter.
The next seven oligonucleotides (no. 12, 23, 34, 45, 56, 67, and
78) were constructed to overlap the first eight oligonucleotides
in order to account for any binding sites that may be disrupted
by linker design. The ability of these linkers to compete with
the32P-labeled vrg6 promoter DNA for RisA-binding was eval-
uated with gel-shift assays (Fig. 4). Four linkers (no. 2, 5, 12,
and 56) were identified that competed with the32P-labeled vrg6
probe (Fig. 4). Linkers 2 and 12 overlap each other, as do
linkers 5 and 56. However, linkers 2 and 12 do not overlap with
linkers 5 and 56 (Fig. 5B). Our results, therefore, define two
FIG. 3. Binding of RisA to the vrg6 promoter. Gel shifts were
performed with32P-labeled vrg6 promoter DNA and purified recom-
binant RisA protein as described in Materials and Methods. (A)32P-
labeled vrg6 promoter DNA was added to the gel-shift reaction mix-
ture containing either no RisA, undiluted RisA, or serial fivefold
dilutions of RisA. (B)32P-labeled vrg6 promoter DNA was added to
the gel-shift reaction mixture containing either no RisA protein, RisA
protein with no DNA competitor, or RisA protein with excess amounts
of double-stranded competitor DNA bearing either the vrg6, vrg18,
asd, or sodB promoter sequences.
TABLE 3. Sequences of the double-stranded DNA linkers used for
competition of RisA binding to the vrg6 promoter
1654O ´ CRO ´INI´N ET AL.J. BACTERIOL.
distinct regions of approximately 25 bp each, which contain a
RisA binding site.
Sequence analysis of putative RisA binding site. An exam-
ination of the sequences of the two 25-bp regions that were
shown to bind RisA revealed a conserved 7-bp sequence (5?-
AAATG/TTA-3?; Fig. 5B). A search of the 500 bp upstream of
the start of translation of the vrg6, vrg18, vrg24, and vrg73 genes
identified three matches to this sequence in the vrg6 promoter
region, two matches to this conserved sequence in the vrg18
promoter region, five matches in the vrg24 promoter region,
and a single match in the vrg73 promoter region (Fig. 5C).
Effect of bvgR and modulation on RisA expression and sta-
bility. The observation that the presumed repressor-binding
site in vrg6 does not contribute to repression of gene expres-
sion compelled us to consider the alternative mechanisms by
which BvgR may repress its target genes. We examined the
possible role of BvgR in the expression and stability of RisA.
The risA and bvgR promoters were inserted into the promoter
assay vector pSS2809. The promoter constructs were crossed
into the wild-type, ?bvgR, and ?risA backgrounds, and the
?-galactosidase activity of each reporter strain was determined
after growth in the presence or absence of 50 mM MgSO4(Fig.
6A and B). The expression and regulation of the bvgR pro-
moter were the same in the wild-type, ?bvgR, and ?risA back-
grounds, indicating that bvgR does not regulate its own expres-
sion, nor is its expression regulated by risA (Fig. 6A). The
expression of the risA promoter was the same in the wild-type
and ?risA backgrounds but higher in the ?bvgR background,
indicating that risA does not regulate its own expression (Fig.
Polyclonal antiserum to recombinant RisA was raised in
mice and was used to probe immunoblots containing cell ly-
sates prepared from wild-type, ?bvgR, and ?risA strains after
growth in the presence or absence of 50 mM MgSO4(Fig. 6C).
As expected, no RisA could be detected in the ?risA mutant
strain. The amounts of RisA protein present in cell lysates
were the same in both the wild-type and ?bvgR backgrounds
regardless of the growth conditions. These results indicate that
the level of expression and the stability of RisA are not affected
by BvgR- or bvg-mediated regulation.
