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Previous Article | Next Article 
Journal of Bacteriology, November 1999, p. 6832-6835, Vol. 181, No. 21
Department of Biological Sciences, University
of Alberta, Edmonton, Alberta, Canada T6G 2E9
Received 18 May 1999/Accepted 20 August 1999
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 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- 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).
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
ABSTRACT
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Abstract
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References
10 region of the promoter. How BldD
binds to its promoter and the effect this binding has on the expression
of BldD are discussed.
TEXT
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Abstract
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References
-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).
TABLE 1.
Oligonucleotides used for PCR amplification
View larger version (49K):
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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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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%
H2O2 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-N11-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-N11-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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ACKNOWLEDGMENTS |
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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 |
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* 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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REFERENCES |
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Pleiotropic morphological and antibiotic deficiencies result from mutations in a gene encoding a tRNA-like product in Streptomyces coelicolor A3(2).
Genes Dev.
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| 8. | Nodwell, J. R., K. McGovern, and R. Losick. 1996. An oligopeptide permease responsible for the import of an extracellular signal governing aerial mycelium formation in Streptomyces coelicolor. Mol. Microbiol. 22:881-893[Medline]. |
| 9. |
Pope, M. K.,
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| 10. | Sambrook, J., E. F. Fritsch, and T. Maniatis. 1989. Molecular cloning: a laboratory manual, 2nd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y |
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