Insecticidal Crystal Proteins of Bacilllus thuringiensis

MICROBIOLOGICAL REVIEWS, Jlune 1989. p. 242-2550146-0749/89/02024'-14$0'2.00/0)Copyright 1 1989. American Society for Microbiology

Insecticidal Crystal Proteins of Bacilllus thuringiensisHERMAN HOFTE' AND H. R. WHITELEy

Plitit Genetic Sy stemns N. V., B-9000 Geunt, Belgium, l(1/1t1 Departmient )fIMicrlobiology, SC-42, Unievesity of Washington.Seattle, Washington 981952

INTRODUCTION ................................................................. 242

DIVERSITY AND CLASSIFICATION OF CRYSTAL PROTEIN GENES .......................................242

cryl Genes ................................................................. 243

Distribution of Cryl proteins among different B. thuringiensis strains ........................................244

cryII Genes ................................................................ 246

cryill Genes ................................................................. 246

Diptera-Active Crystal Protein Genes (crylV and cytA) ..............................................................247

EXPRESSION OF cry GENES ................................................................. 248

MODE OF ACTION ................................................................ 249

Factors That Determine Specificit ..................................................................249Conserved Features of cry Genes ................................................................. 250

USE OF B. THURINGIENSIS CRYST1AL PROTEINS IN PLAN PROTECTION ............................250

Transgenic Plants ................................................................. 250

Resistance Development ................................................................. 251

CONCLUSIONS AND PERSPECTIVES ................................................................ 251

ACKNOWLEDGMENTS ................................................................. 252

LITERATURE CITED ................................................................. 252

INTRODUCTION

Bacillus thmrin!giensis a gram-positive soil bacterium

characterized by its ability to produce crystalline inclusions

during spor-ulation. TIhese inclusions consist of proteinsexhibiting a highly specific insecticidal activity (reviewed in

references 4 and 97). Many B. thuriuigiensis strains withdifferent insect host spectra have been identified (9). TIheyar-e classified into different serotypes or subspecies based on

their flagellar antigens. Most strains are active against larvaeof certain members of the Lepidoptera, but some showtoxicity against dipterian (reviewed in refer-ence 22) or co-

leopteran (53) species. For several cryst.al-producing strains.no toxic activity has yet been demonstrated.

B. tlhiringiensis crystalline inclusions dissolve in the larvalmidgut, relcasing one or more insecticidal crystal proteins(also called 6-endotoxins) of 27 to 140 kilodaltons (kDa). Asdescribed in the following section, most crystal proteins aLre

protoxins that are proteolytically conver-ted into smallertoxic polypeptides in the insect midgut. TFhe activclted toxininteracts with the midgut epitheliLum cells otf susceptibleinsects. Electrophysiological (32) and biochemical (49) evi-

dence suggests that the toxins genercate por-es in the cellmembrane, thus disturbing the osmotic balance. Conse-quently, the cells swell and lyse. The larva stops feeding Candeventually dies. For several B. tlhuringiensis toxins, specifichigh-affinity binding sites have been demonstrated to existon the midgut epithelium of susceptible insects (37. 38). Thiscould, at least in part. explain the extr-eme specificity ofthese proteins.

Formulations of B. thuringiensis have been ulsed for morethan two decades as biological insecticides to control agri-

cultural pests and, more recently, insect vector-s of a varietyof human and animal diseases. Recently, the cloning ofinsecticidal crystal protein genes (97) alnd their expression in

plant-associcated microorgalnisms (72) or transgenic plants (5.

Corresponding author.

23. 90) hCas provided potentially powerful alternative strate-gies for- the protection of crops against insect damage.These applied caspects are to a large extent responsible for

an increased interest in this bacterium and its crystal pro-teins in recent years. Extensive screening progranms are

being carried out by various groups to search for B. tulij-,0iensis strains with new insecticidal spectra. Numerouspublications repor-t the identification of crystal proteins andthe cloning and sequencing of cr-ystal protein genes. Oneproblem related to this is the lack of a unitorm nomenclaturefor these genes and their products. which makes the literca-ture rather confusing.

In this review, we will present an update of the currentknowledge of B. thuringienNsis crystal proteins Cand theilrgenes. We will also propose a nomenclaiture and classifica-tion scheme for cr-ystal proteins based on their structure(deduced from the deoxyribonucleic acid IDNA] sequence)as well as their host ralnge.

DIVERSITY AND CLASSIFICATION OF CRYSTALPROTEIN (GENES

To date, nucleotide sequences have been reported for 42B. tlhuriagiensis crystal protein genes. Severcal sequences arceidentical or nearly identical cand thus represent the same geneor slight variants of the same gene. Taking this into account,14 distinct crystal protein genes remain. Several lines ofevidence, summarized below, suggest thfat 13 of thesegenes-the so-called crY (crystcal protein) genes-specify a

family of related insecticidal proteins (Cry proteins). These13 genes hCave been divided into a minimum number of majorclasses (four) aind several Subclasses charaicterized by boththe structural similalrities and the insecticidal spectra of theencoded proteins. The four ma jor classes atre Lepidoptera-specific (I). Lepidoptera- and Diptera-specific (II), Co-leoptera-specific (III), aind Dipteral-specific (IV) genes. Ad-dition(al classes cOuld be proposed and may be added later,for example. to include genes coding for nontoxic crystal

242

Vol. 53. No. 2

INSECTICIDAL CRYSTAL PROTEINS OF B. THURINGIENSIS 243

TABLE 1. Insecticidal crystal protein genes of B. thiuringiensis"

No.of PredictedRerncGene type Host range" io ods mol mass Other gene designations hotyramino acid (kDa) hltp)

cryIA(a) L 1,176 133.2 4.5-kb gene (56). c'/'-I (85) 79crx'IA(b) L 1,155 131.0 5.3-kb gene (56). kuirltldl (27), bt2 (39), (-cl-2 (85) 92coIA(c) L 1,178 133.3 6.6-kb gene (56) 3cryIB L 1,207 138.0 (ryA4 (7), type B (41) 7(rvIC L 1,189 134.8 Type C (41), BTVI (42). Bt (77) 42crylD L 1,165 132.5 Hofte, unpub-

lishedurvllA L/D 633 70.9 P2 gene (17), (7-sB! (98) 17crvIIB L 633 70.8 c-rvB2 (98) 98cl-NIIIA C 644 73.1 (TvC (18) 35Ct-IVA D 1,180 134.4 130-kDa endotoxin gene (96), 125-kDa protein gene (6), 96

ISRH3 (81)crWIVB D 1,136 127.8 130-kDa endotoxin gene (10)6), ISRH4 (82), Bt8 (13), 13

135-kDa protein gene (6)crvIVC D 675 77.8 ORFI (88) 88crvIVD D 643 72.4 (rvlD (16) 16CtA D/cytol. 248 27.4 27-kDa toxin gene (91) 91

See text for description and gene designations; designations approved by the Nomenclatiure Committee (D. J. Nierlich, Chairman) of the Publications Boardof the American Society for Microbiology.

