Characterization of DNA Binding Sites of the ComE Response Regulator from Streptococcus mutans

JOURNAL OF BACTERIOLOGY, July 2011, p. 3642–3652 Vol. 193, No. 140021-9193/11/$12.00 doi:10.1128/JB.00155-11Copyright © 2011, American Society for Microbiology. All Rights Reserved.

Characterization of DNA Binding Sites of the ComE ResponseRegulator from Streptococcus mutans�†

David C. I. Hung,1‡ Jennifer S. Downey,1‡ Eduardo A. Ayala,1‡ Jens Kreth,2 Richard Mair,3

Dilani B. Senadheera,3 Fengxia Qi,4 Dennis G. Cvitkovitch,3Wenyuan Shi,5 and Steven D. Goodman1*

Department of Molecular and Computational Biology, Division of Biomedical Science, Herman Ostrow School of Dentistry, University ofSouthern California, Los Angeles, California 900891; Department of Microbiology and Immunology, University of

Oklahoma Health Science Health Center, Oklahoma City, Oklahoma 731042; Dental Research Institute,Faculty of Dentistry, University of Toronto, Toronto, Canada3; College of Dentistry, University of

Oklahoma Health Sciences Center, Oklahoma City, Oklahoma 730344; and Department ofOral Biology and Medicine, UCLA School of Dentistry, Los Angeles, California 90095-16685

Received 2 February 2011/Accepted 6 May 2011

In Streptococcus mutans, both competence and bacteriocin production are controlled by ComC and theComED two-component signal transduction system. Recent studies of S. mutans suggested that purified ComEbinds to two 11-bp direct repeats in the nlmC-comC promoter region, where ComE activates nlmC and repressescomC. In this work, quantitative binding studies and DNase I footprinting analysis were performed to calculatethe equilibrium dissociation constant and further characterize the binding site of ComE. We found that ComEprotects sequences inclusive of both direct repeats, has an equilibrium dissociation constant in the nanomolarrange, and binds to these two direct repeats cooperatively. Furthermore, similar direct repeats were foundupstream of cslAB, comED, comX, ftf, vicRKX, gtfD, gtfB, gtfC, and gbpB. Quantitative binding studies wereperformed on each of these sequences and showed that only cslAB has a similar specificity and high affinity forComE as that seen with the upstream region of comC. A mutational analysis of the binding sequences showedthat ComE does not require both repeats to bind DNA with high affinity, suggesting that single site sequencesin the genome may be targets for ComE-mediated regulation. Based on the mutational analysis and DNase Ifootprinting analysis, we propose a consensus ComE binding site, TCBTAAAYSGT.

Bacteria can respond to various environmental stimuli byregulating metabolic pathways through two-component signaltransduction systems (TCSTS) (7, 29, 31, 39). TCSTS havebeen shown to control many diverse processes in bacteria, suchas sporulation in Bacillus subtilis, chemotaxis, nitrogen assim-ilation, and outer membrane protein expression in Escherichiacoli, virulence in Agrocaberium tumefaciens, Bordetella pertus-sia, and Salmonella enterica serovar Typhimurium, and biofilmformation, acid tolerance, competence development, and bac-teriocin production in streptococci, such as Streptococcus mu-tans, Streptococcus pneumoniae, and Streptococcus pyogenes (7,9, 10, 15, 18, 23, 30, 39). These adaptive systems in theirsimplest form consist of two components: a membrane-boundsignal-transducing protein, referred to as a histidine kinase,and a cytoplasmic transcription factor, referred to as a re-sponse regulator. Upon detection of the external signal, the�-phosphoryl group of ATP is transferred to a conserved his-tidine residue on the histidine kinase and subsequently to aconserved aspartic acid residue in the regulatory domain of the

response regulator, which thereby undergoes a conformationalchange and activates a response (38).

In silico analysis has shown 14 TCSTS in S. mutans UA159(4). Of these, the ComED system has been studied extensivelyand shown to be involved in biofilm formation, competencedevelopment, and bacteriocin production (10, 18, 22, 23, 41).The first study done on the ComED system was reported by Liet al., who identified a genetic locus comprised of three genes,comC, comD, and comE, that encode a precursor to the com-petence-stimulating peptide (CSP), a histidine kinase, and aresponse regulator, respectively (22). S. mutans comCDE ho-mologs were shown to function in competence by the fact thatinactivation of each individual gene resulted in decreasedtransformation efficiency (22). Furthermore, the addition ofchemically synthesized CSP was able to restore transformabil-ity to comC-deficient cells (22). Based on homology to the S.pneumoniae comCDE locus (1, 8, 14, 15, 20, 24, 32, 34), amodel for competence in S. mutans was proposed (23). Whena critical cell density is reached, cslAB (comAB), comC, andcomED are induced, activating competence (13, 23, 33). cslABlikely encodes a CSP-specific secretion apparatus consisting ofan ATP binding cassette (ABC) transporter (CslA) and itsaccessory protein (CslB), which presumably is involved in theprocessing and export of CSP (33). By analogy with otherTCSTS, it is assumed that extracellular CSP binds ComD,induces autophosphorylation, and subsequently transfers thisphosphate group to its cognate response regulator, ComE.Phosphorylated ComE would then directly activate expression

* Corresponding author. Mailing address: Division of BiomedicalScience, Herman Ostrow School of Dentistry, University of SouthernCalifornia, 925 West 34th Street, Room 4108, Los Angeles, CA 90089-0641. Phone: (213) 740-3867. Fax: (213) 740-4981. E-mail: [email protected].

† Supplemental material for this article may be found at http://jb.asm.org/.

‡ D. C. I. Hung, J. S. Downey, and E. A. Ayala contributed equallyto this work.

� Published ahead of print on 20 May 2011.

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of early com genes, which include cslAB, comC, comED, andcomX. In S. pneumoniae, ComE induces expression of comX(also known as sigX), an alternate sigma factor that initiatestranscription of late competence genes required for DNAuptake and recombination (20). A homolog of ComX in S.mutans is postulated to direct transcription of several com-petence-related genes as a result of a CSP-induced signalcascade (2).

