Antirepression as a second mechanism of transcriptional activation by a minor groove binding protein

Article (PDF Available)inMolecular Microbiology 64(2):368-81 · May 2007with18 Reads
DOI: 10.1111/j.1365-2958.2007.05662.x · Source: PubMed
Abstract
Competence for genetic transformation in the bacterium Bacillus subtilis is a bistable differentiation process governed by the minor groove DNA binding protein ComK. No detectable comK transcription occurs in the absence of an intact comK gene, indicating that ComK has auto-activating properties. ComK auto-stimulation, which is dependent on ComK binding to the comK promoter, is a critical step in competence development, ensuring quick and high-level expression of the late-competence genes. Auto-stimulation is also essential for the bistable expression pattern of competence. Here, we demonstrate that ComK acts as an activator at its own promoter by antagonizing the action of two repressors, Rok and CodY. Importantly, antirepression occurs without preventing binding of the repressing proteins, suggesting that ComK and the repressors might bind at distinct surfaces of the DNA helix. DegU, a DNA binding protein known to increase the affinity of ComK for its own promoter, potentiates the antirepression activity of ComK. We postulate that antirepression is primarily achieved through modulation of DNA topology. Although to our knowledge ComK is the only DNA binding protein shown to act in this novel fashion, other minor groove binding proteins may act similarly.
Antirepression as a second mechanism of
transcriptional activation by a minor groove
binding protein
Wiep Klaas Smits,
1
Tran Thu Hoa,
2†
Leendert W. Hamoen,
1‡
Oscar P. Kuipers
1
and
David Dubnau
2
*
1
Department of Genetics, University of Groningen,
Kerklaan 30, 9751NN, Haren, the Netherlands.
2
Public Health Research Institute, 225 Warren St,
Newark, NJ 07103-3535, USA.
Summary
Competence for genetic transformation in the bacte-
rium Bacillus subtilis is a bistable differentiation
process governed by the minor groove DNA binding
protein ComK. No detectable comK transcription
occurs in the absence of an intact comK gene, indi-
cating that ComK has auto-activating properties.
ComK auto-stimulation, which is dependent on ComK
binding to the comK promoter, is a critical step in
competence development, ensuring quick and high-
level expression of the late-competence genes.
Auto-stimulation is also essential for the bistable
expression pattern of competence. Here, we demon-
strate that ComK acts as an activator at its own pro-
moter by antagonizing the action of two repressors,
Rok and CodY. Importantly, antirepression occurs
without preventing binding of the repressing pro-
teins, suggesting that ComK and the repressors
might bind at distinct surfaces of the DNA helix.
DegU, a DNA binding protein known to increase the
affinity of ComK for its own promoter, potentiates the
antirepression activity of ComK. We postulate that
antirepression is primarily achieved through modula-
tion of DNA topology. Although to our knowledge
ComK is the only DNA binding protein shown to act in
this novel fashion, other minor groove binding pro-
teins may act similarly.
Introduction
In bacteria, the activation of transcription typically involves
a DNA binding protein that recognizes a sequence
upstream from the RNA polymerase (RNAP) binding site,
resulting in the recruitment of RNAP to the promoter or
stabilization of RNAP binding. Indeed, the latter mecha-
nism obtains in the case of ComK, the master regulator of
competence for genetic transformation (Susanna et al.,
2004).
Competence, the ability to take up DNA from the envi-
ronment and integrate it into the genome, is the end-point
of a complex developmental process resulting in the
expression of ComK (van Sinderen et al., 1995). This
activator drives the expression of a multitude of genes,
including those encoding the DNA uptake and integration
machinery (Berka et al., 2002; Hamoen et al., 2002;
Ogura et al., 2002). In contrast to the majority of DNA
binding regulatory proteins, ComK activates transcription
by binding in the minor groove of the DNA, upstream of its
target genes (Hamoen et al., 1998).
In B. subtilis there is no detectable comK–lacZ expres-
sion in the absence of functional ComK, demonstrating
that ComK has auto-activating properties (van Sinderen
and Venema, 1994). ComK exerts this auto-activation by
direct binding to its own promoter, PcomK (van Sinderen
and Venema, 1994). This binding is stimulated by the
presence of the priming protein DegU, which exerts a
positive effect on competence development (Hamoen
et al., 2000). Although the transcription of the downstream
comG operon does not occur in vitro or in Escherichia coli
unless ComK is provided, the transcription of comK itself
occurs readily under both conditions in the absence of
ComK (van Sinderen et al., 1995; below and Fig. S1).
These apparent discrepancies suggest that ComK acts in
vivo at PcomK by antagonizing the action of one or more
repressor proteins. Three candidate repressors have
been identified that directly repress transcription from the
comK promoter; the transition state regulator AbrB
(Hamoen et al., 2003), the nutritional regulator CodY
(Serror and Sonenshein, 1996) and Rok (Hoa et al.,
2002).
In this study, we demonstrate that ComK acts as an
antirepressor at its own promoter in vivo as well as in vitro,
Accepted 10 February, 2007. *For correspondence. E-mail dubnau@
phri.org; Tel. (+1) 973 854 03400; Fax (+1) 973 854 3401. Present
addresses:
Department of Microbiology, Faculty of Pharmacy, Uni-
versity of Medicine and Pharmacy of Ho Chi Minh City, 41 Dinh Tien
Hoang St., Dist.1, HCMC, Vietnam;
Institute for Cell and Molecular
Biosciences, The Medical School, University of Newcastle, Framling-
ton Place, Newcastle, NE2 4HH, UK.
Molecular Microbiology (2007) 64(2), 368–381 doi:10.1111/j.1365-2958.2007.05662.x
© 2007 The Authors
Journal compilation © 2007 Blackwell Publishing Ltd
relieving Rok- and CodY-mediated repression. Strikingly,
it does so without preventing binding of the repressors.
The antirepression activity of ComK is potentiated by
DegU, which also binds to the promoter of comK. Inter-
estingly, ComK is able to activate transcription of recA by
reversing the repression exerted by LexA (Hamoen et al.,
2001). ComK thus activates transcription in two distinct
manners; as a classical transcriptional activator at down-
stream competence genes such as comG, and as an
antirepressor at PcomK and PrecA. To our knowledge,
ComK is the only DNA binding protein proven to reverse
transcriptional repression without eliminating the binding
of the repressors, although this novel mechanism may
reflect a property of other proteins that bind through the
minor groove of DNA.
Results
Rationale for the antirepression hypothesis
ComK acts as a transcriptional activator at PcomK and at
downstream competence promoters, such as the pro-
moter of comG (PcomG). ComK is required for in vitro
transcription from PcomG (Hamoen et al., 1998; Susanna
et al., 2004) but when PcomK was incubated with nucle-
otides and RNAP in the absence of ComK, strong tran-
scription was observed (see below). Additionally, a fusion
of the lacZ coding sequence to PcomK was readily
expressed in a heterologous host (E. coli), whereas a
PcomGlacZ fusion was not, unless comK was coex-
pressed (van Sinderen et al., 1995; Susanna et al., 2006).
