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The Bacillus subtilis Competence Transcription Factor, ComK, Overrides LexA-imposed Transcriptional Inhibition without Physically Displacing LexA

2001; Elsevier BV; Volume: 276; Issue: 46 Linguagem: Inglês

10.1074/jbc.m104407200

1083-351X

Leendert W. Hamoen, Bertjan Haijema, Jetta J. E. Bijlsma, Gerard Venema, Charles M. Lovett,

Fungal and yeast genetics research

During the development of competence inBacillus subtilis the recA gene is activated by the competence transcription factor, ComK, which is presumably required to alleviate the transcriptional repression of recA by LexA. To investigate the mechanism by which ComK activatesrecA transcription we examined the binding of ComK and LexA to the recA promoter in vitro. Using hydroxyl radical protection analyses to establish the location of ComK dimer-binding sites within the recA promoter, we identified four AT-boxes in a configuration unique for ComK-regulated promoters. Gel mobility shift experiments showed that all four ComK dimer-binding sites were occupied at ComK concentrations in the physiological range. In addition, occupation of all ComK-binding sites did not prevent LexA from binding to the recA promoter, despite the fact that the ComK and LexA recognition motifs partially overlap. Although ComK did not replace LexA from the recA promoter, in vitro transcription analyses indicated that the presence of ComK is sufficient to alleviate LexA repression ofrecA. During the development of competence inBacillus subtilis the recA gene is activated by the competence transcription factor, ComK, which is presumably required to alleviate the transcriptional repression of recA by LexA. To investigate the mechanism by which ComK activatesrecA transcription we examined the binding of ComK and LexA to the recA promoter in vitro. Using hydroxyl radical protection analyses to establish the location of ComK dimer-binding sites within the recA promoter, we identified four AT-boxes in a configuration unique for ComK-regulated promoters. Gel mobility shift experiments showed that all four ComK dimer-binding sites were occupied at ComK concentrations in the physiological range. In addition, occupation of all ComK-binding sites did not prevent LexA from binding to the recA promoter, despite the fact that the ComK and LexA recognition motifs partially overlap. Although ComK did not replace LexA from the recA promoter, in vitro transcription analyses indicated that the presence of ComK is sufficient to alleviate LexA repression ofrecA. polymerase chain reaction maltose-binding protein α COOH-terminal domain Bacillus subtilis differentiates into cells competent for genetic transformation by synthesizing a complex DNA binding and uptake system and by activating recombination genes. Paramount among the recombination gene products that enable cells to incorporate newly acquired genetic material is the RecA protein, which plays a crucial role in recombination by promoting homologous pairing and DNA strand exchange (1Kowalczykowski S.C. Dixon D.A. Eggleston A.K. Lauder S.D. Rehrauer W.M. Microbiol. Rev. 1994; 58: 401-465Crossref PubMed Google Scholar, 2Lovett C.M. Roberts J.W. J. Biol. Chem. 1985; 260: 3305-3313Abstract Full Text PDF PubMed Google Scholar). RecA is also essential for the regulation of the SOS DNA repair system, which is activated when DNA is damaged or whenB. subtilis cells differentiate to a competent state (3Yasbin R.E. Cheo D.L. Bayles K.W. Mol. Microbiol. 1992; 6: 1263-1270Crossref PubMed Scopus (33) Google Scholar). The SOS system operates in many bacteria and has been the subject of extensive studies in Escherichia coli (for review, see Ref.4Walker G.C. Neidhardt F.C. Escherichia coli and Salmonella. American Society for Microbiology, Wahington, D. C.1996: 1400-1416Google Scholar). Following exposure to DNA damaging treatments, a set of damage inducible (din) genes becomes transcriptionally activated. Expression of B. subtilis din genes is regulated by the products of the lexA (formerly called dinR) andrecA genes (5Raymond-Denise A. Guillen N. J. Bacteriol. 1991; 173: 7084-7091Crossref PubMed Google Scholar, 6de Vos W.M. Venema G. Mol. Gen. Genet. 