Ween et al. showed that S. pneumoniae ComE is a DNAbinding protein that self-regulates its own expression by bind-ing to a region in its own promoter as well as the promoterregion of cslAB (42). In a study by van der Ploeg, it wasdemonstrated that ComED is not only responsible for compe-tence development but is also involved in the regulation ofbacteriocin production in S. mutans. When the promoter re-gions of these bacteriocin genes were aligned, direct repeatelements were found and proposed to be ComE binding sites(41). Recently, we reported identical sequences in the inter-genic region of nlmC-comC located upstream of the codingsequence of comC. Furthermore, we demonstrated that S. mu-tans ComE is also a DNA binding protein that recognizes thesesequences, containing two 11-bp imperfect direct repeats, DRIand DRII, which are critical for specific binding (18). In thisstudy, we provide quantitative DNA binding and footprintinganalyses results to show that ComE indeed binds to DNAcontaining these two imperfect direct repeats. We have fur-ther identified and biochemically characterized 13 geneswith this motif in the vicinity of their promoter regions,including the bacteriocin genes identified by van der Ploeg.Furthermore, we demonstrated that ComE may have twodifferent modes of binding among these different high-affin-ity sites and that ComE only needs a single 11-bp site forhigh-affinity binding.

MATERIALS AND METHODS

Bacterial strains and culture conditions. E. coli strains used in the study arelisted in Table 1 and were maintained in Luria broth (LB) medium at 37°C with100 �g/ml ampicillin, 200 �g/ml spectinomycin, or 1 mM isopropyl �-D-thio-galactopyranoside (IPTG) as needed.

ComE purification. The comE coding region was PCR amplified with primersComE-F and ComE-R (Table 2) from S. mutans strain UA159 and ligated inframe into the expression vector pQE30 (Qiagen) to produce a coding se-quence with a C-terminal His tag. The vector was transformed into E. colistrain DH5� and selected for ampicillin resistance, and the insertion of comEwas confirmed by sequencing. Purification of ComE was performed as pre-viously described (18).

DNA substrates for electromobility shift assay (EMSA) and DNase I foot-printing. For substrates with DRII removed, SG515 containing pFW5::�(nlmCp�DRII-luc) was used as previously described (18). For the substrateswith both DRII and DRII deleted, the comC�� fragment from pFW5::�(nlmCp�DRI�II-luc) (18) was subcloned into pCR 2.1 TOPO to generate SG516

(Table 1). Upon confirmation by sequencing, a sequence variation was observedoutside the deletion region and is reflected below in Fig. 2.

To label the DNA substrates, 1 �M oSG316 (Table 2) was labeled at the 5� endwith 0.85 �M [�-32P]ATP (10 mCi/ml; New Life Science Products, Boston, MA)by using T4 polynucleotide kinase (Promega, Madison, WI). Labeled oSG316was used along with the unlabeled primer, oSG317 (Table 2), in a PCR toamplify comC (204 bp), comC� (deleted for DRII; 192 bp), and comC��

(deleted for both DRII and DRI; 161 bp) products by using purified UA159chromosomal DNA and plasmids isolated from SG515 and SG516 (Table 1) asthe DNA template, respectively. All PCR products were purified using aQIAquick PCR purification kit (Qiagen, Valencia, CA) and quantified based onthe optical density at 260 nm (OD260) using a Beckman spectrophotometerDU650 (Fullerton, CA). In addition, oligonucleotide primers (oSG454 andoSG455) and templates (oSG456, -457, -458, -459, -461, -462, -463, -518, -519,-520, and -521) (Tables 3 and 4) were synthesized by Invitrogen (Carlsbad, CA).

DNase I footprinting assays. The upstream regions of comC and cslAB and thebinding site identified within the coding sequence of gtfC were used for foot-printing analysis with purified ComE. Labeled oSG316 and unlabeled oSG317were used to generate a 204-bp comC promoter-containing fragment. LabeledoSG368 and unlabeled oSG369 (Table 2) were used to generate a 173-bp cslABpromoter-containing fragment. Meanwhile, labeled oSG420 and unlabeledoSG421 were used to generate a 188-bp internal gtfC* fragment. ComE wasincubated in footprinting buffer (25 mM Tris-HCl [pH 7.5], 50 mM KCl, 6.25 mMMgCl2, 10% glycerol, and 1 mM dithiothreitol) with 1 nM labeled substrate in afinal volume of 50 �l and incubated at ambient temperature for 30 min. Afterincubation, CaCl2 and MgCl2 were added to 2.5 mM and 5 mM, respectively,vortexed, and incubated for 1 min before the addition of 0.25 U of DNase I(Promega, San Luis Obispo, CA). Reactions were allowed to proceed for anadditional 1.5 min before quenching by the addition of DNase I stop buffer (200mM NaCl, 30 mM EDTA, 1% sodium dodecyl sulfate [SDS], 100 �g/ml tRNA[final concentrations]). The DNA was then extracted with an equal volume ofphenol-chloroform (1:1), ethanol precipitated, and resuspended in 3 �l of se-quence stop buffer (10 mM NaOH, 95% formamide, 0.05% bromophenol blue,0.05% xylene cyanol). The pellet was solubilized on ice for at least 30 min beforeheating at 95°C for 2 min and then stored on ice for at least 2 min prior toloading. Reactions were separated on a 6% sequencing gel run at 40 V/cm forapproximately 3 h. The gels were dried and scanned with a Pharos FX Bio-Radimaging system (Hercules, CA). A sequence ladder for sense and antisensecomC, cslAB, and gtfC* were generated using the fmol DNA cycle sequencingsystem (Promega, San Luis Obispo, CA) according to the manufacturer’s instruc-tions.

EMSA. ComE was incubated in 20 �l reaction buffer (52.5 mM HEPES [pH6.5], 50 �M EDTA, 9.5% glycerol, and 50 �g/ml bovine serum albumin) con-taining 100 ng salmon sperm DNA and 1 nM isotopically labeled DNA substratefor 30 min at ambient temperature. Following incubation, EMSA reaction mix-tures were analyzed on a 6% nondenaturing polyacrylamide gel, run at 10 V/cmfor 3 h, and subsequently dried and visualized with the Bio-Rad PharosFXmolecular imaging system. Competition experiments were performed as de-scribed previously (12). Briefly, for each competition experiment, ComE wasfixed at 1 nM with 1 nM labeled comC� DNA fragment. For competitions withbsmC, nlmAB, immB, and bsmB the template was too similar to amplify sub-strates by PCR from the chromosome of S. mutans. Therefore, to ensure that thecorrect sequence was being used for competition experiments, complementary96-bp oligonucleotides were synthesized and used as templates for PCR (Table4). Unlabeled competitor DNA from the promoter regions of cslAB, vicRKX,gtfB, gtfC, gtfD, comED, comX, ftf, gbpB, bsmC, nlmAB, immB, and bsmB (Table2) was added, and the reduction of ComE bound to comC� DNA fragments wasmeasured. ImageQuant 5.0 (Molecular Dynamics, Sunnyvale, CA) was usedto quantify the shifted complexes and free DNA substrate in each reactionmixture. Each reaction was then plotted using Microsoft excel, and the 50%inhibitory concentration (IC50) was calculated. The IC50 represents theamount of competitor DNA that is required to reduce the ComE-comC�

complex by 50%, which is a good indicator of the equilibrium dissociationconstant.