" Specified host ranges: L, Lepidoptera; D. Diptera; C. Coleoptera: cytol., cytolytic and hemolytic.Reference of the first submitted publication describing the gene type. The number of amino acids and predicted molecular masses are derived from the

holotype sequences.

proteins. However, the relationship of such genes to thosedescribed below is not known, since the DNA sequences ofgenes coding for nontoxic proteins have not yet been deter-mined. One crystal protein gene of B. thiiriingiensis subsp.israelensis codes for a 27-kDa protein that exhibits cytolyticactivity against a variety of invertebrate and vertebrate cells,and this gene is totally unrelated structurally to the cryvgenes. On this basis, we propose a separate designation forthe gene coding for the 27-kDa protein: cytA for cytolyticcrystal protein. Table 1 lists the genes assigned to the fourmajor cry classes and to the cyt class.

cryI Genes

The Lepidoptera-specific crystal proteins are undoubtedlythe best-studied crystal proteins. The 20 cryI sequences thathave been reported are listed in Table 2. Six different genes(Table 1) can be recognized among the 20 sequences. All 20genes encode 130- to 140-kDa proteins, which accumulate inbipyramidal crystalline inclusions during the sporulation ofB. thuringiensis. As stated above, these proteins are protox-ins which solubilize in the alkaline environment of the insectmidgut and are proteolytically converted by crystal-associ-ated or larval-midgut proteases into a toxic core fragment of60 to 70 kDa. This activation can also be carried out in vitrowith a variety of proteases (reviewed in reference 97).The toxic domain is localized in the N-terminal half of the

protoxin. This was demonstrated for the CryIA(b) (39) andCryIC (H. Hofte, unpublished data) proteins through N-terminal amino acid sequencing of the trypsin-activatedtoxin. Both proteins are cleaved at a homologous position inthe sequence (residues 29 and 28, respectively). Nagamatsuet al. (71) determined the N-terminal amino acid sequence ofthe tryptic core fragment of a B. thitiingiensis subsp. deni-drolimius crystal protein. This sequence corresponds to theCrylA sequence starting from residue 29. The proteolyticcleavage site is highly conserved for the other CrylA and theCryID proteins as well, suggesting that for these proteins,the N terminus of the toxic fragment is localized at the sameposition. CryIB, however, is very different from the other

Cryl proteins in this region. It is not known whether thisprotein is also processed at the N terminus. Deletion analy-sis of several cryI genes [cryIA(a) (78), crylA(b) (39, 92),crvIA(c) (3), and crvlC (77)] further confirmed that the 3' halfof the protoxin is not absolutely required for toxic activity.The shortest reported toxic fragment was localized betweencodons 29 and 607 for CryIA(b) (39). Further removal of 4codons from the 3' end (92) or 8 codons from the 5' endcompletely abolished the toxic activity of the gene product.Similar observations were made for the crvIA(W) (78) andcrv'IA(c) (3) genes.The cryI genes can be distinguished from the other cry

genes simply by sequence homology (>50% amino acididentity [Table 3]). Three of these genes, (cryIA(a), crylA(b),and cr/VA(c), show more than 80% amino acid identity andhave therefore been considered as a separate subgroup.These three genes were previously designated as the 4.5-,5.3-, and 6.6-kilobase (kb) genes, respectively, on the basisof the size of the HiidIIl restriction fragment containing the5' end of the genes (56). The nucleotide differences betweenthese three genes are localized mainly in a limited section ofthe region encoding the toxic fragment (97). The recentlyidentified crviB, crivIC, and crvID genes differ much morefrom each other and from the crylA genes (Fig. IA).A crystal protein gene from B. thlalt-ingienKsis subsp. aiza-

wai IC-1 is listed in Table 2 as a crylA(b) gene, despite thefact that the protein has dual host specificity. As described ina later section on specificity, the toxicity of the IC-1 proteinto lepidopteran or to dipteran insects depends on the sourceof the proteolytic enzyme that generates the toxin (29, 31).The decision to include the IC-1 gene in the crylA(b)subclass (rather than creating a new class) was based on thestructure of the gene product: the amino acid sequencediffers from the holotype by only four amino acids.

Interestingly, comparison of the predicted amino acidsequences shows that the C-terminal half is highly conservedfor all cri!l genes (Fig. 1A). It is unclear whether thesequence conservation in this region reflects any functionalsignificance. As shown above, this domain does not seem to

VOL. 53, 1989

244 HOFTE AND) WHITELEY

TABLE 2. Overview of reported crystal protein gene sequences

No. of amnino acidCrystal d.fml~esscifferiences tfromprotein Iholotpe scqLncne'Refei-cncegene SLIbSp. and/or strain

Pr-otoxin Toxin"

crvIA(a) kuistaki HD-1

kurstaki HD-1sotto

enttomlo(cidits

crvIA(b) berliner 1715be'-lite'l 1715kil'ist(lki HD-1kii-Nttiki HD-1ai.(liawai IPL-7kurstaki HD-1kurstaki NRD-12(li1a00ai IC-1

(CiVIC entio/lno(idiis 601ailoawai HD-137enltomo(idIis HD-llt)

cr!ID ai(olawi HD-68

crvIIA kiustaki HD-263ki,'.svtoki HD-1

crvIIB kursAkti H 1)-l

(crY!IIIA si(I/t di(,gotCtCE/)1'iO/ i.5EG2 158

crvI VA is'rc('l('/elsisisra(cl(entsis

crvIVB iSraeielOs isisr'aeil('lnsisXiSraEleslitXIsisraelen,si.s

H31

24l

H2030)

79855283. 8464a

H H 2. 922 39

4 27. 88X4 2 736 2 23

10( 6 334 4 3t)

H H 3

H H 71 1 H6fte. un-

published

H H 42)7" 7 772 H6fte, un-

published

H H Ht'fte. un-published

H 17() 0) 98

H 98

H 350 40. 69. 81

t) t) 18

H H 964 1 82

H H 131 1 893 3 82

97 78 10)6

H H 88

H 16

H 911 1 190) 0) 931 1 24

The fir-st r-eported sequelnce ofla gene type is considcred the holotvpc (H)sequence. Suibsequiently reported amino aicid sequences are defined by thenumber of amino acid changes with r-espect to the holotype sequlencc.

Toxin. The N-terminal half ot the crystal pr-otein, delinecated by theC-terminal amino acid of the conserved amino acid sequjence block 5 (Fig. 2).

` Cor-rections to the sequlcnce in reference 88 (T. J. Pollock, personalcommunication) show that it is identical to that in reftcrencc 27.

' The rcported sequlence r-epresents the fir-st 823 residues (48).

play a role in toxicity. However, it is interesting that almostall cysteine residues are localized in the C-terminal half andthat disulfide bonds have been implicated in the maintenanceand the unusual solubility properties of the crystals (63).Hence, the C-terminal half may be intimately involved incrystal formation, and the conservation of sequences in thispart of' the molecule is apparently sufficient to allow thecoassembly of diff'erent crystal proteins in the same crystals.

Distribution of Cryl proteins among different B. thuringien-sis strains. Traditionally, the protein composition of B.thiiringienisis crystals has been studied through polyacryl-amide gel electrophoresis (10, 46) or immunologically byusing polyclonal antisera against purified crystals (57). Thesemethods showed that some crystals contained more than oneprotein. A genetic approrach became possible with the clon-ing ot crystal protein genes. The use of gene-specific probesled to the discovery thiat various subspecies of B. tliihitrig-ieisis contained one, two, or three closely related genes (54,56). These three genes were found in a number of diff'erentstrains or subspecies (e.g., B. tihuringiensis subsp. kurstakiHD-1 contains all three genes, strain HD-1 Dipel has thecrvIA(a) and (crIA(c) genes, B. thuringiensis subsp. tlhuil-giensis HD-2 contains (crvIA(b) and (crvIA(c), etc.).

Recently, an alternative approach was described in whichmonoclonal antibodies were used to identify single proteinsin crystal preparations. Huber-Lukac et al. (43) described 10monoclonal antibodies generated against purified crystalsfrom B. tltluiiigictisis subsp. kurstaki. Investigation of thecrystals of 14 B. thuringiensis strains with these antibodiesrevealed clear-cut differences in immunoreactivity. How-ever, in this study, no direct correlation was made betweenthe reactivity with certain monoclonal antibodies and thepresence of certain crystal protein types.