The observed expression from PcomK in E. coli could
indicate that ComK acts as an antirepressor in B. subtilis,
antagonizing one or more species-specific repressors
(Supplementary Fig. S1). In fact, the E. coli genome
(Blattner et al., 1997) contains no homologues of the pre-
viously identified repressors of comK (CodY, AbrB and
Rok), as judged by a
BLAST similarity search (http://www.
ncbi.nlm.nih.gov/blast/). The hypothesis of antirepression
makes a strong prediction; that removal of repressors
would render transcription from PcomK in B. subtilis at
least partially independent of ComK. To test this, we intro-
duced mutations in the genes encoding these repressors,
and evaluated the expression of an ectopic comK–lacZ
reporter fusion.
Repressor mutations result in a partial bypass of the
ComK requirement in vivo
The requirement for ComK was not detectably bypassed
by inactivation of codY or abrB, either individually or in
combination (Fig. 1 and W.K. Smits et al., unpubl. obs.).
Rok was recently identified as a major repressor of com-
petence (Hoa et al., 2002). Therefore, we evaluated the
effect of a rok mutation on PcomK expression in wild type
(Fig. 1A) and comK backgrounds (Fig. 2B). In the pres-
ence of ComK, the deletion of rok leads to high levels of
transcription, as was previously reported (Hoa et al.,
2002). Importantly, there is significant comK–lacZ activity
in the DcomK Drok background, although it is about three-
to fourfold lower than wild type and seven- to eightfold
reduced compared with a comK
+
background carrying a
rok mutation. This result indicates that a rok mutation
partially bypasses the in vivo requirement of ComK for its
own expression. Interestingly, the bypass is stronger
when both codY and rok are inactivated (Fig. 1B), despite
the fact that a codY mutation alone does not lead to a
detectable bypass.
Taken together, these results indicate that ComK
reverses the action of Rok and strongly suggests a similar
role for ComK towards CodY, thus supporting the hypoth-
esis that ComK acts as antirepressor at its own promoter
in vivo. The partial nature of the bypass suggests either
that ComK plays a role at PcomK as a classical activator
Fig. 1. Mutation of rok and codY results in a bypass of the ComK
requirement. Strains containing the comK–lacZ reporter were either
comK
+
(A) or DcomK (B). In both panels wild-type expression
levels of comK–lacZ (i.e. in comK
+
background) are indicated using
solid symbols. Time is given in hours relative to the transition
between exponential and stationary growth phase (T
0
). A comK
mutation in an otherwise wild-type strain completely abolishes
comK–lacZ expression (not shown). The apparent delay of a rok
codY double mutant in comK
+
background compared with wild type
was not consistently observed.
ComK reverses Rok and CodY repression 369
© 2007 The Authors
Journal compilation © 2007 Blackwell Publishing Ltd, Molecular Microbiology, 64, 368–381
as well as an antirepressor protein or that an additional
repressor is involved. Such an additional repressor is
unlikely to be AbrB, for reasons presented in the
Discussion.
Rok and CodY repress transcription from PcomK in vitro
In order to further analyse the effects of individual tran-
scription factors, we established in vitro transcription
assays for the promoter of comK using purified B. subtilis
RNAP holoenzyme (a kind gift from M. Salas). A template
for these experiments was constructed by cloning a
186 bp fragment derived from sequences upstream of
comK into plasmid pAN583 (Predich et al., 1992), as
described in Experimental procedures. The resulting
plasmid contains the sequence of the comK promoter that
is required for binding of all known transcriptional regula-
tors of the comK gene (Serror and Sonenshein, 1996;
Hamoen et al., 1998; Hamoen et al., 2000; 2003; Hoa
et al., 2002).
In contrast to the results obtained with pAN-G, a
PcomG-containing derivative of pAN583 (Hamoen et al.,
1998), there is a substantial level of transcription from
PcomK under the conditions used (Figs 2 and 3, inset
lanes marked with X). This level of transcription was not
significantly augmented by the addition of ComK or DegU
over a wide range of concentrations, either alone or in
combination (Fig. 2A and B). A small but consistent
increase (around 1.5-fold) was observed when only
ComK was added to the reaction mixture, but this effect
was not observed in the presence of DegU. In both cases,
a decrease in transcription was evident at higher concen-
trations of ComK protein. A similar decrease with high
ComK concentrations was observed when pAN-G was
used as a template for the in vitro transcriptions (data not
shown). The reason for this is not clear, but it may reflect
non-specific inhibition by this DNA binding protein at high
concentrations. These experiments indicate that strong
auto-stimulation from PcomK is absent when ComK is
added to the mixture, either alone or in the presence of
DegU.
Next, we investigated the effects of Rok and CodY on
the in vitro transcription from PcomK. The addition of Rok
leads to strong repression, with no detectable transcrip-
Fig. 2. ComK reverses the repression by Rok in vitro. In vitro
transcription experiments using circular pAN-K plasmid (see
Experimental procedures) and purified B. subtilis RNA polymerase
in the presence of several transcription factors. All experiments
were performed at least in triplicate and representative examples
are shown in the figure. The experiments were performed in the
presence (open symbols) or absence (closed symbols) of DegU
protein.
A. Results from in vitro transcription experiments with ComK
(4–1100 nM). The inset shows signals from reactions containing no
transcription factors (X), 137.5 nM ComK (K) or 137.5 nM
ComK/500 nM DegU (UK).
B. Results from in vitro transcription experiments with Rok
(12–3000 nM) and DegU (14–3500 nM). The inset shows signals
from reactions containing no transcription factors (X), 750 nM Rok
(R), 750 nM Rok/500 nM DegU (RU) or 500 nM DegU (U).
C. Results from in vitro transcription experiments in the presence of
170 nM Rok, various amounts of ComK protein. The dash-dotted
line indicates the level of transcription under these conditions in the
absence of ComK and DegU. The experiments were performed in
the presence (open symbols) or absence (closed symbols) of DegU
protein (500 nM) and ComK. The inset shows signals from
reactions containing no transcription factors (X), 170 nM Rok (R),
137.5 nM ComK/170 nM Rok (KR), 500 nM DegU/170 nM Rok (UR)
or 137.5 nM ComK/500 nM DegU/170 nM Rok (KUR).
370 W. K. Smits et al.
© 2007 The Authors
Journal compilation © 2007 Blackwell Publishing Ltd, Molecular Microbiology, 64, 368–381
tion at nanomolar concentrations of Rok protein (Fig. 2B).
These values are in good agreement with electrophoretic
mobility shift assay (EMSA) results showing an apparent
K
D
of around 50 nM for the interaction of Rok with PcomK
(Hoa et al., 2002; Albano et al., 2005). The addition of
DegU slightly enhanced the repression by Rok at lower
concentrations, but had no effect when enough Rok was
added to reduce transcription to less than 20% of the
unrepressed state (Fig. 2B).
The binding of CodY to the promoter of comK has been
described previously (Serror and Sonenshein, 1996).
