during Bacillus subtilis Sporulation

Vol. 176, No. 13 JOURNALOFBACTERIOLOGY,JUlY1994,P.3983-3991

0021-9193/94/$04.00+0

Copyright C1994, AmericanSociety for Microbiology

Temporal Regulation and

Forespore-Specific Expression of the Spore

Photoproduct

Lyase Gene

by

Sigma-G

RNA

Polymerase

during

Bacillus subtilis Sporulation

MARIOPEDRAZA-REYES,1 FELIX

GUTIERREZ-CORONA,1

ANDWAYNE L.NICHOLSON2*

InstituteofInvestigation inExperimental

Biology,

FacultyofChemistry, Universityof Guanajuato, Guanajuato, Gto., 36060, Mexico,1andDepartmentsof Microbiology andImmunologyandBiochemistryand MolecularBiology,

UniversityofNorth Texas Health Science Center,Fort Worth, Texas 761072

Received 18 January 1994/Accepted29April 1994

Bacterial spores arehighlyresistantto killing by UV radiation and exhibit unique DNA photochemistry. UV irradiation of spore DNA resultsinformation of sporephotoproduct (SP), the thymine dimer 5-thyminyl-5,6-dihydrothymine.'RepairofSPoccursduring germinationofBaciUussubtiis spores by two distinctroutes, either by the

general

nucleotide excision repair (uvr) pathway orby a novel SP-specific monomerization reaction mediated by the enzyme SP lyase, which is encoded by the spl gene. Repair of SP occurs early in spore germination and is independent of de novo protein synthesis, suggesting that the SP repair enzymes are

synthesizedduring sporulation and are.packaged in the dormant spore. Totestthis

hypothe$is,

theexpression of a translational spl-lacZ fusion integrated at the spl locus was monitored during B. subtilis growth and sporulation.

P-Galactosidase

expressionfrom thespl-lacZ fusionwassilentduring vegetativegrowth andwas notDNA damageinducible, butit was activated atmorphological stage III ofsporulation

specifically

in the forespore comphrtment, coincident with activation of expression of the stage III marker enzyme glucose

dehydrogenase.

Expressionof thespl-lacZfusionwasshowntobedependentupon thesporulation-specificRNA polymerase containing the sigma-G factor(E(G), asspl-lacZexpressionwasabolished in amutantharboring a deletion in the sigG gene and restored by expression ofthesigG genein trans.Primerextension

analysis

of splmRNA revealedamajor extension product initiating upstream fromasmall openreadingframe of unknown functionwhichprecedes spl, and it revealedtwo other shorterminor extension products. All three extension products were present in higher quantities during sporulation and after sigGinduction. The three putative transcripts are all preceded by sequences which share homology with the consensus

EiG-type

promoter sequence,

but

int vitro transcription bypurified sigma-GRNApolymerasewasdetectedonlyfromthe promoter correspondingtothemajor extension product. The openreading frame-spl'operontherefore appears tobean additional member of the sigma-G regulon, which also includes as members the small, acid-soluble spore proteins wh ch are in large part responsible.for spore DNAphotochemistry.Therefore, sporulatingbacteria appeartocoordinately regulate genes whose products not

only

alter spore DNA

photochemistry

but also repair the majorspore-specific photoproduct duringgermination.

Dormant bacterial spores are 1 to 2 orders of magnitude moreresistant to

the

killing effectsof'UV radiation than are vegetativelygrowingcells ofthe same organism (42, 49). The mechanisms underlying this phenomenon have to date been mostextensively studied in the caseofthegram-positive soil bacteriumBacillus subtilis;at present, two related phenomena have been foundtoaccountfor the high resistance of B.subtilis spores toUV

light.

First, DNA from UV-irradiated spores has photochemistry verydifferentfrom that of DNAfrom UV-irradiated vegetative cells (5). In contrast to the cis-syn cyclobutane pyrimidine dimers which are produced in DNA as a result of UV irradiation of vegetative cells, UV irradiation

of

B. subtilis sporesresultsin theproduction of a novel DNA photoproduct informally called spore photoproduct (SP) (5). The chemical structure ofSP has been elucidatedtobe the thymine dimer 5-thyminyl-5,6-dihydrothymine (51). SP has been isolated in natureonly from UV-irradiated bacterial spores (5), although this

photoproduct

can also be formed in vitro (32). During

*

Corresponding

author.

Phone:

(817) 735-2120.

Fax:

(817) 735-2118.

sporulation bacteria produce specific proteins which upon binding to spore DNA determine in large part its unique photochemistry.DNAinside spores is found associated witha group of DNA-binding proteins called small, acid-soluble spore proteins (SASPs) of a class known in the case of B. subtilisasthe

a/4-type (12, 47).

Deletion ofthe genesencoding themajor

a/p-type

SASP in B. subtilisresults in strains whose sporesareverysensitivetoUVradiation(22). Studiesinvitro have demonstrated that

ox/p-type

SASPs

bind

DNA

(31) and promoteaDNAhelicalchangefromtheBconformationtoan

A-like conformation(25);UVirradiationof these SASP-DNA complexesin vitro results informationof SP and suppression ofcyclobutyl dimer formation (32).