1982; 187: 439-445Crossref PubMed Scopus (18) Google Scholar). LexA acts as the repressor of allB. subtilis din genes, including recA andlexA, by binding specifically to DNA sequences located within the putative promoter regions (5Raymond-Denise A. Guillen N. J. Bacteriol. 1991; 173: 7084-7091Crossref PubMed Google Scholar, 7Raymond-Denise A. Guillen N. J. Bacteriol. 1992; 174: 3171-3176Crossref PubMed Google Scholar, 8Haijema B.J. van-Sinderen D. Winterling K. Kooistra J. Venema G. Hamoen L.W. Mol. Microbiol. 1996; 22: 75-85Crossref PubMed Scopus (55) Google Scholar, 9Lovett C.M. Cho K.C. O'Gara T.M. J. Bacteriol. 1993; 175: 6842-6849Crossref PubMed Google Scholar, 10Miller M.C. Resnick J.B. Smith B.T. Lovett C.M.J. J. Biol. Chem. 1996; 271: 33502-33508Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar). Comparison of the DNA sequences of more than 20 din promoter regions that bind LexA revealed a consensus sequence for binding of a LexA dimer, CGAACATATGTTC. 1L. Bothwell, S. Canny, S. Colavito, S. Fuller, E. Groban, L. Hensley Jr., T. O'Brien, T. M. O'Gara, L. Tomm, and C. M. Lovett, unpublished results. Analogous to the situation in E. coli, RecA is required for SOS induction in B. subtilis (6de Vos W.M. Venema G. Mol. Gen. Genet. 1982; 187: 439-445Crossref PubMed Scopus (18) Google Scholar, 11Love P.E. Lyle M.J. Yasbin R.E. Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 6201-6205Crossref PubMed Scopus (61) Google Scholar, 12Lovett C.M. Love P.E. Yasbin R.E. Roberts J.W. J. Bacteriol. 1988; 170: 1467-1474Crossref PubMed Google Scholar). B. subtilis RecA is activated to promote the autocleavage of LexA repressor in vitro when it binds single-stranded DNA and nucleoside triphosphate (10Miller M.C. Resnick J.B. Smith B.T. Lovett C.M.J. J. Biol. Chem. 1996; 271: 33502-33508Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar). Correspondingly, B. subtilis RecA is activated in vivo by binding single-stranded DNA exposed by discontinuous replication past UV-induced lesions and to a lesser extent by the processing of gaps formed during excision repair (13Lovett C.M. O'Gara T.M. Woodruff J.N. J. Bacteriol. 1994; 176: 4914-4923Crossref PubMed Google Scholar). Although the SOS system is induced during competence development by a similar RecA/LexA-dependent mechanism, B. subtilis recA expression is additionally stimulated by a competence-specific mechanism (8Haijema B.J. van-Sinderen D. Winterling K. Kooistra J. Venema G. Hamoen L.W. Mol. Microbiol. 1996; 22: 75-85Crossref PubMed Scopus (55) Google Scholar, 14Lovett C.M. Love P.E. Yasbin R.E. J. Bacteriol. 1989; 171: 2318-2322Crossref PubMed Google Scholar). Competence is a starvation-induced differentiation process that develops optimally at sufficiently high cell densities and in minimal growth medium with glucose as the main carbon source (for review, see Ref. 15Dubnau D. Sonenshein A.L. Hoch J.A. Losick R. Bacillus subtilis and Other Gram-positive Bacteria. American Society for Microbiology, Washington, D. C.1993Google Scholar). The various environmental signals are interpreted by a complex signal transduction cascade and ultimately lead to the activation ofcomK, which encodes the competence transcription factor (16van-Sinderen D. Luttinger A. Kong L. Dubnau D. Venema G. Hamoen L. Mol. Microbiol. 1995; 15: 455-462Crossref PubMed Scopus (194) Google Scholar,17Hahn J. Luttinger A. Dubnau D. Mol. Microbiol. 1996; 21: 763-775Crossref PubMed Scopus (82) Google Scholar). ComK is essential for: (i) the expression of all late competence gene products that assemble the DNA-binding and -uptake system; (ii) the competence-related expression of the recombination genesrecA and addAB (the homologue of E. coli recBCD); and (iii) its own expression (8Haijema B.J. van-Sinderen D. Winterling K. Kooistra J. Venema G. Hamoen L.W. Mol. Microbiol. 1996; 22: 75-85Crossref PubMed Scopus (55) Google Scholar, 16van-Sinderen D. Luttinger A. Kong L. Dubnau D. Venema G. Hamoen L. Mol. Microbiol. 1995; 15: 455-462Crossref PubMed Scopus (194) Google Scholar, 18Haijema B.J. Hamoen L.W. Kooistra J. Venema G. van-Sinderen D. Mol. Microbiol. 