However, the effects of the recently identified cofactors
guanosine triphosphate (GTP) (Ratnayake-Lecamwasam
et al., 2001) and branched chain amino acids (BCAAs)
(Shivers and Sonenshein, 2004) have not been docu-
mented for PcomK. Although binding of CodY occurs in
the absence of cofactors, despite the presence of an
excess of non-specific competitor (poly-dIdC), we found
that BCAAs stimulated binding of CodY to the ComK
promoter (data not shown), in agreement with results
obtained with other promoters (Shivers and Sonenshein,
2004). In contrast, GTP only stimulated binding detectably
in the presence of BCAAs (data not shown). However,
these effects on binding were relatively minor; affinity in
the presence of both effector molecules increased only
approximately twofold under our experimental conditions.
In spite of this, we observed no transcriptional repression
with CodY in the absence of effectors, under conditions
that resulted in a fully retarded PcomK probe in the EMSA
experiments (Fig. 3A). The addition of a mixture of BCAAs
resulted in strong repression, with a cumulative repres-
sive effect in the presence of GTP (Fig. 3A). We conclude
that the effector molecules have a minor effect on the
affinity of CodY for its target promoter, but are required for
the repressor activity of CodY. The concentration of CodY
required to achieve full repression is in good agreement
with both EMSA experiments and with the concentration
used for previously reported footprinting experiments
(Serror and Sonenshein, 1996). As in the case of Rok, we
observed that the addition of DegU to the in vitro tran-
scription reactions exerted only a mild effect on CodY-
mediated repression under the conditions tested (data not
shown).
Rok reduces the affinity of RNAP for PcomK
Many repressors act by masking the -35 or -10 promoter
elements that constitute the RNAP binding site. Indeed,
DNase I footprinting experiments have revealed that AbrB
and CodY bind to PcomK at sequences that partially
overlap these elements (Serror and Sonenshein, 1996;
Hamoen et al., 2003). However, the location of the binding
site for Rok is unknown. Despite repeated attempts, we
were unable to further define the binding site for Rok
using DNaseI footprinting experiments or bioinformatic
approaches (Albano et al., 2005). Using Rok and overlap-
ping PcomK-probes in EMSA experiments, we narrowed
the region encompassing the Rok binding site to 130 bp
(Fig. 4). This segment includes the sequences that are
protected by ComK and DegU in hydroxyl radical footprint
experiments (Hamoen et al., 1998; 2000). Surprisingly,
the Rok-binding site appears to lie upstream of the -35
and -10 sequences, making it unlikely that Rok occludes
Fig. 3. ComK reverses the repression by CodY in vitro. In vitro
transcription experiments using circular pAN-K plasmid (see
Experimental procedures) and purified B. subtilis RNA polymerase
in the presence of several transcription factors. All experiments
were performed at least in triplicate and representative examples
are shown in the figure.
A. Results from in vitro transcription experiments with a titration of
CodY protein, in the presence and absence of effector molecules.
GTP, guanosine triphosphate; ILV, a mixture of isoleucine, leucine
and valine. The inset shows signals from reactions containing no
transcription factors (X), 8 mM of CodY (Y), 2.5 mM GTP/8 mM
CodY (GTP Y), 10 mM ILV/8 mM CodY (ILV Y) or 10 mM
ILV/2.5 mM GTP/8 mM CodY (ILV GTP Y).
B. Results from in vitro transcription experiments in the presence of
8 mM CodY, 10 mM of isoleucine, leucine and valine (ILV) and
various amount of ComK protein. The dash-dotted line indicates the
level of transcription under these conditions in the absence of
ComK or ComK and DegU. The experiments were performed in the
presence (open symbols) or absence (closed symbols) of DegU
protein (500 nM) and ComK. The inset shows the signals from
reactions containing no transcription factors (X), 8 mM CodY (Y),
137.5 nM ComK/8 mM CodY (KY), 500 nM DegU/8 mM CodY or
137.5 nM ComK/500 nM DegU/8 mM CodY.
ComK reverses Rok and CodY repression 371
© 2007 The Authors
Journal compilation © 2007 Blackwell Publishing Ltd, Molecular Microbiology, 64, 368–381
the RNAP binding site in the comK promoter. Considering
its function as a repressor, we were therefore interested to
see whether Rok reduces the affinity of RNAP for PcomK,
despite the location of its binding site.
EMSA experiments using purified RNAP and Rok dem-
onstrated that Rok reduces the binding of RNAP (Fig. 5).
The concentration of Rok required for half-maximal rever-
sal of RNAP binding is comparable to the apparent K
D
of
Rok-binding to PcomK in the absence of RNAP. Although
this reversal would appear to be consistent with direct
competition between RNAP and Rok for binding to PcomK,
we also consistently observed a slightly super-shifted
protein/DNA complex in the presence of both RNAP and
Rok, evident when intermediate concentrations of Rok are
used. This super-shifted band migrates slightly slower than
the complexes of the DNA with RNAP or Rok alone (Fig. 5).
It thus appears possible that, in contrast to AbrB and CodY,
Rok does not compete directly with RNAP for binding to the
DNA but relies on an alternative mechanism to reduce the
affinity of RNAP for PcomK. Although this would be con-
sistent with the unusual location of the Rok repressor
binding site noted above and is not unprecedented (Perez-
Martin et al., 1994; Rojo, 2001), it is a suggestion that
deserves further confirmation.
ComK reverses repression in vitro
These experiments enabled us to directly test the antire-
pression hypothesis by determining whether ComK acts
in vitro to reverse Rok and CodY repression. At 170 nM
Rok, transcription from PcomK was reduced sixfold
(Fig. 2B and C, dash-dotted line). The addition of ComK to
the repressed promoter resulted in a moderate increase
in transcription, although transcription was submaximal
at concentrations greater than ~130 nM ComK protein
(Fig. 2C). The observed decrease in transcription at high
levels of ComK may be non-specific, as noted above, and
was not investigated further. Previously, it was reported
that DegU acts as a priming protein, enhancing the affinity
of ComK for its own promoter (Hamoen et al., 2000).
Interestingly, this phenomenon is independent of protein-
protein contacts, and presumably relies on modulation of
Fig. 4. Schematic representation of the
binding sites of various transcription factors
on the promoter of comK to indicate overlap
and relative positions of the various sites. K,
ComK; U, DegU; Y, CodY; A, AbrB; R, Rok.
-35 and -10 indicate the core promoter
elements. The comK gene is truncated.
Overlapping probes for EMSA experiments
were generated with various combinations of
primers (see Table S2). These probes were
tested for their ability to bind Rok according to
Experimental procedures (indicated in the
column Shift). The inferred area of
Rok-binding is indicated by the shaded box.
Fig. 5. Rok reduces affinity of RNA
polymerase for PcomK. The percentage of
probe in the complex with RNA polymerase
with or without Rok (grey) or Rok only (black)
was determined as described in Experimental
procedures. The lower part of the figure
shows the actual EMSA experiment using a
fragment of the comK promoter. The big
triangle indicates a decrease in Rok
concentration of twofold per lane, ranging
from 1.7 mM to 13 nM. The small triangles on
the image of the gel indicate the shift (closed
symbol) or super-shift (open symbol) caused
by Rok binding. Binding of proteins was
performed as described in Experimental
procedures. Free probe and complex were
resolved on a non-denaturing 6%
polyacrylamide gel. Rp, RNA polymerase
holoenzyme (1.5 mg).
