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JOURNAL OF BACTERIOLOGY,0021-9193/00/$04.0010
Apr. 2000, p. 1916–1922 Vol. 182, No. 7
Copyright © 2000, American Society for Microbiology. All Rights
Reserved.
Purification and Characterization of the DeoRRepressor of
Bacillus subtilis
XIANMIN ZENG,1 HANS H. SAXILD,1* AND ROBERT L. SWITZER2
Department of Microbiology, Technical University of Denmark,
DK-2800 Lyngby, Denmark,1
and Department of Biochemistry, University of Illinois, Urbana,
Illinois2
Received 24 September 1999/Accepted 13 December 1999
Transcription of the Bacillus subtilis dra-nupC-pdp operon is
repressed by the DeoR repressor protein. TheDeoR repressor with an
N-terminal His tag was overproduced with a plasmid under control of
a phage T5 pro-moter in Escherichia coli and was purified to near
homogeneity by one affinity chromatography step. Gel fil-tration
experimental results showed that native DeoR has a mass of 280 kDa
and appears to exist as anoctamer. Binding of DeoR to the operator
DNA of the dra-nupC-pdp operon was characterized by using an
elec-trophoretic gel mobility shift assay. An apparent dissociation
constant of 22 nM was determined for bindingof DeoR to operator
DNA, and the binding curve indicated that the binding of DeoR to
the operator DNA wascooperative. In the presence of
low-molecular-weight effector deoxyribose-5-phosphate, the
dissociation con-stant was higher than 1,280 nM. The dissociation
constant remained unchanged in the presence of
deoxyribose-1-phosphate. DNase I footprinting exhibited a protected
region that extends over more than 43 bp, covering apalindrome
together with a direct repeat to one half of the palindrome and the
nucleotides between them.
In Bacillus subtilis, the dra-nupC-pdp operon encodes
threeenzymes required for deoxyribonucleoside and
deoxyriboseutilization (12). Expression of the operon is induced by
de-oxyribonucleosides and deoxyribose. Transcription of this
op-eron is negatively regulated by the DeoR repressor protein,which
is encoded by the deoR gene located immediately up-stream of the
operon (12, 14). DeoR regulates the expressionof the dra-nupC-pdp
operon by binding to an operator se-quence located in a region
corresponding to 260 to 222 bprelative to the transcription start
point (14). This site containsa palindromic sequence in the region
from 260 to 243 bp anda direct repeat to the 39 half of the
palindrome located betweenthe 235 and 210 regions. Previous studies
with crude DeoRshow that both the palindrome and the direct repeat
are nec-essary for DeoR regulation of dra-nupC-pdp operon
expression(14). Both deoxyribose-5-phosphate (dRib-5-P) and
deoxyri-bose-1-phosphate (dRib-1-P) are suggested to be internal
in-ducers for the expression of the operon, but dRib-5-P seems tobe
the preferred inducer (14).
In Escherichia coli, the expression of the deo operon is
neg-atively regulated by the DeoR repressor protein and dRib-5-Pis
the effector molecule (1, 2, 9). B. subtilis DeoR shows noamino
acid sequence similarity to E. coli DeoR, which belongsto the
LacI-GalR family. Furthermore, there is no similarity inthe DNA
operator sites for these two repressors (14). In thepresent work,
we describe the purification of DeoR of B. sub-tilis and show that
the native DeoR repressor protein mostlikely exists as an octamer
in solution. We also report thespecific binding of DeoR to the
operator DNA of the B. subtilisdra-nupC-pdp operon.
MATERIALS AND METHODS
Bacterial strains, plasmids, and media. The bacterial strains
and plasmidsused in this work are listed in Table 1. B. subtilis
was grown in Spizizen’ssalt-containing minimal medium (13)
supplemented with 50 mg of L-tryptophan
per ml and with 0.4% succinate as a carbon source. L broth
(Difco Laboratories,Detroit, Mich.) was used as a rich medium for
both E. coli and B. subtilis.Culturing of cells was performed at
37°C. For selection of antibiotic resistance,the following
antibiotics and concentrations were used: ampicillin, 100
mg/ml;neomycin, 5 mg/ml; erythromycin, 1 mg/ml; lincomycin, 25
mg/ml; and phleomy-cin, 1 mg/ml. dRib-5-P and dRib-1-P are from
Sigma.