Second, SPwhich accumulates in dormant spores is elimi-nated from spore DNA at the onset of sporegermination in partthroughtheactivityof nucleotideexcisionrepair mediated bytheuvrpathway(28, 29).Inadditiontonucleotide excision repair, however, B. subtilis possesses a unique, SP-specific DNArepairenzyme called SPlyase (26, 28, 29), encodedby thesplgene (7, 26). SP lyaseappears to actby direct in situ monomerization of SP in DNAto two thymines(28, 29, 52). Taken together, the uvr and spl repair pathways probably

TABLE 1. Bacterial strains used in this study

Strain Genotype and/or phenotype Source (reference)'

B.subtilis 168 trpC2 Laboratory stock WN118 sigGAI trpC2 P.Setlow (50) WN119 trpC2spl-lacZCmr pWN116 168 WN126 sigGAI trpC2

spl-lacZ

Cmr pWtN116 WN118 WN127 sigGAI

trpC2 spl-lacZ

Cmr pDG298 WN126 [pDG298; Ps C-sigGKm)1b

YB5176 TnO17dinA76-acZ Cmr R. Yasbin (3) E. coli

JM83 araA(lac-proAB)rpsL4)80 Laboratory stock (53) lacZAM15

GM161 F-thr-lleuB6 dam-4 thi-I hsdSI Laboratory stock (7) lacYItonA421X-supE44

a",

transformation.

bBrackets contain the plasmid and marker.

from DNA duringsporegermination, asit has been observed thatmutantstrains of B. subtilis lacking both repair pathways producesporeswhicharehighly sensitivetoUVradiation (26, 27).

The SP lyase repair system was originally defined by a mutation now called spl-1 (26). To better understand spl-mediated DNA repair, the wild-typesplgenefromB.subtilis

168was recently cloned because of its abilityto restore UV resistance to spores of a B. subtilis strain which harbors mutations in both theuvr andsplpathways (7). The physical organization of thespllocus has been deducedby nucleotide

sequence analysis of the region, and comparison by FASTA

analysis (35) of the deduced amino acidsequencesof SPlyase

and other DNA repair proteins revealed that the carboxy-terminal region of SP lyase shares sequence homology with those ofDNAphotolyases from a numberoforganisms (7). This observation suggests that these enzymes may have

de-scended from a common ancestral protein, even though SP

lyase isnotdependentonvisible light for its function (7). Early indirect studies of SP repair duringB.subtilis

germi-nation indicated that both theuvrandsplrepairsystemswere operationalwhensporesweregerminated inthepresence of eitherchloramphenicolorrifampin, compoundswhichinhibit denovosynthesis of proteinand RNA, respectively (29). The results of these experiments suggest that the componentsof thesplanduwpathways are synthesized during the previous round of either vegetative growth or sporulation and are

packaged in the mature dormant spore. With the recent

molecularcloning of thesplgene, thishypothesiscannowbe

tested by directassayof spl expression. In this articlewereport

the construction ofa transcriptional and translationalfusion between thespl gene and the Escherichia coli lacZgene to

study the regulation of expression of thesplgene during the

developmental cycle of B. subtilis and the characterization of spl transcription both in vitro and in vivo.

MATERIALS ANDMETHODS

Bacterial strains, plasmids,and growthconditions. B. sub-tilisand E. coli strains used in this studyarelisted in Table 1, andplasmids used in this studyaredescribed in Table 2. Media usedwere Difco sporulation medium (DSM) (41) and

Luria-Bertani (LB) medium (24). Preparation of competent B. subtilis cells and theirtransformation with plasmid DNAwere performed as previously described (2). When appropriate, antibioticswereaddedtomediaasfollows: chloramphenicol, 3

p,g/ml;

ampicillin, 50

jig/ml;

and kanamycin, 10 ,ug/ml. Cells weregrowninliquid media with vigorous aerationoronsolid

media at 37°C. The optical density of liquid cultures was monitored with either a Klett-Summerson colorimeter fitted

with a no. 66 (red) filter or a Perkin-Elmer model 565 spectrophotometerset at600nm.

Construction and integration of the spl-lacZ gene fusion.

Construction ofanin-frametranslational fusion between the

spl gene and the E. coli lacZ gene was carried out in the integrative plasmid pJF751 (11) by insertingan846-bp EcoRI-BclIfragment from plasmid pWN87, correspondingtoaregion fromnucleotide(nt) 527to1373 inthepublishedsplsequence

(7), into EcoRI-BamHI-cleaved plasmid pJF751,asillustrated

inFig. 1. Identification of the desired plasmid constructionwas performed by restriction enzyme analysis of small-scale

plas-mid preparations (1) digested with EcoRI and PvuII. The resulting plasmid containing the spl-lacZ fusion, designated pWN116 (Fig. 1),waspropagatedinE. coliJM83.Competent cells ofB.subtilisweretransformed withplasmidpWNl16,and transformantswere selected on solid DSMcontaining chlor-amphenicol. ChromosomalDNAwasisolated(4) fromoneof

thesetransformants, designated strain WN119, and integration of the spl-lacZ fusion at the chromosomal spl locus was

confirmed by Southern blot analysis (48). Briefly, genomic DNAisolated from B. subtilis 168 and WN119was restricted

withHindIII and separated by electrophoresis througha 1% agarosegel. The DNA fragmentsweretransferredto a nitro-cellulose membrane and were probed with an agarose

gel-purified 2.3-kb EcoRI-HindIII fragment from plasmidpWN41, whichencodes the entiresplsequence(Table 2) (7). The probe

waslabeledby the random oligonucleotide priming technique

(10) using digoxigenin-dUTP. Detection of hybridswascarried

TABLE 2. Plasmidsusedin thisstudy

Plasmid Description Source(reference)

pDG298 IPTG-induciblesigGgene;Km' P.Setlow(50)

pJF751 Integrational lacZ fusion vector; Cmr P.Setlow(11)

pPS395 sspA gene cloned inpJH101 P.Setlow(3a)