In another study, monoclonal antibodies were used todistinguish the CryIA, CryIB, and CryIC proteins in crystalpreparations (41). T'able 4 shows the results of a survey of 29strains of 11 serotypes by using 35 monoclonal antibodies.CrylA is the most common crystal protein type and waspresent in all but one strain tested; CryIB and CryIC wereless common. The former occurred in five subspecies.whereas CryIC was present only in B. thluringiensis subspp.(iZUmttli and cettooto(cidts. Certain strains of B. thitring!iensissubspp. galleriae and morrisoni contained proteins thatreacted only with aL limited subset of the antibodies specify-ing a crystal protein type. It will be interesting to see to whatextent these crystaLl proteins differ from the already-knowncrystal protein types with regard to structure and insectspecificity. It should be noted that the monoclonal antibodiesused in the above studies may not have detected all of thetoxins in the crystals. Similarly, subtle variations in aminoacid sequence which might be responsible for differences inhost spectrum might have escaped detection. It is clear,however, that monoclonal antibodies can significantly speedup the identification of crystal proteins produced by newlyisolated B. thitringiensis strains and are therefore a powerfultool in screening for strains with new insecticidal properties.They allow the rapid identification not only of known crystalproteins but, more importantly, also of crystal proteins withas yet unknown structural characteristics (e.g.. as wasshown for strains of B. thutringiensis subspp. galleriae andmol'rrisoni). In this way, strains producing crystal proteinswith unique structural properties-and possibly also uniqueinsecticidal spectra-can be preselected prior to the moretime-consuming bioassays.

In toto, these data clearly demonstrated that many strainsproduce severfal crystal proteins simultaneously and that the

cr',IA(()

crvIB

kirstoki HD-73

tliurintgiensis HD-2Centonoc('idlSs HD-110)

(inVIVCu 'vI VI)

INS'(Il(' ('1lSiS

iNlsl ('I'lN.i.S

(vtA israelensis1m1r1ri.XO11i PCJ-14islrailli' PGism?orr1isonti PG-14

M ICRKOBIo()I .. R F V.


TABLE 3. Percent amino acid identity between (1r-encoded proteins

%/4 Amino aicid identitv" for:Crystal protein

CrI-yA(b) CrvIA(c) CrylB Cr-ylC CrylD CrvIIIA" CryIVA CrylVB CrylVC"

CryIA(a) 90 82 55 67 71 25 27 27 22

CryIA(b) 86 56 66 71 28 27 27 23

CryIA(c) 55 67 70 23 28 24 24

CryIB 58 56 34 26 28 22

CrylC 70 31 26 30 21

Cry1D 23 28 28 22

Cry1llA 23 19 26

CryIVA 54 29

CrylVB 29

For the calculation of this perccntage, pairs of amino acid sequLences were aligned by using the GENALIGN prograni (IntelliGenetics). with a gap penaltyof zero. The number of matched amino acids was divided by the number of residues of the longest sequence of the two, using the entire deduced sequence. Crylland CrylVD are not included, because there is little or no significant amino acid identity between these two proteins and the other-s shown in the table. Because100% matches are not indicated, CryIA(a) has been omitted from the columns and CrylVC has been omitted from the rows.

" The comparison was made by uIsing only the putative toxin fragments (residLie 1 to the C-ter-minal r-esiduLe of conserved sequence block 5 of Fig. 2).

same (or very similar) crystal proteins occur in B. tliiiniigi-etnsis strains of different subspecies. This mobility of crystalprotein genes among strains of B. thinliingienisis subspecies isnot unexpected, since most genes are localized on largeconjugative plasmids (reviewed in reference 11). In addition,the observed association of several crvlA genes (55, 61) andcrvIVB (6) (see below) with IS elements (64) and/or trans-posonlike structures could contribute to their mobility.The insecticidal activity of B. tlilrinigiensis crystal pro-

teins has traditionally been investigated by using crudepreparations of spore-crystal mixtures (9). The results ofthese studies, however, are difficult to interpret. Spore-crystal preparations generally also contain other toxicagents, such as the beta-exotoxin (58) and toxic sporecomponents. In addition, in some cases it is not knownwhether the germination of spores inside the larvae contrib-utes to the observed toxic effects. Hence, these earlierstudies did not yield conclusive data that toxicity was duesolely to the activity of the crystal proteins. In some recentinvestigations, gradient-purified crystals were used in toxic-

ity assays (45, 60). However, since many B. tliniringienLsisstrains simultaneously produce more than one crystal pro-tein, it is still difficult to accurately determine the toxicityspectrum of individual proteins from these studies.

Preparations containing single crystal proteins have beenobtained in the following ways: through protein purification(105), through the introduction (by cloning or conjugation)and expression of the corresponding genes in heterologoushosts (see the references cited in Table 2), or by using B.thiiri-igienisis strains that produce only one crystal protein(41). The toxicity data for individual (crl-specified proteinsobtained so far are summarized in Table 5. The proteins,purifiedfrom B. tIliinigienisis crystals consisting of a single protein[CryIA(c) and CryIB] or from recombinant Eschericlhia coliclones, were solubilized in an alkaline buffer (pH 10) withreducing agents prior to the bioassay. These data clearlydemonstrate that single crystal proteins are highly specificfor certain lepidopteran species. For instance, CryIB andCryID were active against only one of the lepidopteran

FIG. 1. Amino acid sequence comparison of B. thluringientsis crystal proteins. Sequences were aligned by using the GENALIGN program(IntelliGenetics). Gaps that were added for optimal alignments are not indicated. Vertical lines represent amino acids that are conserved for

all lepidopteran crystal proteins (Cryl) (A); all dipteran crystal proteins (CrylV) (B); and all Cryl CrylIl, and CrylV crystal proteins (exceptCrylVD) (C). The positions of the five conserved sequence blocks (see text and Fig. 2) are underlined. The Cryll proteins show significanthomology only to the sequence of block 1. Vertical lines in the C-terminal half (outside the toxin-encoding region) represent amino acids

conserved for the CrylVA and CrylVB proteins in panel B and for all crystal proteins specified by cry genes (except Cryll. Crylll. and

CrylVC and CrylVD) in panel C. Abbreviation: H. hydrophobic transmembrane sequence (20) present in all crystal proteins except Cryll and

CrylVD. Numbers refer to positions in the sequence after alignment (amino acids plus gaps).

VOL. 53, 1989

246 HOFTE AND WHITELEY

TABLE 4. Distribution of three crystal protein types amongdifferent B. tlhiringiensis strains as determined by

reaction with monoclonal antibodies'

Presence of followingFlagellar fB. thlrilltttiet- Strain protein':serotvpe xi.s stUbsp.

CrylA' CrylB CryIC

1 tli/turin gienisis HD-2. Berliner 1715 + +HD-14 +(a) - -4412 - + -

3a ntiesti HD-4 + - -3a3b kiurstuiki HD-1 + - -

HD-73 + (c) - -4a4b (letidroliutixs HD-7 + - -

HD-37 + (a) - -4a4c kenwie HD-5, HD-64. + (c) - -

HD-136HD-551 + + -

Sa5b g/illcriicte HD-8. HD-129 + ? ?6 entotno(idt(s HD-110. 4448 + (a) + +6 siihtoXi(tits HD-10 + + -7 aiz,awai HD-11, HD-68 + - -

HD-127, HD-854. + - +HD-229, HD-133,HD-137

HD-272 + + -HD-847 + - +

8a8b m7orrisonti HD-12 + -9 tolwtorthlii HD-121 + - -

See refer-cnce 41.Proteins that react with only some of the monoclonal antibodies that

specify a crystal pr-otein type are designated by '. All CrvIA-specific antibod-ies recognized CrylA(b); two did not bind to CrylA(a): two other monoclonalantibodies did not react with CrylA(c). Hence, the last two crystal proteinscould be distinguished. provided that no other CrylA proteins were present inthe cr-ystals.

The CrylA subtype (a or c) is indicated when known.

species tested. An interesting observation is that the threeCrylA proteins, which are structurally very closely related.also show largely overlapping activity spectra.

cryIl Genes

The (cryll genes encode 65-kDa proteins which formcuboidal inclusions in strains of several subspecies: B.thiiriingienisis subsp. kurstaki HD-1 and 14 other strains, B.thitiingien1sis subsp. thuirintgiensis Berliner. and B. t/liirinl<gi-en.sis subspp. tolwtordtlii and kentvae (103, 104; W. R. Widner,unpublished results). These crystal proteins were previouslydesignated as P2 proteins, as opposed to the 130-kDa P1crystal proteins present in the same strains (105).