DNA manipulations and genetic techniques. Plasmid DNA was
isolated by thealkaline-sodium dodecyl sulfate method (13).
Transformation of E. coli and B.subtilis was performed as
previously described (13). Treatment of DNA withrestriction enzymes
and T4 DNA ligase was performed as recommended by thesupplier. A
standard PCR was performed as described previously (14).
Construction of plasmids and strains. The deoR gene was
amplified by PCRusing plasmid pHH1002, which carries deoR (12). The
forward and reverseoligonucleotide primers were synthesized with
BamHI and HindIII 59-linkedrestriction sites, respectively (Table
2). The PCR product was digested withBamHI and HindIII and then
ligated to BamHI- and HindIII-digested plasmidpQE-30, generating
pJOY1000. The E. coli TG1 strain harboring pJOY1000 orpQE-30 is
designated strain JOY1000 or JOY999, respectively. For in
vivocomplementation, the deoR gene with six histidine codons at the
59 end fromJOY1000 was amplified by PCR using plasmid pJOY1000 as
template DNA. Theforward and reverse oligonucleotide primers were
synthesized with PstI andHindIII 59-linked restriction sites,
respectively (Table 2). The PCR product wasdigested with PstI and
HindIII, ligated to PstI- and HindIII-digested plasmidpEB112, and
transformed into E. coli TG1, selecting for ampicillin
resistance.Plasmid extracted from E. coli was transformed into B.
subtilis XM25 by selectingfor phleomycin resistance, yielding
XM1000 (Table 1).
Expression and purification of the DeoR repressor protein. E.
coli strain TG1bearing pJOY1000 was grown in 3 liters of Luria
broth. After the optical densityat 600 nm reached 0.5, the culture
was induced with 2 mM IPTG (isopropyl-b-D-thiogalactopyranoside)
for 4 h. All the cells from the 3-liter cultures wereharvested by
centrifugation and stored at 280°C.
All purification procedures were performed at 4°C. The cells
were resus-pended in sonication buffer (50 mM sodium phosphate [pH
7.8], 300 mMNaCl) and disrupted by sonication on ice, and cell
debris was removed bycentrifugation. Streptomycin sulfate (0.11
volume of a 10% solution freshlyprepared in sonication buffer) was
added, and the precipitate was removed bycentrifugation. The
solution was dialyzed against sonication buffer. The entiresample
was loaded onto an Ni-nitrilotriacetic acid (Ni-NTA) agarose
columnthat had been equilibrated in sonication buffer. Ten column
volumes ofsonication buffer was allowed to flow through the column,
and then 10 columnvolumes of washing buffer (50 mM sodium phosphate
[pH 6.0], 300 mM NaCl,10% glycerol) were allowed to flow through
the column. DeoR was elutedusing a 250-ml linear imidazole gradient
from 100 to 500 mM in wash buffer.Fractions from the trailing half
of the DeoR peak, which eluted at a conduc-tivity equivalent to
around 200 mM imidazole, were pooled. The pooledDeoR sample was
then dialyzed against wash buffer containing 0.2 M imida-zole (0.2
M imidazole was included to prevent precipitation of DeoR)
andfrozen at 280°C in 50-ml aliquots. Approximately 40 mg of DeoR
was purifiedfrom 3 liters of culture.
* Corresponding author. Mailing address: Department of
Microbi-ology, Technical University of Denmark, Building 301,
DK-2800 Lyn-gby, Denmark. Phone: 45 25 24 95. Fax: 45 88 26 60.
E-mail: [email protected].
