Proc. Nat!. Acad. Sci. USAVol. 85, pp. 4166-4170, June 1988Biochemistry

Isolation of a Bacillus thuringiensis RNA polymerase capable oftranscribing crystal protein genes

(sporulation-specific RNA polymerase/or factor/sporulation/transcriptional regulation/in vitro transcription)

KELLY L. BROWN AND H. R. WHITELEY*Department of Microbiology, University of Washington, Seattle, WA 98195

Communicated by Earl W. Davie, February 9, 1988 (received for review October 16, 1987)

ABSTRACT We report the isolation of an RNA polymer-ase from sporulating cells of Bacilus thuringiensis subsp.kurstaki HD-4-Dipel that directs transcription from the pro-moter region of an insecticidal crystal protein gene. The corecomponents of this RNA polymerase are associated with apolypeptide that has an apparent mass of35 kDa. Neither RNApolymerase holoenzyme isolated from vegetative B. thu-ringiensis, nor the core derived from this enzyme, is capable oftranscribing from the crystal protein gene promoter region; theaddition of gel-purifiled 35-kDa polypeptide to the core recon-stitutes the specific transcribing capability. The reconstitutedenzyme does not direct transcription from the promoters forthe ck or spoVG genes of Bacilus subtdis; however, this formof RNA polymerase does direct transcription from a promoterfor the 27-kDa crystal protein of B. thuringiensis subsp.israelensis and from a promoter for a 29-kDa polypeptidepresent in cuboidal crystals of B. thuringiensis subsp. kurstakiHD-1. We propose a tentative consensus sequence based on thealignment of the three B. thuringiensis promoters. This con-sensbs sequence is different from consensus sequences reportedfor promoters recognized by enzymes containing other (subunits, suggesting that the 35-kDa polypeptide is an unusualr subunit.

Bacillus thuringiensis is extraordinary among the endospore-fbrming bacteria because of its ability to synthesize aninsecticidal protein during sporulation. This protein is pro-duced abundantly, ultimately accumulating as a parasporalcrystalline inclusion. A number of crystal protein genes,largely plasmid-bome, have been cloned and their DNAsequences have been determined (reviewed in ref. 1). Thegenes encoding proteins lethal to Lepidopteran larvae aretranscribed from dual overlapping promoters. Studies of onesuch crystal protein gene cloned from B. thuringiensis subsp.kurstaki HD-1-Dipel showed that the downstream promoterBt I is used during early to midsporulation, and the upstreampromoter Bt II is activated at midsporulation (2). The -10and - 35 regions ofeach ofthese promoters are different fromthe -10 and - 35 consensus sequence that is recognized bythe predominant vegetative RNA polymerases of Bacillussubtilis and B. thuringiensis.

Extensive studies of sporulation in B. subtilis (reviewed inref. 3) indicate that regulation of this complex processinvolves the temporal control of several gene families at thelevel of transcription initiation. This regulation of transcrip-tion is achieved, at least in part, by the association of differento- subunits with the core components of RNA polymerase.Polymerases containing different ov subunits have been iso-lated from vegetative and sporulating cells of B. subtilis(reviewed in refs. 3-5; see also ref. 6). It is likely thatalternative oa subunits also play a role in the regulation of

transcription during vegetative growth and sporulation ofother bacilli.We have isolated an RNA polymerase from B. thu-

ringiensis subsp. kurstaki HD-1-Dipel that is capable ofdirecting transcription in vitro from the Bt I promoter for acrystal protein gene encoding a 133-kDa peptide and frompromoters for two additional crystal protein genes: oneencoding a 29-kDa peptide present in cuboidal crystals of B.thuringiensis subsp. kurstaki HD-1 and the other encoding a27-kDa crystal peptide in B. thuringiensis subsp. israelensis.This RNA polymerase contains a ov subunit (35 kDa) that isdifferent from the major or subunit present in vegetative cellsof B. thuringiensis (61 kDa; ref. 7) and has a differentspecificity from those isolated from B. subtilis (3-6).