Gel permutation assays. Two substrates, comC� and gtfC*, were chosen to testfor the bending of a DNA substrate by ComE. Each DNA target site had two setsof primer pairs (Table 2) designed to amplify the sequence from S. mutansUA159 chromosomal DNA to place the putative ComE binding site either in themiddle or at the 3� end of the substrate. These substrates were then incubatedwith ComE (10 nM), and the reactions were analyzed under the same conditionsas described above for the EMSA.

TABLE 1. Bacterial strains used in this study

E. colistrain Relevant characteristics Reference

or source

DH5� F �80lacZ�M15 �(lacZYA-argF)U169 deoRrecA1 endA1 hsdR17(rK

mK�) phoA supE44

thi-1 gyrA96 relA1

Invitrogen

SG481 DH5� containing pQE30 � comE (Ampr) This studySG515 Top10 containing pFW5::�(nlmCp�DRII-luc); Specr 18SG516 Top10 containing pCR2.1 � nlmCp�DRI�II-luc;

(Ampr)This study

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RESULTS

ComE protects the comC promoter region. We have previ-ously shown by qualitative EMSA that ComE binds with highaffinity to a specific DNA sequence that contains two directrepeats upstream of comC (18). Here we further characterizedthis interaction by DNase I footprinting. As shown in Fig. 1A,ComE not only binds to the two direct repeats (DRI andDRII) on the sense strand but also further protects regions 5�to each direct repeat. However, it was observed that ComEprotects more nucleotides associated with DRII than withDRI. Moreover, one hypersensitive cleavage was observed atthe 5� edge of each repeat. Such enhanced DNase I cleavage isindicative of greater access of DNase I to the phosphodiesterbackbone and is often the result of local DNA bending.

To test local DNA bending, we performed a DNA bendpermutation experiment. We were able to show that a sub-strate containing only DRI (comC�) in a nucleoprotein com-plex with ComE migrated more slowly when the putative

ComE binding site was placed in the middle of a DNA frag-ment, relative to a similar-length substrate where the bindingsite was placed at the end (see Fig. S1 in the supplementalmaterial). This result is indicative of the induction of a mostlyplanar DNA bend. However, for another single-repeat ComEbinding site (a putative site within gtfC, discussed below), thenucleoprotein complex migrated similarly whether the putativebinding site was placed in either the middle or at the end,indicating no net DNA bending for either substrate. The di-chotomy of this effect on these different substrates is discussedbelow.

Footprints of the antisense strand upstream of the comCcoding sequence (Fig. 1B) showed that ComE protection alsoextends to the 3� end from DRII, but not all 11 bp of eachdirect repeat were protected. A diagram of the protections andenhancements on each strand is shown in Fig. 1C. Again, moreof DRII was protected than DRI, with some protection in thespacer region. The differences in affinity of ComE to DRII

TABLE 2. Primers used for DNase I footprinting, competition EMSA, and mutagenesis of ComE

Primer name Sequence Gene targeta

oSG316 5�-CCCATTTTTAGTTTTTTGTCTG-3� comC (249 to 228)oSG317 5�-GAAAAAATCATGGATTTTCTTG-3� comC (67 to 46)oSG381 5�-CTGAGCGATCACTTAAAGATC-3� gtfB (291 to 271)oSG382 5�-GATTACAAACTCCAACTTTAG-3� gtfB (493 to 471)oSG383 5�-GGATTTACCTATGAAAGGCG-3� gbpB (123 to 104)oSG384 5�-GTCGCAGAACTAAGAGTTAC-3� gbpB (�37 to �56)oSG385 5�-GTTTCCATTAGCAAACCTCC-3� ftf (11 to �5)oSG386 5�-CTTGTTTTTTGGTTCAAGAG-3� ftf (193 to 181)oSG387 5�-CCTTAGCCAAGTTAAACTTG-3� vicRKX (�67 to �48)oSG388 5�-GGTTCTAACATAAAGTTTACTC-3� vicRKX (134 to 113)oSG389 5�-GGTTGTTGTGATGGTAAAAAAG-3� gtfD (175 to 156)oSG390 5�-CCTTGTACATTTTGTAACGTC-3� gtfD (�11 to �31)oSG364 5�-CTGATTAACAGAAAAAAAGCAG-3� comED (449 to 428)oSG365 5�-GTTCTCTAAAACTGTTAACC-3� comED (262 to 243)oSG366 5�-GTATTGAGTTGAATCGGGTAG-3� comX (558 to 538)oSG367 5�-CCAATTCGAGAACAATATCAAC-3� comX (351 to 330)oSG368 5�-GTAAATTATAAATTGGAGCTTGC-3� cslAB (200 to 178)oSG369 5�-GTAAATCTATTATCTTATAATTTTG-3� cslAB (53 to 29)oSG137 5�-TATTATTTATTATTTTTCTAAAAAA-3� gtfC (102 to 128)oSG181 5�-CAGATACTGTCACCCATCTTTT-3� gtfC (�37 to �58)oSG420 5�-GCTATGTCTTAAAAGATCAGG-3� gtfC (�3227 to �3249)oSG421 5�-GCTAATGAAAGCATTTTTAGC-3� gtfC (�3297 to �3419)oSG442 5�-TTTAGTTTTTTGTCTGGCTGC-3� comC (ComE BS middle)oSG443 5�-TTTCTTGAAAAAGTAATATTTTC-3� comC (ComE BS middle)oSG444 5�-GCTTCATTATCCATTACGTTAAA-3� comC (ComE BS 3� end)oSG445 5�-ATCAAAAATGACCGTTTAGGAC-3� comC (ComE BS 3� end)oSG681 5�-GGGACAAATATTTTAGGGCGC-3� gtfC (ComE BS middle)oSG682 5�-AAGGTTATGTTTATTATTCAACG-3� gtfC (ComE BS middle)oSG683 5�-CAAATTTCCACAGGGGTTCCGA-3� gtfC (ComE BS 3� end)oSG684 5�-TTCCTAAATCGTTAGTTAACCCA-3� gtfC (ComE BS 3� end)oSG454 5�-TCTCTAGTATAACAATTTATTTTC-3� Amplify DGS and SSoSG455 5�-GATAGTTTCAGAACATCAAAAATG-3� Amplify DGS and SSComE-F 5�-AGATAAGTAGGGTTATTAAGTTAGTAG-3� Amplify comEComE-R 5�-AGTTAATAAACCATTTGAAAGTATCATTAAG-3� Amplify comEoSG509 5�-CTCTAATATAGCAAATTATTTTTC-3� bsmC (37 to 60)oSG510 5�-ACAGGCATTATAACGTCATTTTTA-3� bsmC (109 to 132)oSG511 5�-TCTTTAGTATAACAATTTATTTTC-3� nlmAB (35 to 58)oSG512 5�-GATAGTTTCAGAACATCAAAAATG-3� nlmAB (107 to 130)oSG513 5�-TCTTTAGTATAACAATTTATTTTC-3� bsmB (35 to 58)oSG514 5�-TTCTAATAGAAATTTGGCATTTTT-3� bsmB (107 to 130)oSG515 5�-TCTTAGTATAACAGAAAATATCCT-3� immB (67 to 90)oSG516 5�-AAGGCACAATTTCTGTGTCTTTTT-3� immB (139 to 162)