The first crVIIA gene was cloned from B. tlilurin£giensissubsp. kiur-staiki HD-263 and expressed in Bacillus inegate-riumt (17). Cells producing the CryllA protein were toxic forthe lepidopteran species Heliottiis virescens and Lymunntriadispar as well as for larvae of the dipteran Aedes aegvpti.Widner and Whiteley (98) cloned two related genes (cryIIAand (-rvIIB) from B. thuringient.sis subsp. kiir-staki HD-1.Both genes encode proteins of 633 amino acids with a

predicted molecular mass of 71 kDa, slightly larger than theapparent molecular mass determined for the P2 proteinsproduced in B. thliringient.sis. Both genes were expressed inE. coli. and the recombinant proteins were purified. Al-though the two proteins are highly homologous (87% aminoacid identity), they differ in their insecticidal spectra. CryllAis active against both a lepidopteran (Mancluca sexta) and a

dipteran (A. (laegpti) species, whereas cvIlIB is toxic only tothe lepidopteran insect. The decision to classify the proteinencoded by the latter gene as Cryll (rather than Cryl) isbased on the structural similarity of CryllA and CrylIB andon the fact that the CrylI proteins show a rather limitedhomology to the other Cry proteins (see the section on

conserved sequences). The DNA sequence further indicatedthat crvIIA is the distal gene of an operon, consisting of threeopen reading frames (o:fl, o,i:2, and crvIIA). The gene

products of o,Jf2 and cryIIA could be detected in cuboidalcrystals in several B. thiniisgietisis subspecies. It is unclearwhether the orfl and crvNIIIB products are also present in thecrystals. The oiJ2 gene product, which is highly immuno-genic, has an unusual repeated structure; the functions of theproteins encoded by oifl and oi;f2 are not known.The reported sequences of the two previously described

crvIIA genes (17, 98) are identical, except that Donovan etal. (17) identified an open reading frame of 590 rather than633 amino acids. This difference is the result of a sequencingerror (the insertion of one additional thymidine residue[W. P. Donovan, personal communication]). As shownbelow, the cryll genes show a rather limited homology to theother cry genes.

cryIlI GenesThree Coleoptera-specific B. tltiitgi-igieitsis strains have

been described so far: B. tlhlriitgieitsis subsp. tenebrionis(53), B. tltluri,tgienisis subsp. sauni diego (36), and B. tliiring-ieitsis EG2158 (18). Each of the strains produces rhomboidalcrystals containing one major protein. Cloning and sequenc-

ing demonstrated the presence of the same crystal proteingene in all three strains. The gene, expressed in E. coli,directs the synthesis of a 72-kDa protein toxic for theColorado potato beetle (Leptiiosarc-sa decemnlineata). Thisprotein is converted into a 66-kDa toxin by spore-associated

TABLE 5. Toxicity of crystal proteins against five Lepidoptera species"

LC,,, of protein" for:Cry B. ttI1ittiifieiNiS sLabsp.

pr-otein and strain Pi'ri,ishro.s.siicoe Mittlo s x.vta Heliothi.slir} sC( tIN Moane str broas.sico, .Spolopterao littoroli.s(rLg/m (ng/cm2) (ng/cm2) (ng/cm2) (ng/cm2)

CrylA(a) aizt,awai HD-68 0.8 5.2 9() 165 >1,350)CrylA(b) berlliner 1715 0).7 8.6 10 162 >1,350CrylA(c) kiil-sttiki HD-73 0.3 5.3 1.6 2.t)00 >1,350CrylB tltt/rittgieitsis 4412 2.8 >625 >625 >1,350 >1,350)CrylC ettotnocidius HD-110 6.0) >128 >256 22 104Cryll) aizotiwti HD-68 >75 5 >256 >1.350 >1,350

Proteins were purified from recombinant E. co/i clones or from B. thutingientsi.s crystal containing a single crystal protein [CrylA(c) from B. tllllttigi1itsi.ssubsp. kursiaki HD-73 and CrylB from B. tlhttringien.si.s sLabsp. tttringiet.si.s 4412) and solubilized prior to the bioassay.

" Data arc from r-eference 41 (for- CrylA and CryI1B) or H. H6fte. uinpublished observations (for CrylC aind CryID). LC,,, St0(l lethal concentration.

MICROBIOI.. REV.


FIG. 2. Conserved amino acid sequence blocks. Amino acid sequence blocks conserved for all crystal proteins encoded by cry genes.except CrylI and CrylVD, are shown. The position of these blocks in the sequence is shown in Fig. 1. Amino acids marked by the shadedarea are identical or conservatively changed for at least 8 of the 10 cry sequences. The region in the CrylIA. CryIIB, and CrylVD amino acidsequence homologous to the block 1 sequence is also shown.

proteases which remove 57 N-terminal amino acids (69). Asdiscussed below, crlIIIA is homologous to the toxin-en-coding domain of the cr1 and c(ryIV genes and lacks a regioncorresponding to the 3' half of these molecules. Deletionanalysis demonstrated that the gene cannot be truncated atthe 3' end without the loss of toxic activity (D. A. Fischhoff,Soc. Invertebr. Pathol. Abstr. Twenty-First Meeting. abstr.60, p. 60, 1988). Interestingly, the 12 C-terminal amino acidsof this protein are highly similar to the sequence at the Cterminus of the smallest toxic fragment of CryIA(b), asdetermined by deletion analysis (described above). In addi-

tion, all but three other crystal proteins contain a block of 12conserved amino acids at a comparable position (Fig. 2,block 5). This makes it reasonable to assume that the Cterminus of the toxic fragment of these crystal proteins willalso be defined by this sequence. Deletion studies on crv-IA(a), crvIA(c) (cited above), and crvIVB (74) at least do notcontradict this assumption.

Diptera-Active Crystal Protein Genes (crylV and cytA)The crvlV class of crystal protein genes is composed of a

rather heterogeneous group of Diptera-specific crystal pro-

VOL. 53, 1989

Yv:

-ee'

248 HOFTE ANI) WHITELEY

tein genes. Four described genes, as well as the (ctA gene.were all isolated from the same 72-MDa plasmid present instrains of B. thurinigiensis subsp. israclensis. The (l IVA.crNIVB, crvIVC, and crvIVD genes encode proteins withpredicted molecular masses of 135. 128. 78. and 72 kDa,respectively. These proteins assemble, together with the27-kDa cvtA gene product, in ovoid crystal complexes. Acrystal complex with the same or a very similar proteincomposition has also been observed in one other strain, B.thliirinigiensis subsp. mnorrisoni PG-14. The biochemicalproperties and the toxicity of the different crystal compo-nents have been extensively reviewed by Federici et al. (22).Here we will summarize only the most recent data.

Toxicity tests with crystal protein preparations. derivedeither from B. tluringicensis subsp. israelensis crystals (12)or from recombinant E. coli (6. 13, 15) or Bacillus (16, 25.106) strains, provide the following picture. All crystal com-ponents are, to various extents, toxic against larvae ofcertain mosquito species. Solubilization of the proteinsreduces the toxicity dramatically (50- to 100-fold). This isattributed not to a real loss of toxicity but rather to reducedlevels of toxin ingestion by larvae owing to their filter-feeding behavior. No single crystal component is as toxic asthe intact crystal complex. One possible explanation for thisis that two or more proteins act synergistically, yielding ahigher activity than would be expected on the basis of thespecific toxicity of the individual proteins. The followinglines of evidence suggest that this is indeed the case. The27-kDa cvtA-specified protein exhibits no or a rather lowdipteran activity (see below). However, subtoxic doses ofthis protein were reported to significantly increase the tox-icity of the ca. 130-kDa (CryIVA and CryIVB) and the ca.70-kDa (CryIVD) protein fractions of B. tliiinigieiisis subsp.isr(lelensis crystals against A. alegypti larvae (102). Similarobservations were described for combinations of CrylVBand CryIVC against larvae of Cld.vx pi)iPens (15).The architectule of crvNIVA and crNlIVB is similar to that of