pWN41 2.3-kbEcoRI-HindIII fragmentofspl frompWN35cloned inpBGSC6 Thisstudy(7) pWN68 1,357-bpEcoRI-SphIfragment of spl gene(fromnt527to1884)clonedinpBGSC6 Laboratory stock(7)

pWN78 839-bpPstI-EcoRI fragmentofspl(fromnt117to956)cloned inpUC18 P.Fajardo-Cavazos,thislaboratory pWN81 678-bpPstI-EcoRIfragmentofspl(fromnt117to795)cloned inpUC18 P.Fajardo-Cavazos,thislaboratory pWN87 pWN68propagatedinE. coliGM161;lacksDammethylation Thisstudy

pWN116 846-bpEcoRl-BclIfragment(fromnt527to1373)of spl frompWN87cloned into Thisstudy' EcoRI-BamHIsite ofpJF751

pWN204 292-bpHindIII-EcoRIfragmentofsspA5' end frompPS395cloned inpUC18 Thisstudy

REGULATION AND EXPRESSION OF THE SP LYASE GENE 3985

Plasmid pWN87 PstI Plasmid pJF751

oA

E

CmR

App

I 5' end ofspi 1 promoterlesslacZ *

BclI SphII

MCS

I22

EcoRI

BeI

'pI

IC

HindM A

relI

EcoRI SinaI

BamEHI

..ATT GAT CAT.. AGA ATT CCC GGG GAT CCC GTC

ile asp his arg ile pro gly asp pro val-lacZ

1.EcoRI,Belldigest 1.EcoRI, BamHIdigest

2.Gelpurify846bpfragment 2.Gelpurifyvector

.Lgate

2. TransformE. coli, select

4p

flr3.Screen PstI

Plasmid

pWN116

Apr onE Cmr spl-laeZ fusion

EcoRIl

A2 BcllIBamHI Bell/BamHI ..ATT GAT CCC GTC..

spl-ile asp pro val-lacZ

FIG. 1. Construction of plasmid pWN116 containing an in-frametranslationalspl-lacZgenefusion. See Materials and Methods for details. MCS, multiplecloningsite; oriE, E. coli origin ofreplication.

outbyusing antidigoxigenin antibodies conjugated to alkaline phosphatase and the lumigen substrate 4-methoxy-4-(3-phos-phatphenyl)spiro(1,2dioxyetane-3,2'-ada-mantane)with the Dig-luminescent Detection kit (Boehringer-Mannheim Biochemi-cals) as described by the manufacturer. The presence of the spl-lacZ fusion integrated at the spl locus was confirmed by visualization of an expected 11.3-kb hybridizing band in the sample from strain WN119, whereas an expected 3.7-kb hy-bridizing band was detected in the sample from strain 168

(datanotshown).

Cellgrowthandenzymatic assays.B.subtilis strains carrying the spl-lacZ fusion were grown and allowed to sporulate in liquid DSM containingchloramphenicol. Samples (1 or 1.5 ml each)werecollectedperiodicallyduringvegetativegrowth and throughout sporulation. Cell samples were washed with cold 0.25 M Tris-HCl buffer (pH 7.5), and the cell pellets were storedat -20°C. Cellextractswereprepared from mother cell andforespore fractions as previously described (21, 33) and wereassayed for

P-galactosidase

(24, 33) and glucose dehydro-genase(GDH) (13, 33) activities. Briefly, washedcell samples werefirst disrupted with lysozyme and subjected to centrifu-gation.

P-Galactosidase

activitypresent in the supernatant was measured and assigned to the "mother cell" fraction (which actually consisted of mother cells plus lysozyme-sensitive fore-spores[21]). Thepellet, which consisted of lysozyme-resistant foresporescontaining spore coats,wassubjected to spore coat removal, washing in buffer, and a second round of lysozyme treatment (21).

1-Galactosidase

activity after this treatment wasassignedtothe"forespore" fraction. Total

3-galactosidase

activity expressedby the cells is thesumof theactivities in the mother cellandforespore fractions.

Becauseof thesmallamountof,B-galactosidase expressed by the spl-lacZ fusion and the endogenous ONPGase activity expressed duringB.subtilissporulation,ineachexperimentthe strainharboringthespl-lacZfusionwasgrownandsporulated inparallel with aculture of thecorrespondingparentalstrain lacking thespl-lacZfusion; the endogenous ONPGase activity oftheparentalstrainwasthen subtracted from the

3-galacto-sidase activity of the fusion-containing strain at each time point.

Induction experiments. Expression of the spl-lacZ fusion followinginduction ofsigGexpressioninB.subtilis WN127was

performedasfollows. Cellsweregrownto anoptical densityat 600 nm

(OD600)

of 0.3. The culture was divided into two subcultures ofequalvolume, and

isopropyl-p-D-thiogalactopy-ranoside (IPTG) was added to one subculture to a final concentration of 1 mM; the second subculture served as a control. Samples of cells harvested before and after IPTG additionwereassayed for

3-galactosidase

and GDH activities as described above. To analyze the DNA damage inducibility of spl-lacZ expression, B. subtilis WN119 was grown in LB medium toan OD600of 0.5, the culturewas divided into two equal subcultures, andmitomycin (0.5-,ug/ml final

concentra-tion)wasaddedtooneof the subcultures. Cellswereharvested and assayed for ,B-galactosidase activity before and after the addition of mitomycin as described above. As a positive control, cells ofB.subtilis YB5176,containing the dinA76-lacZ fusion(kindly provided byR.Yasbin, UniversityofMaryland)

were grown and treated identically with mitomycin. In addi-tion, spl-lacZ expression was assayed as described above for

samples harvested throughout the growth of strain WN119 under conditions which inducegenetic competence (2).