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Gel filtration analysis of DeoR. A column (1-cm diameter, 95-cm
height, and75-cm3 bed volume) of Sephadex G-150 (Pharmacia Biotech
Inc.) was used todetermine the native molecular weight of DeoR. The
buffer used contained50 mM sodium phosphate (pH 6.0), 300 mM NaCl,
0.2 M imidazole, and 10%glycerol. The column was loaded with 0.5-ml
samples of DeoR (concentra-tions varied from 2.5 to 5.0 mg/ml) and
eluted at 4°C. Proteins used toconstruct an Mr standard curve for
the column were myoglobin, chickenserum albumin, yeast hexokinase,
and bovine gamma globulin. The proteinconcentrations in the eluted
fractions were determined from their absorbanceat 280 nm
(A280).
b-Galactosidase assay. b-Galactosidase activity was measured by
the methodof Miller (8). Specific enzyme activities were expressed
in units per milligram ofprotein. One unit is defined as 1 nmol of
substrate converted per minute. Thevalues shown are the means of at
least two different experiments. The variationwas less than 10%.
The concentration of total protein was determined by themethod of
Lowry et al. (7).
Mobility shift assay. The standard PCR mixtures containing 25
mmol of[a-33P]dATP (25 mCi) were used to produce the radiolabeled
operator-contain-ing DNA probes. The following labeled fragments
were generated: primers 4 and5 (111-bp product), 6 and 7 (42-bp
product), 7 and 8 (34-bp product), and 6 and9 (30-bp product). Each
binding reaction mixture contained 10 mM Tris-HCl, 50mM NaCl, 1 mM
EDTA, 1 mM dithiothreitol (pH 7.5), double-stranded poly(dI-dC) (1
U/ml) [1 U of poly(dI-dC) is 1 A260 unit in a 1-cm light path], 250
mg of
bovine serum albumin per ml, and 5% glycerol in a final volume
of 10 ml.Approximately 10 to 100 pM of labeled DNA probe and
various concentrationsof DeoR were used in each binding reaction
mixture. For the binding stoichi-ometry experiment, in addition to
the labeled DNA, 1 mM nonradioactive DNAof the same fragment was
added to each binding reaction mixture. After incu-bation for 20
min on ice, samples were loaded onto a 5% polyacrylamide gel
andelectrophoresed at 7 V/cm for 2 h at 4°C. Dried gels were
visualized and quan-titated with a Packard Instant Imager. Apparent
Kd values were calculated fromisotherms of free DNA at various
repressor concentrations according to the Hillequation. The
repressor concentration was calculated on the basis of the
35-kDasubunit.
DNase I footprinting. The 111-bp DNA probe used for DNase I
footprintingwas similar to the probe used for gel shift assay
except that a single strand was32P labeled at its 59 end by T4
kinase. The DNA fragment was incubated withDeoR as described above
for the mobility shift assay. For DNase I digestion, 1ml of 50 mM
CaCl2 was added to the 10-ml DNA-protein mixture, followed by
theaddition of 1 U of DNase I. Digestion was stopped after 5 min on
ice by theaddition of 10 ml of a stop solution (200 mM NaCl, 30 mM
EDTA, 0.1 mg of yeasttRNA per ml). Samples were precipitated with
ethanol on dry ice for 20 min andcentrifuged. Precipitated DNA was
washed with 70% ethanol, dried, taken up informamide sequencing gel
buffer, and electrophoresed on an 8% polyacrylamidesequencing gel
alongside a Maxam-Gilbert A1G sequencing ladder (11) for thesame
fragment.
TABLE 1. Bacterial strains and plasmids used in this studya
Bacterial strainor plasmid Relevant genotype or description
Source or reference
Bacterial strainsB. subtilis
168 trpC2 C. AnagnostopoulosXM15 trpC2 amyE::dra-lacZ 14XM25
trpC2 amyE::dra-lacZ deoR::erm 14XM251 trpC2 amyE::dra-lacZ
deoR::erm pXM1000 Transformation of XM25
by pXM1000, Plr
E. coliJOY999 TG1(pQE-30) This workJOY1000 TG1(pJOY1000) This
workTG1 Wild type; lacIq Laboratory stock
PlasmidspEB112 Apr (E. coli) Plr (B. subtilis); multiple copy
shuttle vector containing the pBR322 rep. origin for
replication in E. coli and pC194 rep. origin for replication in
B. subtilis6
pJOY1000 BamHI-HindIII PCR fragment containing deoR generated by
primers S3 and S4, ligated to pQE-30digested with BamHI and
HindIII
This work
pXM1000 PstI-HindIII PCR fragment containing deoR generated by
primers S4 and S5, ligated to pEB112digested with PstI and
HindIII
This work
pQE-30 Apr, has a promoter and operator element consisting of
the E. coli phage T5 promoter and two lacoperator sequences, used
for overexpressing deoR
Qiagen
pHH1002 12
a Apr, ampicillin resistance; Plr, phleomycin resistance; rep.,
replication.