1995; 15: 203-211Crossref PubMed Scopus (27) Google Scholar). Purified ComK has been shown to bind to the promoter regions of all these genes, and its transcription stimulating activity has been demonstrated in vitro with the late competence gene, comG (19Hamoen L.W. Van-Werkhoven A.F. Bijlsma J.J. Dubnau D. Venema G. Genes Dev. 1998; 12: 1539-1550Crossref PubMed Scopus (91) Google Scholar). ComK footprinting analyses with a number of ComK-regulated genes established a conserved AT-rich palindromic sequence (called the AT-box) as the ComK-recognition sequence (19Hamoen L.W. Van-Werkhoven A.F. Bijlsma J.J. Dubnau D. Venema G. Genes Dev. 1998; 12: 1539-1550Crossref PubMed Scopus (91) Google Scholar). Since competence-dependent recA induction occurs in RecA-minus cells, deficient in LexA cleavage, and before LexA is cleaved in wild-type cells, 2B. Chaudhuri and C. M. Lovett, unpublished results. the LexA-imposed transcriptional repression of recA is presumably alleviated by the activity of ComK (14Lovett C.M. Love P.E. Yasbin R.E. J. Bacteriol. 1989; 171: 2318-2322Crossref PubMed Google Scholar). To examine whether ComK is able to prevent the association of LexA with therecA promoter region, we analyzed the binding of purified ComK in the absence and presence of LexA. Hydroxyl radical footprinting analysis revealed four possible ComK dimer-binding sites, which was substantiated by the results of gel mobility shift experiments. Surprisingly, the occupation of all ComK-binding sites did not interfere with LexA binding, yet our in vitro transcription analysis showed that ComK is sufficient to overcome LexA repression. All molecular cloning and PCR3 procedures were carried out using standard techniques (20Ausubel F.M. Brent R. Kingston R.E. Moore D.D. Seidham J.G. Smith J.A. Struhl K. Current Protocols in Molecular Biology. John Wiley & Sons, New York1998Google Scholar, 21Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1989Google Scholar). Labeled nucleotides were fromAmersham Pharmacia Biotech. Media for growth of B. subtilisand E. coli have been described by Sambrook et al. (21Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1989Google Scholar) and Venema et al. (22Venema G. Pritchard R.H. Venema-Schroder T. J. Bacteriol. 1965; 89: 1250-1255Crossref PubMed Google Scholar). B. subtilisstrain 8G5 chromosomal DNA used as template for PCR, was purified as described by Venema et al. (22Venema G. Pritchard R.H. Venema-Schroder T. J. Bacteriol. 1965; 89: 1250-1255Crossref PubMed Google Scholar). ComK was purified as an MBP-ComK fusion protein on an amylose resin (New England Biolabs) column and separated from MBP by cleavage with protease Factor Xa, as previously described (16van-Sinderen D. Luttinger A. Kong L. Dubnau D. Venema G. Hamoen L. Mol. Microbiol. 1995; 15: 455-462Crossref PubMed Scopus (194) Google Scholar). After cleavage was complete, Factor Xa was inactivated by the addition of 1 mm phenylmethylsulfonyl fluoride. To separate ComK from MBP and DNA, the protein mixture was loaded onto a DEAE column (Amersham Pharmacia Biotech) equilibrated with 20 mm Tris-HCl, pH 8, 1 mm EDTA, and 0.5 mm dithiotreitol. MBP and ComK were sequentially eluted with a 0 to 50 mm Na2SO4 gradient and a 0 to 1 m KCl gradient (containing 50 mmNa2SO4). Fractions were collected and the Na2SO4 concentration increased to 100 mm to prevent precipitation of ComK. The ComK containing fractions were checked for the absence of contaminating DNA by ethidium bromide-stained agarose gel electrophoresis, aliquoted, and stored at −70 °C. Purification and cleavage of MBP-ComK were followed by SDS-polyacrylamide gel electrophoresis. B. subtilis LexA was purified as described previously (10Miller M.C. Resnick J.B. Smith B.T. Lovett C.M.J. J. Biol. Chem. 1996; 271: 33502-33508Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar). E. coli strain BL21(DE3) containing pET21a-dinR was grown in 1 liter of LB broth containing carbenicillin (50 μg/ml) with shaking until anA600 of 0.6. The culture was induced with 10 ml of 100 mmisopropyl-1-thio-β-d-galactopyranoside and grown for an additional 3 h. Cells were harvested by centrifugation at 4 °C, 5000 × g for 20 min, and resuspended in 5 ml of 20 mm Tris, pH 7.5, 10% (w/v) sucrose, 1 mm EDTA. Cells were lysed with lysozyme (0.2 mg/ml) by incubation on ice for 30 min followed by a 15-min incubation at 37 °C. After 3 times freeze-thawing and