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: imhhs@pop.dtu.dk.

1916

on April 7, 2021 by guest

http://jb.asm.org/

Dow

nloaded from

http://jb.asm.org/

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.

VOL. 182, 2000 B. SUBTILIS DeoR REPRESSOR PROTEIN 1917

on April 7, 2021 by guest

http://jb.asm.org/

Dow

nloaded from

http://jb.asm.org/

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.

1918 ZENG ET AL. J. BACTERIOL.

on April 7, 2021 by guest

http://jb.asm.org/

Dow

nloaded from

http://jb.asm.org/

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.

VOL. 182, 2000 B. SUBTILIS DeoR REPRESSOR PROTEIN 1919

on April 7, 2021 by guest

http://jb.asm.org/

Dow

nloaded from

http://jb.asm.org/

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.

1920 ZENG ET AL. J. BACTERIOL.

on April 7, 2021 by guest

http://jb.asm.org/

Dow

nloaded from

http://jb.asm.org/

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.

REFERENCES

1. Amouyal, M., L. Mortensen, H. Buc, and K. Hammer. 1989. Single anddouble loop formation when DeoR repressor binds to its natural operatorsites. Cell 58:545–551.

2. Dandanell, G., P. Valentin-Hansen, J. E. L. Larsen, and K. Hammer. 1987.Long-range cooperativity between gene regulatory sequences in a pro-karyote. Nature (London) 325:823–826.

3. Harrison, S. C., and A. Aggarwal. 1990. DNA recognition by proteins withthe helix-turn-helix motif. Annu. Rev. Biochem. 59:933–969.

4. Janknecht, R., G. de Martynoff, J. Lou, R. Hipskind, A. Nordheim, and H.Stunnenberg. 1991. Rapid and efficient purification of native histidine-tagged protein expressed by recombinant vaccinia virus. Proc. Natl. Acad.Sci. USA 88:8972–8976.

VOL. 182, 2000 B. SUBTILIS DeoR REPRESSOR PROTEIN 1921

on April 7, 2021 by guest

http://jb.asm.org/

Dow

nloaded from

http://jb.asm.org/

5. Kunst, F., et al. 1997. The complete genome sequence of the Gram-positivebacterium Bacillus subtilis. Nature (London) 390:249–256.

6. Leonhardt, H., and J. C. Alonso. 1988. Construction of a shuttle vector forinducible gene expression in Escherichia coli and Bacillus subtilis. J. Gen.Microbiol. 134:605–609.

7. Lowry, O. H., N. J. Rosebrough, A. L. Farr, and R. J. Randall. 1951. Proteinmeasurement with the Folin phenol reagent. J. Biol. Chem. 193:265–275.

8. Miller, J. H. 1972. Experiments in molecular genetics, p. 352–355. ColdSpring Harbor Laboratory, Cold Spring Harbor, N.Y.

9. Mortensen, L., G. Dandanell, and K. Hammer. 1989. Purification and char-acterization of the DeoR repressor of Escherichia coli. EMBO J. 8:325–331.

10. Pabo, C. O., and R. T. Sauer. 1984. Protein-DNA recognition. Annu. Rev.Biochem. 53:293–321.

11. Sambrook, J., E. F. Fritsch, and T. Maniatis. 1989. Molecular cloning: alaboratory manual, 2nd ed. Cold Spring Harbor Laboratory, Cold SpringHarbor, N.Y.

12. Saxild, H. H., L. Andersen, and K. Hammer. 1996. dra-nupC-pdp operon ofBacillus subtilis: nucleotide sequence, induction by deoxyribonucleosides,and transcriptional regulation by the deoR-encoded DeoR repressor protein.J. Bacteriol. 178:424–434.

13. Saxild, H. H., and P. Nygaard. 1987. Genetic and physiological character-ization of the Bacillus subtilis purT mutants resistant to purine analogs. J.Bacteriol. 169:2977–2983.

14. Zeng, X., and H. H. Saxild. 1999. Identification and characterization of aDeoR-specific operator sequence essential for induction of dra-nupC-pdpoperon expression in Bacillus subtilis. J. Bacteriol. 181:1719–1727.

1922 ZENG ET AL. J. BACTERIOL.

on April 7, 2021 by guest

http://jb.asm.org/

Dow

nloaded from

http://jb.asm.org/