TABLE 2. Oligonucleotides used for the PCR amplifications
Primer 59- or 39-linked restrictionsite sequence Nucleotide
sequencea Coordinatesb or source
Amplification of deoR1 59 BamHI
59-CGCGGATCCATGGATCGGGAAAAACAG-39 4052107–40520882 59 HindIII
59-GCCGAAGCTTTCACAAATCATTAACAAG-39 4051166–40511873 59 PstI
59-GAACTGCAGATTAAAGAGGAGAAATTAAC-39 Qiagen
Mobility shift assay4 59 EcoRI
59-GCCGGAATTCGTGACACGTTCAAACCTT-39 2805 59 KpnI
59-GCCGGGTACCATCCTTCGCACACTTCC-39 1306 59 KpnI
59-CGGCGGTACCCTTTTGAACATATGTAAATTGGTAATTG-39 2197 59 EcoRI
59-GCCGGAATTCTTCAATTACCAATTTACATATG-39 2488 59 KpnI
59-CGGCGGTACCCATATGTAAATTGGTAATTG-39 2279 59 EcoRI
59-GCCGGAATTCCCTTTCATTGAACAAAATTTCAATTACC-39 266
a Italic letters indicate nucleotides of the linker sequences.
Underlined letters indicate nucleotides of the restriction site
sequence.b The number indicates the position of the 59-proximal
nucleotide (14) of the primer except for primers 1 and 2, for which
the nucleotide numbering of genome
sequence (5) has been used.
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RESULTS
Overexpression and purification of DeoR. The B. subtilisdeoR
gene was cloned into pQE-30 to generate plasmidpJOY1000, in which
the expression of deoR was driven fromthe E. coli phage T5 promoter
containing two lac operatorsequences, so that production of DeoR
was induced by IPTG.His-tagged DeoR was overproduced in E. coli
strain TG1 andpurified in a single step by Ni-chelate affinity
chromatographyas described in Materials and Methods. The His-tagged
DeoRprotein was purified because the native DeoR protein
wasrefractory to purification. Although DeoR was an abundantprotein
in cells following overexpression, a substantial fractionwas
insoluble. Nevertheless, DeoR comprised a significantfraction of
the proteins in the soluble cell extract (Fig. 1, lane2). Extract
proteins were absorbed to Ni-NTA-agarose, andthe repressor was
eluted by approximately 0.2 M imidazole.Fractions from the trailing
half of the DeoR peak were pooledto yield a preparation exceeding
95% homogeneity (Fig. 1,lanes 5 and 6). The yield was approximately
40 mg of purifiedprotein from 3 liters of E. coli culture.
In vivo complementation by the six-histidine-tagged
DeoRrepressor. Although the His tag does not usually interfere
withthe structure or function of purified proteins (4), we tested
theHis-tagged DeoR for in vivo complementation of a B. subtilisdeoR
mutant. The deoR gene with six histidine codons frompJOY1000 was
subcloned into pEB112 under the control ofinducible promoter Ptac
as described in Materials and Meth-ods. When transformed with this
plasmid, the deoR strainXM251 was phenotypically DeoR1 in the
presence of IPTG.b-Galactosidase activity showed that dra-lacZ
expression instrain XM251 had a normal, approximately 15-fold DeoR
reg-ulation (14) similar to the wild-type XM15 when grown inminimal
medium succinate containing (Table 3).
