Nucleotide Sequence, Regulation Bacillus Protease ... · DNA from B. megaterium QMB1551wasfirst treated withEcoRI methyl-ase, and the methylated DNA was digested to various degrees

Vol. 173, No. 1

Cloning, Nucleotide Sequence, and Regulation of the Bacillussubtilis gpr Gene, Which Codes for the Protease That

Initiates Degradation of Small, Acid-SolubleProteins during Spore Germination

MICHAEL D. SUSSMAN AND PETER SETLOW*

Department ofBiochemistry, University of Connecticut Health Center,Farmington, Connecticut 06032

Received 3 July 1990/Accepted 9 October 1990

The gpr gene, which codes for the protease that initiates degradation of small, acid-soluble proteins duringspore germination, has been cloned from Bacillus megaterium and Bacilus subtilis, and its nucleotide sequence

has been determined. Use of a translational gpr-lacZ fusion showed that the B. subtilis gpr gene was expressedprimarily, if not exclusively, in the forespore compartment of the sporulating cell, with expression taking placeapproximately 1 h before expression of glucose dehydrogenase and ssp genes. gpr-lacZ expression was abolishedin spoIlAC (sigF) and spolIlE mutants but was reduced only -50% in a spoIJIG (sigG) mutant. However, thekinetics of the initial -50% of gpr-lacZ expression were unaltered in a spolliG mutant. The in vivotranscription start site of gpr has been identified and found to be identical to the in vitro start site on this genewith either ECFF or ECG. Induction of CG synthesis in vivo turned on gpr-lacZ expression in parallel withsynthesis of glucose dehydrogenase. These data are consistent with gpr transcription during sporulation first byErF and then by EYG.

During the first minutes of germination of spores ofBacillus species, 10 to 20% of the dormant spore's protein isdegraded to amino acids (21). The proteins degraded in thisprocess are a group of small, acid-soluble spore proteins(SASP), and their degradation is initiated by an endopro-tease also present in the spore (20, 21). This protease hasbeen purified from Bacillus megaterium spores and is spe-

cific for cleavage within a pentapeptide sequence found in allSASP (9, 23). The protease is synthesized during sporulationwithin the developing forespore as a polypeptide of 46 kDa(P46) (9, 10). Approximately 1 h after its synthesis, P46 is

converted to a polypeptide of 41 kDa (P41), which is the formfound in the dormant spore (10). Early in spore germination,the enzyme is processed again, being converted to a slightlysmaller polypeptide of 40 kDa (P40) (7, 10). The active formof the protease is a tetramer, and P40, P41, and P46 all formtetramers (7, 9). However, only P40 and P41 exhibit enzy-matic activity in vitro (7).

It is clear that the activity of this SASP-specific protease isregulated at a variety of levels. First, the gene coding for theprotease (termed gpr [27]) appears to be expressed only inthe forespore for a defined time in sporulation, presumablyas a result of regulation at the transcriptional level, althoughthis has not yet been proven. Second, since the proteasepolypeptide undegoes multiple processing reactions, at leastone of which appears to activate the enzyme, there is alsoregulation at the posttranslational level. However, nothing isknown of the mechanism(s) or regulation of these transcrip-tional and posttranslational events. As an initial step towarda detailed understanding of the regulation of this sporeprotease, in this communication we report the cloning andsequencing of the gpr gene from B. megaterium and Bacillussubtilis, as well as studies on the regulation of gpr expressionin B. subtilis.

* Corresponding author.

MATERIALS AND METHODS

Organisms and plasmids used and isolation of nucleic acids.The B. megaterium, B. subtilis, and Escherichia coli strainsused are described in Table 1. The sources of Agtll andvarious plasmids have been described previously (6, 26, 27).B. megaterium and B. subtilis chromosomal DNA wasisolated and purified as described previously (2, 6). PlasmidDNA was isolated from E. coli strains grown overnight in Lbroth plus 0.5% glucose with the appropriate drug (ampicillin[50 ,ug/ml], tetracycline [10 ,ug/ml], or chloramphenicol [10,ug/ml]) as needed and purified by CsCl gradient centrifuga-tion if necessary. Xgtll derivatives were grown and phageDNA was isolated as described previously (6). B. subtilisstrains were made competent and transformed as previouslydescribed (2, 11, 12).

Molecular genetic methods. DNA fragments were sepa-rated by agarose gel electrophoresis, and individual frag-ments were isolated as previously described (6). Fragmentsto be labeled for hybridization probes were separated byusing low-gel-temperature agarose and labeled by randompriming. DNA hybridization analyses were carried out afterelectrophoretic separation of fragments and their transfer tonitrocellulose paper as described by Southern (2, 25). Hy-bridization analyses were carried out at 68°C for detection ofhomologous DNA fragments and at 52°C for detection ofheterologous DNA (2, 6).DNA was sequenced either by the method of Maxam and

Gilbert (13) or by the chain termination method, using DNAfragments cloned in pUC vectors (18). All sequences re-

ported were determined completely in both directions, andall restriction sites used in cloning fragments for sequencingwere sequenced across.

Isolation and analysis of enzymes. The B. megateriumspore protease was purified to homogeneity from 40 g of dryspores as described previously (9). Amino acid analysis ofthe purified protein and automated sequenator analysis of

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292 SUSSMAN AND SETLOW

TABLE 1. Bacillus strains used

Strain Genotype or phenotype Source or reference

B. subtilis168 trpC2 Laboratory stockPS766 trpC2(pDG298 spac-sigG 26

Kmr)M0428(PS683) sigGAI trpC2 P. Stragier (8)PS1208 spolIACi trpC2 J. ErringtonPS606 spoIIIB2 trpC2 BGSCaPS607 spoIIIE36 trpC2 BGSCPS605 spoIIID83 trpC2 BGSCPS604 spoIIIC94 trpC2 BGSCPS889 spoIIIA53 R. LosickPS1029 gpr-lacZ trpC2 pMS16-*168bPS1084 gpr-lacZ trpC2(pDG298 pDG298.-*PS1029b

spac-sigG Kmr)PS1190 gpr-lacZ spoIIACi trpC2 pMS16--*PS1208bPS1087 gpr-lacZ spoIIB2 trpC2 pMS16---*PS606bPS1089 gpr-lacZ spoIIIGAl trpC2 pMS16-*PS683bPS1086 gpr-lacZ spoIIID83 trpC2 pMS16--*PS605bPS1085 gpr-lacZ spoIIIC94 trpC2 pMS16-*PS604bPS1088 gpr-lacZ spoIIIE36 trpC2 pMS16--*PS607bPS1394 gpr-lacZ spoIIIA53 pMS16___>PS889b

B. megateriumQMB1551 Laboratory stock

a BGSC, Bacillus Genetic Stock Center.b Arrow represents transformation of plasmid into competent cells, with

selection for the appropriate antibiotic resistance.

the protease were also carried out as previously described(1). RNA polymerase from B. subtilis strains was purifiedthrough the heparin-agarose step and assayed as describedpreviously (26). Specific B. subtilis strains were used toproduce RNA polymerase preparations containing eitherEuG or ECFF or neither EuF nor E(JG was as recentlydescribed (16).

