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J Bacteriol, March 1998, p. 1567-1569, Vol. 180, No. 6
0021-9193/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Hybrid Bordetella
pertussis-Escherichia coli RNA Polymerases: Selectivity of
Promoter Activation
Pierre
Steffen and
Agnes
Ullmann*
Unité de Biochimie Cellulaire, Institut
Pasteur, 75724 Paris Cedex 15, France
Received 24 October 1997/Accepted 9 January 1998
 |
ABSTRACT |
We constructed hybrid Bordetella
pertussis-Escherichia coli RNA polymerases and compared
productive interactions between transcription activators and
cognate RNA polymerase subunits in an in vitro transcription system.
Virulence-associated genes of B. pertussis, in the presence
of their activator BvgA, are transcribed by all variants of hybrid RNA
polymerases, whereas transcription at the E. coli lac
promoter regulated by the cyclic AMP-catabolite gene activator protein
has an absolute requirement for the E. coli 
subunit.
This suggests that activator contact sites involve a high degree of
selectivity.
| |
TEXT |
In Bordetella
pertussis, the causative agent of whooping cough, the
expression of virulence-associated genes including fhaB, cyaA, and ptx, encoding filamentous
hemagglutinin, adenylate cyclase-hemolysin, and pertussis toxin,
respectively, is controlled by the BvgA-BvgS two-component
system (1, 8, 13, 15, 16, 21). BvgA is a global
transcriptional regulator that is phosphorylated by the BvgS sensor
kinase (22, 26-28). Phosphorylation of BvgA increases its affinity for target promoter sequences and confers on it the capacity to function as a transcriptional activator at several virulence-regulated promoters (2, 3, 10, 18, 29).
The degree of BvgA phosphorylation differentially affects its DNA
binding and transcriptional activation properties (2, 3, 10,
18, 29). In addition, differences in the DNA binding sites for BvgA at various virulence-associated promoters (2, 4, 10, 11, 29) suggest that the specificity of protein-RNA polymerase (RNAP) interactions may control transcriptional
activation at these promoters. It has been demonstrated in
vitro, with a set of mutated reconstituted Escherichia coli
RNAPs, that the C-terminal domain of the subunit (-CTD) of
E. coli RNA polymerase is required for BvgA-dependent
transcription at the fha promoter (4). However,
in a recent study we reported that the RNAP 
subunit of
B. pertussis confers enhanced expression of
fhaB in E. coli (19), indicating that
determinants in are important for BvgA-dependent transcription in
vivo.
The and major subunits of the B. pertussis RNAP
have been cloned and characterized (6, 19). These subunits
are the most commonly contacted activator targets on the bacterial RNAP (5). To further characterize the BvgA-RNA polymerase
interactions responsible for transcriptional activation of
B. pertussis virulence genes, we developed an in vitro
transcription system using hybrid B. pertussis-E. coli
RNAPs containing the and/or the subunits of B. pertussis. We demonstrate the use of this system for the study of
RNAP-activator interactions.
Construction of hybrid reconstituted B. pertussis-E. coli RNAPs.
We obtained hybrid reconstituted
RNAPs by the method described by Tang et al. for the E. coli RNAP (23, 24). Briefly, each single subunit of the
RNAP was overexpressed separately in E. coli BL21. Then, 6 M
guanidine hydrochloride extracts containing the different RNAP subunits
were prepared, and the core enzyme-containing fractions were mixed.
Renaturation of the core enzyme and the sigma-containing fraction was
performed by dialysis. The renatured core enzyme and sigma fraction
were mixed, and the reconstituted holoenzyme was purified by using
Ni2+ affinity chromatography, taking advantage of the
fact that the subunit was His tagged. For overexpression
of the E. coli RNAP subunits, we used the vectors described
by Tang et al. (24). Overexpression of the B. pertussis RNAP subunits was performed by using the pET/BL21 system
(Novagen) with the vectors pES80 and pES70 for the subunits and
pE6HNBp for the subunit. pES80 coding for the major sigma
factor, 80, of B. pertussis and pES70
coding for an N-terminally truncated form of 80,
named 72, were described earlier (19).
pE6HNBp is a derivative of pET19 (Novagen) containing the
sequence of the B. pertussis rpoA gene (6).
This construction allowed the obtention of the B. pertussis subunit with an N-terminal extension including a
6-His tag followed by an enterokinase cleavage site.
Reconstitution mixtures containing the and/or 80
or 72 subunit of B. pertussis were
performed by using the same protocol as previously described for the
E. coli RNAP (23). We constructed and purified
the following enzymes:
Bp2(
'70)Eco,
Bp2(')Eco72Bp,
Bp2(')Eco80Bp,
(2')Eco72Bp,
and
(2')Eco80Bp,
and typical purities, as measured by sodium dodecyl
sulfate-polyacrylamide gel electrophoresis (Fig.
1), were about 95%. The transcriptional activities of the hybrid enzymes were very similar to the activity of
the wild-type B. pertussis RNAP, with values of
approximately 50 U/mg (18), thus demonstrating that
heterologous subunits can be successfully assembled to yield functional
hybrid RNAPs.
