Vol. 173, No. 5JOURNAL OF BACTERIOLOGY, Mar. 1991, p. 1696-17030021-9193/91/051696-08$02.00/0Copyright © 1991, American Society for Microbiology

Cloning and Characterization of DNA Damage-InduciblePromoter Regions from Bacillus subtilisDAVID L. CHEO, KEN W. BAYLES, AND RONALD E. YASBIN*

Program in Molecular and Cell Biology, Department ofBiological Sciences,University ofMaryland, Baltimore County, Baltimore, Maryland 21228

Received 31 August 1990/Accepted 2 January 1991

DNA damage-inducible (din) genes in Bacilus sublilis are coordinately reguled and together compose a

global regilatory network that has been termed the SOS-lke or SOB regulon. To elucidate the mechms ofSOB regulation, operator/promoter regios from tree din loci (dHAA, dinB, and dinC of B. subilis were

cloned. Operon fusioms construed with these cloned din promoter regions rendered reporter gene damageinducible in B. subis. Induc of all three din promoters was dependent upon a functional RecA protein.

Analysis of these fusins bas lzed quences required for damage-inducible expression of the dmA, dm8,and dinC promoters to within 120-, 462-, and 139-bp regios, respectively. Comparison of the nucleo

sequeees of these three din promoters with the recA promoter, as well as with the promoters of other lociassociated with DNA repair in B. subiis, has Identified the consensus sequence GAAC-N4-GITC as a putativeSOB operator site.

Inducible DNA repair systems, such as the SOS system,have been most extensively studied with the gram-negativeenteric bacterium Escherichia coli (16, 41). Regulation of theSOS system in E. coli is controlled by the products of therecA and lexA genes. The RecA protein has many functionsin E. coli and is involved in the processes of recombination,DNA repair, and mutagenesis (31, 41, 45). The LexA proteinis a repressor of as many as 20 unlinked, coordinatelyregulated loci which include the recA and lexA genes them-selves (19, 41). Following exposure of E. coli to agents thatalter DNA structure or interfere with DNA replication (suchas UV radiation, mitomycin, nalidixic acid, etc.), an induc-ing signal is generated. The signal (believed to consist, inpart, of single-stranded DNA) reversibly activates the RecAprotein. Activated RecA protein has apoprotease activitywhich facilitates the autocatalytic cleavage of LexA repres-sor (17, 18), certain lambdoid prophage repressors (4, 7, 17,34, 35), and the UmuD protein (2, 39). As levels of LexArepressor decline, damage-inducible loci are derepressed,resulting in expression of'the physiological phenomena thatcompose the SOS response (16, 41).The SOS system of E. coli has served as a model for the

study of similar inducible DNA repair systems in othergram-negative bacteria (14, 36, 37, 43, 44). Similarly, theSOS system has been used as a model to study the SOS-like,or SOB, system of the gram-positive soil bacterium Bacillussubtilis (21). Like E. coli, B. subtilis responds to agents thatdamage DNA or interfere with DNA replication by inducinga coordinately regulated set of diverse physiological phe-nomena (21). Phenomena associated with the SOB responsein B. subtilis include induction of DNA damage-inducible(din) loci, including the recA gene (formerly referred to asthe recE gene), enhanced capacity for DNA repair, en-hanced mutagenesis, Weigle (W) reactivation, prophageinduction, and filamentation (21). While these analogoussystems in E. coli and B. subtilis appear similar, significantfunctional and regulatory differences do exist. For instance,W reactivation in B. subtilis is pyrimidine dimer specific (8)

* Corresponding author.

and essentially error free (7a). This contrasts with W reac-tivation in E. coli, which is capable of repairing a variety ofDNA lesions by an error-prone mechanism (32). Further-more, while induction of all SOS phenomena in E. coli isdependent upon a functional RecA protein, filamentation inB. subtilis is a RecA-independent response (21). Finally, theSOB system in B. subtilis is developmentally regulated. AsB. subtilis differentiates into the physiological state of natu-ral competence (6), SOB phenomena are spontaneouslyinduced in the absence of externally generated DNA damage(20, 23, 47, 49, 50).DNA damage-inducible loci in B. subtilis were first iden-

tified by using transposon-mediated gene fusions (20).Tn917-lacZ transposon insertions within din loci were iso-lated from a library of insertions by selecting those fusionsthat induced expression of the lacZ reporter gene afterexposure to DNA-damaging agents (20). Fifteen indepen-dently isolated din gene transposon insertions were geneti-cally mapped and localized to three loci (dinA, dinB, anddinC) on the B. subtilis chromosome (11). As mentionedabove, induction of all three din loci was demonstrated to bedependent upon a functional RecA protein (20). In order toelucidate the mechanisms that regulate damage-induciblegene expression in B. subtilis, we have cloned and se-quenced DNA fragments that contain the dinA, dinB, anddinC promoter regions. Described here is our initial charac-terization of these cloned din promoter regions and theidentification of a putative SOB operator sequence.

(This research was conducted in partial fulfillment of therequirements for a Ph.D. degree by David L. Cheo at theUniversity of Maryland, Baltimore County, Baltimore, Md.)