372 W. K. Smits et al.
© 2007 The Authors
Journal compilation © 2007 Blackwell Publishing Ltd, Molecular Microbiology, 64, 368–381
DNA topology (Hamoen et al., 2000). In light of these
findings, we were interested to see if the antirepression by
ComK would be enhanced in the presence of DegU.
Results from in vitro transcription experiments indeed
demonstrate that the transcription from PcomK is restored
to approximately 80% of the unrepressed state, when
DegU is present in the reactions (Fig. 2C). It thus seems
that DegU potentiates the antirepressing activity of ComK.
During our in vitro transcription experiments, we
observed that antirepression was strongest at concentra-
tions of Rok that did not fully repress the ComK promoter.
Previous observations of multiple DNA–protein com-
plexes in EMSA experiments using the comK promoter
(Albano et al., 2005) hint at the possibility that at high
concentrations of Rok, the protein occupies multiple sites
and may not be susceptible to antirepression. Thus,
in vivo auto-stimulation might only occur within a narrow
range of Rok levels, implying that the expression of Rok
must be fine-tuned to allow competence development.
Previously, it was reported that a rok-lacZ promoter fusion
is expressed at an essentially constant rate throughout
growth (Hoa et al., 2002). This conclusion is supported by
measurements of fluorescence made with a Prok-gfp
reporter in single cells showing that Prok-driven expres-
sion remains constant in minimal medium and demon-
strates little variability between cells, whereas it increases
in a rich medium (Fig. S2).
The in vivo data strongly suggest that ComK not only
antagonizes repression by Rok, but also by CodY. To test
this, we performed in vitro transcription reactions under
conditions that support repression by CodY, i.e. in a
binding buffer containing BCAAs (Fig. 3A). The addition of
the amino acid mixture did not affect transcription in the
absence of CodY (data not shown). The addition of 8 mM
CodY in the absence, or of 4 mM CodY in the presence of
additional GTP, reduced transcription around 10-fold com-
pared with the unrepressed state (Fig. 3, dash-dotted
line). From Fig. 3B it can be seen that the repression by
CodY in the presence of BCAAs is readily reversed upon
the addition of ComK protein. We consistently observed a
four- to sixfold increase in transcription compared with the
CodY-repressed state. As was the case for Rok, we
observed stronger antirepression in the presence of DegU
protein. Moreover, the concentrations of ComK required
to achieve maximal derepression are comparable for both
CodY and Rok (~130 nM). Taken together, these results
demonstrate that ComK acts as an antirepressor at its
own promoter towards both Rok and CodY.
ComK does not prevent binding of the repressing
proteins at PcomK
The binding site of ComK (partially) overlaps those of the
repressor proteins acting on PcomK as judged by the
results from DNA footprinting experiments (Fig. 4) (Serror
and Sonenshein, 1996; Hamoen et al., 1998; 2000;
2003), raising the possibility that the prevention of repres-
sor binding to the DNA is responsible for the antirepres-
sion observed in this study. In fact, it was recently reported
that CcpA activates transcription of the ilvB operon in
B. subtilis by preventing the binding of the transcriptional
repressor CodY (Shivers and Sonenshein, 2005). There-
fore, EMSA experiments were carried out to test this
hypothesis.
The addition of Rok, DegU and ComK individually,
resulted in significant retardation of a fragment of PcomK
encompassing all three binding sites (Fig. 6A), confirming
earlier findings (van Sinderen et al., 1995; Hamoen et al.,
2000; Hoa et al., 2002). DegU can bind simultaneously
with Rok or ComK, as is evident from the super-shifted
bands (Fig. 6A), and stimulates the binding of ComK
(Hamoen et al., 2000), but probably not Rok. Surprisingly,
the presence of both Rok and ComK results in a super-
shifted band, indicating that there is no obvious competi-
tion between the two proteins for binding to PcomK.In
fact, a difference in mobility of the probe (super-shift) was
Fig. 6. Various transcription factors bind simultaneously to PcomK.
Binding of proteins was performed as described in Experimental
procedures. Free probe and complex were resolved on a
non-denaturing 6% polyacrylamide gel. (X) indicates a lane with
only the radiolabelled probe.
A. Results from an EMSA experiment using purified Rok (R;
50 nM), ComK (K; 100 nM) and DegU (U; 500 nM), or combinations
of those.
B. Results from an EMSA experiment using purified ComK
(100 nM), CodY (2 mM) and DegU (500 nM), or combinations of
those.
ComK reverses Rok and CodY repression 373
© 2007 The Authors
Journal compilation © 2007 Blackwell Publishing Ltd, Molecular Microbiology, 64, 368–381
observed between the lanes with two proteins and the
lane in which all three transcription factors (Rok, ComK
and DegU) were present (Fig. 6A; W.K. Smits et al.,
unpubl. obs.). Moreover, the addition of DegU to a mixture
containing Rok and ComK increased the total amount of
probe shifted. These observations demonstrate that Rok,
ComK and DegU can simultaneously interact with
PcomK, and suggests that these proteins bind at distinct
surfaces of the DNA helix.
Analogous to the situation for Rok, we investigated
whether competitive binding occurs between CodY and
ComK. Strikingly, the addition of CodY to a mixture that
contains ComK resulted in a super-shift as well, both in
the presence and absence of DegU protein (Fig. 6B). This
indicates that ComK, DegU and CodY are also able to
bind simultaneously to the same fragment of the comK
promoter. Although in vitro repression by CodY only
occurred in the presence of BCAAs, the super-shift results
obtained in the presence of BCAAs and/or GTP were
comparable to those in the absence of effector molecules
(data not shown). The presence of ComK appears to
release some CodY from the comK promoter (Fig. 6B,
compare lanes Y and YK), and we cannot exclude the
possibility that these proteins interact directly. This nega-
tive effect of ComK on CodY binding was not observed in
the presence of DegU and does not affect the conclusion
that CodY, ComK and DegU can interact simultaneously
with the same fragment of DNA.
Rok binding is inhibited by major groove binding drugs
ComK is able to reverse Rok- and CodY-imposed repres-
sion at its own promoter, without reversing the binding of
these repressor proteins. Simultaneous binding of pro-
teins to the DNA is possible if they bind to different sur-
faces of the DNA helix. ComK is known to bind through
the minor groove of the DNA (Hamoen et al., 1998) and
based on the published crystal structure, CodY is thought
to be a major groove binding protein (Levdikov et al.,
2006). Because the DNA binding domain of Rok and
its binding characteristics are largely unknown, we
addressed whether Rok binds to the major or minor
groove of the DNA. To this end, we performed gel shift
experiments in the presence of drugs that interfere with
binding in either the major (methyl green) or minor (chro-
momycin A3) groove of the DNA. We found that the addi-
tion of methyl green strongly reduced the affinity of Rok
for the comK promoter, whereas no effect was observed
for chromomycin A3 at relevant concentration for these
drugs (Fig. 7). These results are precisely the opposite of
those obtained for ComK binding to the same promoter
(Hamoen et al., 1998). Similarly, the addition of actinomy-
cin D, another minor groove binding drug, did not interfere
with Rok-binding (data not shown). These data strongly
suggest that Rok is a major groove binding protein, like
CodY, and thus might bind to PcomK without interfering
with ComK-binding through the minor groove of the DNA.