a All primer positions are in reference to the ATG translational start site. BS, binding site; DGS, degenerate sequence; SS, scrambled sequence.

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versus DRI are discussed below. In addition, hypersensitivesites were found at the ends of both direct repeats. Hence, bothsense and antisense strands are similarly protected.

To determine the contribution of each direct repeat forComE binding, we compared the footprints of two types ofdeletion mutants of the upstream comC substrate lacking ei-ther DRII or both DRII and DRI, comC� and comC��,respectively (Fig. 2A). The single deletion of DRII showed thesame protected regions of DRI (Fig. 2B) as in the intact comCsubstrate seen in Fig. 1A. No footprint was observed in themutant, where both direct repeats were deleted (Fig. 2C),suggesting that the specificity of ComE binding for these se-quences is necessary and sufficient.

ComE cooperatively binds the comC promoter region. Pre-viously we had shown that ComE binds the comC promoterregion with high affinity, and we revealed two shifted bands inan EMSA when both direct repeats were present (18). Todetermine if this binding is cooperative, EMSA analyses werecarried out with a fixed amount of comC substrate (0.1 nM)and increasing concentrations of ComE (0 to 64 nM) (Fig. 3).A Hill plot was generated based on the analysis of three inde-pendent EMSAs, and the subsequently derived slope of eachplot was taken as the Hill coefficient (1.7 � 0.2 [mean �standard error of the mean] (see Fig. S2 in the supplementalmaterial), demonstrating positive cooperativity (Hill coeffi-cient of �1) (35).

Binding affinity of ComE for comC�. To estimate bindingaffinity, we performed quantitative EMSAs to determine theequilibrium dissociation constant, Kd. Briefly, a binding iso-therm was created by keeping the concentration of purified

ComE constant (15 nM) and varying the concentration ofisotopically labeled comC� substrate (0 to 80 nM). The con-centration of DNA, which produced the half-maximal amountof shifted complex (ComE bound to comC�), was used as anestimate of Kd. Although this method does not identify theactive protomer (monomer, dimer, etc.), it does indicate theactive quantity of protein, which is identical to the maximalamount of molar equivalents of shifted DNA (Bmax). comC�was used to examine the binding affinity, since it gave a singleshifted complex over a wide range of protein concentrations(18). We calculated the Kd as (3.4 � 0.5) 109 M, indicatingthat ComE binds strongly to comC� (Fig. 4). In addition, aBmax value of 5.9 109 M was observed. This Bmax is roughly40% of the estimated quantity of added ComE, assuming thatComE is binding to the DNA as a monomer, i.e., this suggeststhat only 40% of our protein preparation is active. Alterna-tively, the functional protomer of ComE could be a dimer,implying that 80% of the protein is active. The actual oligo-meric state of ComE in solution and bound to both single andtandem direct repeats will need to be determined.

Identification of other possible ComE binding sites in theupstream region of genes in the S. mutans genome. After dem-onstrating that ComE protected the two direct repeats in theupstream promoter region of comC, we set out to determine ifsimilar direct repeats exist in the promoter regions of cslAB,comED, and comX, as they do in S. pneumoniae. The 500-bpupstream regions from each ATG start site were used with theMultiple Em for Motif Elicitation (MEME) online program(http://meme.sdsc.edu/meme), and a putative consensus se-quence, TCNTAAANGGT-10-TCNTAAANGGT, was identi-

TABLE 3. Oligonucleotides and templates synthesized for degenerate comC sequencesa

Template Sequence

oSG456 (DGSI) .............5�-TCTCTAGTATAACAATTTATTTTCAAAATGGNNNNNNNNNTCCTAAATGGTAGCATTTTGTCCTAAACGGTCATTTTTGATGTTCTGAAACTATC-3�

oSG457 (DGSII)............5�-TCTCTAGTATAACAATTTATTTTCAAAATGGWWWWWWWWWTCCTAAATGGTAGCTATTTTGTCCTAAACGGTCATTTTTGATGTTCTGAAACTATC-3�

oSG458 (DGSIII) ..........5�-TCTCTAGTATAACAATTTATTTTCAAAATGGAGCAAAATATCCTAAATGGTNNNNNNNNNNTCCTAAACGGTCATTTTTGATGTTCTGAAACTATC-3�

oSG459 (DGSIV) ..........5�-TCTCTAGTATAACAATTTATTTTCAAAATGGAGCAAAATATCCTAAATGGTWWWWWWWWWWTCCTAAACGGTCATTTTTGATGTTCTGAAACTATC-3�

oSG461 (SSII) ................5�-TCTCTAGTATAACAATTTATTTTCAAAATGGAGCAAAATAgtaccgtattaAGCTATTTTGTCCTAAACGGTCATTTTTGATGTTCTGAAACTATC-3�

oSG462 (SSI) .................5�-TCTCTAGTATAACAATTTATTTTCAAAATGGAGCAAAATATCCTAAATGGTAGCTATTTTGctagcgctataCATTTTTGATGTTCTGAAACTAT-3�

oSG463 (SSIII) ..............5�-TCTCTAGTATAACAATTTATTTTCAAAATGGAGCAAAATAgtaccgtattaAGCTATTTTGctagcgctataCATTTTTGATGTTCTGAAACTATC-3�

a Underlined portions of primers were used to amplify these templates (oSG454 and oSG455). Boldface indicates putative ComE binding site; lowercase portionsindicate scrambled ComE binding sites. DGS, degenerate sequence; SS, scrambled sequence.

TABLE 4. Template primers used for PCR to obtain bsmC, nlmAB, bsmB, and immB fragments for competition EMSA

Primer name Sequence (5�–3�)

oSG518.................CTCTAATATAGCAAATTATTTTTCAAAATGGAGTAAAATATCCTAAACGGTAGCTATTTTGTCCTAAACGGTTAAAAATGACGTTATAATGCCTGT

oSG519.................TCTTTAGTATAACAATTTATTTTCAAAATAGAGCAAAATATCCTAAATGGTAGCTATTTTGTCTTAAACGGTCATTTTTGATGTTCTGAAACTATC

oSG520.................TCTTTAGTATAACAATTTATTTTCAAAATGGAGCAAAATATCCTAAACGGTAGCTATTTTGTCCTAAACGGTAAAAATGCCAAATTTCTATTAGAA

oSG521.................TCTTAGTATAACAGAAAATATCCTCTAACGGAGTCAAAAATCCCAAATGGTAGCAATTTTGTCCTAAACGGTAAAAAGACACAGAAATTGTGCCTT


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fied. Furthermore, Senadheera et al. also showed that a two-component system, VicRK, affects the expression of gtfBCD,gbpB, and ftf (36) and suggested that VicRK may interact withComED to regulate the expression of these genes. Using MacVector 7.2 software (Accelrys, Cary, NC), the aforementionedconsensus was used to find potential matches in the promoterregion of the following genes: cslAB (GenBank accession num-bers AAN59510.1 and AAN59511.1), comED (AAN59528.1and AAN59527.1), comX (AAN59601.1), gtfB (AAN58705.1),gtfC (AAN58706.1), gtfD (AAN58619.1), gbpB (AAN57811.1),ftf (AAN59631.1), and vicRKX (AAN59168.1, AAN59167.1,and AAN59166.1). To provide biochemical evidence thatthese genes contain bona fide ComE targets, we performedquantitative EMSA on all nine targets. In addition, we alsotested four genes encoding putative bacteriocins (bsmA[AAN59525.1], bsmB [AAN59518.1], bsmC [AAN58177.1],

and nlmAB [AAN57926.1 and AAN57927.1]) and one geneencoding bacteriocin immunity protein (immB [AAN58631.1])that have been found to be regulated by ComED and containputative ComE binding sites (41). The primer sets (Table 2)were used to amplify the putative ComE binding sites of thepromoter region for each of these genes. Each selected regionof DNA was used as competitor DNA with isotopically labeledcomC� in an EMSA, and the IC50 for each competitor wascalculated. The resulting IC50 (2.9 109 M) for comC wildtype was lower than the homologous competition with comC�(3.7 109 M), indicating that DNA containing both directrepeats has a higher affinity for ComE than with a single site.As expected, when the two direct repeats were deleted, ahigher IC50 (52 � 40) 109 M was observed, suggestingcritical binding sequence elements were removed. As shown inFig. 5, each sequence tested was aligned with comC to show the

FIG. 1. DNase I footprint analysis of ComE on the upstream region of comC. (A) Sense strand of comC. Increasing concentrations of ComE(0, 19.5, 39, 97.5, and 195 nM; lanes 1 to 5, respectively) were incubated with labeled probe and subjected to DNase I digestion. Lane 6, 19.5 nMComE without DNase I treatment. The sequence protected by ComE is shown on the right. (B) Antisense comC protected by ComE from DNaseI digestion. Increasing concentrations of ComE (0, 19.5, and 97.5 nM; lanes 1 to 3, respectively) were incubated with labeled probe and subjectedto DNase I digestion. Lane 4, 19.7 nM ComE without DNase I treatment. The sequence protected by ComE is shown on the right. Solid sidelinesare the direct repeat sequences. Nonprotected sequences are shown in lowercase, and hypersensitive sites are shown in boldface. �, hypersensitivesite; dashed line, direct repeat site on the gel. The numbers indicate the position from the translational start (ATG) of comC. (C) The region 136bp upstream from the ATG start of comC. Underlined sequences represent the direct repeats of ComE binding sites. Boldface sequence portionsare strongly protected, and those shown in italics are weakly protected. Lowercase letters indicate hypersensitive sites. The numbers indicate theposition from the translational start (ATG) of comC.


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nucleotide differences, and the sequences were placed in anorder from high affinity to low affinity, based on the IC50s. Outof the nine genes that were identified in this study, only cslAB(2.5 109 M) had a high affinity of binding to ComE similarto that of the wild-type comC. As expected, genes found by vander Ploeg (bsmB, bsmC, nlmAB, and immB) (41) had similarIC50s to wild-type comC, indicating high binding affinities tothose sites.

To examine the cslAB binding site for ComE further, weused DNase I footprinting analysis. Figure 6 shows that ComEprotects the upstream region of cslAB, and as expected theprotected regions cover the consensus binding region. Further-more, two hypersensitive sites were observed and showed sim-ilar spacing for both comC and cslAB, which correlates toalmost two precise turns of the DNA helix. We have alsoexamined the upstream regions of comED, comX, and vicRKXby footprinting analysis; however, none of these sequences wasprotected by ComE (data not shown).