MATERIALS AND METHODS

Strains, plasmids, and bacteriophage. All B. subtilis strainsused in this study are listed in Table 1. Plasmid and bacte-riophage constructs are listed and described in Table 2. Theplasmid pPL703C2 (15) replicates in B. subtilis, maintaining30 to 50 copies per genome (13), and expresses constitutiveneomycin resistance. Promoter fragments subcloned into theEcoRI and BamHI cloning sites of pPL703C2 drive expres-

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TABLE 1. B. subtilis strains

Strain Characteristics Source or

YB886a metBS trpC2 xin-J SP1- amyE 48YBA886b trpC2 xin-l SP[- amyE+ B. M. FriedmanYB1015C metB5 trpC2 recA4 10

din: :Tn9J7-lacZ transposon insertion strainsdYB5076 YB886 dinA76::Tn917-IacZ 20YB5176 YB886 dinA76: :Tn9J7-lacZ: :pTV21A2 This workYB5007 YB886 dinB7::Tn917-1acZ 20YB5107 YB886 dinB7::Tn9J7-lacZ::pTV21A2 This workYB5017 YB886 dinC17::Tn9J7-lacZ 20YB5117 YB886 dinCJ7::Tn9J7-lacZ::pTV21A2 This workYB5001 YB886 dinCl::Tn9J7-1acZ 20YB5101 YB886 dinCI::Tn917-1acZ: :pTV21A2 This workYB5021 YB886 dinC21::Tn917-1acZ 20YB5121 YB886 dinC2l::Tn9J7-lacZ::pTV21A2 This workYB5018 YB886 dinCJ8::Tn9J7-lacZ 20YB5118 YB886 dinCJ8::Tn917-lacZ::pTV21A2 This work

Strains carrying pPL703C2 derivativeseYB5200 YB886(pPL703C2) This workYB5276 YB886(pCATA76) This workYB5376 YB1015(pCATA76) This workYB5207 YB886(pCATB7) This workYB5307 YB1O15(pCATB7) This workYB5217 YB886(pCATC17) This workYB5317 YB1O15(pCATC17) This workYB5218 YB886(pCATC18) This workYB5318 YB1015(pCATC18) This work

amyE::din lacZ fusion strainsfYB5476 YBA886 amyE::pDCA1600 This workYB5576 YBA886 amyE::pDCA1200 This workYB5676 YBA886 amyE::pDCA900 This workYB5776 YBA886 amyE::pDCA280 This workYB5876 YBA886 amyE::pDCA120 This workYB5417 YBA886 amyE::pDCC139 This worka Repair-proficient parent strain.b Amylase-producing transformant of YB886.c Repair-deficient transformant of YB886.d Transposon insertions were originally isolated and described by Love et al. (20). The transposon encodes MLS resistance, which is replaced by Cmr in strains

containing pTV21A2 sequences.edin promoter fusions to the cat-86 reporter gene were constructed in pPL703C2 (15); this plasmid replicates in B. subtilis, maintaining 30 to 50 copies per

genome (13).f din promoter fusions to the lacZ reporter gene were constructed in pAF1 (9), generating the pDC plasmids, which were integrated into the amyE locus.

sion of a chloramphenicol acetyltransferase (CAT) reportergene (cat-86). The plasmid pAF1 (9) contains amyE se-quences which are disrupted by a complete cat gene and apromoterless lacZ reporter gene. Although pAF1 does notreplicate in B. subtilis, chloramphenicol-resistant (Cm')transformants can be isolated after integration of the plasmidinto the bacterial chromosome. Promoter fragments sub-cloned into the EcoRI and HindIII cloning sites of pAF1drive expression of the lacZ reporter gene.Media and growth conditions. B. subtilis strains were

maintained on tryptose blood agar base medium, and liquidcultures were grown in antibiotic medium 3 (Difco Labora-tories, Detroit, Mich.) with aeration at 37°C unless otherwisestated. Chloramphenicol (2.5 to 20 ,ug/ml), kanamycin (5jig/ml), erythromycin (1 ,ug/ml), lincomycin (25 ,g/ml),mitomycin (15 to 500 ng/ml), and 4-methylumbelliferyl 1-D-galactoside (MUG) (20 ,g/ml) (Sigma Chemical Co., St.Louis, Mo.), were added as specified. E. coli strains weregrown on Luria agar or in Luria broth. Ampicillin (50 jig/ml)and 5-bromo-4-chloro-3-indolyl-i-D-galactopyranoside (X-

Gal) (30 jig/ml) (Bethesda Research Laboratories, Gaithers-burg, Md.), were added as required.DNA manipulations. Isolation of chromosomal DNA (48)

and transformation of B. subtilis (50) were performed aspreviously described. Plasmid DNA was isolated by thealkaline lysis method (26), and DNA was sequenced by thedideoxy-chain termination method (33). DNA endonucleases(restriction enzymes) and T4 DNA ligase (Promega, Madi-son, Wis.) were used according to the manufacturer's in-structions.Regions of the B. subtilis chromosome upstream of the

different din::Tn9J7-lacZ transposon insertions were clonedby the methods of Youngman et al. (51-54). Essentially, theplasmid pTV21A2 (53) encodes resistance to chlorampheni-col and contains the origin of replication and ampicillinresistance determinant from pBR322. Flanking these se-quences are the proximal and distal ends of the transposonTn917. Derivatives of strain YB886 carrying din::Tn9J7-lacZtransposon insertions were transformed to Cmr with XbaI-linearized pTV21A2 DNA. The linearized plasmid aligns by

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TABLE 2. Plasmids and bacteriophages