Discussion
The expression of comK in B. subtilis demonstrates an
absolute requirement for ComK. In contrast to comG,
however, the gene is readily expressed in vitro or in a
heterologous host (van Sinderen and Venema, 1994; van
Sinderen et al., 1994). These data led to the prediction
that there are two distinct mechanisms for transcriptional
activation by ComK. The first involves direct activation of
genes that show low (or no) basal levels of transcription in
Fig. 7. Major groove binding drug decreases
the affinity of Rok for PcomK. Quantitative
analysis of and EMSA experiment to assess
the effects of methyl green and chromomycin
A3 on the affinity of Rok for PcomK in
independent experiments. The percentage of
shifted probe is decreased in the presence
of methyl green (grey bars), whereas
chromomycin A3 does not interfere with Rok
binding to PcomK (black bars). The dotted
line indicates the percentage of shift at 1.5 mM
Rok in the absence of DNA binding drugs.
Triangles indicate successive twofold dilutions
of the concentrations of DNA binding drugs.
374 W. K. Smits et al.
© 2007 The Authors
Journal compilation © 2007 Blackwell Publishing Ltd, Molecular Microbiology, 64, 368–381
the absence of ComK, such as comG. Indeed it has been
shown that ComK directly activates transcription from
PcomG, by stabilizing the binding of RNAP (Susanna
et al., 2004). For the second class of genes, which
includes recA (Hamoen et al., 2001), ComK reverses the
repression of promoters that demonstrate a significant
basal level of transcription in the absence of transcription
factors. The present study substantiates this hypothesis
and establishes comK as a representative example of this
second class of activated genes.
Antirepression at the comK promoter
Three repressors of comK have been identified: AbrB
(Hamoen et al., 2003), CodY (Serror and Sonenshein,
1996) and Rok (Hoa et al., 2002). In vitro transcription
experiments demonstrate that ComK can act as an anti-
repressor towards the latter two (Figs 2 and 3). This is
supported by the observation that the in vivo requirement
of ComK for its own expression is partially bypassed by
inactivation of rok, and that this bypass is stronger when
codY is inactivated as well (Fig. 1B). However, the levels
of transcription from PcomK in a codY rok comK triple
mutant do not reach those of a wild-type strain (Fig. 1B).
The residual requirement of ComK may be due to the
small but significant increase in transcription through the
action of ComK itself (Fig. 2A). Alternatively, the action of
another repressor, such as AbrB, might be responsible.
Repeated attempts to construct an abrB codY rok comK–
lacZ strain failed, both in a comK
+
and a DcomK back-
ground, suggesting that knocking out all three pleiotropic
repressors is lethal. AbrB acts both positively and nega-
tively in the competence regulatory cascade (Hahn et al.,
1995) (Hahn et al., 1996), but its positive requirement is
bypassed by a rok mutation (Hoa et al., 2002). When the
effect of a rok abrB comK triple mutation on comK–lacZ
expression was assessed, only a marginal increase in
transcription compared with a rok comK double mutant
was observed (data not shown). Furthermore, in vitro
transcription results suggest that ComK is unable to act as
an antirepressor with respect to AbrB (Hamoen et al.,
2003; W.K. Smits et al., unpubl. results). Although we
cannot exclude the possibility that yet another repressor
of comK which is antagonized by ComK remains to be
identified, this is unlikely to be AbrB.
The priming protein DegU exerts its positive influence
on competence development by stimulating ComK
binding to its own promoter (Hamoen et al., 2000). Muta-
tion of degU reduces the overall expression of comK
(van Sinderen and Venema, 1994; Hahn et al., 1996) and
leads to a decrease in the fraction of competent cells
(W.K. Smits, unpubl. obs.). In this study, we report that
DegU potentiates ComK as an antirepressor (Figs 2 and
3). It is noteworthy that the EMSA results demonstrate
that CodY and Rok do not prevent binding of DegU
(Fig. 6). Had this been the case, antirepression would
have been severely hampered. As might be expected
from its effect on the affinity of ComK-binding to PcomK,
the presence of DegU permits slightly higher levels of
antirepression at lower concentrations of ComK than are
needed in the absence of DegU (Fig. 2).
Previous work has shown that even a slight increase in
the native levels of Rok is sufficient to completely abolish
competence development (Hoa et al., 2002). In addition,
AbrB and SinR, which exert minor negative effects on rok
expression and Rok protein levels are both positively
required for competence development unless rok is
mutated (Hoa et al., 2002). It appears that small increases
in the in vivo concentration of Rok have major effects on
the expression of comK. In the course of this study, we
observed that in vitro we could only observe antirepres-
sion when transcription from PcomK was not fully
repressed, and, our in vivo measurements of Prok-gfp
levels support the notion that under conditions that
sustain competence, rok expression is kept constant.
Together, this evidence suggests that Rok is a critical
determinant for competence development by determining
the potential for auto-activation by ComK. The compe-
tence regulatory system is maintained on a knife-edge in
which minor perturbations of protein concentrations can
drive the system towards dramatically increased expres-
sion, or prevent the initiation of competence altogether.
The importance of Rok in controlling the level of comK
expression is reinforced by observations that complete
reversal of repression in vitro cannot be achieved by the
addition of ComK (Fig. 2) and that a rok knockout mutant
expresses higher levels of fluorescence per competent
cell from comK-cfp than the isogenic rok
+
strain (data not
shown). The rok gene itself is subject to complex regula-
tion (Hoa et al., 2002) and more detailed investigations
are necessary to address how Rok levels are regulated.
A model for the action of ComK early in competence
development
Based on the data presented in this study, the following
sequence of events can be proposed for early events
in the development of competence for genetic
transformation. During the exponential growth phase,
transcription from the comK promoter is prevented
through the repression exerted by AbrB, CodY and Rok
(Fig. 8A). The little ComK that is produced is trapped by
the adaptor protein MecA, and targeted for degradation by
the ClpCP protease (Turgay et al., 1997; 1998). At the end
of the exponential growth phase, quorum sensing stimu-
lates the release of ComK from the proteolytic complex,
after which it is able to exert its function as a transcription
factor in the cell (Fig. 8B). Consistent with this sequence
ComK reverses Rok and CodY repression 375
© 2007 The Authors
Journal compilation © 2007 Blackwell Publishing Ltd, Molecular Microbiology, 64, 368–381
Fig. 8. Model for the regulatory events early
in competence development. Thickness of the
lines indicates the strength of the regulatory
interaction. Positional information is not
conserved in this representation. A, AbrB; K,
ComK; P, proteolytic complex; R, Rok; Rp,
RNA polymerase holoenzyme; U, DegU; Y,
CodY.
A. The comK promoter is repressed by Rok,
AbrB and CodY. DegU is present. Residual
ComK is trapped by the proteolytic complex.
B. Quorum sensing events cause limited
release of ComK. In the presence of DegU,
ComK acts as an antirepressor towards Rok
and CodY, without preventing their binding.