sonication, debris was removed by centrifugation at 18,000 × g for 15 min, and the supernatant was used for further purification. The supernatant (0.5–2 ml) was filtered and applied to a 5-ml heparin-agarose column, equilibrated in 20 mm Tris, pH 7.5, 10 mm NaCl, and subjected to fast protein liquid chromatography. Following elution with a 40-ml linear NaCl gradient (10 mm to 1 m), fractions containing LexA protein were pooled, concentrated 10-fold by centrifugation in a Centricon 10 unit (Amicon), and diluted to the original volume in 20 mm Tris, pH 7.5, 10 mmNaCl; concentration and dilution was then repeated twice. Sample was applied to a Mono-S column and chromatographed as described for the heparin-agarose column, and fractions containing LexA were pooled and dialyzed against 20 mm sodium phosphate buffer, pH 7.5, 200 mm NaCl, and concentrated by centrifugation in a Centricon 10 unit. Hydroxyl radical protection analyses were performed as described by Tullius and Dombroski (23Tullius T.D. Dombroski B.A. Proc. Natl. Acad. Sci. U. S. A. 1986; 83: 5469-5473Crossref PubMed Scopus (476) Google Scholar) with the modifications described by O'Halloran et al. (24O'Halloran T.V. Frantz B. Shin M.K. Ralston D.M. Wright J.G. Cell. 1989; 56: 119-129Abstract Full Text PDF PubMed Scopus (196) Google Scholar). The DNA probes were obtained by PCR amplification using primers R1 (5′-TACGGCTGCCATTTAATG-3′) and R2 (5′-CTGCCTGACGATCACTC-3′), complementary to the sequence at nucleotide position −184 and +32, relative to the transcriptional start of recA. Primers were end-labeled with T4-polynucleotide kinase using [γ-32P]ATP. Binding reaction conditions were as for the gel mobility shift experiments described below, except that glycerol was omitted. DNA fragments, containing ∼30,000 cpm, were added to each 50-μl reaction mixture. After 20 min at room temperature, 1 μl of 5.6 mm(NH4)2Fe(SO2)2·6H2O was mixed with 1 μl of 11.2 mm EDTA, and added to the incubation mixture, followed by the addition of 2 μl of 3.36% H2O2 and 2 μl of 112 mmNa-ascorbate. Reactions were incubated for 1 min at room temperature and terminated with 44 μl of stop solution (10 μl of 3m Na-acetate, 6 μg of yeast tRNA, and 10 μl of 320 mm thiourea) and the subsequent addition of 300 μl of ethanol. Samples were extracted with phenol-chloroform, and ethanol precipitated. The precipitates were resuspended in 3 μl of loading buffer. Analysis of DNA products was carried out by electrophoresis on a 6% polyacrylamide urea gel. Maxam-Gilbert G + A reactions were run with each experiment to locate sequence positions and protected regions (21Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1989Google Scholar). Gel mobility shift experiments were carried out essentially as described (16van-Sinderen D. Luttinger A. Kong L. Dubnau D. Venema G. Hamoen L. Mol. Microbiol. 1995; 15: 455-462Crossref PubMed Scopus (194) Google Scholar). The recApromoter fragment was isolated by PCR, using the primer set as described for the hydroxyl radical protection analyses, and end-labeled with T4-polynucleotide kinase using [γ-32P]ATP. The purified proteins and probes were premixed on ice in binding buffer (20 mm Tris-HCl, pH 8, 100 mm KCl, 5 mmMgCl2, 0.5 mm dithiotreitol, 10% (v/v) glycerol, 0.05 mg/ml poly(dI-dC), and 0.05 mg/ml bovine serum albumin). After 15 min incubation at 37 °C, the samples were loaded on a nondenaturing 4% polyacrylamide gel. The following modifications were applied to increase the resolution of retardation. Electrophoresis was performed with the Bio-Rad Mini-protein cell system, using a spacer thickness of 0.75 mm. A voltage gradient stacking was established by using 1 × TAE buffer (40 mm Tris acetate, pH 8, 2 mm EDTA) for the nondenaturing 4% polyacrylamide gel, 0.5 × TAE buffer for the cathode compartment, and 2 × TAE buffer for the anode compartment. Gels were run at 50 V, dried, and autoradiographed in the absence of intensifying screens. Autoradiograms of the dried gels were digitized and analyzed by densitometry using an Alpha Innotech IS-1000 imaging system. To determine the fraction of DNA molecules with exactly idimers bound, Ø i (i = 0, 2, 3, or 4), the total OD of all bands in each lane was quantified. Ø i was calculated for each band from