Plasmid or Chtti Source orbacteriophage aractenstics reference

PlasmidspTV21A2 Ampr and pBR322 replicon; Cmr and pE194 replicon 53pDINA76a Plasmid rescue from a partial EcoRI digest of YB5176 DNA This workpDINB7a Plasmid rescue from a Hindlll digest of YB5107 DNA This workpDINC17a Plasmid rescue from an EcoRI digest of YB5117 DNA This workpDINC1a Plasmid rescue from an EcoRI digest of YB5101 DNA This workpDINC21a Plasmid rescue from an EcoRI digest of YB5121 DNA This workpDINC18a Plasmid rescue from an EcoRI digest of YB5118 DNA This workpPL703C2 Neor, pUBilO replicon, promoterless cat-86 15pCATA76 pPL703C2 with 2,500-bp EcoRl fragment from pDINA76 This workpCATB7 pPL703C2 with 753-bp Sau3AI-BamHI fragment from pDINB7 This workpCATC17 pPL703C2 with 461-bp EcoRI-BamHI fragment from pDINC17 This workpCATC18 pPL703C2 with 836-bp EcoRI-BamHI fragment from pDINC18 This workpUC18 Ampr and pMB1 replicon 46pUCDINA120 pUC18 with 120-bp PvuII-Sau3AI dinA fragment This workpAFl Ampr and pBR322 replicon; Cmr, amyE::lacZ fusion vector 9pDCA120 pAFI with EcoRI-HindIII fragmeit from pUCDINA120 This workpDCA280 pAFl with EcoRI-HindIII fragment from M13DINA280 This workpDCA900 pAFl with 900-bp HindIII fragment from M13DINA900 This workpDCA1200 pAFl with 1,200-bp HindIII fragment from M13DINA1200 This workpDCA1600 pAFl with 1,600-bp HindIII fragment from M13DINA2900 This workpDCC139 pAFi with EcoRI-HindIlI fragment from M13DINC138 This work

BacteriophagesM13mpl9 Filamentous E. coli bacteriophage 27M13DINA2900 M13mpl9 with 2,900-bp EcoRI-BamHI fragment from pDINA76 This workM13DINA2500 M13mpl9 with 2,500-bp EcoRl fragment from pDINA76 This workM13DINA1200 M13mpl9 with 1,200-bp HindlIl fragment from pDINA76 This workM13DINA900 PstI-digested and religated M13DINA1200 This workM13DINA280 M13mpl9 with 280-bp PstI-HindIII dinA fragment This workM13DINB7 M13mpl9 with EcoRI-BamHI fragment from pCATB7 This workM13DINC139 M13mpl9 with 168-bp TaqI fragment from pDINC17 This workM13DINC17 M13mpl9 with 461-bp EcoRI-BamHI fragnent from pDINC17 This workM13DINC1 M13mpl9 with 514-bp EcoRI-BamHI fragment from pDINC1 This workM13DINC21 M13mpl9 with 655-bp EcoRI-BamHI fragment from pDINC21 This workM13DINC18 M13mpl9 with 836-bp EcoRI-BamHI fragment from pDINC18 This worka The pDIN plasmids were isolated using the method of Youngman et al. (53) to clone chromosomal DNA flanking Tn917-lacZ transposon insertions by using

pTV21A2. Plasmids were generated from intramolecular ligations of chromosomal DNA digested with either EcoRI or HindUI and used to transform E. coli.

homology to the proximal and distal ends of the Tn9J7-lacZtransposon and integrates into the transposon by doublehomologous recombination. This event replaces transposonsequences encoding macrolide-lincosamide-streptogramin B(MLS) resistance with plasmid sequences resulting in Cmr,MLS-sensitive transformants that maintain the damage-in-ducible lacZ phenotype. Chromosomal DNA from theseinsertion strains was digested with either EcoRP or Hindllland was ligated under conditions favoring intramolecularligation. The ligated DNA was used to transform E. coliJM109 to ampicillin resistance by electroporation with aBio-Rad Gene Pulser, as specified by the manufacturer. Theresulting pDIN series of plasmids is described in Table 2.A 2.5-kb EcoRl fragment from pDINA76, a 753-bp

Sau3AI-BamHI fragment from pDINB7, a 461-bp EcoRI-BamHI fragment from pDINC17, and an 836-bp EcoRI-BamHI fragment from pDINC18 were subcloned intopPL703C2, resulting in pCATA76, pCATB7, pCATC17, andpCATC18, respectively. These plasmids were then used totransform the B. subtilis strains YB886 (recA+) and YB1015(recA4) to neomycin resistance (Neor). The resulting trans-formants (Table 1) were each assayed for CAT activity in thepresence and in the absence of mitomycin as describedbelow. DNA fragments that generated damage-inducible

promoter activity were subcloned into M13mpl9 and se-quenced.