the crlI genes. They also encode ca. 130-kDa proteins,which are proteolytically converted into smaller toxic frag-ments. There is some controversy about the exact molecularmasses of the toxic core fragments, which vary from 53 (12)to 78 (13) kDa in different studies. The 3' halves of thesegenes are almost identical to each other and ar-e highlysimilar to the 3' halves of the cr l genes (Fig. 1). Thissuggests that the toxic fragment olt CrylVA and CryIVB isalso localized in the N-terminal half. This was confirmed forthe cryIVB gene product through deletion analysis (13, 15,74).The cry'IVC gene encodes a protein with a predicted

molecular mass of 78 kDa. A second open reading frame(ORF2) is localized 45 base pairs downstream from the stopcodon of this gene (ORFI). ORFI shows homology to the 5'half of the other crv genes, whereas ORF2 corresponds tothe remaining 3' part (88). 'IThis gene configuration hasprobably evolved through the insertion of a DNA fragmentinto a gene that otherwise would encode a ca. 130-kDapolypeptide. When introduced into B. subtilis or into a curedB. thuringicensis subsp. israclcensis str-ain, crvNIVC directs theexpression of a toxic protein of ca. 58 kDa, presumably aproteolytic fragment of the ORFI gene product. Minoramounts of this 58-kDa protein have also been found in B.tlhuringicnsis subsp. isralenisis crystals (25). The regionspecifying the active toxin in crlIVA. (ryIVB. and (crIVC ishighly divergent. Conserved amino acids ar-e restrictedmainly to five sequence blocks, which are also conser-ved forthe Cryl and CryllIA proteins (sec below).

I'he crYIVD gene encodes a 72-kDa protein (16), which isa major component of the B. thlurinigiensis subsp. israclkesiscrystals (22). This crystal protein, unlike all other knowncr.y-encoded proteins, is proteolytically converted into anactive fragment of ca. 30 kDa (12, 44, 75). The exactlocalization of this fragment in the intact protein is notknown. Sequence comparisons reveal a rather limited ho-mology to the other crystal proteins in a short region of themolecule (between codons 45 and 174).The 27-kDa protein encoded by c tA shows no sequence

homology to the other crystal protein genes. In addition, thisprotein, purified from B. thuringiensis subsp. israelenisiscrystals or from a recombinant B. sub tilis clone, showsunique functional features. It is cytolytic for a variety ofinvertebrate and vertebrate cells, including mammalianerythrocytes (87. 95); however, its in vivo insecticidal activ-ity is uncertain. Some authors (68, 95) have shown that thisprotein has a weak toxicity to A. acgypti larvae (50% lethalconcentration, 110 to 125 p.g/ml, compared with ca. 1 ng/mlfor intact crystals), whereas others have failed to demon-strate any insect toxicity (22).

EXPRESSION OF cry GENES

The structure of the (cvIA(a) promoter region has beenreviewed previously (97). In brief, in B. thliniiiigiensis,(cvIA(a) is transcribed from two start sites, located ca. 16base pairs apart: Bt 1, which is activated early in sporulation(t, to t, [where t, indicates the number of hours after theonset of sporulation]), and Bt II, which is activated atmidsporulation (t4 to t5 (101). In vitro transcription from BtI is catalyzed by a specific B. thluiin,-igiecisis ribonucleic acid(RNA) polymerase containing a new sigma subunit of Cca. 35kDa (8); vegetative genes are transcribed by the predominantRNA polymerase, which contains a sigma subunit of 61 kDa.T'ranscription from Bt II requires a second RNA polymerasecontaining another new sigma subunit of ca. 28 kDa (K. L.Brown and H. R. Whiteley, unpublished observations). ThecrvlA(b) and crYIA(c) genes are reported to have the samepromoter structure cis (crIA(a) (2, 88), and transcription ofseveral other genes (cr,xIB, crvIIA, and cYtA) requires thesigma-35-containing RNA polymerase (Brown and Whiteley,unpublished). Lastly. many crystal protein genes have astrong terminator. Wong and Chang (100) showed that thepresence of the terminator significantly enhances the stabil-ity of crystal protein messenger RNA (mRNA).

In Spo' Bacillus subtilis, transcription of civlA(a) ispredominantly from the Bt I start site (H. E. Schnept, W. R.Widner. K. L. Brown, and H. R. Whiteley, unpublishedobservations), and preliminary evidence (Brown and White-ley, unpublished) indicates that in vitro transcription re-quires a B. siihtilis RNA polymerase containing a sigmasubunit of ca. 35 kDa. The identity of the RNA polymeraseresponsible for the much weaker transcription from Bt II inSpo' cells has not been investigated. Transcription mappingshowed that Bt I is utilized in the spoliC, spolIAC, andspolIlE sporulation mutants of B. subtilis, but not in the.spoII41 mutant. Assays of chloramphenicol acetyltransfer-ase fused to the crv'lA(a) promoter showed little or noutilization of the promoter (<5% activity) in spoOA, spoOB,spo(F. spoOH, spoIE, and spoIlAA mutants and pacrtialactivity (40%) in spoOJ mutants. However, the dependenceon sporulaltion may be related to the vector used in cloning,since Shivakumar et al. (86) reported that a crystal proteingene cloned into a different plasmid was not regulated bysporulation.

MICROBI()I-.REV.

INSECTICIDAL CRYSTlAL PROTEINS OF B. THURINGIENSIS 249

In E. (oli, (rvIA(a) is transcribed from a site at or very

near Bt II; the identity of the polymerase responsible for thistranscription has not been established. Unexpectedly.expression of the crvIA(a) gene in E. coli, but not in B.silbtilis, is regulated negatively by a region of DNA locatedat about position -87 to -258 relative to Bt I. Deletion or

interruption of this region enhances gene expression approx-

imately 10-fold (80). Deletion of this region also yieldsincreased expression of cryIA(b) (88). The biochemical basisfor this regulatory mechanism has not been investigated.There is one additional regulatory mechanism that should

be mentioned. Full expression of the cytA gene in E. coli(i.e., abundant synthesis of CytA, toxicity to mosquitolarvae, and haemolytic activity) requires the presence of a

segment of B. t/lOliillgiecnsis subsp. israelen/sis DNA locatedabout 4 kilobases upstream from the c(tA gene (68). Thisregion is not required for expression of the (ctA gene in B.si,btilis (95). The upstream DNA that is involved in fullexpression encodes a 20-kDa peptide (1), which acts in trans

at the posttranscriptional level to increase the amount ofCytA produced. This effect occurs after the initiation oftranslation; possibly the 20-kDa peptide is involved in pro-

tecting CytA from degradation. The gene encoding the20-kDa peptide is apparently transcribed as part of an operon

which contains crYIVD, and its product is synthesized con-

currently with CytA, beginning at about t. Interestingly, the20-kDa protein is present in B. t/huringielSis subsp. isra/eln-sis crystals; thus, crystals may contain not only mixtures ofseveral insecticidal proteins, but also regulaltory proteins or

proteins such as the oif2 product (part of the (crIIA operon)which may have some structural function.

MODE OF ACTION

Most studies of the histopathology and mode of action

have been carried out on lepidopteran larvae with oftenrather ill-defined toxin preparations derived from whole B.t/muringiensis crystals. In summary, these investigations (re-viewed in detail in reference 63) showed that the crystalproteins dissolve in the larval insect midgut and are prote-

olytically converted into a toxic core frcagment. The epithe-lial cells of the midgut, or cultured insect cells, rapidly swelland burst after toxin treatment. The absence of a lag periodbefore the appearance of the first symptoms suggests that thetoxin does not need to be internalized to mediate cytotoxic-ity. Recent investigations shed more light on the mechanismof action. Knowles and Ellar (49) studied the effect of B.tl/liurligiesliis toxins on the permeability of cultured insect

cells by using radioactively labeled small molecules. Aftertoxin treatment, they observed a rapid release of the smallmolecules, which leaked out of the cell before the largerones. In addition, cytolysis was inhibited or delayed byosmotic protectants. On the basis of these and other obser-vations, the authors proposed that all four of the B. t/lil-giensis toxin preparations they tested (derived from B.t/111iniensiel.$is subspp. kurstaki HD-1, aiz(lawai H D-249, th/ir-ingienisis HD-50, and israelensis IFS-73) provoke a colloid-osmotic lysis as described for- mellitin. According to thismodel, these toxins induce the formation of small, nonspe-

cific pores (0.5 to 1 nm) in the membrane ot susceptible cells,resulting in a net influx of ions and an accompanying inflowof water. As a result, the cells swell and lyse.