1-Galactosidase produced from the gpr-lacZ fusion as wellas glucose dehydrogenase were assayed in sporulating cellsafter direct lysozyme treatment as previously described (11).In some cases, samples were first treated with urea andsodium dodecyl sulfate to remove spore coat proteins andrender maturing spores sensitive to lysozyme (11).

Analysis of gpr mRNA by primer extension and in vitrotranscription. The 5' end of B. subtilis gpr mRNA in vivowas determined by primer extension analysis as describedpreviously (3, 16). The primers used were 20- and 30-residuesynthetic oligonucleotides from the amino-terminal codingregion of the gpr gene (see Fig. 3). The RNA for analysis wasisolated from sporulating cells as previously described (16).The extended products were analyzed on a 6% polyacryl-amide sequencing gel adjacent to the four lanes of a DNAsequencing ladder obtained from the cloned gpr gene by the

MboBM

Hind I INsi

dideoxy-chain termination procedure, using the 5'-32P-la-beled 20- and 30-residue oligonucleotides noted above as theprimer.

In vitro transcription of plasmid DNA containing the gprpromoter was carried out with (x-32P-labeled UTP and RNApolymerase preparations containing various sigma factors;transcription products were analyzed as described previ-ously (16). Analyses of the efficiency of various forms ofRNA polymerase on a gpr template in vitro were measuredrelative to the efficiency on an sspE template, a knownrQ-dependent gene. These in vitro transcription reactionscontained 2 F.g of total template: either pPS591 cut withEcoRI, which yields an EuG or EuF sspE transcript of 193nucleotides (16), or the 500-bp HindIII-BclI fragment carry-ing the gpr gene's amino-terminal and 5' region clonedbetween the HindlIl and BamHI sites of pUC18. The latterplasmid, termed pPS1101, was routinely linearized withEcoRI, but in some experiments it was linearized with KpnI.

Cloning procedures. The strategy we used to isolate the B.megaterium gpr gene was to clone DNA fragments in theEcoRI site of the fusion vector Xgtll (29) and detect positiveclones with spore protease antibody (9). DNA from B.megaterium QMB1551 was first treated with EcoRI methyl-ase, and the methylated DNA was digested to variousdegrees of completion with either AluI, HaeIII, or RsaI,using five different amounts of each enzyme, each acting on50 V*g of DNA. The highest amount of enzyme used pro-duced complete digestion. Each digest was electrophoresedon a 1% agarose gel, and DNA fragments of 0.5 to 2 kb wereisolated. The pooled fractionated DNA was mixed with a30-fold molar excess of EcoRI linkers; this linker mixturecontained an equimolar mixture of 8-, 10- and 12-mer EcoRIlinkers (New England BioLabs). The mixture was treatedwith T4 DNA ligase and then digested with EcoRI. Thedigested DNA was refractionated by agarose gel electropho-resis; 0.5- to 2-kb fragments were isolated and ligated withEcoRI-digested, calf intestinal phosphatase-treated XgtllDNA. The ligation mixture was packaged by using a com-mercially obtained packaging extract and used to infect E.coli Y1090 (ATCC 37197); approximately 40% of the pack-aged phage contained inserts.We screened for the gpr gene by analyzing phage plaques

for protease antigen by the procedure of Young and Davis(29), using antiserum to the B. megaterium spore proteaseprepared as described previously (9) and alkaline phos-phatase-conjugated second antibody (Promega). Screeningof _ 105 plaques resulted in isolation of one phage whichsubsequent analysis showed to carry an insert from the gprgene. The DNA insert in this phage was then used as a probein colony hybridization to detect other fragments of the genecloned in plasmid vectors. The B. megaterium gpr gene was

Clal Mbo XbaI I Hindilll BamHI

Pstl Hindl 11DC I I

EcoRI HindilI I PstI

OObpFIG. 1. Restriction maps of the B. megaterium (BM) and B. subtilis (BS) gpr genes. The open boxes are the coding regions, and each arrow

denotes the amino terminus of the protein.

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ANALYSIS OF THE B. SUBTILIS gpr GENE 293

S

TGTGGCTGATATTGCGCCCTTCCTTCCACTMCATTTCTTMAIOCAIMTCTGCCAGTCCTCGGTGM&ATTTCMCAAAGATTG100

TCTAGAGGTAMMGAA ATG GAA AAA GAA CTT GAT TTA AGT CM TAT A1C GTG aC ACG GAT TTA CTMet Gk Lys Glu Lu Asp La Ser Gn Tyr Ser Val Arg Thr Asp Lt Ala

200GTC GAA CA AAA GAC ATA G0 TTA GAA AAT CAG CCT AAA COOAMT AAC CAG TCA GAA ATC AAAVal Gku Ala Lys Asp lie Ala Leu Gu Asn Gin Pro Lys Pro Asn Asn Ghi Ser Gku lie Lys

G GTA ATT GTA AAA GAA AAA GAA GM CM GGT GTC AAA ATC TCA ATG GTA GAA ATT AC GAAGly Val lie Val Lys Glu Lys Glu Gu Gin Gly Val Lys lie Ser Met Val Gku lie Thr Gk

3004G; GX GCA GM GCA ATC GT AAG AAA AAA GGr OGA TAT GTA ACC CTT GAA TCOG GTA G3 ATTGlu Gly Ala Glu Ala lie Gly Lys Lys Lys Gly Arg Tyr Val Thr Lau Glu Ser Val Gly lie

1 400aC GAG CG GAT ACT GAA AAG CM GAA G0G GC ATG GAA GAA GTA TTT GCT AAA GAG CTG MTArg Glu Gln Asp Thr Glu Lys Gn Gu Glu Ala Met Glu Glu Val Phe Ala Lys Glu Leu Asn

TTT TTT ATT AAA AC CTC MC ATT OCA GAT GAT OCT AGC TGT CTT GTA GTG GT CTT MCPhe he le Lys Ser Lou Asn lie Pro Asp Asp Ala Ser Cys Leu Val Val Gly Lau Gly Asn

500TTA AGT GTT ACA CCA GAT G CTT CCA AAG CA GTA GAT MT TTG TTG ATT ACA AGA CATLou Ser Val Thr Pro Asp Ala Lou Gly Pro Lys Ala Val Asp Asn Lou Leu lie Thr Arg His