MATERIALS AND METHODSPurification of RNA Polymerase. B. thuringiensis subsp.

kurstaki HD-1-Dipel (a derivative of strain HD-1) was grownto the stage showing optimum in vivo transcription from Bt I(2)-i.e., stage III-IV of sporulation (phase dark prespores)in M/2 medium (8) containing 0.1% glucose. Cell growth wasmonitored both spectrophotometrically and by phase-contrast microscopy. Approximately 100 g of cells wasdisrupted by two passages through a French pressure cell; theresulting crude extract was subjected to phase partitioningand chromatography on Bio-Gel A 0.5m as described (9).Bio-Gel fractions containing the highest RNA polymeraseactivities were pooled and fractionated by chromatographyon a DNA-cellulose column with a linear salt gradient [0.4-1.5 M NaCl in 20 mM Tris buffer (pH 7.9)]. Transcriptionallyactive fractions from this column (eluting at 0.6-0.8 M NaCl)were pooled and loaded onto a BioRex-70 column (10), andproteins were eluted with a linear salt gradient [0.1-0.6 MNaCl in 10 mM Tris (pH 7.9)]. Trace amounts of vegetativepolymerase were eluted by 0.2 M NaCl, whereas Bt I-specificactivity began to elute at 0.35 M. The same procedure wasused to purify RNA polymerase from early stationary-phaseB. subtilis and midlogarithmic phase B. thuringiensis (thelatter were eluted from the BioRex-70 column at 0.2M NaCI).None of the transcriptionally active polymerases obtainedfrom the latter column contained the 8 subunit. Fractionswere analyzed by electrophoresis on NaDodSO4/polyacryl-amide gels (11) and were stained with Coomassie brilliantblue.RNA Synthesis. The activities of fractions obtained prior to

chromatography on DNA-cellulose were measured as de-scribed (9) by following the incorporation of [3H]UTP intotrichloroacetic acid-precipitable material with B. subtilisphage SP82 DNA as template. All fractions were assayed forthe ability to use a crystal protein gene promoter in reactionmixtures containing 2 gg of template DNA, 6.0% (vol/vol)glycerol, 250 mM NaCI, 10 mM MgC12, 40 mM Tris HCl (pH

*To whom reprint requests should be addressed.

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7.9), and 1 mM each ATP, GTP, and UTP, in a total volumeof 18 ,.d. After incubation at 37°C for 30 min, 2 ,ul of a solutioncontaining [32P]CTP (0.5 ,uCi; 1 Ci = 37 GBq) and 0.1 mMunlabeled CTP was added and the reaction mixtures wereincubated an additional 5 min at 37°C. Specific transcriptioncould also be detected at 22°C, provided the concentration ofNaCl in the reaction mixtures was decreased to 150 mM.Vegetative RNA polymerase activity was assayed in thesame way except the reaction mixtures contained either SP82DNA or a template containing a crystal protein gene and thesalt concentration was 50 mM NaCl. After adding 10 ,ul of"stop" mixture (10 M urea/0.02% bromophenol blue), 20 ,ulof the reaction mixture was electrophoresed on a 4% or 6%polyacrylamide slab gel containing 7 M urea and TBE buffer(89 mM Tris/89 mM sodium borate/2.5 mM EDTA, pH 8.5).The gels were dried and transcripts were detected by auto-radiography.

Reconstitution of Specific Transcriptional Activity. The ofsubunit was removed (12) from RNA polymerase isolatedfrom vegetative cells of strain HD-1-Dipel; the core prepa-rations contained the 8 polypeptide (9). To obtain the 35-kDapolypeptide, high specific activity fractions from the BioRex-70 column were electrophoresed on a preparative NaDod-S04/10% polyacrylamide gel; 3-mm regions of the gel wereexcised, eluted, and renatured according to Hager andBurgess (13). Renatured materials were added to the core-8preparation and incubated first at 22°C for 15 min, then at37°C for 15 min, and finally at 22°C for 30 min with thecomponents of the in vitro RNA synthesis reaction mixtureas described above except the glycerol concentration wasincreased to 15%. Specific transcription was then assayed asindicated above.Template DNAs. The template for measuring transcription

