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Journal of Bacteriology, November 1999, p. 6832-6835, Vol. 181, No. 21
0021-9193/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
The BldD Protein from Streptomyces
coelicolor Is a DNA-Binding Protein
Marie A.
Elliot and
Brenda K.
Leskiw*
Department of Biological Sciences, University
of Alberta, Edmonton, Alberta, Canada T6G 2E9
Received 18 May 1999/Accepted 20 August 1999
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ABSTRACT |
Gel mobility shift assays with His-tagged BldD isolated from
Escherichia coli have illustrated that BldD is capable of
specifically recognizing its own promoter region. DNase I and hydroxyl
radical footprinting assays have served to delimit the BldD binding
site, revealing that BldD recognizes and binds to a site just upstream from, and overlapping with, the 
10 region of the promoter. How BldD
binds to its promoter and the effect this binding has on the expression
of BldD are discussed.
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TEXT |
The life cycle of the multicellular
bacterium Streptomyces coelicolor is complex by prokaryotic
standards, progressing through a series of structurally and
metabolically differentiated states. Morphological
differentiation begins with a spore, which germinates through the
outgrowth of vegetative hyphae and then produces aerial mycelium, upon
which mature spores will develop (reviewed in references 2 and 5). This morphological
differentiation has been found to be dependent on two main classes of
genes: the bld genes, which are required for the erection of
aerial hyphae (1, 7), and the whi genes, which
are needed for the formation of mature spores (3).
Physiological differentiation involves a shift from primary metabolism
to secondary metabolism, where antibiotic production is observed. The
developmental and metabolic cycles are not completely exclusive; the
timing of aerial mycelium formation and that of antibiotic production
coincide, suggesting that they may share regulatory elements. In
support of this, it has been found that mutations in many of the
bld genes not only result in the failure to form aerial
hyphae but also adversely affect the production of secondary metabolites.
A number of the bld genes have been cloned, and of these,
bldA, bldK, bldB, and bldD
have been sequenced and at least partially characterized. It appears
that while the bld genes have similar phenotypic
characteristics, they play very different roles in the regulation of
differentiation. bldA was found to encode a leucyl-tRNA,
which is responsible for recognizing the rare UUA codon in mRNA
(6), and bldK comprises a gene cluster coding for
an oligopeptide permease (8). bldB and
bldD encode small proteins possessing helix-turn-helix
motifs, characteristic of DNA-binding proteins, and as such may
function as transcription regulators (4, 9). To gain an
understanding of how the bldD gene exerts its regulatory
effects, the BldD protein was overexpressed and purified from
Escherichia coli and then examined for its ability to bind DNA.
Overexpression and purification of BldD.
The bldD
coding region was PCR amplified from chromosomal DNA of S. coelicolor J1501 and was cloned downstream of the six-His tag in the plasmid pQE9 (Qiagen) to yield pQE9BldD+.
E. coli JM109 was transformed with
pQE9BldD+, ampicillin-resistant transformants were
selected, and the integrity of the recombinant plasmids was confirmed
by DNA sequencing. Induction of the BldD fusion protein with 0.5 mM
isopropyl-
-D-thiogalactopyranoside (IPTG) resulted in
the production of 50% soluble and 50% insoluble protein. The soluble
protein fraction was then purified per the manufacturer's
recommendations (Qiagen).
DNA binding by BldD.
Previous analyses comparing mutant and
wild-type bldD transcription had suggested that functional
BldD may negatively regulate its own expression (4), so the
bldD promoter region was selected as a target for the
examination of BldD DNA-binding activity. A 257-bp probe (MAE11-12)
(Table 1) was amplified by PCR, end labeled, and used to assay the binding of BldD at 30°C in a buffer consisting of 10 mM Tris-HCl (pH 7.8), 150 mM NaCl, 2 mM
dithiothreitol, 1 µg of poly(dI-dC), and 10% glycerol. Two retarded
fragments were seen when the DNA-protein complexes were run out on an
8% polyacrylamide gel (data not shown). The smaller of the two
complexes was formed at low BldD concentrations and was followed by the appearance of a higher-molecular-weight complex as the amount of BldD
was increased. The two retarded fragments were present in approximately
equivalent amounts when maximal binding was attained, suggesting that
there may be some equilibration between the two forms. Furthermore, the
absence of a complete shift to the higher-molecular-weight complex
suggested a single site for BldD binding on this 257-bp fragment.
Attempts were then made to delimit the site recognized and bound by
BldD, first using a 100-bp fragment (MAE1-4) and then using a 52-bp
probe (MAE1-15) and a 77-bp probe (MAE16-4) (Table 1), overlapping by
22 bp, obtained by subdividing the 100-bp fragment. It was found that
BldD was capable of binding to MAE16-4 (Fig.