Molecular mass of the DeoR repressor protein. By compar-ing the
mobility of purified repressor on sodium dodecyl
sul-fate-polyacrylamide gels with those of several other proteins
ofknown molecular weight, the mass of the His-tagged DeoRsubunit
was found to be 35 kDa (Fig. 1). This agrees with the
molecular mass of 34 kDa calculated from the derived aminoacid
sequence of the deoR gene (12).
The native molecular mass of DeoR repressor in wash
buffercontaining 200 mM imidazole, as determined from its
elutionprofile from a Sephadex G-150 gel filtration column, is 280
610 kDa. This estimate was based on a comparison with theelution
pattern of several other proteins of known molecularweight.
Assuming that the ratio of Stokes radius to mass of theDeoR protein
and the size standard proteins is the same, thismeans that the
native protein is most likely an octamer.
DNA binding to DeoR. An electrophoretic gel mobility shiftassay
as described in Materials and Methods was used to mea-sure the
binding of DeoR to labeled operator DNA. In mostcases, purified
DeoR was used. The radioactive oligonucleo-tide used for the
characterization of binding was a 111-bpfragment corresponding to
nucleotides 280 to 130 relative tothe dra-nupC-pdp operon
transcription start point (14). This111-bp fragment contains the
operator for DeoR and wasshown in preliminary studies to bind well
to crude DeoR (14).The specificity of the interaction between DeoR
and operatorDNA was tested in two ways. First, to demonstrate that
theDNA was bound specifically by DeoR, gel shift assays
wereperformed using the 111-bp DNA fragment and crude extractsfrom
either E. coli JOY1000 which overexpresses DeoR orJOY999 which
carries the vector plasmid only. The crude ex-tract containing
overexpressed DeoR clearly contained a pro-tein that binds to DNA:
increasing amounts of this extractincreased the amount of DNA bound
(Fig. 2, lanes 2 to 5). In
FIG. 1. Purification of DeoR. Sodium dodecyl
sulfate-polyacrylamide gelelectrophoresis results are shown. Lane
1, molecular mass standards (from top tobottom, 94, 67, 43, 30, 20,
and 14 kDa); lane 2, soluble cell extract; lane 3,flowthrough from
an Ni-NTA agarose column; lane 4, wash from an Ni-NTAagarose column
with wash buffer; lanes 5 and 6, pooled fractions eluted by
theimidazole gradient and dialyzed pooled fractions,
respectively.
TABLE 3. b-Galactosidase level of B. subtilis wild-type anddeoR
strains carrying a dra-lacZ fusiona
Strain RelevantgenotypeInduceradded
Enzyme activity(nmol/min/mg)
XM15 Wild type None 15Deoxyribose 262
XM25 deoR::erm None 107Deoxyribose 110
XM251 XM25(pXM1000) None 14b
Deoxyribose 189b
a Cells were grown in minimal medium containing succinate.
Inducer wasadded to a final concentration of 1 mg/ml.
b IPTG was added to a final concentration of 1 mM to induce
deoR.
FIG. 2. Binding of the 111-bp dra-nupC-pdp operator DNA by crude
extracts(15 mg/ml) of E. coli cells in which DeoR was overexpressed
(JOY1000 [lanes 1to 5]) and a control strain bearing the plasmid
vector only (JOY999 [lane 6]).Lanes: 1, free DNA fragment (no
extract); 2 to 5, JOY1000 extract dilution of1:50, 1:20, 1:10, and
1:5, respectively; 6, JOY999 extract dilution of 1:5.
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contrast, crude extracts from cells that carried the vector
onlycontained no protein that bound to DNA (Fig. 2, lane 6).These
results indicate that DeoR protein binds to DNA andrule out the
possibility that an impurity in the DeoR prepara-tion binds to the
DNA instead.
To demonstrate that DeoR binds specifically to the operatorDNA,
quantitation of binding affinity was studied with purifiedDeoR,
which was not possible in earlier experiments withcrude extracts.
We have determined the apparent dissociationconstant for binding of
the purified repressor to the operatorregion of the dra-nupC-pdp
operon. Binding isotherms werecalculated from the increase in the
levels of bound DNA, andapparent Kd values represented the DeoR
concentration re-quired for 50% saturation of the control site DNA.