C. In stationary growth phase and upon
nutrient limitation AbrB and CodY repression
are relieved (see text for details).
D. Phosphorylation reduces the affinity of
DegU for PcomK, causing it to dissociate.
E. ComK antirepression with respect to Rok
still occurs in the absence of other factors.
376 W. K. Smits et al.
© 2007 The Authors
Journal compilation © 2007 Blackwell Publishing Ltd, Molecular Microbiology, 64, 368–381
of events is the observation that disruption of the pro-
teolytic complex leads to an altered timing of competence
development, whereas deletion of a repressor does not
markedly alter the timing (Maamar and Dubnau, 2005).
Low levels of ComK, through the priming action of DegU,
are able to initiate the reversal of repression by Rok and
CodY (Figs 2, 3 and 8B). Additionally, ComK might stimu-
late its own transcription directly. Levels of activated
Spo0A rise in late exponential growth phase, thereby effi-
ciently downregulating the transcription of AbrB (Fujita
and Sadaie, 1998; Fujita et al., 2005) and this is the sole
reason for the Spo0A requirement for the development of
competence (Hahn et al., 1995). Therefore, reversal of
repression by AbrB in vivo occurs independently of ComK.
Nutritional repression by CodY is relieved when depletion
of nutrients occurs, affecting the energy status of cells
(Serror and Sonenshein, 1996; Ratnayake-Lecamwasam
et al., 2001). This may explain why mutation of codY has
a minor effect in the bypass of the ComK requirement in
vivo, because it is already partially inactivated by this
physiological mechanism. This notion is also consistent
with the strong effect of Rok, because no downregulation
of rok expression in minimal medium has been docu-
mented (Hoa et al., 2002 and Fig. S2). Together, antire-
pression and derepression at the comK promoter (Fig. 8B
and C) lead to a strong increase in ComK levels, allowing
transcription of the ComK regulon (Berka et al., 2002;
Hamoen et al., 2002; Ogura et al., 2002).
The mechanisms of antirepression
Antirepression in bacteria can occur by at least three
distinct mechanisms. In one, the antirepressing protein
binds to the repressor preventing its binding to DNA. This
mechanism has been documented in a number of cases,
for instance in the regulation of SinR activity in B. subtilis
(Bai et al., 1993) and MogR regulation in Listeria by GmaR
(Shen et al., 2006). A second mechanism of antirepression
in bacteria involves occlusion of the repressor binding site
by other DNA binding proteins. For example, in B. subtilis,
the binding of CcpAto the ilvB promoter prevents binding of
the nutritional repressor CodY (Shivers and Sonenshein,
2005). In E. coli, several instances have been identified
where antirepression involves interactions between mul-
tiple proteins, resulting in the release of the repressors
(Browning et al., 2004; 2005; 2006). Here, we demonstrate
a third mechanism in which ComK does not interfere with
binding of the repressor proteins Rok and CodY. How does
ComK act as an antirepressor?
The fact that ComK (Hamoen et al., 1998) and the
repressors (Levdikov et al., 2006 and Fig. 7) bind through
different grooves of the DNA may permit simultaneous
binding by placing the proteins on distinct surfaces of the
DNA helix even when they bind to the same area of
the promoter. Minor groove binding proteins are fre-
quently architectural proteins that sometimes bind non-
specifically to DNA (Bewley et al., 1998). It has been
noted (van Sinderen et al., 1995) that ComK demon-
strates limited similarity to the male sex determining factor
SRY and T cell factor 1 (TCF-1), activators belonging to
the high mobility group of minor groove binding proteins
(Bewley et al., 1998). Binding of SRY to DNA induces
strong bending, due to insertion of a hydrophobic wedge
of the protein into the helix, forcing it from the B-form to
the A-form (Bewley et al., 1998). Virtually all minor groove
binding proteins, including ComK (Hamoen et al., 1998),
bend or distort the DNA. This change in topology has
been implicated in the assembly of higher-order DNA–
protein complexes such as the transcriptional machinery
(Bewley et al., 1998; Carey, 1998).
We suggest that this known ability of ComK to bend
DNA is at the heart of its action as both an antirepressor
and an RNAP recruitment protein. In the case of the comK
promoter, we have suggested above that Rok may reduce
the affinity of RNAP for PcomK through a mechanism
other then occlusion of the core promoter elements
(Fig. 5). If Rok alters the DNA topology such that the
RNAP–DNA interaction is weakened, ComK-induced
bending of the DNA might counteract these effects without
preventing the binding of the repressor. If instead Rok
represses by competing for RNAP binding, then ComK
may reverse this effect by inducing a bend in DNA that
reduces steric clashes between Rok and RNAP. A similar
mechanism may obtain in the case of CodY.
In a detailed investigation of the mechanism of tran-
scriptional activation of PcomG, it was reported that
ComK does not seem to interact directly with the alpha
subunit of RNAP (Susanna et al., 2004). Instead, it
was postulated that ComK-induced bending wraps the
upstream DNA around RNAP, thus stabilizing the interac-
tion (Bewley et al., 1998; Susanna et al., 2004). We thus
propose that ComK acts as an activator at both types of
promoters by modulating DNA topology.
Other minor groove binders may act similarly to ComK
as antirepressors. For instance, the phage protein p4
G
that presumably recognizes specific AT-tracts in the minor
groove of the DNA has also been postulated to act as
an antirepressor, antagonizing an unknown repressor
protein (Horcajadas et al., 1999). Another interesting case
involves regulation of the aceBAK operon by the integra-
tion host factor (IHF) (Goodrich et al., 1990; Resnik et al.,
1996). IHF, a minor groove binding architectural protein
that binds to an AT rich consensus sequence (Yang and
Nash, 1989; Sun et al., 1996), interacts with two sites
upstream of PaceBAK, and relieves repression by IclR.
Because these two proteins bind to distinct sites, it is
possible that IHF, like ComK, can relieve repression
without prevention of repressor binding.
ComK reverses Rok and CodY repression 377
© 2007 The Authors
Journal compilation © 2007 Blackwell Publishing Ltd, Molecular Microbiology, 64, 368–381
Perspective on ComK-mediated antirepression
ComK also antagonizes the repression by LexA on PrecA
without directly displacing the repressor (Hamoen et al.,
2001) and indeed other cases of antirepression by ComK
may exist. Recently, it was reported that the ComK-
activated gene rapH (Berka et al., 2002; Hamoen et al.,
2002; Ogura et al., 2002) is under control of the repressor
RghR (Hayashi et al., 2006). As with PrecA and PcomK,
expression from PrapH occurs readily in the absence of
ComK when RghR is absent (Hayashi et al., 2006). In
addition, we found that RghR and ComK can bind simul-
taneously to a promoter fragment of rapH (W.K. Smits, C.
Bongiorni, J.W. Veening, L.W. Hamoen, O.P. Kuipers and
M. Perego, submitted for publication). It seems likely that
further characterization of ComK-activated genes will
reveal additional examples of antirepressed genes.