Mapping of the 5' ends of spl mRNA. The 5' ends of spl mRNA were mapped by primer extension (23) of spl tran-scripts produced either during sporulation or in response to induction of sigG expression, as follows. Total RNA was isolated from sporulating cells of B. subtilis WN119 or from either IPTG-induced oruninduced cells of strain WN127 as previously reported (20) by using the expression of ,-galacto-sidaseactivity directed by the spl-lacZ fusion to determine the time at which to harvest the cells to obtain the maximum amount of spl transcripts (20, 34). The synthetic 17-mer oligonucleotide5'-dGCTGCGGAACAAATGGG-3', comple-mentary tospl mRNAbetween9and 26 ntdownstream from the putative spl translation initiation codon (7), was end labeled by using [_y-32P]ATP and phage T4 polynucleotide kinaseand was hybridized to 10 ,g of total RNA from each sample.The primerwas extended with Moloney murine leu-kemiavirus reverse transcriptase, and the extended products were separated by electrophoresis through a 6% polyacryl-amide DNAsequencing gel. The positionsof the radioactive products relative to a DNA sequencing ladder (40) generated from plasmid pWN41 (Table2) (7) weredetermined by using theoligonucleotide describedabove as the sequencing primer with theSequenase version 2.0 kit (U.S. Biochemical Corp., Cleveland,Ohio).

RNA polymerase purification and in vitro transcription. RNApolymerasecontainingthesigma-Gfactor wasisolatedas follows. B. subtilis WN127 was cultivated in 250 ml of LB medium containing chloramphenicol and kanamycin to an

OD600

of 0.25. IPTG was added to the culture to a final concentrationof 5 mM, and theculturewasincubatedfor 2 h. During this time period, samples were withdrawn from the culture andassayed for

3-galactosidase

activity encoded bythe spl-lacZ fusionasdescribedabove,toensureinductionofsigG expression.Cellswereharvestedby centrifugationandwashed, and RNApolymerasewasisolatedasdescribedpreviously (34, 50) through the heparin-agarose column step. The resulting proteinpreparationwasassayed for sigma-GRNApolymerase activity by runoff

transcription

utilizing the sspA promoter carried onplasmid pWN204cleaved with EcoRI. To assay in vitrotranscriptionfrom theputativespl promoters, the RNA polymerasepreparationwasused in runofftranscription exper-iments with EcoRI-linearized plasmidspWN78 andpWN81.In vitro transcription reactions were performed as previously described

(34),

with theexceptionthat thelabelednucleotide usedwas

[a-35S]UTP (Dupont,

NewEngland

Nuclear).

Tran-scription

productswereseparated

by

electrophoresis through

a

6% polyacrylamide sequencing gel, and the dried

gel

was

subjected to

autoradiography.

Sizes of observed

transcripts

wereestimatedby comparisonof theirmobilitieswithaDNA

sequencing ladder generated by

using

the

phage

M13 DNA template supplied with the Sequenase version 2.0 kitand

by

correction for the

slightly

decreased

mobility

ofRNArelative to that of DNA

(approximately

3% difference under the electrophoresis conditions used

[38a]).

RESULTS

Temporal and compartmental expression of an spl-lacZ fusion. B. subtilis WN119, containing a single copy of the spl-lacZ fusion

integrated

atthespllocus,wasusedtostudythe expression of spl throughout growth and sporulation. The kinetics of appearance of

P-galactosidase

activity directly ex-tractedfrom the cellsbylysozymetreatment revealed thatspl

was not expressed during vegetative growth; ,-galactosidase

activity

first

appeared

4 h after the onset of the

stationary

I

I

40 ofs q p MD.t Time(h)

FIG. 2. Forespore-specific expression of spl-directed ,B-galactosi-dase activity during stage III of B. subtilis sporulation. B. subtilis WN119 was grown and induced to sporulate in liquid DSM as described in MaterialsandMethods(circles). Samples collectedatthe indicated times were treated with lysozyme, and the extracts were assayeddirectly for,3-galactosidase activity (open squares) or GDH

(triangles). I-Galactosidase activitypresent in thelysozyme-resistant forespore fraction (solid squares)wasassayed asdescribed in Materi-als andMethods.

phase(i.e.,atT4) and peakedatT5, and then the enzyme levels subsequently decreased (Fig. 2).

Sporulating B. subtilis cells undergo an asymmetric cellular division which results in the establishment of two cell compart-ments, a forespore envelopedwithin a mother cell (36). The observed increase andsubsequent decline ofspl-lacZ-encoded

,B-galactosidase activity directly

extractable

by lysozyme

(Fig.

2) were reminiscent of genes which are expressed in the developing forespore compartment at an intermediate stage of development (44), as it has been observed previously that during the sporulation process theforespore becomes resistant to direct disruption by lysozyme as a result of deposition of spore coat proteins on its surface (21). To test this notion, cellular fractionation experiments were performed to assay compartment-specific expression of ,-galactosidase directed by the spl-lacZ fusion (Fig. 2). During sporulation of strain WN119,

P-galactosidase

activity extracted from the lysozyme-resistant forespore compartment was detected at

T5,

and forespore ,3-galactosidase activity continued to accumulate until at least

T1o

(Fig. 2). The increase in forespore enzyme activity was paralleledbyadecrease in,B-galactosidaseactivity extracted from the lysozyme-sensitive mother cell fraction, characteristic offorespore-specificgeneexpression (Fig.2)(9, 13, 21).