The ap-parent dissociation constant determined from this data was
22nM (Fig. 3A). The binding of DNA to DeoR was described bya
sigmoid curve (Fig. 3B), which suggested that the binding ofDeoR to
the operator DNA is cooperative. In other words, thebinding of
operator DNA to DeoR enhances the binding ofadditional operator DNA
to the same DeoR molecule.
Binding stoichiometry. In order to determine the DeoRbinding
stoichiometry for operator DNA of the dra-nupC-pdpoperon, gel shift
assays using high concentrations of operatorDNA (DNA concentration
much greater than Kd) were per-formed as described in Materials and
Methods. The bindingstoichiometry was approximately four DeoR
molecules per
111-bp DNA fragment, assuming all the DeoR protein wasactive
(Fig. 4). This suggested that four DeoR subunits wereneeded for
total binding to the operator DNA.
Three palindromic halves are required for DeoR binding. Ithas
been shown that both the palindrome and the direct repeatare
necessary for the binding of DeoR to the operator DNA ofthe
dra-nupC-pdp operon (14). To investigate the roles of thesethree
palindromic halves, the binding affinity was quantitatedwith three
DNA fragments containing different parts of theoperator site and
the apparent dissociation constant for DeoRbinding was determined
(Fig. 5). The apparent Kd value de-termined from these data was 20
nM for a DNA fragmentcontaining the palindrome and the direct
repeat (Fig. 5). No orvery weak binding was found for DNA fragments
containingeither only the palindrome (Fig. 5) or containing the 39
half ofthe palindrome and the direct repeat (data not shown).
Thisresult indicated that binding of the DeoR repressor to
theoperator DNA operon required both the palindrome and thedirect
repeat. In other words, three palindromic halves areneeded for
tight binding.
Effect of dRib-5-P on DeoR binding to the operator. In anearlier
gel shift assay with crude DeoR, dRib-5-P was able torelease DeoR
from the DNA-protein complex (14), but nosimilar experiment has
been performed with dRib-1-P in vitro.Here we have determined the
apparent dissociation constantfor binding of the purified repressor
to the 111-bp fragment in
FIG. 3. Binding of the DeoR repressor protein to the 111-bp
operator DNA of the dra-nupC-pdp operon. A profile of a gel shift
assay (A) and the calculated bindingisotherm for DeoR with the
operator DNA (B) are shown. DeoR concentrations (in nanomolar) are
given.
FIG. 4. DeoR binding stoichiometry for the 111-bp operator DNA
of the dra-nupC-pdp operon. DeoR concentrations (in micromolar) are
given at the top of thegel. Nonradioactive 111-bp DNA fragment (1
mM) was added to each binding assay in addition to the radiolabeled
DNA.
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the presence of dRib-5-P or dRib-1-P (Fig. 6). The resultsshowed
that Kd was increased 60-fold when 100 mM dRib-5-P(Kd . 1,280 nM)
was present in the assay mixture, whereasalmost no change was
observed for Kd when 100 mM dRib-1-P(Kd 5 25 nM) was present. These
results confirmed the resultsof a previous report that dRib-5-P
binds to DeoR in vitro andacts as an internal inducer for the
expression of the dra-nupC-pdp operon (14). In contrast, dRib-1-P
binds only very weakly,if it binds at all, to DeoR under the in
vitro conditions tested.
DNase I footprinting analysis of the interaction betweenDeoR and
DNA. DNase I footprinting was used to identify theprecise locations
of the DeoR binding sites. The same 111-bpDNA fragment that was
used for the measurement of DeoRbinding affinity (except that a
single strand was end labeled)was used for the DNase I footprinting
experiment. The labeledDNA fragment was incubated with or without 5
mM DeoR andpartially digested with DNase I. The pattern of
protection andhypersensitivity is shown in Fig. 7. In the absence
of repressor,DNase I cleavage produced a distinct pattern of bands
(Fig. 7,lane 3). Upon addition of the DeoR repressor, a
protectedregion of 43 bp appeared covering most of the palindrome,
thedirect repeat, and all the nucleotides between them (Fig. 7,lane
4). This confirms the previous reports about the locationsof DeoR
binding sites from work with mutagenesis and gelshift assays
(14).