Because ComK can act as either an antirepressor or as
a more classical activator protein, it might appear that
some common promoter sequence element determines
the mode of action of ComK in each case. Inspection of the
various ComK-dependent promoters does not suggest any
obvious explanatory feature, and perhaps the relevant
difference is that some promoters like PcomG have rela-
tively weak affinities for RNAP and need ComK to recruit
this enzyme while PcomK and PrecA do not. Unlike
PcomG, both of the latter promoters instead bind repres-
sors and have therefore evolved to permit antirepression
by ComK. It may be then that the two classes of promoters
are distinguished by their relative affinities for RNAP, and
by the presence or absence of repressor binding sites.
Promoters subject to antirepression by ComK would be
further characterized by the precise disposition of the
repressor binding sites with respect to the ComK boxes so
that the ComK-induced bending of DNA will prevent the
repressors from interfering with the action of RNAP.
In conclusion, we propose that ComK exerts its function
as an antirepressor without preventing binding of the
repressors, based on its ability to induce bending of DNA,
thereby stabilizing the binding of RNAP. We suggest that
a remarkable choreography of DNA bending at PcomK,
mediated by the binding of multiple repressors, activators
and RNAP, regulates comK expression in response to
upstream signal transduction pathways. Most minor
groove binding proteins induce bending of DNA (Bewley
et al., 1998) and it is tempting to speculate that antirepres-
sion or direct activation by other minor groove binding
proteins relies on similar mechanisms.
Experimental procedures
Bacterial strains and media
All Bacillus subtilis strains are derivatives of the reference
strain B. subtilis 168 (Kunst et al., 1997), are isogenic
derivatives of strain BD630 (his leu met) or 168 (trpC2) and
are listed in Table S1. Transformation, as well as selective
and growth media are described or referenced in Albano
et al. (1987) and Hamoen et al. (2002). Plasmids were
maintained in E. coli strains as indicated in Table S1. Strain
ProkG was constructed as follows. The promoter region of
the rok gene was amplified by polymerase chain reaction
(PCR) using primers Prok-F-new-KpnI and Prok-R-new-
EcoRI and chromosomal DNA of strain 168 as a template.
The 1543 bp amplified fragment was digested with KpnI and
EcoRI and cloned into similarly digested pSG1151 (Lewis
and Marston, 1999), yielding plasmid pSG1151-Prok(NN).
This plasmid was used to transform B. subtilis, and trans-
formants were selected after overnight growth at 37C on TY
with chloramphenicol. Campbell-type integration of the
plasmid was verified by PCR (data not shown). Strain
KGFP (comK-gfp) was obtained by transformation of
plasmid pcomK-gfp (Haijema et al., 2001) into B. subtilis
strain 168. Multiple loci were combined by transformation
with chromosomal DNA.
Expression and purification of proteins
Rok-his6 (Albano et al., 2005), ComK (Susanna et al.,
2006) and DegU-his6 (Hamoen et al., 2000) were purified
as described previously. CodY-his6 was isolated from E. coli
strain KS272 harbouring the pKT1 plasmid (Kim et al.,
2003) as follows. An overnight culture was diluted 1:100 into
fresh TY with appropriate antibiotics. Growth was continued
until OD 0.70 with continuous shaking (250 r.p.m., 37°C).
At that moment, expression of CodY-his6 was induced
by the addition of 0.1% arabinose, and continued for 1 h.
Subsequently, cells were pelleted by centrifugation (10 min,
8000 r.p.m., 4°C), and stored at -80°C. The pellet was
resuspended in 5 ml of buffer A (20 mM Tris-HCl pH 8.0,
0.2 M NaCl, 10 mM MgCl
2
, 7% glycerol, 1 mM
b-mercaptoethanol), supplemented with Complete Mini Pro-
tease Inhibitor (Roche), and cells were disrupted by soni-
cation. Cellular debris was removed by centrifugation
(10 min, 14 000 r.p.m., 4°C), and the supernatant fraction
was incubated with 2 ml of equilibrated Superflow NiNTA
resin (Qiagen) in a total volume of 15 ml of buffer A for 2 h
with continuous mixing. The column material was packed in
a Poly Prep Chromatography Column (Bio-Rad) and
washed by gravity flow with 10 column volumes buffer A
and 10 column volumes buffer B (identical to buffer A, but
with 20 mM of imidazole). The protein was eluted from the
column with buffer C (identical to buffer A, but with 500 mM
imidazole), and 0.5 ml fractions were collected. Fractions
were checked for protein content and purity by SDS-PAGE.
Protein was quantified using the RC/DC protein determina-
tion kit (Bio-Rad), using a commercial bovine serum
albumin solution (New England Biolabs) as a standard. The
presence of a C-terminal 6xHis-tag on the DegU protein
does not interfere with its function in competence develop-
ment (Hamoen et al., 2000) and CodY-his6 is functional in
vivo (Ratnayake-Lecamwasam et al., 2001), but the effects
of the tag on Rok are not documented. Although the pres-
ence of the 6xHis-tag might interfere with protein–protein
interactions, it does not affect the binding of the Rok to the
DNA or its ability to repress transcription.
378 W. K. Smits et al.
© 2007 The Authors
Journal compilation © 2007 Blackwell Publishing Ltd, Molecular Microbiology, 64, 368–381
Electrophoretic mobility shift assays
Primers used to generate fragments for use in this EMSA
experiments are listed in Table S2. EMSAs were performed
as described (Albano et al., 2005), except for samples con-
taining RNAP polymerase. For these samples, the protocol
was adapted as follows. Reaction mixtures containing 5000
cpm labelled double stranded DNA were incubated at 37°C
for 45 min before loading. Free probe and DNA/protein com-
plexes were separated on a 6% non-denaturing polyacryla-
mide gel prepared with 1¥ TAE (40 mM Tris acetate [pH 8.0],
2 mM EDTA), in a 0.5–2.0¥ TAE gradient, for 2 h at 70 V.
Subsequently, gels were dried and signals were recorded as
described previously (Albano et al., 2005). To quantify
signals, unprocessed captured images were analysed using
Quantity One software (Bio-Rad). Data were imported into
Microsoft Excel for further analysis and graphs for publication
were prepared in Corel Graphics Suite 11.
In vitro transcription assays
In vitro transcription experiments were carried out as
described before (Hamoen et al., 1998), using 150 ng B. sub-
tilis RNAP holoenzyme (a kind gift of M. Salas) and non-
linearized pAN-K plasmid as a template. The pAN-K plasmid
was constructed as follows. Plasmid pAN583 (Predich et al.,
1992) was cut with PvuII and EcoRI. The fragment carrying the
T7-terminator was blunted, and cloned into the PvuII site of
pUC19, yielding pAN583-inverse. The comK promoter was
isolated by PCR using primers pCK2 and Kprom1 (Table S2)
and chromosomal DNA of BD630 (his leu met) as a template.
The product was ligated into the SmaI site of pAN583-inverse,
resulting in plasmid pAN-K. The resulting plasmid allows the
generation of a terminated transcript derived from the comK
promoter. Radiolabelled RNA from the in vitro transcription
experiments was loaded onto a 6% denaturing polyacrylamide
gel (Sequagel-6, National Diagnostics) and run in 1¥ TBE
buffer (100 mM Tris borate [pH 8.3], 10 mM EDTA) for 1 h at
150 V. Signals were recorded from unprocessed gels using
phosphorscreens and a Cyclone PhosphorImager (Packard).