Various restriction fragments of the cloned dinA76 pro-moter region were subcloned into the E. coli bacteriophageM13mpl9 (Table 2). These subclones were then used toisolate the 1,600-, 1,200-, and 900-bp HindlIl fragments,which were cloned into pAF1, generating pDCA1600,pDCA1200, and pDCA900, respectively. In addition, a280-bp PstI-HindIII fragment was cloned into M13mpl9,generating M13DINA280. This fragment was reisolated bydigestion of M13DINA280 with EcoRI and HindIlI and thencloned into pAFl generating pDCA280. Similarly, a 120-bpPvuII-Sau3AI fragment was cloned into the SmaI andBamHI sites of pUC18, resulting in pUCDINA120. Thisfragment was reisolated by digestion of pUCDINA120 withEcoRI and HindIII and then cloned into pAF1, resultingin pDCA120. The pDCA1600, pDCA1200, pDCA900,pDCA280, and pDCA120 plasmids were each used to trans-form YBA886 (the amyE+ parental strain) to Cmr, generat-ing YB5476, YB5576, YB5676, YB5776, and YB5876, re-spectively. Integration at the amyE locus was identified byplating on tryptose blood agar base medium containing 2%starch (soluble potato starch; J. T. Baker Inc.) and screeningfor the inability to hydrolyze starch. Southern analysis (data

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not shown) was used to verify that the plasmids had inte-grated at the amyE locus and not at the dinA locus. Simi-larly, a 168-bp TaqI fragment from pDINC17 was cloned intoM13mpl9 and then reisolated on an EcoRI-HindIII fragmentthat was cloned into pAF1, resulting in pDCC139. Thisplasmid was used to transform YBA886 to Cmr, and anamyE transformant (YB5417) was selected for further study.CAT assay. Qualitative CAT activity (resistance to chlor-

amphenicol) was assayed by observing growth on solidmedium consisting of nutrient broth 2 and purified agar(Oxoid Ltd., Basingstoke, United Kingdom) supplementedwith 10 to 20 ,ug of chloramphenicol per ml with or without15 ng of mitomycin per ml. CAT specific activity wasquantitated from cultures grown in nutrient broth 2 supple-mented with 0.1% yeast extract. Cells were grown withaeration at 37°C to early exponential phase (50 Klett units[KU]; Klett-Summerson colorimeter; filter no. 66; 1 KU = 1x 106 CFU/ml) and then divided. Mitomycin (500 ng/ml) wasadded to one sample, and the cultures were incubated asbefore for 2 h. The cultures were then centrifuged at 4,000 xg for 10 min at 4°C. The cell pellets were resuspended inCAT buffer (0.1 M Tris hydrochloride [pH 7.8], 0.1 mMdithiothreitol) and centrifuged as before. The cells were thenresuspended in CAT buffer containing 1 mg of lysozyme perml and 0.75 mg of phenylmethylsulfonyl fluoride per ml,incubated at 37°C for 30 minutes, and sonicated (three 5-sbursts at maximum setting) on ice. The cell extracts werecentrifuged at 10,000 x g for 15 min at 4°C to remove debrisand were assayed spectrophotometrically by the procedureof Shaw (38). Protein determinations were performed by themethod of Bradford (la). CAT specific activity was ex-pressed as micromoles of chloramphenicol acetylated perminute per milligram of protein.

,I-Galactosidase assay. Qualitative ,-galactosidase activitywas assayed by observing fluorescence of bacteria (underlong-wave UV light) grown on solid medium consisting ofnutrient broth 2, purified agar, and 20 ,ug of MUG per mlwith or without 50 ng of mitomycin per ml. ,-Galactosidasespecific activity was quantitated from cultures grown innutrient broth supplemented with 0.1% yeast extract. Thecultures were grown with aeration at 37°C to early exponen-tial phase (50 KU), an aliquot was removed from eachculture, and the cultures were divided. Mitomycin (500ng/ml) was added to one sample, and aliquots were takenfrom each culture after 1 and 2 h of further incubation. Thecells were centrifuged at 4,000 x g for 10 min at 4°C, washedfor Z buffer (28), resuspended in Z buffer containing 1 mg oflysozyme per ml, and incubated for 30 min at 37°C. The cellextracts were centrifuged at 10,000 x g for 15 min at 4°C toremove debris and assayed for ,-galactosidase activity asdescribed by Miller (28).

RESULTS

Cloning of damage-inducible promoter regions. Three DNAdamage-inducible loci (dinA, dinB, and dinC) of B. subtilishad previously been identified and were genetically mapped(11). In order to elucidate the mechanisms that controldamage-inducible regulation in B. subtilis, the operator/promoter regions of these three din loci were cloned andcharacterized. Regions of the bacterial chromosome up-stream of din::Tn917-lacZ transposon insertions were clonedin E. coli by the strategy of Youngman et al. (53, 54). Theresulting pDIN series of plasmids is diagrammed in Fig. 1.To determine whether the cloned regions of B. subtilis DNAcontained damage-inducible promoters, DNA fragments

1.0 Kb

R H Sp H P H R T SmBS R

pDINA76 L E i 'H Sa T SmBS R H

pDINB7 a

RT T SmBS R

pDINC 1 7

RT T SmBS R

pDINC1 I L aRT T SmBS R

pDINC21 a

RT T SmBS R

pDINC1 am

FIG. 1. Restriction maps of B. subtilis chromosomal DNA up-stream of din::Tn917-IacZ transposon insertions cloned in E. coli.The arrows represent Tn917-lacZ sequences and the orientation ofthe promoterless lacZ gene. Vector sequences derived frompTV21A2 are not drawn to scale and include the origin of replication(A) and ampicillin resistance (amp) determinant from pBR322 and achloramphenicol resistance determinant (cat). DNA regions markedby thick lines represent subcloned fragments that generate damage-inducible promoter activity in the pCAT plasmids. Restriction sites:R, EcoRI; H, HindIII; P, PstI; B, BamHI; S, Sall; Sa, Sau3AI; Sm,SmaI; Sp, SphI; T, TaqI.