The mechanism by which the expression of the bvg-repressed
genes is regulated is unknown. We demonstrated previously
that, in B. pertussis, the expression of the vrg6 and vrg73 genes
is repressed by the bvgAS-activated protein BvgR (21). Jung-
nitz et al. identified a locus in B. bronchiseptica, which they
designated as the risAS locus, that was required for survival of
B. bronchiseptica in eukaryotic cells and contributed to persis-
tence of the bacterium in a mouse infection model (15). Sten-
son et al. independently identified the risAS locus in B. pertussis
and demonstrated that it was required for the expression of
two distinct bvgR-repressed surface antigens (31, 32). In this
study, we examined the roles of BvgR and RisA in the regu-
lation of four bvg-repressed genes in B. pertussis (vrg6, vrg18,
vrg24, and vrg73). Our results demonstrate that the repression
of expression of all four of these genes is dependent upon
BvgR and the activation of expression of these genes is depen-
dent upon RisA (Fig. 1). The observation that the expression
of all of the bvg-repressed genes examined to date (vrg6, vrg18,
vrg24, vrg73, and the bvg-repressed surface antigens described
by Stenson et al.) is dependent upon RisA is striking. It sug-
gests that there is a large, if not complete, overlap of the BvgR
and RisA regulons. This might be expected if, for example,
BvgR represses expression of the bvg-repressed genes by con-
trolling the activity of RisA. Microarray and proteomic ap-
proaches should be undertaken to define the BvgR and RisA
regulons, in order to evaluate the extent to which they overlap.
Parkhill and colleagues first determined that in B. pertussis
the risS gene is disrupted by a frameshift mutation (25). We
have confirmed the presence of the same frameshift in the risS
gene in four randomly selected clinical isolates. The isolation
of these four strains was separated both geographically and by
time (separated by nearly 50 years). It is clear from our results
that the risA locus is required for expression of the bvgR-
repressed genes; yet it is equally clear that the risS gene does
not encode a functional RisS protein. Given these facts, we
conclude that either RisA is able to activate transcription at its
target promoters in the unphosphorylated state or RisA is
phosphorylated by a sensor kinase other than RisS.
FIG. 4. Identification of RisA binding regions. Gel-shift assays were performed with32P-labeled vrg6 promoter DNA and purified recombinant
RisA protein as described in Materials and Methods.32P-labeled vrg6 promoter DNA was added to gel-shift reaction mixture containing either no
RisA protein or undiluted RisA protein and excess double-stranded linker DNA derived from the vrg6 promoter sequence. The sequence of each
of the linkers is provided in Table 3. The position of each linker sequence within the vrg6 promoter is shown schematically in Fig. 5. (A) Com-
petition of RisA binding to32P-labeled vrg6 promoter DNA by linkers 1 to 8. (B) Competition of RisA binding to32P-labeled vrg6 promoter DNA
by linkers 12 to 78.
VOL. 187, 2005RisA ACTIVATION OF vrg61655
Once we demonstrated that RisA activates the expression of
the bvg-repressed genes, we focused on identifying the cis-
acting sequences that are required for RisA activation of the
bvg-repressed genes. We noted that Beattie et al. had per-
formed a deletion analysis of the vrg6 promoter (2) and dem-
onstrated that as progressively larger fragments of the pro-
moter were deleted from the upstream side between positions
?428 and ?221, relative to the transcription start site, almost
a fourfold drop in vrg6 promoter activity was observed. Beattie
et al. also observed a fivefold drop in promoter activity when
the deletions were extended further to position ?186, and
activity was further diminished as the sequence was deleted
even further toward the transcription start site. The activity of
all of the constructs generated by Beattie et al. appeared to be
FIG. 5. Putative consensus RisA binding site. (A) Schematic dia-
gram of the vrg6 promoter sequence. The black box marks the vrg6
coding region, and the gray box marks the putative repressor-binding
site. The arrow marks the transcription start site (?1). (B) Schematic
diagram showing the positions of the double-stranded linkers used as
competitors in the gel-shift assays. The linkers that inhibited binding of
RisA protein to the32P-labeled vrg6 promoter DNA are circled, and
the sequences of the regions of overlap between the two sets of linkers
that inhibited binding of RisA protein to the vrg6 promoter DNA are
shown. A conserved sequence found in both overlapping regions is
shaded. (C) Alignment of sequences from the vrg6, vrg18, vrg24, and
vrg73 promoter regions that were found to contain the conserved
element (putative RisA binding site). The conserved element in each
sequence is shaded. Because the transcription start site for four of the
five genes is unknown, the upstream and downstream boundaries of
each sequence relative to the translation start site (ATG) of each gene
are shown in parentheses.