TTG TTT G-G CTA CAG CCT GAA AC GrG CM GAT G: TTT AGA OOC GTT AW IC ATT GTT COGLeu Phe Gu Lou Gan Pro Glu Ser Val Gan Asp Gly Phe Arg Pro Val Ser Ala lie Val Pro600GA GTA ATG GOA ATG AG GGT ATT G0G ACA AGT GAT ATT ATC m - GTT GTA AAG AAA GIGGly Val Met Gly Met Thr Gly lie Gk Thr Ser Asp lie lie Phe Gly Val Val Lys Lys Val

700MT OCA GAT TTT ATC ATT GCA ATC GAT GOG CTT 0OC GCT CG TCOG ATT GAA 00 GTA MT GCTAsn Pro Asp Phe lie le Ala lie Asp Ala Lau Ala Ala Arg Ser lie Glu Arg Val Asn Ala

ACC ATT CM ATT TCA GAT TCA GGT ATT CAT OOG G3G TCT OUGTT A MC AAG OGT AAA GMThr lie Gan lie Ser Asp Ser Gly lie His Pro Gly Ser Gly Val Gly Asn Lys Arg Lys Gli

800ATC AGT TAT GAA ACA CTT ATT OCA GTT ATT GCA ATA GT ATT OCA AOG GTA GTG GAT OCTle Ser Tyr Glu Thr Lwu Gly lie Pro Val lie Ala lie Gly lie Pro Thr Val Val Asp Ala

900GTT TCC ATT ACA A GOAT ACA ATT GAC TTT ATC TTA AAA CAT TTC GC CCA GAA ATO AAA G0OVal Ser lie Thr Ser Asp Thr lie Asp Phe lie Lwu Lys His Phe Gly Arg Glu Met Lys Gli

CAG GGA AAG OOG TCT AAG TCOG CTG CTTU OG TCC GOT ATG ACG TTT GOA GAA AAA AAG AAG CTCain Gly Lys Pro Ser Lys Ser Lwu Lw Pro Ser Gly Met Thr Phe Gly Glu Lys Lys Lys Lw

1000ACG GAG GAT GA@ TTA CCC MT GAA G0G C0 COGT CM ACG TAT CTC GGT ATG ATT G: ACC CTTThr Gan Asp Asp Lwu Pro Asn Glu Gu Gan Arg Gn Thr Tyr Lwu Gly Met lie Gly Thr Lau

I 1100CCA GAT GAA GAA AAA AGA CGT CTT ATT CAT GAA GTG CTT GCA OCT TTA G: CAT MT TTG ATGPro Asp Gu Glu Lys Arg Arg Lw lle His Glu Val Lwu Ala Pro Lwu Gly His Asn Lwu Met

GTT AOG OCA AAA GAA GrG GAT ATG TTT ATT GAA GAT ATG GCT MT GTA GTT CA GGA GGA CTTVal Thr Pro Lys Glu Val Asp Met Phe lie Glu Asp Met Ala Asn Val Val Ala Gly Gly Lu

1200MC GCT GCT TTG CAT CAT GAA GTG GAT CM G0G AAC TTT OGGA TAT ACT CAT TMAsn Ala Ala Lu His His Glu Val Asp Gan Gu Asn Phe Gly Ala Tyr Thr His * *

1300TACGCTTTTAGGTGAATGAGTTCTTCACMTATAGTCATMTGGTTAGTACTACAGAAGATAAAGAAGTMGGGGCCTMTMTATGATTA

1400MTTACTTMAAAATCAGTAGTGCTMCCATITITMACTGTGCGTCGTAITTUTAGGGATGCAGACGGCTCAGCMGGGTTGATTAAAATG

MGGGATATMTGATCCTTCGATTGAACATGTTATTMTGTGTCTAAAACATCAGATGGTCAGGTGGAAGCTGCTGTTTTAGGTTCMCM1500

MGTCGTGAACATTGATGAAAMCAGCAGTCTAGAMAACGAAAGGCTTTTAACITTIICCCCTCGCTAGGTCAMAGTCTAGCGGMGG

FIG. 2. Nucleotide sequence of the B. megaterium gpr gene. Nucleotides are numbered every 100 residues, with the first number over thedesignated nucleotide. The two singly underlined regions denote the -35 and -10 regions of the gene's promoter as determined by homologywith those of the B. subtilis gpr gene. The doubly underlined region is a putative ribosome binding site, exhibiting significant complementaritywith the 3' end of 16S rRNA. The dot over residue 81 denotes the putative transcription start as determined by homology with that for theB. subtilis gpr gene. The horizontal arrow at amino acid 18 denotes the amino terminus of P40 as determined by protein sequence analysis.Vertical arrows denote the two HaeIII sites, cleavage at which gave the initial 700-bp fragment cloned.

used similarly as a hybridization probe to clone the B. RESULTSsubtilis gene in plasmid vectors.

Nucleotide sequence accession numbers. The B. megate- Cloning and sequence of B. megaterium and B. subtilis gprrium and B. subtilis gpr sequences have been assigned genes. Use of the fusion vector Xgtll for cloning fragments ofGenBank accession numbers M-55262 and M-55263. B. megaterium DNA allowed isolation of one phage carrying

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TABLE 2. Automated sequenator analysis of B. megaterium p4aCycle Residue identifiedCycle ~~~~~~~~~~~~~(pmol)

1 ...................................... Ala (1,560)2 ...................................... Val (1,183)3 ........ .............................. Glu (1,275)4 ........ .............................. Ala (1,228)5 ........ .............................. Lys (1,309)6 ...................................... Asp (1,388)7 ........ .............................. Ile (1,283)8 ........ .............................. Ala (947)9 ........ .............................. Leu (1,045)10 ......... ............................. Glu (1,082)11 ......... ............................. Asn (1,029)12 ......... ............................. Gln (893)13 ......... ............................. Pro (996)14 ......... ............................. Lys (1,071)15 ......... ............................. Pro (915)a About 2 nmol of purified P40 was subjected to automated sequenator

analysis as described in Materials and Methods.

a 0.7-kb insert which expressed spore protease antigen. Useof this initial fragment as a hybridization probe allowedisolation of a 1.8-kb MboI fragment and a 1.6-kb ClaI-BamHI fragment in pUC vectors. Together, these twofragments encompass the complete gpr gene (Fig. 1). DNAsequence analysis of the cloned fragments showed the pres-ence of a long open reading frame in this region (Fig. 1 and2) and identified the initial 0.7-kb fragment cloned in Agtll asa HaeIII fragment (vertical arrows in Fig. 2). To prove thatthe gene we had cloned did indeed code for the sporeprotease, we determined the amino-terminal 15 amino acidsof purified P40 from B. megaterium (Table 2). Gratifyingly,this sequence was in perfect agreement with amino acidresidues 12 to 26 in the predicted coding sequence (Fig. 2).Subsequent use of the 1.2-kb NsiI-XbaI fragment from the