from the pDIN plasmids were used to construct operonfusions to the cat-86 reporter gene in pPL703C2. The result-ing pCAT series of plasmids is listed and described in Table2. Promoter activity generated from each pCAT construct inB. subtilis YB886 (recA+) and YB1015 (recA4) was assayedby monitoring the expression of the cat-86 reporter gene inthe presence or absence of mitomycin. Each of the pCATconstructs rendered expression of the cat-86 reporter genedamage inducible in YB886. The dinA, dinB, and dinCpromoter regions were thus localized to the DNA fragmentsthat are indicated in Fig. 1.CAT specific activities generated in B. subtilis strains

carrying each of the pCAT constructs are reported in Table3. Treatment with mitomycin induced CAT specific activity

TABLE 3. CAT specific activity in B. subtilis YB886 (recA+)and YB1015 (recA4) carrying pCAT plasmids

CAT sp actarecA InductionStrain allele Plasmid Without With ratiob

mitomycin mitomycin

YB5200 recA+ pPL703C2c 0.012 0.012 1.0YB5276 recA+ pCATA76 0.287 1.364 4.8YB5376 recA4 pCATA76 0.185 0.282 1.52YB5207 recA+ pCATB7 0.020 0.274 13YB5307 recA4 pCATB7 0.050 0.040 0.80YB5217 recA+ pCATC17 0.018 0.434 23YB5317 recA4 pCATC17 0.026 0.022 0.85YB5218 recA+ pCATC18 0.035 1.39 36YB5318 recA4 pCATC18 0.040 0.038 0.95

a Cell extracts were prepared and assayed after a 2-h exposure to 500 ng ofmitomycin per ml. CAT specific activity is reported as micromoles ofchloramphenicol acetylated per minute per milligram of protein. The CATspecific activities reported for each strain are results from one experiment.Comparable induction ratios were obtained for each strain from repetitions ofthe experiment.

b Induction ratios are expressed as induced activity/uninduced activity.c pPL703C2 (15) is the parent vector used to generate the pCAT series of

plasmids, which maintain 30 to 50 copies per genome (13).

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-3 5 ' -10 RBS1 GATTGTCGATTCTTCATTTTTTACTATIACAGATCAGATGAAGAATCCCTG&AAQ G

Damage-Induction 61 TTTTTGATTACAAAAGCCGTTTTTGCATTGTTTTTCCCTTTTATGCTTGTTGTTCTATTT

M I T K A V F A L F F P F M L V V L F

+ 121 ACTAGAGTCACCTTTAATCATTATGTGGCGATCGCTTTAACAGCTGCATTGCTGTTTGCCT R V T F N H Y V A I A L T A A L L F A

+

+RSsK(SnVPv) (Sa/B)SXPSpH

pDC120 U +

FIG. 2. Restriction map of the B. subtilis chromosome upstreamof the dinA76 transposon insertion. The thick line indicates B.subtilis chromosomal DNA, and the arrow represents the Tn917-lacZ transposon. DNA fragments subcloned into the lacZ fusionvector pAF1 (11) are diagrammed below the restriction map follow-ing their respective plasmid designations. Vector sequences are notshown (see Materials and Methods). Each plasmid was integratedinto the B. subtilis chromosome at the amyE locus, and promoteractivity was assayed by quantitating lacZ expression. Also indicatedare the results of P-galactosidase activity assays done in the pres-

ence and in the absence of mitomycin. A plus sign indicates thatdamage-inducible promoter activity was detected; a minus signindicates that it was not. Restriction sites: R, EcoRI; Ss, SstI; K,KpnI; Sm, SmaI; B, BamHI; S, Sall; X, XbaI; P, PstI; Sp, SphI; H,Hindlll; Pv, PvuII; Sa, Sau3AI.

in cultures of YB886 (recA+) carrying the dinA (pCATA76),dinB (pCATB7), and dinC (pCATC17 and pCATC18) pro-

moter constructs by 4.8-, 13-, 24-, and 36-fold, respectively,over untreated cultures. Damage-inducible CAT activityfrom these same din promoter constructs was abolished inthe recA4 (YB1015) genetic background. In addition, thepCAT plasmids were isolated from the YB1015 derivativesand reintroduced into YB886, where they once again gener-ated damage-inducible CAT activity. Induction of eachcloned din promoter is thus dependent upon a functionalrecA+ gene product.The dimA promoter region. In order to localize the dinA

promoter, various restriction fragments from pDINA76 were

used to construct operon fusions to the lacZ reporter genewithin the B. subtilis integration vector pAF1. The resultingpDCA plasmids (Fig. 2) were then integrated into the amyE+locus of B. subtilisYBA886 (amyE+ recA+), and the result-ant amyE transformants were assayed for damage-inducibleexpression of the lacZ reporter gene. The pDCA1600,pDCA1200, pDCA280, and pDCA120 dinA promoter fusionconstructs all generated similar levels of damage-inducible3-galactosidase activity after a 2-h exposure to 500 ng of

mitomycin per ml (Fig. 2). pDCA900 generated relativelyweak promoter activity and did not significantly induce3-galactosidase activity after the same exposure to mitomy-

cin. The DNA sequences required for damage-inducibleexpression from the dinA promoter have thus been localizedto a 120-bp PvuII-Sau3AI fragment.The nucleotide sequence of the dinA promoter region was

determined (Fig. 3). A putative ribosome binding site andsequences similar to sigma A promoter elements (29, 30)were identified. Two open reading frames flank the dinApromoter region. Immediately downstream of the dinA pro-moter are 57 codons of an open reading frame that isdisrupted by the Tn917-lacZ transposon insertion. Theamino acid sequence of this open reading frame is 50%