FIG. 6. Regulation of bvgR and risA expression. Fusions of the
bvgR and risA promoters to the E. coli gene encoding ?-galactosidase
were constructed and crossed onto the chromosome of wild-type strain
BP536, strain BP536::? bvgR, and strain BP536::?risA as described in
Materials and Methods. The ?-galactosidase activity expressed by each
of the bvgR-lacZ reporter strains (A) and each of the risA-lacZ re-
porter strains (B) was determined after growth on BG plates in the
presence (black bars) or absence (gray bars) of 50 mM MgSO4. (C) Im-
munoblot analysis of samples prepared with protein extracts obtained
from wild-type strain BP536, strain BP536::?bvgR, and strain
BP536::?risA after growth on BG plates in the presence (?) or ab-
sence (?) of 50 mM MgSO4. The blots were probed with anti-RisA
polyclonal antibody. The statistical significance for selected compari-
sons was determined by Student’s t test analysis. The mean activity
from either the bvgR or risA promoter, in the presence or absence of
MgSO4in each mutant background, was compared to that of the same
promoter in wild-type bacteria grown under the same environmental
conditions. Statistically significant differences are indicated with an
asterisk (P ? ? 0.05) or a double asterisk (P ? 0.01). Each result
reported is the mean of at least four independent assays. Error bars
represent the standard deviation from the mean.
1656O ´ CRO ´INI´N ET AL.J. BACTERIOL.
repressed upon growth under modulating conditions. These
data suggested that an important promoter element lay be-
tween positions ?221 and ?186. However, it also appeared
that sequences upstream of position ?221 contributed to pro-
moter activity. We noted that Beattie and colleagues per-
formed their analysis with promoter fusions carried on a plas-
mid present at a concentration of five to seven copies per cell.
We hoped a deletion analysis performed with fusions main-
tained in single copy would yield a clearer picture of the up-
stream boundary of the vrg6 promoter. We also sought to take
advantage of the ability to examine the activity of each fusion
in both a ?bvgR background and a ?risA background. Our
deletion analysis of the vrg6 promoter demonstrated that all of
the sequences required for regulated expression of vrg6 were
downstream of position ?271 relative to the transcription start
site (Fig. 2). All of the deletion fragments with upstream
boundaries either at or further upstream than position ?271
retained 100% of promoter activity. This activity was depen-
dent upon RisA and was repressed by BvgR. All of the up-
stream deletions that extended into the promoter to position
?159 or beyond lost all RisA-dependent promoter activity, and
the basal level of expression observed in those fusions was not
repressed by BvgR (Fig. 2). Our results are in general agree-
ment with those of Beattie et al. Our results indicate that the
upstream boundary of an important promoter element lies
between positions ?271 and ?159 relative to the transcription
start site. However, our results clearly demonstrate that no
sequences upstream of position ?271 contribute to promoter
activity. Finally, we did not see any evidence of BvgR-mediated
repression of promoter fusions that had 5? deletions that ex-
tended beyond position ?159.
We extended the promoter deletion analysis by generating
deletions from the 3? end (Fig. 2). Our results demonstrate that
promoter fragments with deletions from the 3? end that ex-
tended as far as position ?24, relative to the transcription start
site, retained 100% of promoter activity. The activity of all
three of these promoter fusions was dependent upon RisA,
and, most significantly, despite the lack of the putative repres-
sor-binding site in two of the constructs, BvgR repressed the
activities of all three of these fusions. As expected, deletions
from the 3? end that extended as far as positions ?63 and
?130, relative to the transcription start site, lost all RisA-
dependent promoter activity and BvgR did not repress the
basal level of expression observed in those fusions (Fig. 2).
Therefore, we have concluded that sequences downstream of
the start of transcription are not required for repression of the
vrg6 gene and probably are not required for repression of any
of the bvg-repressed genes. In retrospect, reexamination of the
data that led to the identification of the repressor-binding site
within the coding region of vrg6 suggests that what was inter-
preted previously as a loss of repression of the vrg6 promoter
constructs (2, 3) was in fact a loss of vrg6 promoter induction.