B. megaterium gpr gene (Fig. 1) as a hybridization probeallowed the determination of the restriction map of the B.subtilis gpr gene (Fig. 1) and the cloning of two fragments(0.8-kb HindIII and 1.1-kb EcoRI-PstI) encompassing theentire coding sequence. Surprisingly, it proved impossible toclone the HindIII fragment by itself in pUC vectors; in thesevectors it was only cloned together with a second B. subtilisHindIII fragment. However, the 0.8-kb HindIII fragmentwas cloned alone in pBR325. DNA sequence analysis of theB. subtilis gpr gene (Fig. 3) showed that the protein codedfor was extremely homologous to that of B. megaterium,showing -68% identity (Fig. 4).The coding sequences of both the B. megaterium and B.

subtilis gpr genes are preceded by sequences homologous tothe 3' end of 16S rRNA from Bacillus species (Fig. 2 and 3).The termination codon is not followed by any obviousstem-loop structure which might be a rho-independent tran-scription termination site. However, there is no open readingframe of more than 40 amino acids which begins either at orshortly after the 3' end of the gpr gene coding sequence.Comparison of the B. subtilis gpr amino acid sequence

with those of the B. subtilis serine proteases (24) or metal-loprotease (28) revealed no significant homologies (data notshown). This suggests that the gpr gene did not evolve froma common ancestor of the subtilisin gene or, if it did, that ithas undergone much divergence. Since the properties of thespore protease (tetrameric, highly sequence specific require-ment for substrate) are very different from those of otherknown B. subtilis proteases (monomeric, relatively nonspe-

cific sequence specificity for substrate), the lack of homol-ogy between the spore protease and other B. subtilis prote-ases is possibly not surprising.

Expression of the gpr gene during sporulation. Previousexperiments using antisera against the B. megaterium sporeprotease have shown that this protein appears only duringsporulation, at or before the time of appearance of its SASPsubstrates (9, 10). We have determined these kinetics moreprecisely for the B. subtilis gpr gene by measuring itsexpression through use of a translational lacZ fusion. A0.32-kb EcoRI-HincII fragment from within the coding se-quence of the B. subtilis gpr gene (Fig. 3) was cloned in E.coli in pJF751 (4) cut with EcoRI and SmaI, giving pMS16,in which the gpr and lacZ coding sequences are in frame.The resulting plasmid was then used to transform B. subtilisto chloramphenicol resistance as a result of the integration ofpMS16 into the chromosome at the site of the gpr gene.Southern blot analysis (not shown) of the chromosomalDNA of one transformant showed that the integrant con-tained the lacZ gene preceded by the amino terminus andcomplete 5' upstream sequence of the gpr gene. The lacZregion and remainder of the plasmid was followed by a 5'truncated gpr gene as expected. Although this strain carriesan interrupted gpr gene, it grew and sporulated normally(Fig. 5). P-Galactosidase was synthesized only during sporu-lation, with much of the synthesis preceding expression ofthe glucose dehydrogenase gene, a known forespore-specificgene (Fig. 5) (22). The fall in apparent levels of both,-galactosidase and glucose dehydrogenase as sporulationproceeded is due to the resistance of maturing spores todirect lysozyme extraction, as has been seen previously withother forespore-specific genes (11). Indeed, comparison ofthe ratios of gpr-lacZ to glucose dehydrogenase in directlylysozyme extracted cells with maximum levels of theseactivities and in mature spores extracted with lysozyme afterremoval of spore coats showed that the two ratios werealmost identical (data not shown). Since glucose dehydroge-nase is made only in the forespore (5, 22), this findingsuggests that the gpr gene is also expressed only in theforespore. A similar conclusion was reached previously in B.megaterium when gpr gene product levels were measureddirectly in forespores and whole sporulating cells (9, 10).The gpr-lacZ fusion was also integrated into strains car-

rying various asporogenous mutations, and maximum lacZ(as well as glucose dehydrogenase [gdh]) expression wasmeasured during sporulation. As found previously with anumber of forespore-specific genes (11, 22), gpr-lacZ expres-sion was not blocked by a mutation in a gene which regulatesmother cell gene expression, spoIIIC, nor by a mutation inthe spolIIB or spoIIID locus (Table 3). However, gpr-lacZexpression was blocked by a mutation in the spoIIAC locus(which codes for the RNA polymerase sigma factor uF) andby a mutation in the spoIIIE locus (Table 3). In contrast toresults with other forespore-specific genes, gpr-lacZ expres-sion was decreased only -60% by the spoIIIA53 mutationand only 40 to 70% by a deletion in the spoIIIG locus (Table3), which codes for the forespore-specific sigma factor a' (8,26). Examination of the kinetics of gpr-lacZ expression inthe spoIllG mutant indicated that the initial synthesis of,-galactosidase was relatively little affected, although laterexpression was greatly reduced (Fig. 6). This experimentwas repeated three times with similar results. However, themaximum level of P-galactosidase attained in the spoIllGmutant varied between 30 and 60% of wild-type levels (datanot shown).

Transcription start site on the gpr gene in vivo and in vitro.

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TGTTTGI I I I TGTACGIIMTCGCTGATTMTAATGMCATGTGTTTCACCTCCTMGATACMCCTGTAGCACAGTGTCTTMGGTTM100