One has to remark here that results from investigations on

cultured insect cells should always be confirmed by studieson larval gut tissue for the following reasons: (i) the availablecell lines are derived from tissues other than the midgut and

are therefore not a primary target of B. thuringienisis toxinsin vivo; (ii) toxic effects on insect cell lines are observed onlyat much higher toxin concentrations (100-fold) than thoserequired for in vivo toxicity; and (iii) the specificity for celllines derived from different insect species does not strictlycorrelate with the in vivo host range of crystal proteins (48,99).

Biochemical studies on isolated midgut membranes lead toslightly different conclusions. Sacchi et al. (76) studied theK -amino acid cotransport into brush border membranevesicles (BBMVs) prepared from midguts of Pieris brassicielarvae. Treatment with the toxin (i.e., purified, solubilized,and activated crystals from B. thiuringiensis subsp. ki,r-stikiHD-1 and B. thiuringiensis subsp. th/ulringiensis 4412) imme-diately inhibited the tr-ansport of labeled histidine driven bya K' gradient. As they observed no influence of the toxin onthe K '-amino acid cotransporter itself, they reasoned thatthe K' gradient must be dissipated through formation ofchannels in the brush border membrane upon toxin treat-ment. 'I'he amino acid transport in this tissue can also bedriven by a Na' gradient, although less efficiently. How-ever, the authors failed to demonstrate any influence of thetoxin on the Na '-driven amino acid cotransport. From this,they concluded that the channels might be K' specific.

In conclusion, these studies suggest that B. thuiringiensistoxins induce the formation of pores in the membrane ofboth midgut epithelial cells and cultured insect cells. Directpermeability measurements on BBMVs with radioactivelylabeled ions are needed to sort out whether the initiallyformed pores are K' specific. In relation to this, it will beinteresting to see whether the toxin induces pore formationindirectly through interaction with resident membrane pro-teins or directly through insertion into the membrane.The mechanism of the cytotoxic activity has been ad-

dressed in several other studies summarized in the discus-sion section of reference 49. A rather unusuLal observationwas made by English and Cantley (21), who demonstratedthat toxin preparations from B. tlll illtii elnSis Subsp. Akurstakicrystals nonspecifically inhibited a K '-adenosine triphos-phatase from vari'OuS Sources (insect cells, human erythro-cytes, and dog kidneys). However, the inhibition occurredonly at very high toxin concentrations (100 Fg/ml). and itseems unlikely that it plays a significant role in insecttoxicity.

Factors That Determine SpecificityIn the previous sections, we presented an overview of the

variation in insect host range among individual crystalproteins. One of the most intriguing questions relates to themolecular basis for this extreme insect specificity. The mostobvious factors that may influence the host range of a crystalprotein are (i) differences in the larval gut affecting thesolubiliziation and/or processing efficiency of the protoxinand (ii) the presence ot specific toxin-binding sites (recep-tors) in the gut of different insects. It has been shown thatcertain insects have a low susceptibility toi- B. t/hurinigiensiscrystal proteins owing to the inefficient Solubilization of thecrystals. In vitro, Solubilization of crystals significantlyenhances the toxic activity (45, 59). However, dramaticdifferences in specificity are maintained after Solubilizationof the crystal proteins (Table 3).

Most experiments in which the toxic activities of protoxinand activated toxin were compared suggest that susceptibil-ity for certain crystal proteins is independent of the activa-tion (45, 63). However, for one crystal protein, evidence was

Vol. 53, 1989

250 HOFTIE AND WHITELEY

provided that a protoxin can be activated into either adipteran or a lepidopteran toxin, depending on the source ofthe proteolytic enzymes (31). In brief, Haider et al. (29, 31)cloned a gene coding for a 130-kDa protein from B. tliirin-giensis subsp. aizaiiwai into E. coli. Treatment of the 130-kDaprotein with trypsin yielded a 55-kDa peptide that was toxicto cultured lepidopteran cells (Choristoneura(limilecrana)but not to cultured dipteran cells. When the trypsin-acti-vated toxin was exposed to mosquito gut proteases, a53-kDa peptide was detected, and the preparation was toxiconly to cultured mosquito cells. Binding studies showed thatthe trypsin-activated 55-kDa peptide bound to a 68-kDaprotein present in the membranes of cultured lepidopterancells. A receptor for the Diptera-specific 53-kDa peptide hasnot been identified.Recent experiments (37. 38) suggest that the interaction

with high-affinity binding sites on the insect midgut epithe-lium may, to a large extent, determine the host spectrum ofB. tlhiiringiensis crystal proteins. In a first set of experi-ments, binding studies were carried out with two 125 I-labeledtoxins [CryIA(b) and CryIB] on BBMVs prepared from thelarval midgut of M. sexat and P. brassicae. respectively. TheCryIA(b) toxin is active agcainst both insects, whereas theCryIB toxin acts only against P. brassicae (Table 5). Inter-estingly, the activated CryIA(b) toxin binds with high affinityto BBMVs derived from both insects, whereas the CrylBtoxin shows saturable binding only to P. brassicae vesicles.Heterologous competition experiments with other toxinsprovide further evidence for a correlation between toxicityand specific binding. CryIA(a) and CrylA(c). which areequally toxic for M. sexta (Table 5). competed with thelabeled CrylA(b) toxin for the sarme binding site, whereas thedipteran-specific CryIVB and the coleopteran-specific Cry-IIIA did not compete. Unexpectedly, competition experi-ments with labeled and unlabeled CrylA(b) and CryIB toxinsshowed that they recognize distinct binding sites on the sameBBMVs from P. brassicae.Very little is known so far abOut the biochemical n1ature of

these specific toxin-binding sites. Binding studies onBBMVs after treatment with various proteases suggest thatthe binding sites consist at least of a proteinaceous compo-nent (37; H. van Mellaert, personal communication).Knowles and Ellar (48) identified a 146-kDa membiraneglycoprotein from Choristoneura faimiferania cclls as a pos-sible toxin receptor. Knowles et al. also repor-ted thatN-acetylgalactosarnine protected these cells against B. tliii-iie,,insis subsp. kairistaiki HD-1 preparations. suggestingthat this sugar is a part of the binding site (51). However.N-acetylgalactosalnmine did not interfer-e with the specificbinding of 251-labeled CryIB toxin to P. brassicac BBMVsor with the binding of '25I-labeled CrylA(b) toxin to Al. s(.vtavesicles (van Mellaert, personal communication). Theseresults also suggest that the effect of B. tilhuringiensis toxinson cultured cell lines is not directly comparable to the in vivoactivity.

Conserved Features of cry Genes

Tlhe crx genes described so faLr share the following char-acteristics. They encode insecticidal proteins, either of 130to 140 kDa or of ca. 70 kDa, containing a toxic fragment of60 + 10 kDa. One exception is the crYIVD protein, whichcontains a ca. 30-kDa active core component (12). For the130- to 140-kDa proteins, the toxic segment is localized inthe N-terminal half of the protoxin. The C-terminal p.art ofthe c.a. 130-kDa proteins (Cryl, CryIVA, and CrylVB) is not

essential for toxicity, but is the most highly conserveddomain of these crystal proteins (Fig. 1).