of the crystal protein gene from strain HD-1-Dipel wasobtained by cloning a 554-base-pair (bp) EcoRI/BamHIfragment of DNA from plasmid pPLZ422 (14) into pUC13(15) to generate plasmid pKLB1. The cloned fragment in-cluded sequences 429 bp upstream from the start site for theBt I promoter and 117 bp downstream extending 48 bp intothe coding region of the crystal protein gene; for in vitroassays, this plasmid was linearized by digestion with eitherSal I or HindIII. Transcription of the 29-kDa cuboidal crystalprotein gene (16) was assayed with a 1.0-kilobase fragmentcontaining the promoter region cloned into pUC8 (15) pro-ducing pWRW41, which was then linearized with either SalI or HindIII. The template for transcription of the 27-kDagene of B. thuringiensis subsp. israelensis was a 2.3-kilobasePvu II/BamHI fragment from plasmid pKM1 (17) containingthe promoter region for the 27-kDa polypeptide and 96 bp ofthe coding region cloned into pUC119 (18), producingpHES57; this template was linearized by digestion with eitherBamHI or HindIII. Transcription of the B. subtilis sporula-tion genes ctc and spoVG was measured with plasmidspUC8-31 (19) and pCB1291 (20) provided by Charles P.Moran, Jr. (Emory University). Plasmid pUC8-31 was lin-earized by digestion with HindIII and pCB1291 was digestedwith EcoRI. Transcription of pUC8-31 and pCB1291 wasassayed by described reaction conditions (19, 20) and our

conditions for measuring the activity of E-oT-35 in the pres-ence of 50, 100, and 150 mM NaCl.

Transcription Mapping. An oligonucleotide primer com-plementary to nucleotides between positions 538 and 552 ofthe DNA sequence encoding the 133-kDa peptide gene (seeref. 2) was 5'-end-labeled with polynucleotide kinase and[y-32P]ATP (21), annealed (670C for 5 min followed by 370Cfor 15 min) to RNAs synthesized either in vivo or in vitro, andextended with Moloney murine leukemia virus reverse tran-scriptase under conditions recommended by the manufac-turer (Bethesda Research Laboratories). RNA was purifiedfrom sporulating cells of B. thuringiensis as described (2). Invitro RNA was synthesized by using BioRex-70-purifiedpolymerase as described above except that 1 mM CTP wassubstituted for the [32P]CTP mixture. Base-specific markersfor determining the lengths of the extended primers wereobtained by sequencing the promoter region of the crystalprotein gene by using the dideoxy method as described earlierwith the oligonucleotide described above (2). The DNAsequencing reactions and the extended primers from theRNA were analyzed by electrophoresis on an 8% polyacryl-amide/7 M urea gel buffered with TBE.

Miscellaneous Methods. Published methods were used forthe isolation of plasmid DNA and digestion with restrictionenzymes (21). Protein concentration was measured by themethod of Bradford (22) with bovine immunoglobulin as astandard.

RESULTS

Isolation of an RNA Polymerase Capable of Transcribingfrom a Crystal Protein Gene Promoter. RNA polymerase waspurified from B. thuringiensis subsp. kurstaki HD-1-Dipelharvested at stage III-IV of sporulation by phase partitioningfollowed by successive chromatography on Bio-Gel, DNA-cellulose, and BioRex-70 columns. Transcription from thepromoter region of the gene encoding the 133-kDa crystalprotein was determined by electrophoresis of RNAs pro-duced in vitro from a template containing the Bt I and Bt IIpromoters. Table 1 indicates the sizes of RNAs expected fortranscription initiating from either of these promoters whenpKLB1 is digested with either Sal I or HindIII.

Fig. 1 presents the results of analyses of polypeptidecomposition (Fig. LA) and in vitro RNA synthesis (Fig. 1B) offractions eluted from a BioRex-70 column. The size of thetranscript (141 bp) detected in reaction mixtures containingfractions 5-19 (Fig. 1B) was in reasonably good agreementwith the size expected for transcription from Bt I. Fig. LA

shows that fractions 7-15 contained readily detectableamounts ofthe core components ofRNA polymerase and twoadditional polypeptides of 95 and 35 kDa; fractions 17 and 19lacked the 95-kDa peptide. None of the fractions containedthe vegetative B. thuringiensis oc subunit. Comparison of Fig.1 A and B indicates that transcription from the Bt I promoterwas correlated with the presence of the 35-kDa polypeptide.The polypeptide composition ofanother preparation ofpolym-erase capable of transcribing from the Bt I promoter andlacking the 95-kDa polypeptide is shown in Fig. 2 (lane 2). In

Table 1. Summary of crystal protein gene promoter templates and lengths of expectedrun-off transcripts

Run-off transcript length in bases

Template when template digested withCrystal protein plasmid Promoter BamHI Sal I HindIII

Bipyramidal (133 kDa) pKLB1 Bt I 128 143Bt II 144 159

Cuboidal (29 kDa) pWRW41 Pcb -729 -743Bti (27 kDa) pHES57 PBti 367 397

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4168 Biochemistry: Brown and Whiteley