To further investigate the timing offorespore-specific

ex-pression of thespl-lacZ fusion, the same cellextractsused in the,-galactosidaseassays(Fig.2)werealsoassayedfor GDH activity encodedby the stage III, forespore-specificgdh gene (13, 21, 30). GDH activity and ,B-galactosidase from the spl-lacZfusionwere expressedwithessentially identical kinet-ics (Fig. 2), strongly indicating that spl gene expression is activated at stage III ofsporulation specifically in the devel-opingforesporecompartment.

Sigma-G dependence ofspl-lacZ fusion expression. It has been reported that the gdh operon (30) and several other sporulation-associated genesincludingthe sspfamily (21), the spoVA operon (6, 21),and thegerA operon(9), amongothers, form aforespore-specific regulon of genes whose coordinate expression is controlled bothtemporallyandcompartmentally byRNApolymerasecontainingtheforespore-specific sigma-G

REGULATION AND EXPRESSION OF THE SP LYASE GENE 3987

1 ~~~~~~~~~~15

~ioo

~

-2_0

60 4

6A

FI. 3.1Exrsino p-ietd-alcoiaeatvt Qtin

~20

0

-2 0

-2

4 6 8

Time(h)

FIG. 3.

Expression

of

spi-directed

13-galactosidase

activity

(trian-gles) during growth and sporulation (circles) of wild-type strain WN119 (closed triangles) andsigGAl mutant strain WN126 (open triangles) in DSM. Samples were harvested and assayed as described in Materials and Methods. Cell extracts were also assayed for GDH activity,with essentially identical results (data not shown).

factor

(EBG)

(34, 44).Thepattern ofexpression ofthe spl-lacZ fusion(Fig. 2)stronfysuggested thatspl maybe anadditional member of the Eu regulon. Two approaches were usedto address this question directly.

First, thespl-lacZfusion was introduced by transformation into strainWN118,aB. subtilis strainharboringa deletionof thesigG gene, which is unable to transcribe EoG-dependent genesand in whichsporulationisarrestedatstageIII(14,50). The resulting strain, WN126, was grown in liquid DSM con-taining chloramphenicol, andsampleswereremoved from the culture throughoutgrowth and the stationaryphase for assay of ,-galactosidase and GDH activity. Expression both of ,-galactosidase directed by spl-lacZ (Fig. 3) and of GDH (data notshown)wasalmostcompletelyabolished instrain WN126, consistent withspl expressionbeing eitherdirectlyorindirectly underthe control ofECrG.

Second,to directlytest

EBG

dependence ofsplexpression, strainWN126wastransformedwithplasmid pDG298carrying the wild-type sigG gene under the control of the IPTG-inducible Pspac promoter (50), resulting in strain WN127 (Table 1). IPTG induction ofsigG expression invegetatively growing cells resulted in activation ofthe expression of both spl-directed ,-galactosidase andGDHactivities(Fig. 4). Mea-surements of the maximum induction of

f-galactosidase

ex-pression indicated that strain WN127 expressed levels of

P-galactosidase

threetofourtimes higher than thoseexpressed bythewild-type strain,WN119(compare Fig.3 and4).Taken together, these results stronglysuggest that splexpression is directly dependenton

EuG

RNApolymerase.

Mapping the5' ends of invivospl mRNA.Examination of thenucleotidesequencesurroundingthe spl gene revealed the presence of a small open reading frame (ORF) situated upstream from spl, which could potentially encode a 9-kDa polypeptide (7). Both the ORF and spl are preceded by sequenceshomologoustoribosomebinding sites,butthe80bp of DNA intervening between the two cistrons lacks inverted repeat sequences

characteristic

ofrho-independent

transcrip-tional terminators, suggesting that the two genes may be cotranscribed (7). In contrast, examination of the nucleotide sequences preceding and following the ORF-spl region re-vealed that ptsI, the genepreceding the ORF, and spl itself are both followed by sequences that could provide

rho-indepen-0

u

ra

04

1 2 3 4 5 6

Hours after IPTG addition

aQ

-O 0

pi

FIG. 4. Growth(circles) and

P-galactosidase

(triangles) or GDH (squares) activityin B.subtilis WN127 with(closed symbols)orwithout (open symbols) theadditionof IPTG. Cells were grown in LBmedium. At zerohours, the culturewasdivided equally and IPTGwasadded to one subculture. Cell samples collected at the indicated times were treated with lysozyme and assayed for ,B-galactosidase and GDH activities asdescribed inMaterials and Methods.

dent transcriptional termination signals (7). To gain further insightsinto thetranscriptional organizationofthespl region, primer extension analysis was used to map the 5' ends of mRNAs originating upstream from thespl coding sequence. Experimentswerecarriedoutbyusinga17-nt-long spl-specific primer (see Materials and Methods) and total RNA isolated fromsporulatingcells of B.subtilisWN119,harvestedateither

To,

the onset of the stationary phase, or at T5, the time of maximum expression of,-galactosidase activityfrom the spl-lacZ gene fusion. Primer extension analysis indicated the existence ofa major primer extensionproduct corresponding tocoordinates 597to 598 of thepublishedsequence (7) (Fig. 5), located 15to16bpupstreamfrom the initiation methionine codon of the small ORFprecedingspl,immediatelypreceding the ORFribosomebindingsite(Fig. 5).Inaddition,twoweak primer extension productswere detected, at coordinates 809 (within the ORF) and 906 (9 bp preceding the spl ribosome binding site). All threeprimer extension productswerepresent in higher amounts in the RNA sample obtained from strain WN119 cells harvested at

T5,

the time of maximal spl-lacZ expression;asmallamountof themajortranscriptatpositions 597to598wasalsodetectedin RNA fromcells harvestedat

To

(Fig. 5). To determine if these primer extension products resulted from transcription by Eu RNA polymerase occur-ringwithin theORF-splregion, primerextensionmappingwas also performed on total RNA isolated from cells of strain WN127which had beencultivated in LB medium either with orwithout IPTG induction ofsigG expression from plasmid pDG298 (Fig.5).Induction ofsigGexpression byIPTG during vegetativegrowth of strainWN127also resulted inproduction of the same three primer extension products seen during sporulation,inapproximatelythesamerelative amounts(Fig. 5).Themajorprimerextension productatpositions 597to598 wasalsodetectedat areduced level inuninduced cells of strain WN127(Fig. 5).