In the amino acid sequence corresponding to the toxicdomain, five highly conserved sequence blocks can bedistinguished in all but three (cv-specified proteins (Fig. 1and 2). Within these conserved sequence blocks, no (orrelcatively few) gaps were needed for the alignment of theidentical or related armino acids. These blocks are separatedby highly variable sequences of various lengths for thediflerent crystal proteins. Exceptions are the Cryll proteinsand CryIVD, which show significant homology only to theother cuI proteins in a region corresponding to block 1.Another recurring feature for all crystal proteins exceptCryll and CryIVD is the presence of a stretch of hydropho-bic amino acids at a comparable position within the 120N-terminal amino acids. This amino acid stretch shows theproperties of a predicted transmembrane sequence accord-ing to Eisenberg et all. (20) (Fig. 1). Remarkably, within thisregion only the hydrophobic character and not the identity ofthe amino acids is conserved, strongly supporting a func-tional significance. It h1as been proposed that the conservedhydrophobic region plfays a role in an interaction of the toxinwith the membrane of midgut epithelial cells (79), but directexperimental evidence for such interactions is lacking.

USE OF B. THURINGIENSIS CRYSTAL PROTEINS INPLANT PROTECTION

B. thuringiensi.s has proven to be a valuable alternative toconventional insecticides. It is highly active and harmless tothe environment owing to its specificity. Formulations of B.thijt,igienisis spore-crystcal mixtures are commercially avalil-able for use as biological insecticides in agriculture andforestry. B. thIiirin,u,,iensis subsp. israelensis, active againstlarvCae of mosquitoes and blackflies, is being used to controlvectors of a variety of human and animal diseases. Animportant disadvantage is the rather restricted host range ofthe existing B. thlaringiensis spriays. Scr-eening samples fromdifferent environments may yield B. thuiingiensis strainswith broader host ranges or new' specificities. The host rangeof strains used commercially can be expanded through theintroduction of new crystal protein genes. This can beachieved through conjugation (reviewed in reference 11) ofplasmids from other B. thutriuigiensis str-ains or throughdirect transformation of crystal protein genes cloned in a B.tiuraingiensis replicon (34). In this respect, the recent devel-opment of an efficient transformation system involving theuse of electroporation (J. Mahillon, personal communica-tion) will greatly facilitate futui-e manipulaktion of B. tliiirinl-g,iensis strains.

Another problem related to commercial B. thuringiensisprepcaraltions is the limited field stability. Here, the introduc-tion of crystal protein genes into plant-associated microor-ganisms might provide a valuable alternative. One pro-posal is to introduce these genes into endophytic bacteria(14). Another approach is the Tn5-medliated insertion of a(crIA(b) gene into the chromosomes of Pseulonionas fluo-rescens and Agrobacteriatn ratliobacter strains that colonizecor-n roots (72). Tralnsfor-mants of the P. fluores(ens and A.ra(lioba(ter strains expressed the crystal protein and weretoxic against M. sexta.

Transgenic Plants

Recently, the feasibility of generating insect-resistanttransgenic crops by using B. thitringiensis crystal proteins

MICROBIOL.. RFV.


was demonstrated. In all previously described experiments(5, 23, 90), an Agrobacteriimn tinmefaciens-based transfor-mation system (28) was used. Modified crystal protein geneswere placed under the control of a promoter and a 3' end ofa plant gene. Vaeck et al. (90) used derivatives of a (ryIA(b)gene under control of the mannopine synthase TR2' pro-moter from the octopine Ti plasmid of A. tuiine ciens. Threegroups of toxin gene cassettes were used: the intact gene; 3'deletion derivatives containing the toxin-encoding half of thegene; and fusions between the toxin-encoding part of thegene and ieo, a selectable marker gene derived from Tn5which confers kanamycin resistance (neomycin phospho-transferase activity) to transformed plant cells. These fusiongenes expressed chimeric proteins with both insecticidal andneomycin phosphotransferase activities. This allowed theselection of plant transformants with high insecticidal activ-ity through selection on high doses of kanamycin. Trans-formed tobacco plants toxic against M. sexta larvae wereobtained, and the amount of crystal protein detected immu-nologically in the plant tissue correlated well with the levelof insect resistance. It is remarkable that levels of crystalprotein that killed insects were obtained only when thetruncated or fusion genes were used. It is not known why theintact gene is not expressed in the plant cells. Transgenictobacco plants tested in field trials were fully protectedagainst M. sextci and H. virescens damage (G. Warren,personal communication). Fischhoffet al. (23) used a similarapproach with 3'-deleted derivatives of a cryIA(b) geneunder the control of the 35S promoter of cauliflower mosaicvirus. They transformed tomato plants and obtained plantsthat were toxic for M. sextai. Barton et al. (5) used a3'-deleted cryA(a) gene under the control of the 35S pro-moter. They also inserted the 5' untranslated leader of thecoat protein gene (gene 4) of alfalfa mosaic virus upstream ofthe toxin gene. This leader might enhance the efficiency oftranslation initiation. Tobacco plants resistant against M.sexta larvae were obtained.

Similar approaches are now being used for other commer-cial crops with important lepidopteran or coleopteran pestsand for which a transformation system is available (potato,cotton, etc.). A recent important development is the use ofhigh-velocity microprojectiles (47, 65), making it possible totransform other crops, especially monocots (e.g., corn),which cannot be transformed by the Agrobacteriu,n system.

Resistance Development

Despite the wide use of B. tliliritngienIsis formulations overthe last 20 years, no cases of resistance development in thefield have been reported. An important factor that may havecontributed to this is the low persistence of B. thliiingienisisin the environment. The situation might change dramaticallywhen the use of insect-resistant transgenic plants becomeswidespread, since several generations of insects per year willbe continuously exposed to crystal proteins, providing anideal environment far the development of resistance. In thisrespect, it is interesting to mention the only reported case ofresistance development in the laboratory.McGaughey (66) demonstrated that the Indian mealmoth

(Plodia interpuinctella), a lepidopteran pest of stored grainand grain products, can develop resistance against Dipel(Abbot Laboratories), a commercial spore-crystal formula-tion of B. tliiliigienisis subsp. ku,rstaki HD-1. A colony ofPlodia interplunctella was reared on a diet containing Dipelat a dose expected to produce 70 to 90% larval mortality. Intwo generations, resistance increased ca. 30-fold, and after

15 generations, a plateau 100 times higher than the controllevel was reached. The resistance remained stable in theabsence of selective pressure and was inherited as a reces-sive trait. An interesting observation was that other B.thutr-inlgientsis strains were still highly active against theresistant insects (67). An attractive hypothesis is that theselected Plodia interpuinctella strain is resistant only toCrylA toxins present in the Dipel preparation, but not toother types of toxins. If this is so, then the simultaneousexpression in transgenic plants of two or more toxins that actindependently against the same insect (perhaps through therecognition of distinct binding sites) should reduce theprobability that resistant insect populations will develop.Overall, the study of the molecular basis of resistanceagainst selected toxins deserves intensive study and mayprovide important clues for the understanding of the basis ofthe insect specificity.

CONCLUSIONS AND PERSPECTIVESIt has become clear in recent years that B. thurinigiensis is

provided with a surprisingly large and variable family ofinsecticidal proteins. In this review, we have presented aclassification of crystal protein genes based on insect spec-ificity and the primary structure of the proteins. Knowlesand Ellar (50) have also pointed out that crystal proteins canbe grouped according to host range. They proposed fivepathotypes: four of these correspond to the four classesshown in Table 1, and the fifth contains nontoxic proteins.We have not included the fifth class, because the structuresof nontoxic crystal proteins have not been determined. Todate, 14 gene types can be distinguished in our classification.We have assigned these genes to the minimum number ofcategories: the 13 related cry' genes have been placed in fourclasses according to structure and host range. A totallyunrelated gene (cytA) has been placed in a separate, fifthcategory. Additional gene types will undoubtedly be discov-ered in the current intensive screening efforts to isolatecrystal proteins with different host ranges. We hope thatTable 1 will provide a useful framework for the classificationof these additional genes.