A

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4"3

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20

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B-2c _ Cle LO I' )^-C M0o C') LO o'- CN bp

506,* 396S 344- 298

_ 220-221

ift *t * 4,do *D w.0 154

75

FIG. 1. Polypeptide composition and transcriptional activity ofB. thuringiensis RNA polymerase fractions eluted from a BioRex-70column. (A) Photograph of a NaDodSO4/polyacrylamide gel stainedwith Coomassie blue. Lane M, molecular mass markers with sizesindicated in kDa (in descending order: phosphorylase b, bovineserum albumin, ovalbumin, carbonic anhydrase, soybean trypsininhibitor, and a-lactalbumin). (B) Autoradiogram of an RNA geldisplaying run-off transcripts produced by in vitro transcription froma template containing promoters for the crystal protein gene (pKLB1linearized with HindIll). Lane M, HinfI fragments of pBR322 withsizes indicated in bp. Numbers above lanes indicate fraction num-

bers.

accord with current terminology, this polymerase is referredto as E-cr-35. In contradiction to previous reports (23, 24), noobvious proteolytic modifications of the core polypeptidescould be detected in stained gels when E-a-35 was comparedto the vegetative core peptides (Fig. 2).

Crystal Protein Gene mRNAs Synthesized in Vivo and inVitro Have the Same 5' Termini. The 5' terminus of RNAsynthesized in vitro by E-oa-35 was compared to the 5'terminus of crystal protein-specific mRNA purified fromsporulating cells ofB. thuringiensis by primer extension usingreverse transcriptase. Fig. 3 (lanes 1, 2, 7, and 8) shows thatthe lengths of the extended primers for the in vitro- and invivo-synthesized RNAs were identical. When allowance ismade for the differences in the phosphorylation of theprimers (the primer used for sequence determination was nottreated with kinase prior to use), it is seen that the in vivo andin vitro RNAs were both initiated from the G residueindicated in Fig. SB. Our previous S1 nuclease mapping data(2) indicated that transcription was initiated from the two Tresidues immediately preceding the G. This discrepancy isprobably due to the use of different methods for determiningtranscriptional start sites in the two studies.

FIG. 2. Polypeptide composition of RNA polymerases purifiedfrom vegetative cells and sporulating cells of B. thuringiensis.Photograph of a NaDodSO4/polyacrylamide gel stained with Coo-massie blue after electrophoresis. Lanes: 1, RNA polymerase (DNAcellulose fraction) purified from vegetative cells (in descending orderthe peptides are A, 145 kDa; (3', 130 kDa; o-, 61 kDa; a, 44 kDA); 2,RNA polymerase containing o-35 (BioRex-70 fraction) purified fromsporulating cells; M, molecular mass markers with sizes indicated inkDa (in descending order: phosphorylase b, bovine serum albumin,ovalbumin, carbonic anhydrase, soybean trypsin inhibitor, anda-lactalbumin).

Reconstitution of Bt I-Specific Transcribing Activity. Afraction similar to that shown in Fig. 2 (lane 2) was subjectedto preparative gel electrophoresis and sections ofthe gel fromregions above, below, and including the 35-kDa band wereeluted and renatured. The renatured materials were added tocore RNA polymerase purified from vegetative cells of strainHD-1-Dipel (this polymerase preparation contained the 8peptide but no detectable o-61). Fig. 4 shows that addition ofmaterial eluted from the gel above (lanes 3 and 4) or below(lanes 7 and 8) the 35-kDa band resulted in very little or notranscription from the template; addition of material elutedfrom the area of the gel containing the 35-kDa band (lanes 5and 6) resulted in the synthesis of run-off transcripts havingthe length expected for transcription from the Bt I promoter(Table 1). These observations suggest that the 35-kDa poly-peptide functions as a sigma subunit. In a separate experi-ment we found that addition of a-43 from B. subtilis to corepolymerase prepared from E-o-35 resulted in transcription ofphage SP82 DNA but not pKLB1 DNA (data not shown).E-o-35 Does Not Transcribe from the B. subtilis ctc and

spoVG Promoters. B. subtilis RNA polymerases containing

12 3 4 5 6 7 8

-

*e,.-_4-

FIG. 3. Autoradiogram showing primer-extension mapping of the5' ends of in vivo- and in vitro-synthesized crystal protein genemRNA. Lanes 3-6, DNA sequence analysis (G, A, T, and Creactions, respectively) of the promoter region of the crystal proteingene from HD-1-Dipel; lanes 1 and 7, extended primers for in vivoRNA; lanes 2 and 8, extended primers for in vitro-synthesized RNA.Lanes 7 and 8 contain approximately twice the amount of sample as

lanes 1 and 2. The position of these extended primers is indicatedwith an arrow; the corresponding initiating nucleotide is indicatedwith an asterisk in Fig. SB.