Thethreeprimerextensionproducts observed could actually representthree distinct transcriptsorcouldalternativelybe (i) invivo-processedproducts ofalongersingletranscriptor (ii) incomplete primerextension productsofalongersingle

tran-script. Examination of the nucleotide sequences upstream from each of the three extension productsrevealed the pres-enceof sequenceswithsimilaritytopromotersprecedinggenes VOL.176,1994

A 597- G* +1 C T A G A

GI

I

\,-'1'

A 809- A* +1 A T 1'F/ C\ 906- A*+1 G A A 1 2 G A T C 1 2 G A T C 1 2 G A T C

'I_

FIG. 5. Primer extension mappingof invivosplt

indicate the positions of the primer extension prodi nucleotide sequence (lanesG, A, T, and C) gener; pWN41 (Table 2) (7)by usingthesameprimer.Ast

positions of the primer extension products relativ

sequencecoordinatesofFajardo-Cavazosetal.(7) reactionswereperformedontotal RNAisolated fro]

To(lane 1), strain WN119 at T5 (lane 2), strainV

(lane 3), and strain WN127 without IPTG(lane 4).

which are members of the sigma-G regulon

quences were designated putativepromoters

(Fig. 6). Of the three putative promoters pi most closely matches the consensus sigma-( quence(34),exhibitingaperfect match in the 6). The -35 region of P1 also matched the sig:

-35 -10

eIgG-type TgAAA---17 or 18 bp----C&TACTA

P1 562-TTCATAkGTAkGGGTATAQAKGGACACAATAI

:1I: I I I I: II I

P2 768-CGCAGAGGACGATGCTGTGTTTG-CAAGCTAT

: :III 1:1II

P3 870-CTCATATCCTTTCCGCCTAGTGA-AAAAGTAA FIG. 6. Comparison of theconsensusE(JGprom

(top line) with the putative promoter sequences

precedingspl (bottom 3 lines),deduced from theprii of Fig. 5. Underlined bases are highly or absolu

EaG-type promoters (34). Perfect matches are in( lines.Matches in lesshighlyconservedregionsoft

promoter sequence are indicated by colons. Aste

quences denote thepositions of the

corresponding

products (Fig. 5).

promoter very well, containing only a single mismatch at position -35, where ahighly conservedGresidueisreplaced in Pl by a T (Fig. 6). The -10 and -35 regions ofP1 are separated byadistance of 18

bp,

also in agreementwiththe sigma-G consensus sequence (Fig. 6). Sequence analysis -._-- showed that

putative

promoters

P2 and P3 exhibited a lower overall degree of homology to the consensus sigma-G-type promotersequence and that the "-10"regionof P2 is in fact located at approximately -15 with respect to the primer extensionproduct atcoordinate 809 (Fig.6).

In vitro transcription of spi. To address whether putative promoter sequences P1, P2, and P3 are functional EaG-type 3 4 promoters, plasmids which contained DNA spanning the

ORF-spl region

were

prepared

foruseas

templates

for in vitro runoff

transcription

reactionswith RNA

polymerase

contain-ing sigma-G.

Sporulating

cells of B. subtilis expresstwo

fore-<-

spore-specificforms of RNA polymerase containing either the

sigma-Ffactor or thesigma-G factor,whichhaveverysimilar promoterspecificitiesinvitro(34,50). To ensure the absence ofE(UFinthepreparation,the source ofEfrGRNApolymerase was vegetative cells of strainWN127grown in LB medium with IPTG induction of sigG expression. Induction of sigG expres-sion by IPTG was monitored by measuring activation of 3 4 P-galactosidase synthesisencodedby thespl-lacZ fusion, in a mannersimilarto thatdescribed in the legend to Fig. 4 (data notshown).The RNApolymerase preparationcontained EfG, I as it was capable of correctly transcribing plasmid pWN204 linearized with EcoRI (Table

2),

which contains the known 4UI~ ~ sigma-G-dependent sspA promoter (34), generating the ex-pected 112-nt runoff transcript (datanot shown). To test for

EUG-dependent

runofftranscriptionfrom theORF-spl region, twotemplates wereused, plasmidspWN78 and pWN81, each

transcripts.

Arrows

linearized with

EcoRI

(Table 2).

Transcription

of

plasmid

ucts relative to the pWN78 from P1, P2, or P3 would be expected to generate a ated fromplasmid runoff transcript of 365, 153, or 56 nt, respectively, while terisks indicate the transcriptionofplasmidpWN81 would beexpectedto gener-'e to the ORF-spl ate a runoff transcript of 204 nt only fromP1,asneither P2 nor

.Primerextension P3 is contained on this plasmid. In vitro transcription of

mstrain WN119 at plasmid pWN78 by

EurG

yielded a single runoff transcript of VN127 with IPTG 365 nt, consistent with transcription from P1 (Fig. 7); the shortertranscripts predictedtobegeneratedfrom P2 and P3 were not detected. When plasmid pWN81 was used as the template, a 204-ntrunofftranscriptwas seen, also consistent (34); these se- with transcription from P1. It therefore appears from this P1, P2, and P3 experiment thatP1can functionin vitro as an

EcrG-dependent

receding spl, P1 promoter and that either P2 and P3 are not functional G promoter se- promoters in vitro or transcription from them is too weak to be -10region (Fig. detected under the experimental conditions used.