To quantify signals, captured images were analysed using
Quantity One software (Bio-Rad). Data were imported into
Microsoft Excel for further analysis and graphs for publication
were prepared in Corel Graphics Suite 11.
b-Galactosidase assays
b-Galactosidase reporter assays were carried out as
described previously (Albano et al., 2005).
Fluorescence microscopy
Fluorescence of individual cells was determined by fluores-
cence microscopy and image analysis. Samples were taken
at hourly intervals and cells were prepared for microscopy as
described previously (Albano et al., 2005). Images were cap-
tured using the same setting for all time points. To quantify
fluorescent signals from individual cells, captured images
were imported into Quantity One software (Bio-Rad). A grid
for all cells was generated based on the phase-contrast
image, and subsequently overlaid on unprocessed images
from the GFP-channel. Data were imported into Microsoft
Excel for further analysis and graphs for publication were
prepared in Corel Graphics Suite 11.
Acknowledgements
The authors wish to thank M. Salas and M. Strauch for the
kind gift of B. subtilis s
A
/RNAP holoenzyme and AbrB protein
respectively. In addition, A. Sonenshein and P. Serror are
acknowledged for kindly providing the strain for purification of
CodY-his6 and helpful discussions. A.M. Miron´ czuk is
acknowledged for performing initial experiments with CodY.
The authors wish to thank D. Tomkiewicz for expert technical
assistance with the purification of ComK. We thank members
of the Groningen and Newark labs for helpful discussions.
W.K.S. was supported by Grant 811.35.002 of the Nether-
lands Organisation for Scientific Research (NWO-ALW).
Work in the lab of DD was supported by National Institutes of
Health Grant GM057720.
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Supplementary material
The following supplementary material is available for this
article online:
Fig. S1. Heterologous expression and anti-repression. (A)
Schematic depiction of the behavior of PcomK when ComK
acts as an anti-repressor or an activator. RNA polymerase is
depicted as a square (Rp), ComK as a diamond (K). Expres-
sion in a heterologous host, E. coli, is given in the absence of
functional ComK protein. (B) Expression from PcomK in the
absence of ComK in B. subtilis (KGFPDK) and E. coli
(ED232) (Haijema et al., 2001). Cells harboring a comK-gfp
reporter construct were grown, harvested and visualized
according to Materials and methods.
Fig. S2. Fluorescence from a Prok-gfp reporter fusion. Fluo-
rescence from single cells was quantified as described in
Materials and Methods. Error bars show the standard
deviation. The number of cells (n) on which the calculated
mean and standard deviations are based is shown below
each bar. Time is indicated in hours relative to the transition
between exponential and stationary growth phase (T0).
Table S1. Strains and plasmids used in this study.
Table S2. Oligonucleotides used in this study.
This material is available as part of the online article from
http://www.blackwell-synergy.com
Please note: Blackwell Publishing is not responsible for the
content or functionality of any supplementary materials sup-
plied by the authors. Any queries (other than missing material)
should be directed to the corresponding author for the article.
ComK reverses Rok and CodY repression 381
© 2007 The Authors
Journal compilation © 2007 Blackwell Publishing Ltd, Molecular Microbiology, 64, 368–381
    • "On the other hand, high Spo0A(∼P) concentration represses comK expression when Spo0A(∼P) binds to its low-affinity binding sites overlapping the P comK core elements (Mirouze et al., 2012;Figure 1). Apart from its direct influence on P comK , Spo0A(∼P) downregulates the AbrB expression, thereby it renders P comK free of AbrB repression (Smits et al., 2007). Besides, high level of the AbrB concentration represses the expression of sigH which is important for the expression of phr genes. "
    [Show abstract] [Hide abstract] ABSTRACT: Competence is a physiological state that enables Bacillus subtilis 168 to take up and internalize extracellular DNA. In practice, only a small subpopulation of B. subtilis 168 cells becomes competent when they enter stationary phase. In this study, we developed a new transformation method to improve the transformation efficiency of B. subtilis 168, specially in rich media. At first, different competence genes, namely comK, comS, and dprA, were alone or together integrated into the chromosome of B. subtilis 168 under control of mannitol-inducible P mtlA promoter. Overexpression of both comK and comS increased the transformation efficiency of B. subtilis REG19 with plasmid DNA by 6.7-fold compared to the wild type strain 168. This transformation efficiency reached its maximal level after 1.5 h of induction by mannitol. Besides, transformability of the REG19 cells was saturated in the presence of 100 ng dimeric plasmid or 3000 ng chromosomal DNA. Studying the influence of global regulators on the development of competence pointed out that important competence development factors, such as Spo0A, ComQXPA, and DegU, could be removed in REG19. On the other hand, efficient REG19 transformation remained highly dependent on the original copies of comK and comS regardless of the presence of P mtlA -comKS. Finally, novel plasmid-free strategies were used for transformation of REG19 based on Gibson assembly.
    Full-text · Article · Jan 2016
    • "comGA Bsu promoter [26]) or relieve transcription repression (e.g. comK Bsu promoter [48]). To test whether the elements of the comGA Bco and comK Bco promoters are functionally conserved, we assayed the effect of the ComK Bsu protein on these promoter fragments in the heterologous host, B. subtilis. "
    Full-text · Dataset · Aug 2014 · Theoretical Biology and Medical Modelling
    • "More specifically, DegU binds in between the two ComK dimer binding sites and may possibly facilitate tetramerization of ComK at the comK promoter site by partial unwinding and bending of the DNA helix [29]. Transcription can also occur in the absence of ComK [30]. It is estimated that a cell exhibiting competence has on average 50,000 ComK dimers during stationary phase [2]. "
    [Show abstract] [Hide abstract] ABSTRACT: Background It is a fascinating phenomenon that in genetically identical bacteria populations of Bacillus subtilis, a distinct DNA uptake phenotype called the competence phenotype may emerge in 10–20% of the population. Many aspects of the phenomenon are believed to be due to the variable expression of critical genes: a stochastic occurrence termed “noise” which has made the phenomenon difficult to examine directly by lab experimentation. Methods To capture and model noise in this system and further understand the emergence of competence both at the intracellular and culture levels in B. subtilis, we developed a novel multi-scale, agent-based model. At the intracellular level, our model recreates the regulatory network involved in the competence phenotype. At the culture level, we simulated growth conditions, with our multi-scale model providing feedback between the two levels. Results Our model predicted three potential sources of genetic “noise”. First, the random spatial arrangement of molecules may influence the manifestation of the competence phenotype. In addition, the evidence suggests that there may be a type of epigenetic heritability to the emergence of competence, influenced by the molecular concentrations of key competence molecules inherited through cell division. Finally, the emergence of competence during the stationary phase may in part be due to the dilution effect of cell division upon protein concentrations. Conclusions The competence phenotype was easily translated into an agent-based model – one with the ability to illuminate complex cell behavior. Models such as the one described in this paper can simulate cell behavior that is otherwise unobservable in vivo, highlighting their potential usefulness as research tools.
    Full-text · Article · Apr 2013
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