1 2 M(kDa)

=v. bd 94

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Proc. Natl. Acad. Sci. USA 85 (1988)

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Proc. Natl. Acad. Sci. USA 85 (1988) 4169

(J)I~~~~~~~~~~~~~~~~~~.>) I, () I, 4 > o

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producing pWRW41 and pHES57, respectively, and used astemplates for in vitro transcription. As shown in Fig. 5A, thesizes of the run-off transcripts (see Table 1) were consistentwith the proposal that E-o--35 directs transcription from thepromoters for each of these genes as well as from the Bt Ipromoter from strain HD-1-Dipel. In the reaction mixturescontaining the templates with the 27-kDa gene promoter, wealso observed larger RNA in addition to the predictedtranscripts from the crystal protein gene promoter; furtheranalysis (data not shown) indicated that E-o--35 directs thesynthesis of three additional transcripts from the 10-kilobasecloned fragment from pKM1 (17).Consensus Sequence for E-o-35. The transcriptional start

sites for genes encoding the 27-kDa protein from B. thu-ringiensis subsp. israelensis (28) and the 29-kDa cuboidalcrystal protein from B. thuringiensis subsp. kurstaki HD-1(ref. 16; W. R. Widner and H.R.W., unpublished data) havebeen located by S1 nuclease mapping. Alignment of thepresumed promoter regions for these two genes with the BtI promoter (Fig. SB) revealed that several bases are con-served in the - 10 and - 35 regions of all three promoters. Wepropose the following as a tentative consensus sequence for

A75 a

g 0

FIG. 4. Autoradiogram showing transcripts produced by recon-stituted E-a-35 and B. subtilis RNA polymerase from templatescontaining promoters for the B. thuringiensis crystal protein gene andfor the B. subtilis ctc and spoVG genes. Lanes M, Hinfl fragmentsofpBR322 with sizes indicated in bp. Lanes 1-8 and 9-12, core RNApolymerase containing the a polypeptide isolated from vegetativecells of B. thuringiensis was tested alone (lanes 1, 2, 9, and 11) andin combination with materials eluted and renatured from gel slices ofa polyacrylamide gel cut from regions above, including (labeled"35"), and below the 35-kDa polypeptide band; lanes 1-8, crystalprotein promoter on pKLB1 linearized with either Sal I (S) orHindill (H); lanes 9 and 10, ctc gene promoter on pUC8-31 linearizedwith HindIll (expected transcript length is 95 bases); lanes 11 and12, spoVG gene promoter on pCB1291 linearized with EcoRI (120and 110 bases, respectively, are the predicted lengths of the RNAsinitiating from P1 and P2 promoters) (25); lane 13, B. subtilis RNApolymerase from stationary-phase cells, ctc gene promoter onpUC8-31 linearized with HindIll.

either o-32, oJ-37, cr-29, or or-30 (6, 20, 25, 26) are able totranscribe the spoVG promoter in vitro; the ctc promoter canbe transcribed in vitro by B. subtilis RNA polymerasecontaining either a-37 or o-32 (19, 25). Fig. 4 shows thatneither the core RNA polymerase (lanes 9 and 11) nor thereconstituted E-ar-35 (lanes 10 and 12) was capable of tran-scribing from promoters for either the ctc gene or the spoVGgene. On the other hand, a preparation of RNA polymeraseisolated from stationary-phase B. subtilis synthesized a95-base RNA from the ctc template (lane 13). This would beexpected since RNA polymerases containing o-37 and cr-32,which utilize the ctc promoter, are present in stationary-phase cells (3). Several vegetative promoters are recognizedby a minor form of vegetative B. subtilis polymerase con-taining o-28 (27); these promoters were not tested in thepresent investigation.E-a-35 Directs Transcription from the Promoters for Other