,ma-Gconsensus Expression ofthe spl-lacZ fusion following DNA damage or duringcompetence. It has beenreported that B. subtilis cells exhibit anSOS-like response similar in many aspects to that observedin E. colicells(18). Several DNAdamage-inducible genes,collectivelycalled dingenes, include those whose

prod-**AS!Z-598

uctsareinvolved in DNA

repair,

suchasrecAandgenesofthe

CATGG-598 * uvrsystem(17, 19, 38).InB.

subtilis,

theexpressionof certain

NAAGLkGCTAA-809

din genes such asrecAcan beactivated not only by exposure of *CGTTA-906 cells to DNA-damaging agents but also when cells are grown underconditions which induce genetic competence (17, 19). otersequence (34) Because the

spl

gene is also involved in DNA repair, the (P1

P2,

and P3) possibilitythatexpressionof thespl-lacZgene fusion could be

ier

extension

data induced either by treatment of B. subtilis cells with a

DNA-dicated

by vertical

damaging

agent

or

by

competence

induction was

explored.

;he

Eu0G

consensus

Mid-logarithmic-phase

cells of B. subtilis WN119were

exposed

risks over the se- to the

DNA-damaging

agent mitomycin ata final concentra-primer extension tion of 0.5

,ug/ml,

whichwas

previously

used to demonstrate induction oflacZ reporter genes fused to the

dinA, dinB,

or

REGULATION AND EXPRESSION OF THE SP LYASE GENE 3989

GATC 1 2

_-

365

I-*-- 204

FIG. 7. In vitro runoff transcription ofspl. RNApolymerase

con-taining sigma-Gwaspreparedasdescribed inMaterials and Methods

and used to transcribe the following templates: plasmid pWN78

linearized withEcoRI (lane 1) and plasmid pWN81 linearizedwith

EcoRI(lane 2).Lanesmarked G,A,T,and Cshowtheresultsof DNA

sequencing reactionsof M13 DNA usedasmolecular size standards.

Arrowsdenote the sizes of the runoff transcripts, correctedfor the fact

that under the electrophoresis conditions used in the experiment,

RNAmigrates approximately 3%moreslowlythanDNA(38a).

dinC promoter region (3). Treatment of strain WN119 with mitomycin didnot leadto increased expressionof

P-galacto-sidaseactivity encoded by the spl-lacZ fusion (datanotshown). As a positive control, treatment of strain YB5176, which contains the dinA76-lacZ fusion (3), with mitomycin under identicalconditions did induce ,B-galactosidase expression 20-fold greater than that of untreated cells (data not shown). Likewise, WN119cellsgrowntogeneticcompetence(2) failed to induce spl-directed 3-galactosidase expression (data not shown).

DISCUSSION

Bacterial spores must rely on the expression of specific

mechanisms to correct damage to their DNA induced as a

resultofexposureto UVduringdormancy. To date, it iswell

established that the SP which accumulates in the DNA of dormant spores is corrected very specifically and efficiently

during early germination through the combined activities of the uvrand spl pathways (26, 28, 29). Because repair of SP

takes place at the onset of sporegermination, it isreasonable tospeculate that theproduct of the spl gene could be synthe-sizedduring thepreviousround of sporulation and packaged in the mature spore (7). This speculation is supported by early experiments demonstrating that spl-mediated repair of SP can take place when UV-irradiated B. subtilis spores are germi-nated in the presence of antibiotics such as rifampin and chloramphenicol, which either indirectly or directly block de novoprotein synthesis (29).

The recentcloning and sequencing of spl (7) have allowed directexaminationofthe molecular mechanisms that regulate its expression. This paper describes the utilization of an in-frame transcriptional and translational spl-lacZ fusion to study thetemporalandcompartmentalexpression of thisnovel DNArepair system.Analysisof theexpressionof thespl-lacZ fusion integrated in the spl locus during the developmental cycle of B.subtilis suggestedthat the Spl enzyme is produced at anintermediate time duringsporulationand specifically in the foresporecompartment (Fig. 2). Interestingly, the kinetics of ,-galactosidase expression exhibited by the spl-lacZ fusion were observedto besimilar to those reported for a number of stage III,forespore-specificgenes (44). By using GDH activity as aforespore-specific, stage IIImarker (13, 21, 44),expression ofthe spl-lacZ fusion was observed to be identical to GDH activity with respect to both its kineticsand its compartmen-talization (Fig. 2). This importantpiece of evidence supports thenotion that the Spl protein is synthesized during sporula-tion and ispackagedwithin thedormantspore.

Theexpression offorespore genes duringsporulation in B. subtilis is controlled by the sequential action of two types of sporulation-specific RNA polymerases, harboring either the sigma-F factor or the sigma-Gfactor (reviewed in reference 15). The results in Fig. 2suggestedthat

EcaG

was likely to be the RNApolymerase responsiblefor the transcription of the spl-lacZfusion;however,transcriptionofthespl-lacZfusion by

Ea'F

could not at first be ruled out. Such apossibilitywas tested bymeasuring theexpression of thespl-lacZ fusion in a strain harboring a deletion of the sigG gene, which lacks EUG but contains

Ecru

(34, 50); expression of thespl-lacZ fusion and expression of GDH activitywere found to be almost totally abolished in the sigGAI mutant, consistent with their strict dependenceonsigma-G RNApolymerase (Fig.3 and data not shown). Furthermore, expression of both the spl-lacZ fusion and thegdh operon was completely restored to the sigGAl strain WN126 by the introduction into this strain of the

wild-type

sigG gene on anextrachromosomalelement (Fig. 4). On the basis of these results it is concluded that spl is transcribed during sporulation in theforespore compartment by thedirect action of

E(JG.