Ø i = ODtot, i /ΣODtot, i, summed over all of the bands in a given lane. The equations for the fractions of DNA molecules with i ligands bound in a three-site system have been derived previously using a general statistical mechanical approach (25Senear D.F. Brenowitz M. J. Biol. Chem. 1991; 266: 13661-13671Abstract Full Text PDF PubMed Google Scholar). In our analyses we assumed that the three sites correspond to the binding of a tetramer first and then subsequently by two dimers such that the equations representing the number of dimers bound are given by, φ0=1/ZEq. 1 φ2=K1[ComK]/ZEq. 2 φ3=K2[ComK]2/ZEq. 3 φ4=K3[ComK]3/ZEq. 4 where Z = 1 + K1[ComK] + K2[ComK]2 +K3[ComK]3. Experimental data shown in Fig. 7 were analyzed according to EquationsEq. 1, Eq. 2, Eq. 3, Eq. 4 with Mathematica and KaleidaGraph using nonlinear least squares methods to determine and apply the best curve fits. In vitrotranscription experiments were performed essentially as described by Hamoen et al. (19Hamoen L.W. Van-Werkhoven A.F. Bijlsma J.J. Dubnau D. Venema G. Genes Dev. 1998; 12: 1539-1550Crossref PubMed Scopus (91) Google Scholar). The DNA template, pAN-recA, was constructed by cloning a PCR fragment containing the recApromoter, between the SmaI and XbaI sites of the pAN583 vector (26Predich M. Nair G. Smith I. J. Bacteriol. 1992; 174: 2771-2778Crossref PubMed Google Scholar). The recA promoter fragment was isolated by PCR using the primers R1 and R3 (5′-TGTTCTAGAGCCATATCTAAGG-3′), and subsequent digestion with HindIII (position +49 relative to the transcriptional start of recA) and XbaI (underlined in R3). TheHindIII site was converted to a blunt-end by using a Klenow fill-in reaction, prior to XbaI digestion. The transcription reactions were performed in the binding buffer described for the gel mobility shift experiments (poly(dI-dC) included). DNA templates, purified B. subtilis RNA polymerase, and purified ComK were incubated for 15 min at 37 °C in a final volume of 20 μl, before the addition of 3 μl of a nucleotide mixture (1 mm ATP, 1 mm UTP, 1 mm GTP, 0.5 μl of [α-32P]CTP). After 1 min incubation at room temperature, 2 μl of 0.3% heparin was added to the mixture and incubation resumed for another 10 min at 37 °C. After the addition of 2 μl of 1 mm CTP, incubation was continued for 10 min before terminating the reaction by the addition of 18 μl of formamide containing 0.05% bromphenol blue and 0.05% xylene blue. After heating for 3 min at 90 °C, the samples were loaded on a 8% polyacrylamide-urea gel and run at 300 V. Gels were subjected to autoradiography immediately after electrophoresis without prior drying. In a previous study Haijema et al. (8Haijema B.J. van-Sinderen D. Winterling K. Kooistra J. Venema G. Hamoen L.W. Mol. Microbiol. 1996; 22: 75-85Crossref PubMed Scopus (55) Google Scholar) determined the ComK-binding site at the recA promoter using DNase I footprinting analysis (8Haijema B.J. van-Sinderen D. Winterling K. Kooistra J. Venema G. Hamoen L.W. Mol. Microbiol. 1996; 22: 75-85Crossref PubMed Scopus (55) Google Scholar). They obtained a clear footprint extending from approximately positions −150 to −50, which is the longest ComK-protected region identified thus far. By analyzing ComK footprints of several ComK-dependent promoters, Hamoen et al. (19Hamoen L.W. Van-Werkhoven A.F. Bijlsma J.J. Dubnau D. Venema G. Genes Dev. 1998; 12: 1539-1550Crossref PubMed Scopus (91) Google Scholar) concluded that ComK binds as a tetramer composed of two dimers, where each dimer recognizes the dyad symmetrical sequence AAAAN5TTTT, the so called AT-box (19Hamoen L.W. Van-Werkhoven A.F. Bijlsma J.J. Dubnau D. Venema G. Genes Dev. 1998; 12: 1539-1550Crossref PubMed Scopus (91) Google Scholar). The two putative AT-boxes in the recA promoter appeared to be located at the center of the DNase I protections, yet the region confined by these AT-boxes covers only half the sequence protected by ComK (19Hamoen L.W. Van-Werkhoven A.F. Bijlsma J.J. Dubnau D. Venema G. Genes Dev. 1998; 12: 1539-1550Crossref PubMed Scopus (91) Google Scholar). To examine which nucleotide sequences could be responsible for this peculiarly extended ComK-binding region, we improved the resolution of the ComK footprint analyses by using a hydroxyl