ATCTTCTTCACAATAGAACAAATTGTATTCTATCAACACACCMAGATTUCMTATATGTAGTATIIrMCATTG GTTCTCM200

TrAW,ATGATAlTCAGGC CCTGT ATG AAA MG A3T GA3 CTGMet Lys Lys Ser Gau Lu

GAT GrTG MT CAGAsp Val AMn Gh

MT CAG C OCTAsn Ga Gan Ala

M G ATT AAAGly Gly le Lys

AAA GAA CCLys Gh Gly Arg

CAG GG AAG GMeGin Gau Lys Val

ATC TCA AAA GATlle Ser Lys Asp

OG CTTG OOAla Lau Gly Pro

COG GAA AAC GTAPro Glu A n Val

ATA ACA G ATClIe Thr Gly lle

GTCVal

ATAlie

AAA

Lys

ACAThr

AACAsn

ACTThr

CTTLU

TTALOU

AACAsn

CAC

TAT TTGTyr Leu

GrM CoVal Pro

400ATC CtZlIe Arg

TAT TTATyr Lou

TCT OCTSer Ala

GOG A3CAla Ser

ATG GCAMet Ala

700CAG GAGan G

GAA ACAGlu Thr

ATC GC ATT GATBe Ala Nb AMp

TOG GAT ACA GSer Asp Thr ely

GA AM CTT GAsp Thr Leu Gly

1000ATT GC AC Clie Ala Ser Mp

AM CCA TCA AGAArg Pro Ser Arg1100GMA GA: OAT CTTGa Asp Asp Lea

CAA GM OAT GAAGln Gu Asp Gu

ATG GTT GAA GMet Val Thr Pro

13003 TTA MC ACA

Gly Leu Asn Thr

ATA C0Ole Arg

ACA AAAThr Lys

ACA GTTThr Val

AC_ CTTThr Lea

GTC UTVal Phe

TGT TTGCy. Lou

GTA GAAVal clu

GlGT TACGly Tyr

AGT AsCSer Asp

GOAla

ATTne

Val

Thr

TOGSer

OCCPro

AAA

Lys

GATLys

GMAla

TTGLUC

CACHls

OOmPro

GMVal

CTTLou

GATAsp

COGArg

TCAGau

CUT

AM OAT TTGThr Asp Lau

G ATT AAAGU le Lys

OAT err ACAAsp Val Thr

GAA GCA GAGGlu Ala GinZG Me GMGM3 GOO GAAAla Gau Gau

ATC Gllle Val Gly

AAT CT TTGUAMn Lau Leu

M3 ccT GTCArg Pro Val

ATT ATT AAAbe Be Lys

GMAla

00CPro

GMGVal

Asp

GTTVal

CAAGin

CGGiSn

TTTVal

CATHb

GMAla

Gly

ATT

TACTyr

COO

Pro

MGLys

CTCIJU

ATCAsp

GM

Gu

GA1Ala

ey

MGLys

Gly

TTC

CTC

GrAVal

prSer

ely

0 G

Arg Ala900

TCT MSer Gly

GCT ATCAla Be

ATA TTANe Lou

Ala ely

Gin Arg1200

ATT C.tCBe Hils

GAC GATSer

AAA GMeLys Val

GM GAA AM MAGVal Gau Thr Lys

TTT ATT G AAAPhF NeGU Lys

GM G: GA GGu Gly Ala Ga

ATC A GAG MTNe Arg Gau Aen

TCASer

Gly

ACAThr

GcrAla

GMVal

GMeVal

GrVal

Gly

AMALys

ATGMet

CMGn

GAG

ACA

TCASer1400

GCT UTAla Phe

MC TGAen Trp

Arg His

m GCPFl Ala

ATT GA

GAAGu

G3Gly

GTTVal

CAT

ADOThr

TOOSer

GrGVal

COOGAsp

CMGn

Arg

MTAMn

COO

Pro

Ph

m

TTC

CTTCMU

AAGAp

GAAGka

CTT

MCAen

TT<3LouUe

Pro

CMGn

VGerCVal

AAALys

ACAThr

Gly

Gly

CTALa

TOSer

ATGMet

MCAen

GMGu

GAGGaU

CTTLei

OATAsp

GAAGaU

GMeVal

mPhe

ely

TOOSer

MCAsn

Arg

GTAVal

Arg

AAALys

Gly

Pro

Ala

AAALyse

GCA ATGAla Met

CGFT OTArg Asp

TOO GGSer Gly

500TCO GAASer Gu

GOCOAla

CACHis

AAALys

ATGmet

MC CTU MTMn Leu Aen

ACA COT OATThr Pro Asp

MeA CTT C/ALys Leu Gn

GTA ATG G3Val Met Gly

MG CCT OATLys Pro Asp

ACA

Thr

AALys

GTVol

GMAGa

Lys

ATT

TTA

MTAMn

ey

AC ATTThr N.

OAT TTAAsp La

GAT GCAsp Ala

ATG AAGMet Lys

AAA GrGLys Val

eITC GDVal Gly

Goa C/Cely Ils

GTC TTAV.1 Ula

TOC TACSer Tyr

Asn His**I

FIG. 3. Nucleotide sequence of the B. subtilis gpr gene. Nucleotides are numbered every 100 residues, with the first number over the

designated nucleotide. The two singly underlined residues denote the -35 and -10 regions, and the dot over the nucleotide at position 226

denotes the in vivo transcription start site as determined by primer extension analysis. This residue was also the site of initiation of in vitro

transcription by EaF and Ea'. The doubly underlined residues show good complementarity to the 3' end of 16S rRNA and undoubtedlycomprise a ribosome binding site. The horizontal arrow at amino acid 19 denotes the putative amino terminus of the mature protease by

homology with that from B. megaterium. The vertical arrows denote the EcoRI and HincIl sites used to isolate a fragment for construction

of the gpr-lacZ fusion plasmid, pMS16. The singly overlined residues from residues 258 and 277 and 279 to 308 denote the regions used to

construct the complementary 20- and 30-residue oligonucleotides for primer extension analysis.

Since the gpr gene is expressed only during sporulation and

only in the developing forespore, we determined the 5' endof the B. subtilis gpr transcript in sporulating cells by primerextension analysis, using both 20-residue (Fig. 7) and 30-residue (data not shown) primers. With both primers, this

work localized the 5' end of gpr mRNA to an A residue justupstream of the ribosome binding site (Fig. 3). Examinationof the sequences centered 10 and 35 residues upstream ofthis point indicated significant homologies between these-10 and -35 sequences with the consensus sequences for

TTTPh.

CAAGin

Ser

eTGVal

GATAsp

CTC

ACCThr

AACAsn

GaAla

MT l'AAAC A A-rA AI' *-

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BS: M K K S E L D V N Y L I R T D L A V E T K E A M A N 0 A V PBM: .E* - * - - L S * - S V * - - - - - A * D I A L EN * P K

- - TKE I K G F I E K E R D H G G I K I R T V D V T KE G A EN N O S * * * * V * V * K E E * V * - S M * E I* E . . . .

L S G K K E G R Y L T L E A Q G I R E N D S E MQ EK VS - A VA I * * * K * * * V * * * S V * * * * C * T * K ^ * E A M E E -

F A E E F S A F L E N L N I S K D A S C L I V G L GN WN V T P* * K * L N F * I K S * * * P D * * * * V * * ** L S * * -

D A L G P M A V E N L L V T R H L F K L P E N V 0 E G Y R P V* * * * * K * * D * * - I * - * * - E * * * * S * - D * F * - -

S A F A P G V M G I T G I E T S D I I K G V I E Q SK PD F V I* - I V * - - - - M-* * * * * * * - - F * - V K K V N **- I -

A I D A L A A R A V E R V N T T I Q I S D T G I H PG SG V G N.--- - SI.*** * *S * *- *

K R K D L S K D T L G V P V I A I G V P T V V D A VT IA S D T* - - E I * Y E * * - I * - * - - - I * - - * - * - S * T * * -

V D Y I L K H F G R E M K D N - R P S R S L V P A G M T F G - KI * F * - - - - - - - - - E CQ G K * * K * - L * S * * -*- E -

K K V L T E D D L P D Q K Q R Q S F L G I V G T L Q E D E K R O* - - * - * - * - * N E E * * - T Y * - M I * * * P D E * * - R

L I H E V L S P L G H N L M V T P K E V D S F I D DMMAN V L A. . . . .* A * * * * * * * *M* *E* * * *M* V

N G L N T A L H E K V S Q E N K G S Y N HG * * * A * * * H E * D * * * F * A * T -

FIG. 4. Comparison of the amino acid sequences of the B. megaterium (BM) and B. subtilis (BS) spore proteases. The amino acidsequences were taken from Fig. 2 and 3 and are given in the single-letter code. Residues with an asterisk in the B. megaterium sequence areidentical to those in the B. subtilis protein. Dashes indicate positions where gaps have been introduced to give maximum sequence alignment.The vertical arrow denotes a site of processing to give mature protease as determined directly in B. megaterium and inferred in B. subtilis.