1A) as strongly as to the original
MAE11-12 fragment; however, there was absolutely no shift seen when the
shorter MAE1-15 fragment was used as the binding target (Fig. 1B).
Competition gel shift assays were also conducted, in which the addition
of excess unlabeled, nonspecific DNA (BKL41-MAE5) (Table 1) from within
the bldD coding sequence did not abolish binding by BldD and
in which the addition of a 500-fold excess of unlabeled probe completely abolished BldD binding to the labeled fragments (Fig. 1A).
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FIG. 1.
Gel mobility shift assays of BldD binding to a 77-bp
region (MAE16-4) of the bldD promoter. One to two nanograms
of DNA end labeled with  -32P was incubated with
different amounts of BldD (0 to 40 pmol). The ability of BldD to shift
its own promoter at increasing protein concentrations is seen in panel
A. Specificity of binding is also illustrated in panel A by the
addition of ~500 ng of unlabeled, nonspecific competitor DNA
(BKL41-MAE5) to the penultimate lane and ~500 ng of unlabeled,
specific competitor DNA (MAE16-4) to the last lane. Panel B illustrates
the binding of BldD to MAE1-15 (left) and MAE18-4 (right) (1 to 2 ng of
each probe) with increasing amounts of BldD (0 to 40 pmol).
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The region contained within MAE16-4 was then further analyzed, and it
was found that while the 52-bp MAE1-15 was not recognized
by BldD (Fig.
1B), the addition of 9 bases to its 3' end to yield
MAE1-35 (Table
1;
Fig.
2C) resulted in complete shifting
(data
not shown), indicating that these 9 bases are important for BldD
binding. The region extending from MAE18-4 (Table
1; Fig.
2C),
which
included these 9 bases, showed defined but lower-affinity
binding (Fig.
1B), suggesting that these 9 bases are required
but are not sufficient
for complete BldD binding. Gel shift experiments
carried out with a
fragment from MAE36-4 (Table
1) showed no
shift (data not shown).
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FIG. 2.
Localization of the BldD binding site. (A) DNase I
footprints of the bldD promoter region, with different
concentrations of BldD (0 to 80 pmol). Areas of strong protection are
shown with large brackets, while weakly protected areas are indicated
with small brackets. (B) Hydroxyl radical footprinting of the
bldD promoter in the absence () or presence (+) of BldD
(80 pmol). Large asterisks indicate strong protection by BldD, while
small asterisks show sites of weak protection. (C) Summary of DNase I
protection, hydroxyl radical protection, and gel mobility shift
experiments. Areas protected from DNase I and the hydroxyl radical are
as indicated in panels A and B. Fragments used for gel shift assays are
outlined at the bottom, along with their relative abilities to shift
DNA (+++, strong binding [100% bound]; +++,
90% bound; +, 20% bound; , no binding).
Potential BldD binding sites are indicated above the sequence, with the
top set of arrows illustrating a direct repeat and the bottom set
indicating an inverted repeat. Downstream of the transcription start
site is a sequence that corresponds to a half site of the inverted
repeat.
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DNase I and hydroxyl radical footprinting experiments were carried out
to more precisely localize the site of BldD interaction
with its
promoter. DNase I footprinting was conducted by using
the 257-bp
bldD promoter fragment (MAE11-12), which had been
specifically
labeled on either its upper or its lower strand. The
reaction
conditions were essentially the same as for the gel shift
assays;
however, the binding buffer was modified to include
MgCl
2 and
CaCl
2, and the salt concentration was
changed to 50 mM KCl. In
the presence of BldD, a single area spanning a
32-base segment
of the DNA was found to be strongly protected from
DNase I cleavage
on both strands (Fig.
2A and C), and this area was
found to encompass
the
bldD 10 promoter region and the
transcription start site.
A region of much weaker protection was also
observed just downstream
from the strongly protected area (Fig.
2A and
C) on both
strands.
A finer analysis of the BldD binding site with hydroxyl radical
footprinting was then undertaken (
12). The upper and lower
strands of the 100-bp promoter fragment (MAE1-4; Table
1) were
independently end labeled and were incubated in binding buffer,
both
alone and with BldD, in the absence of glycerol. Three microliters
each
of 0.5 mM Fe(EDTA)
2
, 10 mM Na-ascorbate, and 0.3%
H
2O
2 was added to each reaction
mixture, and
reactions were allowed to proceed for 2 min before
being stopped with 5 µl of 0.1 M thiourea plus 3 µl of 100% glycerol.
The resulting
products were then applied to an 8% native polyacrylamide
gel to
separate any unbound probe from that bound by BldD, thereby
reducing
the background that would result from cleavage of the
unbound fraction.