Crystal Protein Genes. We tested promoters from two addi-tional crystal protein genes that are expressed during early tomidsporulation: the gene encoding the 27-kDa polypeptidepresent in crystals of B. thuringiensis subsp. israelensis (17,28) and a gene encoding a 29-kDa polypeptide present incuboidal crystals of B. thuringiensis subsp. kurstaki HD-1(16). The promoter regions for these genes were cloned intothe Escherichia coli cloning vectors pUC8 and pUC119,

133 KDa

H S bp'506

396344

- 298

-220

* 154

75

B-35

29 kDa B .. 27 kDo

H S bp B H bp

* 1631 .3_0 __

517 33*506

396 .w. w 50E344 *39

298 * 34~* 291

* 22C* 220

6

4

E

-10

133 kDa ;TDTTTCATAAGATGAGT r TGT [TTAAATTG

29 kDa rM.X=A;ACCGTTTACTCCCCA r;1@GAAJ;GTGCAGABt; 27 kDa r =C;TTCGAACTATAGCG- .GAAaACTA

FIG. 5. Autoradiogram displaying in vitro transcripts producedby E-o'-35 holoenzyme from three templates, each containing thepromoter region for a different crystal protein gene; also shown aresequences of the presumed promoter regions for the three genes. (A)Run-off transcripts produced from the 133-kDa bipyramidal crystalprotein gene cloned from B. thuringiensis subsp. kurstaki HD-1-Dipel, the 29-kDa cuboidal crystal protein gene from B. thuringiensissubsp. kurstaki HD-1, and the 27-kDa crystal protein gene from B.thuringiensis subsp. israelensis. The templates and the sizes of thepredicted transcripts when the template is digested with Sal I (S),HindIII (H), and BamHI (B) are given in Table 1. Each panel in Ashows a separate experiment; sizes of marker DNA fragments in bpproduced by digesting pBR322 with Hinfl. (B) DNA sequences ofregions upstream from the start sites of transcription determined bySI nuclease mapping or by primer extension with reverse transcrip-tase (17, 28, 29, 30); the initiating nucleotide(s) is indicated with anasterisk and the consensus sequence for E-o-35 is outlined in black.

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4170 Biochemistry: Brown and Whiteley

B. thuringiensis RNA polymerase containing the 35-kDa ocsubunit: 5' GCAT9T(N)14 or 15CATA--T 3'.

DISCUSSIONAn RNA polymerase has been isolated from sporulating cellsofB. thuringiensis subsp. kurstaki HD-1-Dipel that is capableof directing transcription in vitro from the Bt I promoter of acrystal protein gene encoding a 133-kDa insecticidal poly-peptide. The core components of this polymerase are asso-ciated with a 35-kDa polypeptide that is not found in RNApolymerase isolated from vegetative cells. When a prepara-tion of gel-purified and renatured 35-kDa polypeptide wasadded to core polymerase from vegetative cells of strainHD-1-Dipel, transcription from the Bt I promoter was ob-served; this suggests that the 35-kDa polypeptide is a ocfactor. Genetic experiments will be needed to determinewhether the 35-kDa polypeptide is essential for the in vivotranscription of crystal protein genes. For example, it hasbeen reported (29) that r-37 directs transcription of the B.subtilis spoVG promoter in vitro but not in vivo: in contrast,the in vivo and in vitro transcription from B. subtilis promoterG4 is dependent on or-29 (31).To date, DNA sequences have been published for several

homologous crystal protein genes cloned from lepidopteran-specific subspecies of B. thuringiensis (1) and for twounrelated genes (27 and 72 kDa) from the dipteran-specificsubspecies israelensis (28, 30, 32). The genes from thelepidopteran-specific subspecies have two independentlyactivated overlapping promoters and have virtually identicalpromoter sequences. As shown in the present report, E-or-35transcribes in vitro from Bt I promoter that is activated fromearly to midsporulation. Bt II is activated from midsporula-tion to the completion of spore formation. In view of thesequence differences between Bt I and Bt II (2), it isreasonable to assume that an RNA polymerase with adifferent a subunit will be required for transcription from BtII; in fact, our preliminary experiments (unpublished data)with polymerase isolated from a later stage of sporulatingcells support this expectation.We found that E-a-35 does not transcribe from the B.