181 TCTTATTTAAAAGGCTATACAGAAACGTATTTTATTGTAGGATTGGATGTTGTGTCTCTTY L K G Y T E T Y F I V G L D V V S L V

PstI241 GTGGCTGGCGGACTGTATATGGCCAAAAAAGCGCAGAGAAAAAAGAAGAATAAATCGGA

S A G G L Y M A K K A A E K K E E <

301 CATAATGAATATAAAGACTGAATACCTGCTTTTACGTTTTAAAAGCAGGTTTTTTATACA

PvuII -35361 CAAAAACAGCTGGAAATAAAAAACCACCGAACTTAGTTCGTArTTTTAGTGATTTTGCT

-10 RBS421 TTCCATTGTGTTACTATATCTATAGGAAGATTTCGTTAAAGAAACGGAGGCTTATTTTT

480 GTGAAAGATCGCTTTGAGTTAGTCTCGAAATATCAGCCCCAGGGAGATCAGCCGAAAGCCM K D R F E L V S K Y Q P Q G D Q P K A

HindIII EcoRI540 ATTGAA&AG=GTGAAAGGAATTCAGGAGGGCAAGAAGCATCAGACTCTGCTGGGTGCA

I E K L V K G I Q E G K K H Q T L L G A

600 ACAGGAACTGGGAAAACATTTACGGTGTCCAATTTGATTAAAGAAGTCAAT dinA76T G T G K T F T V S N L I K E V N

FIG. 3. DNA sequence of the dinA76 promoter region beginning650 nucleotides upstream of the dinA76 Tn917-lacZ transposoninsertion. Two open reading frames flank this promoter, as indicatedby the single-letter amino acid code. Putative sigma A promoterelements, ribosome binding sites (PBS), and relevant restrictionsites are marked and underlined. The arrows indicate regions ofdyad symmetry. The consensus sequence discussed in the text isindicated in boldface type.

identical and 70% similar to the first 57 amino acid residuesof the E. coli UvrB protein (Fig. 4). Within this N-terminalsequence is a conserved Walker type A adenine nucleotidebinding domain (42).The dinB promoter region. The nucleotide sequence of the

dinB promoter fragment from the Sau3AI restriction site tothe Tn9J7-lacZ transposon was determined (Fig. 5). Thissequence contains 28 codons of an open reading frame whichis disrupted by the transposon insertion. A putative ribo-some binding site and sequences similar to sigma A promoterelements were also identified.The dinC promoter region. The dinC17, dinCI, dinC21,

and dinC18 Tn9J7-lacZ insertions represent four differentinsertions within one open reading frame (Fig. 6). Thenucleotide sequence of the dinC18 promoter fragment (Fig.7) contains 125 codons of an open reading frame which isdisrupted by the transposon insertion. A putative ribosomebinding site and sequences similar to sigma A promoterelements were also identified. The pDCC139 lacZ fusionconstruct (see Materials and Methods) generated damage-inducible promoter activity when integrated at the amyE

B. subtilis DinA MKDRFELVSKYQPQGDQPKAIEKLVKGIQI... 1.1 I1: 1111.11 :1 .1::

E. coli UvrB MSKPFKLNSAFKPSGDQPEAIRRLEEGLE

B. subtilis DinA EGKKHQTLLGATGTGKTFTVSNLIKEVN:1 11111:11:11:

E. coli UvrB DGLAHQTLLGVTGSGKTFTIANVIADLQ

FIG. 4. Alignment of amino acid homology between amino acids1 through 57 encoded by the dinA open reading frame and aminoacids 1 through 57 of the E. coli UvrB protein. Within this N-ter-minal 57-amino-acid sequence there are 50%o identical and 70%osimilar residues between the two proteins. Shown in boldface typeare amino acids that are identical to conserved Walker type Aadenine nucleotide binding domains (42).

1.0 kb

R H Sp H P H R SmB S__ M %"

H P H R Sm BXSPSpHpDC1600 '

H P HpDC1 200

H PSpH

pDC900 II

RSsKSmBSXP H

pDC280 1I

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DAMAGE-INDUCIBLE REGULATION IN B. SUBTILIS 1701

Sau3AlGATGCGCTCTTATGTACATTTTGGTACATGGCCAGGCTGCCGGGAGAGGTTGGGCCCTT1

61 GACTTTTAAAATTTTCAACAGGTCTGCCTGTTCCCTTGAGATATGCTTGAAGACATCCTG121 ATTGATTTCACGATGCACAAATTCTGTCCCTTCAAGCAGGACTTCCACAAACAGGTTTAT181 CGCCTGACATATCTTTTGTTGATTCAATATCTTCCCCTCCTCATTATAGTTTACCCCGCT241 AAACTTTATGTTCAATGTCAATTAGTTTATTCCTCTAAACTATATATGTCAACAAATTTT