10Ec0 80

t_ 6

ax 4-

02)

3CD

100 >

80 ,,o

60 03

40

-M

20 >

* t.0 2 4 6 8 10 12 24

Time in HoursFIG. 5. Kinetics of synthesis of glucose dehydrogenase and

gpr-lacZ-driven P-galactosidase during sporulation of B. subtilis.Strain PS1029 was grown and sporulated, and glucose dehydroge-nase and ,B-galactosidase were assayed after direct lysozyme extrac-tion as described in Materials and Methods. The maximum P-galac-tosidase activity found during sporulation of the gpr-lacZ fusion was-55 Miller units. Symbols: O, optical density; 0, P-galactosidase;0, glucose dehydrogenase. The numbered arrows denote the timesof isolation of cells for extraction of RNA for primer extensionanalyses (see Fig. 7).

RNA polymerase containing either UF or a' (16; see Discus-sion). Consequently, we tested gpr transcription in vitrowith both EurF and EaG (Fig. 8). Strikingly, both EaF andEa' initiated transcription on a gpr template at approxi-mately the same point as the in vivo start site (Fig. 3), sinceboth gave a 181-nucleotide runoff transcript with the EcoRI-cut template (Fig. 8) as well as a 165-nucleotide transcriptwith KpnI-cut gpr template (data not shown). When theefficiency of EaF and EUG on a gpr template was measured

TABLE 3. Levels of gpr-lacZ and gdh expression in variousasporogenous mutant strainsa

Maximum level ofAsporogenous expression relative

mutation Strain to wild typegpr-lacZ gdh

spoIIACI PS1190 <5 <3spoIIIA53 PS1111 40 <3spoIIIB2 PS1087 89 94spoIIIC94 PS1085 88 103spoIIID83 PS1086 104 89spoIIIE36 PS1088 <5 <3spoIIIGAI PS1089 30-60b <3

a Cells were sporulated in 2x SG medium and assayed after direct lyso-zyme extaction as described in Materials and Methods. Values presented arethe maximum values found relative to the wild-type values, which are set at100%. For P-galactosidase assays, the low level of activity in strains lackingthe lacZ fusion has been subtracted.

b Varied significantly from experiment to experiment.

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8

Ec

O 6

4,

in 4c

0. 2uZ;O

-

0

0 2 4 6Time in Hours

1000

80 )x m

3 N

60 3 3D

cn 0

40 q

<

>20 'ct

r.I l<

8 10

FIG. 6. Kinetics of expression of a gpr-lacZ fusion in a wild-typeand a spoIIIG mutant strain. Strains PS1029 (wild type) and PS1089(spoIIIGAI) were grown and sporulated, and P-galactosidase was

assayed after direct lysozyme extraction. Symbols: O, opticaldensity, strain PS1029; *, optical density, strain PS1089; 0, n-ga-

lactosidase, strain PS1029; *, ,-galactosidase, strain PS1089. Val-ues for ,B-galactosidase in parallel cultures of parental strains lackinglacZ fusions have been subtracted from the data. These values were

less than 4 Miller units.

in vitro relative to their efficiency on the sspE gene, a knownEcrG-dependent template (26), EUF was found to be 10 timesas efficient as EuG on a gpr template relative to an sspEtemplate (Fig. 8 and data not shown). Note that the apparentweak absolute level of sspE transcription by E(JF and EurGwas due to the use of [ot-32P]UTP at a low concentration as

the labeling nucleotide and the presence of a U residue as thesecond nucleotide in sspE mRNA (16).Although EuF was more efficient than EuG at gpr rela-

tive to sspE transcription in vitro, a spoIIIG mutation didreduce gpr-lacZ expression significantly. Consequently, we

tested whether induction of uG synthesis in vivo would inturn induce gpr-lacZ expression, as was observed previous-ly with many forespore-specific genes (26). Indeed, induc-tion of uG synthesis in vegetative cells of B. subtilis resultedin substantial induction of gpr-lacZ expression comparedwith uninduced control cultures (Fig. 9). The kinetics ofinduction of gpr-lacZ expression were similar to those ofglucose dehydrogenase, a known crG-dependent gene (15, 22,26).

DISCUSSION

As was suggested earlier on the basis of analysis of thelevel of gpr gene product in isolated forespores, the gpr geneis expressed primarily, if not exclusively, in the forespore (9,10). Analysis of the effects of asporogenous mutations on

gpr-lacZ expression is consistent with gpr expression in theforespore; gpr expression was not affected by mutations(spoIIIB, spoIIIC, and spoIIID) that abolish mother cell butnot forespore gene expression but was abolished by muta-tions (spoIIIA, and spoIIIE) that selectively block foresporegene expression. In this respect the gpr gene behaves muchas do other forespore-specific genes or operons such as gdh,gerA, spoVA, and ssp (3, 22, 26). However, gpr regulationdiffers from that of these other forespore-specific genes intwo respects. First, gpr expression precedes glucose dehy-drogenase synthesis; other known forespore genes are ex-

A A C 2 1

A

G

FIG 7.Pie xesinaayi fth -edofgrmN

made .

A K

T

A~~~~~~~~~~~~~~4"....'.,

prmreteso.Tepieexninprodct wee u o

MteilanMehd(lnsGA,TadC.Tenmed lanes

T

A ~

correspondAto the times in sporulation denoted by the numberedA i F h t d h..ranscriptionstartsite.