subtilis ctc and spoVG promoters but that it does recognizetwo additional crystal protein gene promoters: the majorpromoter for the gene encoding a dipteran-specific polypep-tide (the 27-kDa polypeptide) and a promoter for a geneencoding a 29-kDa polypeptide present in cuboidal crystalsfound in B. thuringiensis subsp. kurstaki HD-1. The tran-scriptional start sites and the DNA sequences for both the27-kDa gene (28) and the 29-kDa gene (ref. 16; W. R. Widnerand H.R.W., unpublished data) have been determined. Com-parison of the Bt I promoter with the promoters for the 27-and 29-kDa genes revealed that 5 bases are conserved in the- 10 region and 5 are conserved in the - 35 region, leading tothe tentative E-o--35 consensus promoter sequence proposedin this report.We found that E-a-35 can also transcribe from three sites

located on a cloned fragment of plasmid DNA containing the27-kDa gene of B. thuringiensis subsp. israelensis (unpub-lished data); it is known that this fragment encodes at leastthree other peptides (17). Possibly, E-a-35 can also recognizethe promoter for the gene encoding the 72-kDa peptide fromthe same subspecies. Thorne et al. (32) noted a sequenceupstream from the ribosome binding site that matched (14 of16 bases) the sequence around and including the -10 regionof the Bt I promoter in a lepidopteran-specific gene. Thematching sequence includes the 5 conserved bases in the -10region of the proposed E-c-35 consensus sequence. Also, itis known (33) that the crystal protein gene from HD-1-Dipelcan be expressed in B. subtilis during sporulation. It will beinteresting to determine how many of the conserved bases ofthe proposed consensus sequence will be found in the as yet

unidentified promoters on the plasmid encoding the 27-kDapeptide and also to determine whether E-cr-35 can recognizeany promoters on the chromosomes of either B. thuringiensisor B. subtilis.We thank Masaru Tahara for expert technical assistance and H. E.

Schnepf and George Bolton for helpful discussions. This researchwas supported by Public Health Service Grant GM-20784 from theNational Institute of General Medical Sciences and by Grant PCM-8315859 from the National Science Foundation. H.R.W. is therecipient of Research Career Award K6-GM-442 from the NationalInstitute of General Medical Sciences.1. Whiteley, H. R. & Schnepf, H. E. (1986) Annu. Rev. Micro-

biol. 40, 549-576.2. Wong, H. C., Schnepf, H. E. & Whiteley, H. R. (1983) J. Biol.

Chem. 258, 1960-1967.3. Losick, R., Youngman, P. & Piggot, P. (1986) Annu. Rev.

Genet. 20, 625-669.4. Reznikoff, W. S., Siegele, D. A., Cowing, D. W. & Gross,

C. A. (1985) Annu. Rev. Genet. 19, 355-387.5. Doi, R. H. & Wang, L.-F. (1986) Microbiol. Rev. 50, 227-243.6. Carter, H. L., III, & Moran, C. P., Jr. (1986) Proc. Natl. Acad.

Sci. USA 83, 9438-9442.7. Achberger, E. C., Tahara, M. & Whiteley, H. R. (1982) J.

Bacteriol. 150, 977-980.8. Kolenbrander, P. E., Hemphill, H. E. & Whiteley, H. R.

(1972) J. Virol. 9, 776-784.9. Spiegelman, G. B., Hiatt, W. R. & Whiteley, H. R. (1978) J.

Biol. Chem. 253, 1756-1765.10. Lowe, P. A., Hager, D. A. & Burgess, R. R. (1979) Biochem-

istry 18, 1344-1352.11. Laemmli, U. K. (1970) Nature (London) 227, 680-685.12. Hyde, E. I., Hilton, M. D. & Whiteley, H. R. (1986) J. Biol.

Chem. 261, 16565-16570.13. Hager, D. A. & Burgess, R. R. (1980) Anal. Biochem. 109,

76-86.14. Schnepf, H. E., Wong, H. C. & Whiteley, H. R. (1987) J.

Bacteriol. 169, 4110-4118.15. Norrander, J., Kempe, T. & Messing, J. (1983) Gene 26,

101-106.16. Whiteley, H. R. Widner, W. R., & Schnepf, H. E. (1988) in

Genetics and Biotechnology ofBacilli, eds. Ganesan, A. T. &Hoch, J. A. (Academic, New York), in press.

17. McLean, K. M. & Whiteley, H. R. (1987) J. Bacteriol. 169,1017-1023.

18. Vieira, J. & Messing, J. (1988) Methods Enzymol., in press.19. Tatti, K. M. & Moran, C. P., Jr. (1985) Nature (London) 314,

190-192.20. Moran, C. P., Jr., Lang, N., Banner, C. D. B., Haldenwang,

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