RNA polymerase containing the sigma-G factor activates expression of a large regulon of forespore-specific genes including sspA-E,gdh,gerA,spoIVB,andspoVA,among others (forreviews seereferences15 and 44).Transcriptmapping and in vitromutagenesis

experiments

performedonmany of these genes have resulted in the identification of conserved nucle-otide sequences characteristic of

EcG-dependent

promoters

(8,

9,30, 34, 37).Results obtainedasdescribed above with the

spl-lacZ

gene fusionstrongly suggestthatsplis a newmember of the

EcuG

regulon, and results from primer extension map-ping of the 5' ends of spl mRNA (Fig. 5) support this conclusion. Three primer extension productswereidentifiedin the regionpreceding spl,amajor productprecedingthe small ORF upstream from spl and two minorproducts apparently originating from within the ORFand immediately preceding the spl ribosome binding site (Fig. 5). Nucleotide sequence analysis identified three putative promoter sequences (here

called P1, P2, and P3) preceding these primer extension products, all of which exhibit homology to the sigma-G con-sensus promoter sequence (34) (Fig. 6). The P1 promoter was found to be the most similar to the sigma-G consensus promoter sequence, differing from it by only a single base, the highly conserved G at position -35 being replaced in the P1 promoter by a T (Fig. 6). The great similarity betweenP1 and theErG consensus promoter perhaps explains why most of the primer extension products are associated withP1 in vivo (Fig. 5).At present it is unclear whether P2 and P3 actually function invivo as promoters or whether the primer extension products atthese positions represent in vivo-processed mRNA species or incomplete in vitro extension products. The latter two possibilities appear to be supported by results from the in vitro transcription experiment, in which an

Eor'-dependent

runoff transcript was detected fromP1 only (Fig. 7).

Onthe basis of the observed absence of aputative transcrip-tionaltermination sequence between the ORF andspl, it was previously proposed that these two genes may form a bicis-tronic operon(7); this suggestion is supported by the mapping ofthe major 5' end of the spl mRNA upstream fromthe ORF (Fig. 5) and by thedemonstration that theP1 promoter which precedes this transcript is apparently utilized byE0G in vitro (Fig. 7). Two interesting questions arise from these observa-tions. (i) Is there a structural relationship between the two proteins potentially encoded by this operon? (ii) What is the reason for their spatial arrangement and their coregulation? The deduced Splprotein sequence has beenobserved to share local amino acid homology with a number of microbial and fungal DNAphotolyases(7), and it is interesting to note that in E. coli, the phr gene which encodes DNA photolyase is also preceded by a small open readingframe(called ORF169) (39) which is cotranscribed withphr but whose product is appar-ently not needed for photolyase function (16). Indeed, at present a function has not beenassigned to eitherthe E. coli ORF 169 product orthatof the ORFprecedingthe B. subtilis spl gene. Although the possible structural, functional, or regulatory role of the ORF preceding spl is at present un-known, experimentsthat willaddress theseissuesarecurrently inprogress.

Finally, the possible induction of spl expression by factors which are able totrigger the B.subtilis SOS-likeresponse (18) was investigated. No induction ofspl-lacZ fusion expression was observed under two conditions reported to induce DNA damage-inducible (din) genes, i.e., treatment of cells with mitomycin (3, 18) and growth of cells togenetic competence (19). A probableexplanation for theseresults is theabsence in the ORF-spl region of the cis-acting din-regulatory operator sequenceGAAC-N4-GTTC (3). Rather, the resultspresented in this paper support a model in which the majority of spl expression isactivateddirectlyduring sporulationby Eu0.

Most livingorganisms must handle DNAdamage in which-ever foim it is presented to them. What causes sporulating bacteria to be sounique is thesurprisingfact thatin response to selection for high UV resistance they have evolved both alternative DNA photochemistry and a repair system dedi-cated to the correction of the resulting spore-specific DNA lesion. In B. subtilis, spore UV resistance is governed bytwo typesof gene products whichplaymajorroles in(i) determin-ingspore DNAphotochemistryduring dormancy(i.e., SASPs) and(ii) repair of spore DNA damage duringgermination(i.e., Spl). The ssp genes encoding SASP are transcriptionally acti-vated in the forespore by EuG RNA polymerase, and SASPs bind to sporeDNAand causeit to undergo structural changes which alter its UV photochemistry to favor SPproduction in thedormant spore(reviewed inreferences 42through 47). As

this reportdemonstrates, the spl gene is alsotranscriptionally activated during sporulation in the forespore by Ea' RNA polymerase and the Spl protein is packaged in the mature spore to correct SP during subsequent spore germination. Becausethesuccessfulrepair of SP by the Spl protein during germination of UV-irradiated spores is tightly linked to the prior activity ofSASPson sporeDNA,itseemsreasonableto expectthat theexpression ofthegenesfor all of theseproteins is controlled by a commonregulatory circuitry.

ACKNOWLEDGMENTS

We thankPeter Setlow and RonYasbin for generous donation of strains and plasmids and Tony Romeo andPatriciaFajardo-Cavazos for criticalreading of themanuscript.

This work wassupported bygrantsfrom theNational Institutes of Health (GM47461), the American Cancer Society (JFRA-410), and the TexasAdvanced ResearchProgram(009768-034)toW.L.N.andby adoctoralfellowshiptoM.P.-R.fromtheConsejo Nacional de Ciencia yTecnologia(Mexico).

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