It is worth mentioning that the adenine residue at the 59
end
of the palindrome 59-ATTGAACAAAATTTCAAT-39 wasfound to be not
protected or only weakly protected. Previousmutagenesis studies of
this palindrome showed that this ade-nine residue had no effect in
DeoR regulation in vivo (14).Moreover, the adenine residue at the
39 end of the directrepeat 59-TTCAA-39 was only weakly protected,
too.
FIG. 5. Binding of the DeoR repressor to DNA fragments
containing differ-ent operator sites. Results with a 43-bp fragment
containing the palindrome andthe direct repeat and a 34-bp fragment
containing only the palindrome are shown.
FIG. 6. Binding of DeoR to the 111-bp operator DNA of the
dra-nupC-pdpoperon in the presence of 100 mM dRib-5-P or
dRib-1-P.
FIG. 7. DNase I footprinting of DeoR binding sites. Lane 1, G1A
sequenc-ing ladder; lane 2, T1C sequencing ladder; lanes 3 and 4,
no DeoR (lane 3) and5 mM DeoR (lane 4) was added. The nucleotide
sequence of the sense strand ofthe operator DNA between nucleotides
260 and 222 relative to the transcrip-tion start site is given to
the left of the gel. The palindrome and the direct repeatare marked
by the vertical lines.
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DISCUSSION
We conclude from our studies that the purified B. subtilisDeoR
protein is an octamer composed of 34-kDa subunitswhich binds
cooperatively to dra-nupC-pdp operator DNA.The affinity of DeoR for
operator DNA is greatly reduced bybinding of dRib-5-P. These
conclusions are based on studies ofthe DeoR protein bearing an
N-terminal six-histidine tag,which was used because we were unable
to purify the nativeDeoR protein. Thus, it is reasonable to ask
whether the prop-erties of the His-tagged DeoR are the same as
those of thenative DeoR. We believe that they are for the following
rea-sons. Binding to dra-nupC-pdp operator DNA by overex-pressed
recombinant native DeoR and His-tagged DeoR incrude E. coli
extracts was essentially the same with respect toaffinity for DNA
and the effect of dRib-5-P. Also, a plasmid-borne copy of the gene
for the His-tagged DeoR protein couldcomplement a B. subtilis deoR
mutant just as well as the nativedeoR gene. Finally, the properties
of the purified His-taggedDeoR account very well for previously
described observationson the repression of the dra-nupC-pdp operon
in vivo (12, 14).
A comparison of the primary structure of the B. subtilisDeoR
with protein sequences in the database showed that B.subtilis DeoR
has significant similarity to several regulatoryproteins which
belong to the SorC family of transcriptionalregulators from
different organisms. Interestingly, the proteinswith the highest
degree of similarity can be divided into twogroups. SorC
(Klebsiella pneumoniae) (GenBank accession no.X66059), DalR (K.
pneumoniae) (accession no. AF045245),SmoC (Rhodobacter sphaeroides)
(accession no. AF018073),and EriD (Brucella abortus) (accession no.
U57100) show highdegree of similarity to the amino-terminal part of
DeoR, whichcontains the DNA-binding domain. Much less similarity
isfound in the rest of the primary sequence. SorC, DalR, SmoC,and
EriD all regulate the transcription of genes involved insugar
alcohol catabolism. The second group consists of
GapR(Staphylococcus aureus) (accession no. AJ133520), YgaP
(Ba-cillus megaterium) (accession no. M87647), YvbQ (B.
subtilis)(accession no. Z99121), and ClyR (Leuconostoc
mesenteroides)(accession no. Y10621). This group of regulators
shows simi-larity to the carboxy-terminal region of DeoR. GapR,
YgaP,and YvbQ encode regulators of operons containing gap,
whichencodes the glycolytic enzyme glyceraldehyde-3-phosphate
de-hydrogenase. The ClyR protein is involved in control of
citricacid cycle gene expression. Hence, this group of proteins
reg-ulates genes involved in glucose metabolism. We have foundthat
DeoR most likely binds dRib-5-P, and we speculate thatthe binding
site may include parts of both the amino-terminalregion (perhaps
overlapping the DNA-binding region) and thecarboxy-terminal region.