Effects on ComE affinity by either scrambling the directrepeats or randomizing the sequences adjacent to the directrepeats. As shown in Fig. 1, there are two direct repeats in theupstream region of comC that were protected in a DNase I

FIG. 3. EMSA to determine if ComE binds cooperatively. EMSAanalyses were carried out with a fixed amount of labeled comC sub-strate (0.1 nM) and increasing concentrations of ComE. Lanes 1 to 9,0, 0.5, 1, 2, 4, 8, 16, 32, and 64 nM ComE. The amount of shiftedcomplex was determined using ImageQuant software to generate a Hillplot. The experiments were repeated three times with similar results. Arepresentative gel image is shown.

FIG. 2. Footprinting of deletion mutant sequences. (A) Wild-type comC sequence from 126 to 181 from the translational start site. Bothdirect repeats are indicated in bold. For both comC� and comC��, the deleted sequences are indicated with dashes. (B and C) Footprint assaywith comC� (B) and comC�� (C). ComE was added to 0, 19.5, or 195 nM or 19.5 nM ComE was added without DNase I treatment (control) (lanes1 to 4, respectively). �, hypersensitive site; dashed line, ComE putative binding site on the gel.


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footprinting analysis. In addition to these two protected re-gions, the 5� end of the second direct repeat and the spacerregion between the first and second direct repeats were alsoprotected and contained hypersensitive sites. In order to un-derstand how these two regions affect ComE binding affinity,we designed four templates each with 96 bases, as seen inTable 3, with random sequences in either of these two regions,and performed competition EMSAs with comC�. The firstdegenerate sequence (DGSI; see Table 3 for more details) has9 random base pairs at the 5� end immediately upstream of thesecond direct repeat, while the rest of the sequence is unal-tered, whereas DGSII has 9 As or Ts in the same region.DGSIII has 10 Ns that replace the spacer region between thefirst and second direct repeats, whereas DGSIV has 10 As orTs in place of the spacer. As shown in Table 5, these fourtemplates have IC50s of 2.9 109 M, 3.9 109 M, 3.1 109 M, and 3.0 109 M, respectively, which were compa-rable to the 96-bp wild-type template (3.7 109 M). Theseresults indicate that even though the 5� upstream sequence

immediately adjacent to the second direct repeat and thespacer were protected by ComE against DNase I cleavage, theydo not play a measurable role in ComE binding affinity.

We also synthesized templates where we scrambled the se-quences of the first, second, or both direct repeats, DNA sub-strates SSI, SSII, and SSIII, respectively, making sure thatthere were no bases that matched the original wild-type se-

FIG. 4. EMSA analysis of ComE with comC� substrate. Increasingconcentrations of comC� substrate were incubated with 15 nM ComE.Lanes 1 to 11: 0, 0.5, 1, 2, 4, 8, 10, 20, 40, 50, and 70 nM comC�. Theexperiments were repeated 5 times with similar results. A representa-tive gel is shown.

FIG. 5. Possible matches to the ComE consensus. A compilation ofsequences with the best matches to the putative ComE binding site ofcomC is shown. The underlined sequences are the putative ComE bindingsite. Lowercase nucleotides are variations in the repeats compared to thecomC sequence. The list is arranged in order of IC50s from low to highwhich indicates binding affinity from high to low. IC50s were tested indi-vidually in at least four different experiments.

FIG. 6. DNase I footprint analysis of ComE on the upstream re-gion of cslAB. Increasing concentrations of ComE (0, 19.5 and 97.5nM; lanes 1 to 3, respectively) were incubated with labeled probe andsubjected to DNase I digestion. Lane 4 contained 19.5 nM ComEwithout DNase I treatment. The sequence protected by ComE isshown on the right. Solid lines are the putative ComE binding sites,“less-than” symbols denote hypersensitive sites, and dashed lines indi-cate direct repeat sites on the gel. Nonprotected sequences are inlowercase letters, and hypersensitive sites are in bold. The numbersindicate the positions from the translational start (ATG) of cslAB.

TABLE 5. Binding affinities of randomized comC sequences

Sequence IC50a (109 M)

comC wt (96 bp) ...................................................................... 3.7 � 0.1comC wt (204 bp) .................................................................... 2.9 � 0.4DGSI ......................................................................................... 2.9 � 0.2DGSII........................................................................................ 3.9 � 0.1DGSIII ...................................................................................... 3.1 � 0.1DGSIV ...................................................................................... 3.0 � 0.2SSI.............................................................................................. 4.6 � 0.3SSII ............................................................................................ 6.1 � 0.4SSIII...........................................................................................120 � 12

a Values are means � standard errors of the means.


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quences. A competition experiment with these scrambled di-rect repeats showed that ComE bound to DRII with higheraffinity than to DRI, as shown in Table 5, which was consistentwith the footprinting analysis results indicating that DRII ismore extensively protected by ComE (Fig. 1). When the twobinding sites were scrambled, the IC50 (120 109 M) was thehighest observed, indicating that the repeats represent thestrongest determinants of DNA specificity.

Use of a defined single direct repeat consensus sequence toidentify other possible ComE binding sites. Since ComE wasable to bind to a single direct repeat and had a higher bindingaffinity for DRII of the comC promoter sequence (Fig. 2 andTable 5), we used DRII alone as the query sequence to identifyother possible ComE binding sites in the S. mutans genome.From this search, we found two putative ComE binding siteswithin (�3293 and �3575) the coding region of gtfC, gtfC*, andgtfC** (the previously identified putative ComE biding site wasupstream [120] of the gtfC start codon and has both directrepeats), a gene that is regulated by another TCSTS (VicRK)in S. mutans (36). To further analyze gtfC*, DNase I footprint-ing and binding affinity assays were performed. As seen in Fig.7, we observed that the putative binding site was both pro-tected by ComE and created four hypersensitive sites. In ad-dition, an IC50 of around 15 109 M was observed (Fig. 5).Thus, our data indicate five sequences yielding a footprint withComE: upstream regions of comC and cslAB along with thesite within the coding sequence of gtfC (gtfC*). Assuming that

ComE can bind to a single direct repeat independently and stillhave biological consequences, alignment of these five se-quences generated a consensus single binding site, TCBTAAAYSGT (see Fig. S3A in the supplemental material). Usingavailable online software, Regulatory Sequence Analysis Tools(http://rsat.ulb.ac.be/rsat/), we were able to find this consensusComE binding site at 13 sites either in the upstream region orwithin the coding region of various genes (see Fig. S3B).