The cleavage products were excised and purified
per reference
10 and then electrophoresed on a 6% denaturing
polyacrylamide gel. BldD was determined to afford strong protection
to
bases at positions
16 to
13 and
23 to
24 relative to the
transcription start point (tsp). On the lower strand, BldD was
seen to
confer strong protection at positions
16 to
12 and
5
to
4 and
much weaker protection at positions +9 to +12 (Fig.
2B and C). Taken
together, the DNase I and hydroxyl radical footprinting
results seem to
suggest a site for BldD binding centered just
upstream from the
10
region of the promoter. Examination of the
sequence flanking this site
has revealed an inverted repeat and
a direct repeat (Fig.
2C) which
overlap significantly and either
of which may be the site recognized
and bound by BldD. When the
repeats were segmented and the gel shift
assays were conducted,
no binding was seen to the left half of the
repeat, represented
by MAE1-15 (Fig.
1B and
2C), while there was a
defined single
shifted band seen with MAE18-4 (Fig.
1B), which
contained the
entire right half of the repeat (Fig.
2C). This suggests
that
BldD binds preferentially to the right half of the repeat and
then, after binding to the right, is capable of recognizing and
binding
to the
left.
One possible interpretation of the presence of two shifted bands in the
gel shift assay when both half sites are present is
that the first
shift represents binding to the higher-affinity
right half site, and
then a conformational change permits binding
to the left half, which is
seen as the second shift. Alternatively,
the first shift may represent
binding to the entire repeat, and
upon the addition of more protein,
some higher-order structure
may be seen due to protein-protein
interactions between excess
BldD and BldD already complexed to the DNA.
Finally, it appears
that there is a single half site present just
downstream from
the
bldD tsp (Fig.
2C), and it is possible
that binding of BldD
to the full inverted repeat may allow weak binding
of BldD at
this half site, either through protein-protein interactions
or
through a change in the DNA conformation. This would explain the
weakly protected region observed immediately downstream from the
tsp in
the DNase I and hydroxyl radical footprints. Gel shift
assays done by
using a fragment with only this downstream region
as the potential
binding site (MAE36-4) showed no binding by BldD,
which is not
surprising if binding at the full repeat is required
for stabilization
of the weak binding
downstream.
Attempts were made to clarify whether the inverted repeat or the direct
repeat was the preferred binding motif by using mutagenic
oligonucleotides. Perfect direct and indirect repeats were created
(Fig.
3) and then subjected to gel shift
assays; however, no difference
from the results obtained with the
wild-type fragment could be
detected (data not shown). This suggests
that subtle changes in
the repeats cannot distinguish between binding
to one or the other,
although other factors suggest that it is the
inverted repeat
rather than the direct repeat that BldD recognizes. The
pattern
of protection observed with the hydroxyl radical footprinting
showed that binding by BldD occurred symmetrically on both strands,
supporting recognition of the inverted repeat. Prior analyses
of the
BldD sequence had revealed the presence of a C-terminal
helix-turn-helix domain weakly similar to that of the LysR-like
family
of DNA-binding proteins (
4). These LysR-like proteins
recognize and bind to a consensus sequence containing a
T-N
11-A
motif, usually within a 15-bp partially dyadic
sequence (reviewed
in reference
11). This would also
be true for the BldD binding
site if BldD bound to the imperfect
inverted repeat, within which
was contained a T-N
11-A
motif. It is possible that other genes
controlled by BldD may possess
sequences and/or spacings different
from that of the
bldD
promoter, and the determination of a consensus
binding sequence for
BldD awaits their identification.
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FIG. 3.
Mutagenesis of the bldD promoter region. The
promoter region of the wild-type bldD gene is shown,
together with portions of the fragments containing mutations within
this region. MAE46-4 possesses a perfect direct repeat, and MAE47-4 has
a perfect inverted repeat. The mutagenized bases are indicated in bold
type.
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ACKNOWLEDGMENTS |
This work was supported by the Alberta Heritage Foundation for
Medical Research and the Natural Sciences and Engineering Research Council of Canada.
We thank Karen Anthony for her helpful advice regarding the
overexpression and purification of BldD and for her gift of E. coli JM109, Gerald Stemke for his gift of pQE9, and Cyril Kay for
his suggestions on how to maintain BldD in a soluble form. We are also
very grateful for helpful discussions with Laura Frost, Linda
Reha-Krantz, and Mark Glover and for critical reading of the manuscript
by Laura Frost.
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FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Biological Sciences, CW405 Biological Sciences Bldg., University of
Alberta, Edmonton, Alberta, Canada T6G 2E9. Phone: (780) 492-1868. Fax: (780) 492-9234. E-mail: brenda.leskiw{at}ualberta.ca.
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Journal of Bacteriology, November 1999, p. 6832-6835, Vol. 181, No. 21
0021-9193/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
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