EA cant.ethol or f N plmeae. hc

copetl by a pIA.Cuainad .rytei

FiG.7drierexenionanalythsisofainvgthtie5'ends of gpr mRNAlimaesut invivoeRNAvwasioltdfroexrstrionPo02gp-atZtwontimesigrwtnaddpoution(fcsnumbered arrowsint EFig5 and analyzedobyprimera exteniaonsTenupnmroextenso produects wervirunon6)anofthe gprtgene,-usingdtherePioabeoedtprimer asedescfribe inMabteials and B.metaeiuhods(lneo,oTadC)gThenmberedit lanescorrspondu toquthetiesin.spoulaioIdenote by thmoe numberedttanscrprtion startipteo nvtota s eaiet h

prse nprle ihguos eyrgns 2)Scnexprssinoothr fresorespecficgens isaboishd baspolliG(uG)mutaion; gpr expressio is only reduced 3to70%.Since u synthesis itself precedes gdh expressionby~~~~~~~5

only30to 60 mm (8), these~~~~~~~~~~~~~~~gdata coletveyinict ta

trncIbes7gPrmr intevivon Sincesgsroexpressionnisoabolishedrompltelyn byrltin(umeearrowsCuFmtaionFg )and aFasyntesisy

pitself begiensio2 Thbeforiergd expression (14),ct wersuggest tatE5 carrenies out siognificath thpranscptio of gpruening vecivoInssupportal ofd MthisdsuggaestionA,iThasrcntly bheenumfound thateinductiond tof sytheiesis oforuFatinovegetatiedcell ofeBnumbtelesaresuls in hig.Therlvshofionaexpresowdnofe gpr nuleoidZ athandesirnducritionoftart synthesi. 1a ohEu n u eo

nizessa similaraconensitguspomoer sequroensein2vitr (16)ond,texputative 10 ande -35espregiospeofitc pgenes fromlihe bysutoil isndeB'megatheriumishow gcdeagrheem rentsith thi

EcGcnsnsus sequhen oe(Fg 10). IneeENA slymorae efficin

atrgprreousinfcttranscriptioninvtrohnfsE grelatviveo. the

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2 3 4

,.1

FIG. 8. In vitro transcription of the sspE and gpr genes by EaGand ErF. RNA polymerase containing either EcrG or EaF was usedto transcribe linearized sspE (2 ,ug) or gpr (2 ,ug) templates or amixture of each template (1 ,ug each) as described in Materials andMethods. The products were analyzed on a 6.5% sequencing gelwith reference to RNA markers of known size. The sizes given forthe sspE and gpr transcripts were either those known from previouswork (193 nucleotides; sspE) (16) or those estimated with respect toRNA size markers as previously described (16) (gpr). Lanes: 1, sspEwith ECrF; 2, gpr with EaF; 3, gpr and sspE with EUF; 4, sspE withErG; 5, gpr with EoGr; 6, gpr and sspE with EGG.

activity of these holoenzymes on sspE, a known cG-depen-dent gene (26). The reason(s) for having the gpr genetranscribed in vivo by two similar forms ofRNA polymeraseis not clear. However, its transcription by EurF early in gprexpression suggests that this form of RNA polymeraseholoenzyme may have a role in forespore-specific geneexpression, although we have not yet shown that uF-driven

Eo0'IO

U)0.

c-2

4

-35

Consensus {F/ G:-10

TGAATA 17-18nt CATACTA

BM : TGCATG 18nt GACAATA

BS gQ: AG CAT. 17nt AACAATAFIG. 10. Comparison of promoter sequences of gpr genes and

other cuF- and (uG-dependent genes. Promoter sequences were takenfrom Fig. 2 and 3 and reference 16.

synthesis of ,-galactosidase from a gpr-lacZ fusion is con-fined to the forespore. Similarly, we have not yet proven thatsignificant .F.-dependent transcription of gpr takes place inwild-type cells, although this clearly takes place in a spoIIIGmutant. The fact that gpr expression does not begin untilwell after aF synthesis indicates that the activity or speci-ficity of (F must somehow be regulated early in sporulation.While this regulation is not yet completely understood, itappears to involve the product of one or both of the othertwo cistrons of the spolA operon (19).Although the transcription start site for the gpr gene has

been determined, its transcription stop site is unclear. Noobvious rho-independent terminator is located closely down-stream of the gpr gene, but no other significant open readingframe follows the coding sequence either. Since strainPS1029 grows and sporulates normally, despite havingpJF751 sequences interupting the gpr transcription unit,there is no gene essential for sporulation which is down-stream of, and cotranscribed with, gpr. Perhaps Northern(RNA) blot analysis of the size of gpr mRNA would providesome information on possible transcription termination sitesfor gpr.

,100"<3

80

60 3~4a)360 33

3

20 ,c-

'OC

C:

1 2 3 4 1 2 3 4Time in hours

FIG. 9. Induction of glucose dehydrogenase and gpr expression by induction of cyG synthesis. Strain PS1084 was grown at 37°C in 2x YTmedium. At an optical density of 0.3, the culture was split in half; one half received no addition (a), and isopropyl-P-D-galactopyranoside(IPTG) was added to 0.5 mM to the other (b). At various times, samples (1 ml) were harvested, extracted with lysozyme, and assayed for1-galactosidase and glucose dehydrogenase. The values presented are enzyme activities per milliliter of culture not corrected for the opticaldensity of the culture. The maximum specific activity of ,B-galactosidase reached in this experiment was 40 Miller units. Symbols: O, opticaldensity; *, p-galactosidase; 0, glucose dehydrogenase.

J. BACTERIOL.

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While regulation of gpr expression presents some fea-tures not previously seen with other forespore-specificgenes, of possibly greater interest is the nature and regula-tion of spore protease processing. Previous work has indi-cated that the initial product of the gpr gene, P46, is proc-essed at least twice to lower-molecular-weight forms andthat the timing of these processing steps (i) is regulated and(ii) in turn regulates the activity of the protease (7, 10). Theidentification of at least one processing site has been made inthis work, since the mature B. megaterium P40 lacks the 18amino-terminal residues coded for in the gene. This region ofsequence is highly conserved in B. megaterium and B.subtilis and removes -2 kDa in molecular size from thecomplete protein. However, it is not clear whether thiscleavage site represents P46-*P41 or P4!-P40 processing, noris it clear what enzyme(s) is carrying out this cleavage.However, the amino acid sequence around this processingsite exhibits no homology with the SASP sequence recog-nized and cleaved by the spore protease, thus indicating thatthe spore protease does not process itself, as was suggestedearlier on other grounds (9, 17, 20). Recently, we have beenable to use the cloned B. megaterium gpr gene to overpro-duce P46 in E. coli (26a). This should provide a substrate foridentification and characterization of enzymes catalyzingspore protease processing. These processing reactions takeplace at key periods of development, i.e., very late insporulation as the spore becomes dormant (P46-'P41) andvery early in spore germination (P41-3P40). It is possible thatanalysis of the processing enzymes, in particular how theyare regulated, will provide insight into physiological changestaking place with the spore during these periods in develop-ment.