DISCUSSION

The main objective of this study was to characterize ComEinteractions with specific DNA targets. Both EMSA andDNase I footprint experiments were used to determine bindingaffinities and specific interactions with putative ComE bindingsites of S. mutans.

A previous study by van der Ploeg demonstrated thatComED in S. mutans regulated the expression of bacteriocinsin addition to genetic competence (41). When the upstreampromoter sequences of four genes encoding putative bacterio-cins and one gene encoding a bacteriocin immunity proteinwere aligned, two 9-bp direct repeats were identified as puta-tive ComE binding sites for S. mutans (41). Further analysisusing these two 9-bp direct repeats identified a putative bind-ing sequence in the intergenic region IGS1499, which isflanked by nlmC (also known as bsmA; mutacin V) and comC(CSP) (18). The putative ComE binding site found in thisintergenic region matches the sequences found by van derPloeg (41). In addition, we were able to expand the two 9-bpdirect repeat to 11-bp direct repeats, because two additionalbases at the beginning of each direct repeat were consistentlyidentical for each site identified.

Previously we showed by qualitative EMSA that the twodirect repeats, DRII (the one most distal from the ATG ofcomC) and DRI (the one most proximal to the ATG of comC),were critical for specific binding (18). Specifically, we observedthat with increasing concentrations of ComE, one shifted andone supershifted complex were observed on the wild-typecomC substrate. A similar titration with a DRII-deleted sub-strate (comC�) yielded only one shifted complex, while noshift was observed in the DRI and DRII double-deleted sub-strate (18). This result demonstrated that ComE does notrequire two repeats for high-affinity binding.

The response regulator OmpR from E. coli has three bind-ing sites in the upstream region of the ompC promoter (25).The most upstream OmpR binding site was essential for ompCactivation and was able to function independently of the othertwo binding sites (25). In addition, it was shown that when themost upstream site was absent, the efficiency of OmpR bindingto the downstream sites was reduced significantly, which wouldsuggest cooperative binding for OmpR (25). Herein, we dem-onstrate cooperative binding for ComE, although whether ei-ther or both direct repeats are essential for transcriptionalactivation has yet to be determined.

In addition, we demonstrated the ability of ComE to bendDNA as a result of binding to comC� (see Fig. S1 in thesupplemental material). DNA bending plays a crucial role inmany biological processes, including gene expression (6, 11, 28,40). In our DNA bend permutation experiment, we only ob-served changes induced in gel electrophoresis migration con-

FIG. 7. DNase I footprinting of ComE on the binding site within thecoding region of gtfC. Increasing concentrations of ComE (0, 19.5, 97.5,195, and 292.5 nM; lanes 1 to 5, respectively) were incubated with labeledinternal gtfC* probe (oSG420/oSG421) and subjected to DNase I diges-tion. The sequence protected by ComE is shown on the right. Solid linesare the putative ComE binding site. Hypersensitive sites are shownin boldface on the sequence and by � symbols on the gel; thedashed line indicates a direct repeat site on the gel. The numbersindicate the positions from the translational start (ATG) of gtfC.


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sistent with the bending induced by ComE on comC� and notto gtfC* (see Fig. S1). A possible interpretation of the latterresult is that binding is distributive and depends on the affinityof each sequence (37). For a region with a higher-affinity bind-ing sequence, such as comC�, the binding of ComE resulted ina planar bending with slower mobility in the permutation assay.The functional significance of bending by ComE has yet to bedetermined; however, it is possible that the role of DNA bend-ing is to allow a better interaction between ComE and RNApolymerase that would not be otherwise possible with a linearpromoter (43).

We started our search for additional ComE binding sites byusing an in silico approach, and we generated a consensusComE binding site from the promoter regions of comC, cslAB,comED, and comX. With this consensus as a query sequence,we were able to find similar ComE binding sites at the pro-moter regions of gtfB, gtfC, gtfD, gbpB, ftf, and vicRKX. Fromquantitative EMSA experiments of each sequence, we foundthat only the upstream sequence of cslAB had comparableaffinity to comC, and none was found to bind more stronglythan comC (Fig. 5). Furthermore, footprinting analysis showedthat ComE not only protected the region upstream of cslAB atthe predicted ComE binding site but also that the hypersensi-tive sites were conserved compared to wild-type comC (Fig. 6).Recently, Martin et al., suggested that the S. mutans ComEDare orthologs to the S. pneumoniae BlpRH (TCSTS that con-trols the bacteriocin-like peptides) and, therefore, the S. pneu-moniae competence cascade is not a suitable model for S.mutans competence (26). One argument used was that therewas no detected ComE binding site at the upstream region ofcslAB (26). Here we have shown that ComE does indeed bindthe promoter region of cslAB, which would indicate that duringcompetence, ComE plays a role in regulating the expression ofthe ABC transporter, as suggested in the S. pneumoniae com-petence model (22).

We also analyzed a few of the other putative ComE bindingsites that did not have IC50s that were as low (comED, comX,and vicRKX) in DNase I footprinting, and no protection wasobserved, but hypersensitive sites were found in the upstreamregion of vicRKX (data not shown). One possible reason forwhy ComE did not bind as well to these sequences comparedto comC and cslAB is that there is too much disparity in thebinding sequence from our proposed consensus. As shown inFig. 5, an alignment of the putative direct repeats of cslAB tothose of comC identified only two bases that differed betweenthe two sequences, whereas in all other sequences, there was aminimum of three differences in each direct repeat that possi-bly prevented ComE protection of these sites. Another possi-bility is that ComE is not fully active unless phosphorylatedand therefore does not recognize possible sequences with mul-tiple differences. For this study, we did not focus on the phos-phorylation state of ComE and the role it plays, if any, onComE binding to the various sequences; however, this is def-initely an area to investigate in future studies.

For ComX there may also be another possible explanation.Recently, Mashburn-Warren et al. discovered that competencein Streptococcus is controlled by two different quorum-sensingsystems, ComCDE and ComRS (27). Uniquely, S. mutans hasboth of these systems, explaining why mutations in comE donot completely abolish competence in S. mutans (27). Mash-

burn-Warren et al. proposed a model where the ComRS sys-tem is the proximal regulator of comX and ComDE is anupstream regulator that may be connected to the ComRS sys-tem, although this connection remains unknown (27). The pu-tative ComE binding site upstream of comX (IC50, 8.6 109

M) that was tested is located from 453 to 420 upstream ofthe comX start codon, whereas the sigX conserved promoterstructure (P1) is located from 79 to 27 (27). Interestingly,looking upstream of comR and comS in S. mutans revealed noidentifiable ComE binding sites.