Our promoter deletion analysis demonstrated that all of the
sequences required for the regulated expression of the vrg6
gene lay between position ?271 and ?24, relative to the tran-
scription start site. Since all of the promoter fragments that
demonstrated activity were repressed by BvgR, we concluded
that the sequences required for activation of the promoter by
RisA, and repression of the promoter by BvgR, lie within that
295-bp region. We attempted to overexpress and purify both
BvgR and RisA for use in an in vitro DNA-binding assay.
Although the purification of RisA was relatively straightfor-
ward, multiple strategies to purify soluble BvgR were unsuc-
cessful. A gel-shift analysis clearly demonstrated that purified
RisA binds to the vrg6 promoter (Fig. 3). Binding of RisA to a
32P-labeled vrg6 promoter fragment was blocked by excess
unlabeled DNA fragments containing the vrg6 or vrg18 pro-
moters but not by unlabeled fragments containing the asd or
sodB promoters. This result indicated that binding of RisA to
the vrg6 promoter was sequence specific and also demon-
strated that RisA binds to the vrg18 promoter. By evaluating
the ability of a collection of 15 overlapping 45-bp double-
stranded oligonucleotides bearing sequences from the vrg6
promoter, to block binding of RisA to the
promoter fragment, two distinct RisA-binding regions were
identified (Fig. 4). Although none of the linkers completely
eliminated the gel mobility shift, four linkers clearly inhibited
RisA binding to the vrg6 probe, resulting in a reduced shift in
mobility. Interestingly, these four linkers consisted of two pairs
of overlapping linkers. This observation suggested that the
RisA binding regions are located on the two 22-bp regions
defined by the overlap between linkers 2 and 12 and linkers 5
and 56. A comparison of these two sequences revealed a con-
served 7-bp sequence that is present in both regions (5?-AAA
TT/GTA-3?) (Fig. 5B). Although close approximations of this
sequence are found in the vrg18, vrg24, and vrg73 promoter
sequences (Fig. 5C), it should be noted that a nearly perfect
match to this sequence (5?-AAATTTG-3?) is found at a third
position in the vrg6 promoter, and is incorporated into linker
34, which did not compete with the vrg6 promoter probe for
binding of RisA. Although it is tempting to focus on this
sequence element, definition of the specific sequences that
contribute to the binding of RisA will require a more rigorous
analysis of RisA binding to the risA-activated promoters.
To date, attempts at identifying specific cis-acting sequences
involved in repression of expression of the vrg6 gene have not
been successful. This has led us to consider alternative models
for BvgR-mediated repression of vrg6 promoter activity (i.e.,
models that do not require direct binding of BvgR to the
bvg-repressed promoters). One possibility is that BvgR re-
presses the expression of RisA or reduces the stability of RisA
once it is synthesized. To address this question, we examined
the expression of risA-lacZ fusions in the wild-type, ?bvgR, and
?risA backgrounds (Fig. 6B). Our results indicated that BvgR
does not repress risA transcription. It also indicated that risA
transcription is not autoregulated. This conclusion was con-
firmed by immunoblot analysis which demonstrated the pres-
ence of steady-state levels of RisA in wild-type, ?bvgR, and
?risA strains (Fig. 6C). The observation that the amount of
RisA present in cells is constant in the wild-type and ?bvgR
genetic backgrounds, after growth in the presence or absence
of MgSO4, demonstrates that BvgR neither regulates the tran-
scription or translation of RisA, nor does it mediate its stability
once the protein is expressed. It seems unlikely that BvgR acts
independently of RisA at the vrg6 promoter since BvgR does
not repress the residual RisA-independent activity of the vrg6
promoter. We speculate that if BvgR acts by binding to the vrg6
promoter, its binding may interfere with the binding of RisA
rather than interfere with the binding of the polymerase. Al-
ternatively, BvgR may not directly repress expression of the
VOL. 187, 2005RisA ACTIVATION OF vrg61657
bvg-repressed genes, but rather, it may exert its effect indirectly
through RisA either by binding RisA and preventing its inter-
action with the promoter or by modifying RisA so that the
protein is no longer active. Ongoing work in our laboratory is
directed toward distinguishing between these alternative mod-
We thank Gopa Raychaudhuri, Michael Schmitt, and Scott Stibitz
for their critical reading of the manuscript.
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