-35 -10301 ATTTTCGAGCACTCTACATAAG -TCATGlTC-GTGTAT GAAAGCTATACACACGAA

RBS361 AGGGGGAATTTTAACATGTCAGATTTTGCATTCAAATTGTATGAATATAACGTCTGGGCC

M S D F A F K L Y E Y N V W A421 AATCAACAAATATTCAACCGATTAAAAGAGCTTCCAAA dinB7

N Q Q I F N R L K E L P K

FIG. 5. DNA sequence of the dinB7 promoter region from theSau3AI restriction site to the Tn917-lacZ transposon insertion. Anopen reading frame encoding 28 amino acids that extends intotransposon sequences is indicated by the single-letter amino acidcode. Putative sigma A promoter elements, a ribosome binding site(RBS), and the Sau3AI restriction site are marked and underlined.The arrows indicate a small region of dyad symmetry. The consen-sus sequences discussed in the text are indicated in boldface type.

locus of B. subtilis (data not shown). This construct containsa 168-bp TaqI fragment from pDINC17 which includes 29 bpof the transposon (Fig. 1). Sequences required for damage-inducible expression of the dinC promoter have thus beenlocalized to a 139-bp region (nucleotides 30 to 168 [Fig. 7]).

Identification of a putative SOB operator sequence. Thenucleotide sequences of the dinA, dinB, and dinC promoterregions were examined and compared with the nucleotidesequence of the recA promoter region. Conserved sequenceswere identified within all of the din promoter regions thus farexamined. The consensus sequence GAAC-N4-GTTC ispositioned at -50 within the dinA and dinC promoters and at-20 within the dinB promoter, relative to putative transcrip-tion initiation sites. Similar sequences were also identified at-102 (AAAC-N5-GTTC) within dinB; at -20 (GAAC-N4-GTTT) within dinC; and at -110 (AAAC-N4-TTTC), -50(GAAT-N4-GAAC), and +80 (GAAA-N4-GTTC) withinrecA (40). A comparison of the consensus sequences foundwithin each of the promoter regions described above isshown in Fig. 8. Included in this list are similar sequencesidentified within the promoter regions of the B. subtilis uvrBgene (3) and dnaX operon (1) which are known to expressproteins involved in DNA repair. Damage-inducible expres-

500bp

E8 z

dinC17 -- In974c ' Z. X ---

_ E

8 adinCl8 -I- a+ ---z IE~~~~~8 m

dinC21 --YZZZ_ 17-f-

Er E8 X

dinC18 T--_- -

FIG. 6. Insertion maps of the four dinC Tn917-lacZ transposoninsertions designated dincl7, dinCi, dinC21, and dinC18. TheTn917-lacZ transposon is represented by the open arrows, and thehatched boxes represent an open reading frame.

EcoRI TaqIGAATTCAAGCCAAAATTTATTGAATTTCAz9AhAATTTAATAAGCTAAATGATGACACT

-10>A -3561 TGTTCAAAACAGAACAAGTGTTCTTTTTTCTAIT§GM

RBS dinC17121 TGTAATTAGTTGCTTATGGCAGCAAATAATAATAGAGGTAAATTTATGAGAAAA

M R K KdinCl

181 GAACTGTTTGATTTCACCAACATCACGCCAAAATTATTTACTGAACTACGTGTAGCAGACE L F D F T N I T P K L F T E L R V A D

241 AAAACCGTACTTCAATCATTCAATTTCGATGAGAAAAACCACCAAATTTATACAACCCAAK T V L Q S F N F D E K N H Q I Y T T Q

301 GTCGCAAGTGGATTAGGAAAAGACAACACCCAAAGCTATCGCATAACTCGTCTATCTCTTV A S G L G K D N T Q S Y R I T R L S LdinC21

361 GAAGGTTTACAATTAGACAGCATGCTGTTGAAACATGGAGGTCATGGTACAAATATTGGAE G L Q L D S M L L K H G G H G T N I G

421 ATGGAAAACCGTAATGGCACCATTTATATTTGGTCTCTATACGATAAACCAAACGAAACAM E N R N G T I Y I W S L Y D X P N E T

481 GATAAAAGTGAATTAGTTTGTTTCCCATATAAAGCAGGCGCGACTTTAGATGAAAATTCTD K S E L V C F P Y K A G A T L D E N S

541 AAA dinC18K

FIG. 7. Nucleotide sequence of the dinC18 promoter region fromthe EcoRI restriction site to the Tn9J7-lacZ transposon. An openreading frame encoding 125 amino acids that extends into transpo-son sequences is indicated by the single-letter code. Putative sigmaA promoter elements, a ribosome binding site (RBS), and relevantrestriction sites are marked and underlined. Also marked are thepositions of the four dinC Tn917-lacZ transposon insertions imme-diately following nucleotides 168 (dinCI 7), 221 (dinCI), 362(dinC21), and 543 (dinC18). A region of dyad symmetry is indicatedby the arrows. The consensus sequences discussed in the text areindicated in boldface type.

sion of the uvrB and dnaX loci of B. subtilis remains to betested.