radical protection assay (Fig. 1). As shown in Fig.2, the hydroxyl radical footprint fits well within the borders of the DNase I footprint. The central hydroxyl radical protections mark the two previously assigned AT-boxes in a pattern comparable with that found in a hydroxyl radical ComK footprint of the addAB promoter (19Hamoen L.W. Van-Werkhoven A.F. Bijlsma J.J. Dubnau D. Venema G. Genes Dev. 1998; 12: 1539-1550Crossref PubMed Scopus (91) Google Scholar). A closer inspection of the hydroxyl radical protections at the extremities of the ComK-binding region revealed two additional AT-boxes: a perfect AT-box around position −140, and one AT-box, with two replacements in the thymine tract, around position −55.Figure 2Summary of the ComK DNase I and ComK hydroxyl radical footprints of the recA promoter.Sequences protected from DNase I nuclease and hydroxyl radical activity are marked by bars and dots, respectively.Thin bars and small dots represent weak protection. Hypersensitive sites are indicated byarrowheads. AT-boxes are boxed, the SOS-box is marked with open bars, and the −35 promoter sequence isunderlined. Base pair positions are indicated relative to the transcriptional start site.View Large Image Figure ViewerDownload (PPT) The presence of four ComK-protected AT-boxes in the recApromoter suggests that this promoter is able to accommodate a total of four ComK dimers. Since it has been shown that ComK binds as a single tetramer to the promoter regions of the ComK-regulated genes studied so far (19Hamoen L.W. Van-Werkhoven A.F. Bijlsma J.J. Dubnau D. Venema G. Genes Dev. 1998; 12: 1539-1550Crossref PubMed Scopus (91) Google Scholar), we were interested in determining the oligomerization state of ComK on the recA promoter with its two additional dimer-binding sites. Specifically, we wondered if the detection of partial saturation of the recA promoter at low ComK concentrations might provide clues regarding the nature of ComK binding. Although previous gel mobility shift assays with therecA promoter and purified ComK yielded a single retarded band, the retarded band displayed in the autoradiograms was diffuse and may have obscured the presence of multiple bands (8Haijema B.J. van-Sinderen D. Winterling K. Kooistra J. Venema G. Hamoen L.W. Mol. Microbiol. 1996; 22: 75-85Crossref PubMed Scopus (55) Google Scholar). To increase the resolution of the gel mobility shifts we omitted the use of intensifying screens, reduced the thickness of the nondenaturing polyacrylamide gels, and applied a voltage gradient using buffers with various ionic strengths (for details, see “Experimental Procedures”). These alterations resolved the single diffuse band into three separate bands (Fig. 3). Repeating these high resolution gel mobility shift assays with other ComK-regulated promoters, such as addAB and comK, yielded only a single retarded band (data not shown) consistent with the binding of a single ComK tetramer (19Hamoen L.W. Van-Werkhoven A.F. Bijlsma J.J. Dubnau D. Venema G. Genes Dev. 1998; 12: 1539-1550Crossref PubMed Scopus (91) Google Scholar). Gel mobility shift experiments at low ComK concentrations with these ComK-regulated promoter fragments never revealed intermediate retarded bands that would correspond to the binding of discrete ComK dimers. Based on its relative mobility, we assume that the least retarded recAfragment is bound by a single ComK tetramer, and that the two further retarded bands are due to successive binding of two additional ComK dimers. The LexA-binding site of the recA promoter partially overlaps with the downstream AT-box (AT-box 4) suggesting that ComK binding to this AT-box could reduce the affinity of LexA for the recASOS-box. Thus, a plausible mechanism for competence-dependent recA induction could be that ComK precludes stable binding of LexA to its site. Arguing against this simple mechanism, Haijema et al. (8Haijema B.J. van-Sinderen D. Winterling K. Kooistra J. Venema G. Hamoen L.W. Mol. Microbiol. 