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FIG. 1.
Sodium dodecyl sulfate-polyacrylamide gel
electrophoresis analysis of the purified RNAPs. Samples of the enzymes
were separated on a 7.5% gel and stained with Coomassie blue. The
compositions of the enzymes are indicated at the top of the figure.
Lane M, protein standards (sizes in kilodaltons are shown at left).
Subunit assignments for the different RNAPs are at right.
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Transcriptional activities of the reconstituted hybrid RNAPs
on the trc and lac promoters.
In
vitro transcription assays of the strong, artificial
trc promoter and of the cyclic AMP-catabolite gene
activator protein (cAMP-CAP)-dependent lac promoter, both
present on superhelical templates, were performed as described
previously (12, 18). As shown in Fig.
2A, the trc promoter is as
efficiently transcribed by the hybrid RNAPs as by the wild-type
B. pertussis and E. coli enzymes
(PhosphorImager measurements indicated that the relative amounts of
trc transcripts varied from 70 to 100% compared to the
amount yielded by the B. pertussis wild-type RNAP).
This result demonstrates the functionality of the hybrid polymerases in
an in vitro transcription system. Next we tested their properties on
the cAMP-CAP-activatable lac promoter.
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FIG. 2.
In vitro transcription of the trc and
lac promoters. All reactions were carried out as described
earlier (12, 18). Samples were analyzed on denaturing
6% polyacrylamide-6 M urea gels. (A) The trc
promoter is activated by all tested RNAPs. The trc
transcript is indicated. Lane 1, Bp2('70)Eco; lane
2, Bp2(')Eco72Bp;
lane 3, (2')Eco72Bp;
lane 4, Bp2(')Eco80Bp;
lane 5, (2')Eco80Bp;
lane 6, wild-type B. pertussis; lane 7, wild-type
E. coli. (B) The subunit of the E. coli RNAP
is necessary for activation of the lac promoter. The
lac and RNA1 and RNA2 control transcripts are indicated. The
RNAP assignments are shown at the top of the panel. The presence (+) or
absence ( ) of cAMP-CAP is indicated.
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The
lac promoter was not recognized by the
B. pertussis wild-type RNAP in the presence of cAMP-CAP (Fig.
2B,
lane 9), whereas
the
E. coli wild-type RNAP and
(
2')
Eco80Bp
efficiently transcribed the
lac promoter in the presence of
cAMP-CAP (Fig.
2B, lanes 7 and 3, respectively). The transcription
efficiencies were compared to two constitutive transcripts generated
by
the plasmid vector, RNA1 and RNA2 (
12). None of the hybrid
RNAPs containing the
subunit of
B. pertussis RNAP
[
Bp2(
'
70)
Eco and
Bp2(
')
Eco80Bp]
allowed transcription of the
lac promoter, whether in the
absence or in the presence of cAMP-CAP, whereas RNA1 and RNA2
transcripts were efficiently produced (Fig.
2B, lanes 1 and 5).
This
result confirms earlier reports (
7,
9) that the
subunit
of
E. coli RNAP is indispensable for
transcription of the cAMP-CAP-dependent
lac promoter. It
also suggests that different specificities regarding
activator contact
sites exist between the
subunits of
B. pertussis and
E. coli, despite their striking sequence homologies (see
below).
In vitro transcription of bvg-regulated genes.
Previously we found that, compared to the B. pertussis
RNAP, the E. coli RNAP was much less, if at all, efficient
in the transcription of the fha, cya, and
ptx promoters in the presence of phosphorylated BvgA
(BvgA-P) (18). We therefore performed in vitro transcription assays similar to those performed earlier (18) on the
fha, cya, and ptx promoters with the
purified hybrid RNAPs. As shown in Fig.
3, all hybrid polymerases allowed
transcription at the fha, cya, and
ptx promoters in the presence of BvgA-P (lanes 2, 4, 6, 8, and 10), and virtually no transcripts were observed in
the absence of BvgA-P (lanes 1, 3, 5, 7, and 9). The
Bp2('70)Eco enzyme
transcribed these promoters almost as efficiently as the wild-type
B. pertussis RNAP (Fig. 3, lanes 2 and 12), whereas the
80-containing hybrid enzymes exhibited lower
activities (Fig. 3, lanes 8 and 10). Strikingly, B. pertussis 80-containing hybrid enzymes
were particularly inefficient in transcribing the ptx
promoter (lanes 8 and 10). This may be due to specific features in
ptx promoter architecture (20) and/or to an
inhibiting effect of the N-terminal extension present in the
B. pertussis major sigma factor. Indeed, we
frequently noted that our wild-type B. pertussis
RNAP preparations contained, in part, N-terminally truncated
proteolyzed forms of 80 (18, 19).
Nevertheless, when the subunit is 80, stringent
ptx promoter interactions might require B. pertussis wild-type RNAP architecture to correctly position the
N-terminal extension of 80. The reduced transcription
effect observed with 80 could be relieved by using
hybrid E. coli-B. pertussis RNAPs containing
72, an N-terminally truncated form of
80
[(2')Eco72Bp
and
Bp2(')Eco72Bp].