DISCUSSION

In response to certain types of DNA damage, E. coli andB. subtilis each coordinately induce a similar set of diversecellular phenomena that collectively compose the SOS (16,41) and SOB (21) systems, respectively. Regulation of theSOS-like systems of a variety of gram-negative bacteria isvery similar to the E. coli regulatory paradigm (14, 36, 37, 43,44). In contrast, relatively little is known about the regula-tion of SOS-like systems among gram-positive bacteria. Toelucidate the molecular mechanisms of SOB regulation, we

dinA AAATAAAAAACCACCLL2CTTTA@eX2DGTATTTTTAGTGATT-102

dinB (1) ATAGTTTACCCCGCThO&CTTTATO%PPnAATGTCAATTAGTTT-20

dinB (2) GAGCACTCTACATAALLETCAT§eXEGTGTATAATGAAAAA-50

dinC (1) CACTTGTTCAAAACALLCAAGTO%DCTTTTTTCTATTGAAI-20

dinC (2) TTTCTAZTfTGAACCLLAGTAT§eXEGCTTTAATGTAATTA-110

recE (1) ATTATCCTCCTAAGAL2CATGA¶FMCTCTGATACATTATGA-50

recE (2) GCCAAGAAAAAATCCEaEATGCO@XCGCTTTTTTC,TTGGCA+80recE (3 ) GCTCTTAAACAAATAaAa&AACAWIRCGGCAAAGGTTCCATT

Consensus &C-N4 -9FIRC-20

uvrB GTGAACCA12QAQAA& AAC§eXAGTGTTA-AACTGGAAAHenry Paulus

-20dnaX AGAGCTTZTTGGATCO&MCAAG§X2ATGTATAATGGGAATJuan Alonso

FIG. 8. Consensus sequence found common to all din promoterregions thus far examined in B. subtilis. Also listed are the promoterregions from the B. subtilis uvrB gene and dnaX operon. Positionsrelative to putative transcription initiation sites are indicated. Puta-tive sigma A promoter elements are underlined.

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1702 CHEO ET AL.

have cloned and characterized three DNA damage-induciblepromoters from B. subtilis.The cloning and characterization of operator/promoter

regions that control DNA damage-inducible expression in B.subtilis has provided important information about the natureof SOB regulation. Sequences required for damage-inducibleregulation in B. subtilis have been localized to 120 bp of thedinA promoter region, 460 bp of the dinB promoter region,and 139 bp of the dinC promoter region (Fig. 3, 5, and 7,respectively). Operon fusions constructed with each of thesepromoter fragments rendered reporter genes damage induc-ible in B. subtilis. Induction of each din promoter wasdependent upon a functional RecA protein (formerly calledRecE protein). The following observations support the hy-pothesis that this regulation is controlled at the level oftranscription. The dinC17 insertion, which is damage induc-ible, separates the entire open reading frame from the dinCpromoter; also, mRNA transcripts from the dinC17 pro-moter construct in pCATC17 are induced in B. subtilis afterexposure to mitomycin (unpublished results).Sequence comparisons of din promoters in B. subtilis have

identified the consensus sequence GAAC-N4-GTTC. Thisconsensus sequence is positioned within din promoter re-gions such that a regulatory molecule bound at these sitescould interfere with the initiation of transcription by RNApolymerase. We propose that this consensus sequence func-tions as an SOB operator site to regulate expression of dingenes in B. subtilis. This sequence is centered at position-50 within the dinA and dinC promoters and at -20 withinthe dinB promoter, relative to putative transcription initia-tion sites. Similar sequences that match the consensus in atleast six of eight positions have been identified at otherlocations within the dinB, dinC, and recA promoter regions(Fig. 8). Recent work in Guilldn's laboratory has determinedthat the rec-70 locus of B. subtilis is also DNA damageinducible and contains a sequence that is similar (sevenmatches out of eight positions) to the putative SOB operatorsite and is positioned at -20 relative to the putative tran-scription initiation site (13a). Sequences similar to the con-sensus (seven matches out of eight positions) were alsoidentified at the -20 position within the uvrB gene and dnaXoperon, which are loci in B. subtilis that are known toexpress gene products involved in DNA repair processes.The binding of a repressor to slightly different operatorsequences with different affinities might serve to fine-tunethe SOB response. Furthermore, the presence of multipleoperators at distant sites within one promoter region sug-gests a cooperative loop model of repression such as hasbeen described for the lac, gal, and ara systems of E. coli(12).The putative SOB operator sites are positioned between

the -35 and -10 promoter elements of dinB and dinC andpositioned upstream of the -35 promoter element withindinA and recA (Fig. 8). We speculate that a repressormolecule bound upstream of the -35 promoter elementmight not interfere with the initiation of transcription byRNA polymerase as much as a repressor bound between the-35 and -10 promoter elements. This hypothesis is con-sistent with the relatively low levels of expression generatedby the uninduced dinB and dinC promoters and the 10-fold-higher basal level of expression generated by the dinApromoter (Table 3). It is also consistent with the relativelyhigh basal level of RecA expression that has been observedon Western immunoblots (24).

Overexpression of E. coli RecA protein can complementthe deficiency in din gene induction in strains of B. subtilis

carrying the recA4 mutation (5, 22). In addition, RecA of B.subtilis can facilitate the inactivation of LexA repressor invitro (25). These results demonstrate a high degree offunctional conservation between RecA of E. coli and RecAof B. subtilis and suggest that a LexA-like repressor exists inB. subtilis. B. subtilis din promoters within both the pDINand pDC plasmids produce constitutive 0-galactosidase ac-tivity in E. coli. This activity was observed as a dark bluecoloring on solid media containing X-Gal and is consistentwith the lack of LexA-binding sites within these promoterregions. The putative SOB repressor thus recognizes andbinds to an operator sequence that is different from the LexAbox of E. coli. It will be interesting to determine whether theSOB consensus sequence has been conserved among othergram-positive bacteria. Further molecular characterizationof these cloned din promoters is under way.

ACKNOWLEDGMENT

This research was supported by Public Health Service grantR01DE08506 from the National Institutes of Health.

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