1996; 22: 75-85Crossref PubMed Scopus (55) Google Scholar) showed that both proteins can bind simultaneously to the recA promoter. However, it is possible that in the presence of LexA the downstream AT-box was not occupied (i.e. only three ComK dimers bound) since the unresolved mobility shift experiment could not distinguish between partial and complete occupancy of al ComK-binding sites. To examine whether ComK is capable of displacing LexA from therecA promoter, we repeated the high-resolution gel mobility shift assays with ComK in the presence of purified LexA. Prior to testing retardation with both proteins, we tested the binding of LexA alone to determine the saturating LexA concentration. TherecA promoter contains a single SOS-box, and only a single shifted band was observed in a gel mobility shift assay (Fig.4). Graphical analysis of these data gave a K d of 5 nm for LexA binding to therecA SOS-box (data not shown). Using a LexA concentration (13 nm) sufficient to ensure binding to all recApromoter molecules, we incubated the recA promoter with increasing concentrations of ComK in the absence and presence of LexA. As indicated in Fig. 5, the presence of LexA added to the electrophoretic mobility shift of all three ComK retarded bands. Apparently, despite partially overlapping recognition sites, ComK does not exclude LexA from binding to the recApromoter. The alternative possibility, that LexA-induced supershift resulted from a specific interaction between ComK and LexA, could be refuted. In a gel mobility shift assay with a ComK-dependent promoter which does not contain a SOS-box, the presence of LexA did not result in an additional retardation in electrophoretic mobility of ComK-bound promoter fragments (data not shown).Figure 5Gel mobility shift titration using32P-labeled recA promoter fragment incubated with increasing concentrations of ComK, in the presence (+) and absence of LexA (13 nm). ComK was increased in 2-fold increments from 7 to 900 nm. The left lane (0) contains no protein.View Large Image Figure ViewerDownload (PPT) We quantified our mobility shift data by densitometry and analyzed the data graphically to determine equilibrium binding constants. Our objective was to assess the extent to which LexA alters the ability of ComK to bind the recA promoter. Any changes in ComK binding constants at saturating LexA concentration would suggest that saturating concentrations of ComK could alter the binding of LexA to the recA SOS-box. Fig. 6 shows graphical analyses of our ComK titration data in the absence and presence of LexA at a saturating concentration of 13 nm. Assuming the stoichiometry indicated by our footprinting data (i.e. a maximum occupancy of four ComK dimers per recA promoter), we analyzed the average number of ComK dimers bound per recA promoter as a function of free ComK concentration. The data for the binding of ComK in the presence and absence of LexA fit a binding isotherm with a dissociation constant, K d, of 84 nm. The value of 84 nm was determined from a Scatchard plot of the same data (Fig. 6, middle graph). It is noteworthy that the Scatchard plot is linear suggesting that the thermodynamics of binding is not cooperative despite the existence of multiple contiguous binding sites. Consistent with the linear Scatchard plots, Hill plots of the data (Fig. 6, lower graph) had slopes of exactly 1.0. These results do not rule out the possibility that the kinetics of ComK binding is cooperative, which we suspect is the case for binding of the ComK tetramer since we never detect binding of a single dimer. It is also possible that the kinetics of dimer binding is cooperative. In any case, the significant result is that the presence of LexA does not appear to affect the affinity of ComK for the recApromoter. Our ability to resolve different ligation states for ComK binding makes it possible to analyze individual binding constants to better assess cooperative interactions that might occur between sites. Senear and Brenowitz (25Senear D.F. Brenowitz M. J. Biol. Chem. 1991; 266: 13661-13671Abstract Full Text PDF PubMed Google Scholar) demonstrated the validity of this approach for the Lac, Gal, and λcI repressors (25Senear D.F. Brenowitz M. J. Biol. Chem. 1991; 266: 13661-13671Abstract Full Text PDF PubMed Google Scholar). As described above, our gel mobility shift results can best be explained by a three-site system where one site is bound by a ComK tetramer and the other two sites are bound by ComK dimers. The fraction of DNA molecules