Hybrid RNAPs containing 72 transcribed all three
promoters with efficiencies similar to that of the B. pertussis wild-type RNAP (Fig. 3, lanes 4, 6, and 12). Under
similar conditions, the transcription efficiencies obtained with
wild-type E. coli RNAP for the cya and
ptx promoters were about 10 to 15% and for the
fha promoter it was about 80% of that obtained with
wild-type B. pertussis RNAP (data not shown). Unexpectedly, hybrid RNAPs containing B. pertussis factors efficiently transcribed all three
virulence-associated promoters in vitro, which was not case when the
B. pertussis factor replaced a thermosensitive E. coli factor in vivo (19).
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FIG. 3.
In vitro transcription of bvg-regulated
promoters in the presence or absence of BvgA-P. All reactions were
carried out as described earlier (18) with slight
modifications: the reactions were carried out in 20-µl volumes,
BvgA-P was added prior to RNAP, and the reactions were stopped by the
addition of 10 µl of formamide loading buffer (17). A
total of 10 µl of each reaction mixture was then analyzed on
denaturing 6% polyacrylamide-urea gels. RNAP assignments are shown at
the top, and the fha, cya, and ptx
transcripts are indicated. The presence (+) or absence () of BvgA-P
is shown. Relative amounts of transcripts were evaluated by
PhosphorImager measurements and normalized for the various RNAPs by the
respective activities obtained on the trc promoter. Note
that levels of transcripts initiated at different promoters are not
comparable because of different compositions of messenger RNAs and
different film exposure times.
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|
In conclusion, all of the tested hybrid RNAPs,
Bp2 (
'
70)
Eco,
Bp2(
')
Eco72Bp,
Bp2(
')
Eco80Bp, (
2')
Eco72Bp,
and
(
2')
Eco80Bp,
were able, in the presence of BvgA-P, to initiate transcription
at
the
fha,
cya, and
ptx promoters with
good efficiencies. This
suggests that these polymerases contain all the
determinants necessary
for in vitro BvgA-dependent transcriptional
activation. The high
levels of transcription activity obtained with
hybrid RNAPs compared
to that obtained with wild-type
E. coli RNAP could, in part, be
accounted for by more than
stoichiometric amounts of different
factors present in the hybrid
RNAP preparations (see Fig.
1).
Recently, Boucher et al. (
4)
used the wild type and a set of
truncated as well as mutated
reconstituted
E. coli RNAPs to study
BvgA-RNA polymerase
interactions at the
fha promoter. They reported
that under
these conditions BvgA-P contacts the
-CTD, especially
residues R265
and N268, for transcriptional activation. It has
been shown that the
same residues are crucial for cAMP-CAP-dependent
activation of the
lac promoter (
14,
25). It is most intriguing
that
the
subunit of
B. pertussis RNAP, in spite of the
high
overall sequence homology with the corresponding
E. coli protein
(61% identity), cannot mediate cAMP-CAP-dependent
activation of
the
lac promoter. This result is even more
striking because the
residues important for
E. coli -CTD
and cAMP-CAP interaction
at the
lac promoter (
14,
25) are all conserved, with the exception
of one homologous
replacement in the
B. pertussis -CTD (Fig.
4). This suggests that the
protein-protein contacts between transcriptional-activating
regions and
the corresponding RNAP targets display a high degree
of selectivity.
These contacts might be highly optimized and thus
specific for one
given promoter but completely nonfunctional for
other promoters despite
the high degree of conservation between
the transcriptional
machineries. Therefore, the results obtained
with the use of
heterologous RNAPs to study transcriptional activation
of specific
promoters should be interpreted with caution.
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FIG. 4.
Comparison of amino acid sequences of the -CTDs of
E. coli and B. pertussis. The sequence
between residues 249 and 329 of the E. coli subunit was
compared with the corresponding region of the B. pertussis subunit. The conserved residues are shaded, and the
residues important for cAMP-CAP-dependent activation of the
lac promoter are marked by asterisks (14, 25).
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ACKNOWLEDGMENTS |
We thank Nick Carbonetti and Roy Gross for kindly providing
pNMD120, Hong Tang and Richard Ebright for kindly providing the E. coli RNAP subunits containing plasmids, Annie Kolb for
her kind gift of purified CAP and a lac promoter-containing
template, Sophie Goyard and Gouzel Karimova for helpful
discussions, and Bríd Lefévère-Laoide for
critically reading the manuscript.
Financial support came from the Institut Pasteur, Centre National de la
Recherche Scientifique (URA 1129), and the Human Frontier Science
Program Organization. P.S. was supported by a fellowship from the
Fondation pour la Recherche Médicale.
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FOOTNOTES |
*
Corresponding author. Mailing address: Unité de
Biochimie Cellulaire, 28 rue du Dr Roux, 75724 Paris Cedex 15, France.
Phone: 33 (1) 45 68 83 85. Fax: 33 (1) 40 61 30 19. E-mail:
ullmann{at}pasteur.fr.
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J Bacteriol, March 1998, p. 1567-1569, Vol. 180, No. 6
0021-9193/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
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