ACKNOWLEDGMENTS

We are grateful to Barbara Setlow for construction of the B.megaterium DNA library in Xgtll and to Susan Goldrick forassistance with purification of the spore protease. Ruth Schmidt alsowas extremely helpful in the analysis of expression of the gpr-lacZfusion in spolIIG mutants.

This work was supported by grants from the National Institutes ofHealth (GM19698) and the Army Research Office.

REFERENCES

1. Cabrera-Martinez, R. M., J. M. Mason, B. Setlow, W. M.Waites, and P. Setlow. 1989. Purification and amino acid se-quence of two small, acid-soluble proteins from Clostridiumbifermentans spores. FEMS Lett. 61:139-144.

2. Connors, M. J., and P. Setlow. 1985. Cloning of a small,acid-soluble spore protein gene from Bacillus subtilis and deter-mination of its complete nucleotide sequence. J. Bacteriol.161:333-339.

3. Feavers, I. M., J. Foulkes, B. Setlow, D. Sun, W. Nicholson, P.Setlow, and A. Moir. 1990. The regulation of transcription of thegerA spore germination operon of Bacillus subtilis. Mol. Micro-biol. 4:275-282.

4. Ferrari, E., S. M. H. Howard, and J. Hoch. 1985. Effect ofsporulation mutations on subtilisin expression, assayed using asubtilisin-p-galactosidase gene fusion, p. 180-184. In J. A.Hoch and P. Setlow (ed.), Molecular biology of microbialdifferentiation. American Society for Microbiology, Washing-ton, D.C.

5. Fujita, V., R. Ramaley, and E. Freese. 1977. Location andproperties of glucose dehydrogenase in sporulating cells andspores of Bacillus subtilis. J. Bacteriol. 132:282-293.

6. Hackett, R. H., B. Setlow, and P. Setlow. 1986. Cloning andnucleotide sequence of the Bacillus megaterium gene coding

for small, acid-soluble spore protein B. J. Bacteriol. 168:1023-1025.

7. Hackett, R. H., and P. Setlow. 1983. Determination of theenzymatic activity of the precursor forms of the Bacillus mega-terium spore protease. J. Bacteriol. 153:375-378.

8. Karmazyn-Campeili, C., C. Bonamy, B. Savelli, and P. Stragier.1989. Tandem genes encoding sigma factors for consecutivesteps of development in Bacillus subtilis. Genes Dev. 3:150-157.

9. Loshon, C. A., and P. Setlow. 1982. Bacillus megaterium sporeprotease: purification, radioimmunoassay, and analysis of anti-gen level and localization during growth, sporulation, and sporegermination. J. Bacteriol. 150:303-311.

10. Loshon, C. A., B. M. Swerdlow, and P. Setlow. 1982. Bacillusmegaterium spore protease: synthesis and processing of precur-sor forms during sporulation and germination. J. Biol. Chem.257:10838-10845.

11. Mason, J. M., R. H. Hackett, and P. Setlow. 1988. Regulation ofexpression of genes coding for small, acid-soluble spore pro-teins of Bacillus subtilis: studies using lacZ gene fusion. J.Bacteriol. 170:239-244.

12. Mason, J. M., and P. Setlow. 1987. Different small, acid-solubleproteins of the cx/, type have interchangeable roles in the heatand UV radiation resistance of Bacillus subtilis spores. J.Bacteriol. 169:3633-3637.

13. Maxam, A. M., and W. Gilbert. 1980. Sequencing end-labelledDNA with base specific cleavages. Methods Enzymol. 65:499-560.

14. Moran, C. P., Jr. 1989. Sigma factors and regulation of tran-scription, p. 167-184, In I. Smith, R. A. Slepecky, and P. Setlow(ed.), Regulation of procaryotic development. American Soci-ety for Microbiology, Washington, D.C.

15. Nakatani, Y., W. L. Nicholson, K.-D. Nietzke, P. Setlow, and E.Freese. 1989. Sigma-G RNA polymerase controls foresporespecific expression of the glucose dehydrogenase operon inBacillus subtilis. Nucleic Acids Res. 17:999-1017.

16. Nicholson, W. L., D. Sun, B. Setlow, and P. Setlow. 1989.Promoter specificity of ac-containing RNA polymerase fromsporulating cells of Bacillus subtilis: identification of agroup of forespore-specific promoters. J. Bacteriol. 171:2708-2718.

17. Postemsky, C. J., S. S. Dignam, and P. Setlow. 1978. Isolationand characterization of Bacillus megaterium mutants contain-ing decreased levels of spore protease. J. Bacteriol. 135:841-850.

18. Sanger, F., S. Nicklen, and A. R. Coulson. 1977. DNA sequenc-ing with chain-terminating inhibitors. Proc. Natl. Acad. Sci.USA 74:5463-5467.

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21. Setlow, P. 1988. Small, acid-soluble spore proteins of Bacillusspecies: structure, synthesis, genetics, function, and their deg-radation. Annu. Rev. Microbiol. 42:319-338.

22. Setlow, P. 1989. Forespore-specific genes of Bacillus subtilis:function and regulation of expression, p. 211-221. In I. Smith,R. A. Slepecky, and P. Setlow (ed.), Regulation of procaryoticdevelopment. American Society for microbiology, Washington,D.C.

23. Setlow, P., C. Gerard, and J. Ozols. 1980. The amino acidsequence specificity of a protease from spores of Bacillusmegaterium. J. Biol. Chem. 255:3624-3628.

24. Sloma, A., G. A. Rufo, Jr., C. F. Rudolph, B. J. Sullivan, K. A.Theriault, and J. Pero. 1990. Bacillopeptidase F of Bacillussubtilis: purification of the protein and cloning of the gene. J.Bacteriol. 172:1470-1477.

25. Southern, E. M. 1975. Detection of specific sequences amongDNA fragments separated by gel electrophoresis. J. Mol. Biol.98:503-517.

26. Sun, D., P. Stragier, and P. Setlow. 1989. Identification of a new

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sigma factor which allows RNA polymerase to transcribe thesspE gene and other forespore specific genes during sporulationof Bacillus subtilis. Genes Dev. 3:141-149.

26a.Sussman, M. D., and P. Setlow. Unpublished data.27. Sussman, M. D., P. S. Vary, C. Hartman, and P. Setlow. 1988.

Integration and mapping of Bacillus megaterium genes whichcode for small, acid-soluble spore proteins and their protease. J.

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neutral protease gene of Bacillus subtilis and the use of thecloned gene to create an in vitro-derived deletion mutation. J.Bacteriol. 160:15-21.

29. Young, R. A., and R. W. Davis. 1983. Yeast RNA polymerase IIgenes: isolation with antibody probes. Science 222:778-782.


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Nucleotide Sequence, Regulation Bacillus Protease ... · DNA from B. megaterium QMB1551wasfirst treated withEcoRI methyl-ase, and the methylated DNA was digested to various degrees

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