The interaction between these two systems remains un-known. A recent report by Lemme et al. investigated the com-petence state of individual cells in a population of CSP-treatedS. mutans (21). They found that within the CSP-treated culturethere were two subpopulations, one that became competentand another that lysed (21). A model for this bifurcation stepwas proposed that shows that when there are low levels ofComE, the cells are not competent, and only when there arehigh levels of ComE does the individual cell become compe-tent (21). It is also possible that the phosphorylation state ofComE plays a major role in the integration of these two sys-tems. This will need to be investigated further to determine theexact mechanism for the integration of the ComR/S andComDE systems.

In addition to the various genes directly affected by ComEbinding, we are as yet unable to determine the minimum bio-logical sequence requirements for an active ComE site. It ispossible that the sequences that are protected by ComE aremore biologically significant than the unprotected sequences.van der Ploeg showed decreases in �-galactosidase reporteractivity when mutations in either direct repeat were introducedat the promoter region of nlmAB (41). These mutations in DRIled to an approximately 10-fold reduction in �-galactosidaseactivity, whereas mutation in DRII resulted in a 40-fold reduc-tion. Removal of both repeats, and the region in between,abolished expression nearly completely (41). So, while it isclear that the direct repeats are biologically significant, it isunclear how much relative affinity is required to constitute truebiological importance.

From the footprinting analysis with comC (Fig. 1) and gelshift analysis with scrambled sequences (Table 5), we showedthat neither the 5� extended region of DRII nor the spacerbetween the two repeats significantly influenced ComE bind-ing, clearly demonstrating that the extended region protectedby ComE does not influence ComE binding in a sequence-specific manner. Furthermore, it was clear that ComE boundto DRII with higher affinity than DRI (Table 5). Our obser-vation that a deletion of both direct repeats (comC��) has ahigher binding affinity than a scramble of both direct repeats(SSIII) (Table 5) is interesting. When both sequences werealigned with comC, as shown in Fig. 8, there was an equalnumber of matched sequences for SSIII and comC�� with thenative site of comC; however, for SSIII these identities were allwithin the spacer region, a region that we have demonstrateddoes not affect the affinity of ComE binding. As for comC��,there are seven matched sequences within the two direct re-peats and three in the spacer region, which may allow ComE torecognize the binding site better than the SSIII, resulting in thehigher affinity observed.

Since ComE was able to bind to a single direct repeat and


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DRII has a higher binding affinity, we set out to search forother possible ComE binding sites by using this sequence. Wefound one binding site that was interesting to us within thecoding region of gtfC. This gene encodes a glucosyltransferasethat S. mutans uses to convert sucrose into both water-solubleand water-insoluble glucan for initial attachment to the toothsurface. It has been shown that these water-insoluble glu-cans contribute to the virulence properties of S. mutans in arat model. In addition, in vivo, S. mutans lacking gtfC is lesscariogenic than in animals infected with the parental strain(16, 44).

DNase I footprinting analysis was performed with this pu-tative ComE binding site within the coding region of gtfC*(Fig. 7). ComE was able to protect this sequence with thepredicted hypersensitive sites. Two additional proteins havebeen shown to regulate expression of gtfC in S. mutans. Aglobal regulator, CovR (also known as GcrR), binds to a regionfrom �125 to 132 (�1 is the transcriptional start site) andnegatively regulates the expression of gtfC (5). In addition,VicR, a response regulator of the TCSTS VicRK, was shown tobind DNA containing a consensus sequence at the 26 to 10region and activate gtfC expression (36).

In a previous study, based on the different phenotypic ob-servations caused by comC, comD, and comE mutants on bio-film formation, Li et al. proposed a model that the signalpeptide (CSP) encoded by comC can simultaneously interactwith multiple cognate receptors, one encoded by comD and atleast one other encoded by an unknown gene (23). Since bothComED and VicRK have been shown to be involved in geneticcompetence development and biofilm formation in S. mutans(22, 23, 36), it is possible that there may be interactions be-tween these two systems that regulate expression of compe-tence development and biofilm formation. Currently, we aretrying to determine if there are conditions that allow phospho-transfer between ComD and VicR or VicK and ComE. Ingeneral, cross talk must be kept to a minimum in order toensure that an organism is able to detect a specific stimulus toevoke a specific response (19). However, cross talk betweenTCSTS is not restricted to only phosphotransfer. It has beenshown that cross talk can be regulated at the level of transcrip-tion, for example, as described recently for EnvZ-OmpR andCpxA-CpxR systems in E. coli (3, 17). Although there is min-imal cross talk at the level of phosphotransfer in these systems,certain genes, such as ompR and csgD, are directly regulated byboth OmpR and CpxR (3, 17). It is possible that both ComEDand VicRK cross-regulate the expression of gtfC at the tran-scriptional level, in which VicRK activates gtfC expression andComE binds to the coding region of gtfC and occludes RNApolymerase from completing transcription, thus aborting the

expression of gtfC. However, further genetic experiments needto be performed to test this hypothesis.

In summary, we have biochemically defined the ComE bind-ing site by using EMSA and DNase I footprinting. Based onthe footprinting analysis of comC and cslAB, we suggest theComE binding consensus sequence, TCBTAAAYSGT, is suf-ficient for high-affinity binding. Although necessary, it is stillnot clear whether a single match to this consensus is sufficientfor biological activity in any endogenous S. mutans system.Further investigation is required on these findings in the con-text of understanding competence regulation in S. mutans andthe kinetics of ComE phosphorylation and dephosphorylation.

ACKNOWLEDGMENTS

This work was supported by NIH grants 5R01DE013230 (to D.G.C.and S.D.G.), RO1-DE014757 (to F.Q.), 4R00DE018400 (to J.K.), and1R01DE020102-01 (to W.S.).

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FIG. 8. Alignment of ComE binding sites on comC with comC��(deletion of both direct repeats) and SSIII (scramble of both directrepeats). Underlined sequence portions represent direct repeats, andboldface represents matches to the original comC sequence region.


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Characterization of DNA Binding Sites of the ComE Response Regulator from Streptococcus mutans

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