The DeoR amino-terminal part issimilar to proteins that bind sugar
alcohol phosphates as ef-fector molecules, and the carboxy-terminal
part is similar toproteins that most likely bind
glyceraldehyde-3-phosphate,which is the product of dRib-5-P
cleavage catalyzed by deoxyri-boaldolase. Domains capable of
binding sugar alcohol phos-phates and glyceraldehyde-3-phosphate
may have been incor-porated into the DeoR structure in order to
create a dRib-5-P-specific binding domain.
Although they both appear to contain an a-helix–turn–a-helix
domain of the type commonly found in DNA-bindingproteins (3, 10)
and appear to exist as octamers in the native(DNA-free) state, B.
subtilis and E. coli DeoR repressor pro-teins share little sequence
similarity and the DNA sequences towhich they bind are dissimilar.
The E. coli DeoR is thought tobind simultaneously to two or three
operators of the 16-bppalindrome, which are separated by hundreds
of base pairs.
There is no evidence that B. subtilis DeoR binds to more thanone
operator site, although the operator site to which it bindshas a
complex structure, as noted in the next paragraph. Fur-thermore,
DeoR repression of the deo operon in E. coli ischaracterized by
long-range cooperative regulation (2), where-as no more than 141 bp
of DNA is enough for complete DeoRrepression of the dra-nupC-pdp
operon of B. subtilis (14).
Previous molecular genetic studies with the dra-nupC-pdpoperon
indicated that a palindromic sequence located betweennucleotides
260 and 243 relative to the start of transcriptionand a direct
repeat of the 39 half of the palindrome locatedbetween the 235 and
210 regions were both required forrepression of the operon by DeoR
(14). The results of thepresent studies directly demonstrate that
these DNA elementsare required for binding to DeoR in vitro.
Furthermore, thecorresponding segment of DNA was protected by DeoR
inDNase I footprinting studies. This is a highly unusual
structuralrequirement for a DNA-binding protein. Typical operator
se-quences consist of palindromes only, and typical repressors
aredimeric proteins in which each subunit binds to one of thehalves
of the palindrome. Repressor proteins that are tet-rameric or
larger sometimes bind to multiple palindromic op-erators, as with
E. coli DeoR. In the case of B. subtilis DeoR,our titration studies
indicate that four subunits bind to a singlesegment of operator DNA
(Fig. 4). Assuming that all theDeoR protein was active in the
titration studies, this stoichi-ometry suggests to us that each
subunit binds to one half of thepalindromic sequence, so that three
of the four subunits arebound to DNA in the DeoR-operator complex.
DeoR is anoctamer in solution but may dissociate to a tetrameric
formupon dilution to the concentration used in the gel shift
exper-iments. Cooperativity as observed in the DeoR binding
curves(e.g., Fig. 3 and 4) could reflect differences in the
affinity of thesubunits for the slightly different half-palindromic
sequences.
High-affinity B. subtilis DeoR binding to DNA takes place inthe
absence of effector molecule. dRib-5-P is most likely theeffector
that modulates B. subtilis DeoR binding to DNA, act-ing as an
inducer to inhibit the binding of a repressor proteinto a control
site. Although dRib-1-P has also been reported asan alternative
inducer (12, 14), no effect on DeoR binding toDNA is observed in
the presence of dRib-1-P in vitro. InE. coli, dRib-5-P but not
dRib-1-P induces the expression ofthe deo operon (9), but no
information is available with respectto protein-effector molecule
interaction. More detailed studiesof B. subtilis DeoR are needed to
locate the inducer-bindingdomain.
ACKNOWLEDGMENTS
We thank Eric Bonner for helpful discussions of protein
purificationand the gel shift assay.
This research received financial support from the Plasmid
Founda-tion for Xianmin Zeng as a visiting scholar in University of
Illinois fora period of 4 months. Novo Nordisk Foundation and
Saxild FamilyFoundation also provided financial support.
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