Progress has been made in the understanding of thebiochemical mechanism of toxicity and the factors thatdetermine the extreme specificity. Data from in vitro exper-iments strongly suggest that activated toxins induce theformation of pores in the membrane of susceptible cells andthat they recognize high-affinity binding sites (putative re-ceptors) on the midgut epithelium of susceptible insectsonly. Several interesting questions relating to the structure-activity relationship of B. thliirinigiensis toxins have not yetbeen answered. It is not known whether the active toxin, likeseveral other protein toxins (70), consists of two or moredomains that mediate different steps in the toxic action (e.g.,receptor binding, pore formation). The comparison of thededuced amino acid sequences revealed a number of se-quence elements conserved for most crystal proteins; how-ever, it is not known whether these conserved elements alsohave any functional role in the toxic activity. In vitromutagenesis techniques (94) and/or the functional in vitroassays for receptor binding (37, 38) and membrane perme-ability (76) will shed more light on these matters. Also,monoclonal antibodies can be used to map functional do-mains on the toxin molecule. Finally, two recent reportsdescribe the in vitro crystallization of CryllIA and somepreliminary X-ray diffraction data (26, 62). This approachwill provide the necessary structural data for a rational studyof the molecular basis of the insecticidal activity.

VOL. 53, 1989


The discovery of putative receptor sites for B. tlhurina£icn-sis toxins provides promising avenues for futul-e research.Binding studies involving the use of different toxins and a

variety of insect species will show whether toxicity is alwaysassociated with high-affinity binding to midgut membranes.Interesting results can also be expected from the isolationand characterization of the putative receptor molecules. Dothese receptors have any physiological role in healthy in-

sects, and can one identify related molecules in the midgutepithelium of nonsusceptible insects'? Answers to thesequestions might provide strategies for the development ofnew generations of insecticides.New insights on the regulation of crystal protein gene

expression were provided by the identification of two sporu-lation-specific sigma factors, the finding of negative regula-tion of crystal protein gene expression in E. (oli, and thedescription of a protein affecting the expression of the CytAgene at a posttanslational level in E. (coli. The recentdevelopment of transformation systems for B. tllhuriniisliswill facilitate the study of crystal protein gene regulation in

its original host. The results from these studies are essentialfor the development of new B. thuiringiensis strains express-

ing combinations of crystal proteins with interesting insecti-cidal spectra.The expression of dipteran-active crystal proteins in other

microorganisms, such as B. sphlacri(cus or cyanobacteria (W.ChungJatupornchai, personal communication). may allowmore effective control of medically important dipteran insectvectors. Finally, it may be possible to produce a number ofagriculturally importalnt crops from transgenic plants thatexpress B. tluriiigicaisis crystal proteins. This will provide a

potentiallly powerful alternative to chemical insecticides in

agriculture.

ACKNOWLEDGNIENTS

We thiank D. Burges. B. Federici. A. Aronson. P. B3tauimann. D.Fischhoff. and others for their comments and helpful discussion ofthe gene classificcation scheme at the 1988 meeting of the Society forInvertebrate Pathology. University of Califor-nia at SaIn D)iego.H.R.W. thanks Jonathan Visick and H. Ernest Schnepf for assis-

tance in prepai-ation of the manuscript: H.H. thanks Vera Ver-maercke, Stefaian van Gyseghem, and Kcarel Spruyt for the figures.The wor-k by H.R.W. reported in this review, was supported by

Public Health Service grant GM 20784 fr-om the Nation al Institutesof Health.

LITERATURE CITED

1. Adams, L. F., J. E. Visick, and H. R. Whiteley. 1989. A20)-kilodalton protein is requi-ed for- efficient produIction of theBacillus thturingL,icnsis subsp. israeleasis 27-kilodialton crvstalpr-otein in Escherichia (cli. J Bateriol. 171:521-53t).

2. Adang, M. J., K. F. Idler, and T. A. Rocheleau. 1987. Struc-tural and antigenic relationships imong three insecticidal crvs-

tal proteins of Bacillus thiurinagiesi.s subsp. kurstaki. p. 85-99.Il K. Maramorosch (ed.). Biotechnology in invertebrate pa-

thology and cell culture. Academic Press, Inc.. San Diego.Calif.

3. Adang, M. J., M. J. Staver, T. A. Rocheleau, J. Leighton, R. F.

Barker, and D. V. T'hompson. 1985. Characterized full-lengthand truncated plasmid clones of the crystal protein of Bacillusthurinigicasis subsp. kurstaki HD-73 and their toxicity to

Muaducaset(a. Gene 36:289-30t).4. Aronson, A. I., W. Beckman, and P. Dunn. 1986. Bacillus

ihuiagicansis and related insect pathogens. Microbiol. Rev. 50:1-24.

5. Barton, K. A., H. R. Whiteley, and N.-S. Yang. 1987. Bacillusthuringiaiesis h-endotoxin expressed in transgenic NicOtiana

tabacuitn provides resistance to lepidopteran insects. PlantPhysiol. 85:1103-1109.

6. Bourgouin, C., A. Delecluse, J. Ribier, A. Klier, and G.Rapoport. 1988. A Bacillus tilhuriiigicnsis subsp. isralc/esisgene encoding a 125-kilodalton larvicidal polypeptide is asso-ciated with inverted repeat sequences. J. Bacteriol. 170:3575-3X583.

7. Brizzard, B. L., and H. R. Whitelev. 1988. Nucleotide se-quence of an additional crystal protein gene cloned fromBacillus thiuritgicnsis subsp. tlula-iagicgieasis. Nucleic Acids Res.16:4168-4169.

8. Brown, K. L., and H. R. Whiteley. 1988. Isolation of a Bacillusthuringiensis RNA polymerase capable of transcribing crystalprotein genes. Proc. NatI. Acad. Sci. USA 85:4166-4170.

9. Burges, H. D. (ed.). 1981. Microbiol control of pests and plantdiseases 197(0-1980. Academic Press. Inc. (London). Ltd.,Iondon.

10. Calabrese, D. M., K. W. Nickerson, and L. C. Lane. 1980. Acomparison of protein crystal subunit sizes in Bacillus tliiuriu-gieasis. Ccan. J. Microbiol. 26:1006-1010.

11. Carlton, B. C., and J. NI. Gonzalez, Jr. 1985. Plasmids anddelta-endotoxin production in different subspecies of Bacillusthiringienasis, p. 246-252. Ia J. A. Hoch and P. Setlow (ed.),Molecular biology of microbial differentiation. American Soci-ety for Microbiology, Washington, D.C.

12. Chilcott, C. N., and D. J. Ellar. 1988. Comparative study ofBacilluts tlhuriagicnsis var. israclcaisis crystal proteins i/l vivOand i/l vitro. J. Gen. Microbiol. 134:2551-2558.

13. Chunjatupornchai, W., H. Hofte, J. Seurinck, C. Angsuthana-sombat, and NI. Vaeck. 1988. Common features of Bacillusthuriagicasis toxins specific for Dipterca and Lepidoptera. Eur.J. Biochem. 173:9-16.

14. Crawford, M. 1988. ARS prodded into the open. Science 239:719.

15. Delcluse, A., C. Bourgouin, A. Klier, and G. Rapoport. 1988.Specificity of action on mosquito larvae of Bacillus thuriagiea-sis israclelasis toxins encoded by two different genes. Mol.Gen. Genet. 214:42-47.

16. Donovan, W. P., C. C. Dankocsik, and M. P. Gilbert. 1988.Moleculakr characterization of a gene encoding a 72-kilodaltonmosquito-toxic crystal protein trom Bacillus thuriagieasissubsp. isra(lca.sis. J. Bacteriol. 170:4732-4738.

17. D)onovan, W. P., C. C. Dankocsik, NI. P. Gilbert, M. C.Gawron-Burke, R. G. Groat, and B. C. Carlton. 1988. Aminoacid sequence and entomocidal activity of the P2 crystalprotein. J. Biol. Chem. 263:561-567. (Erratum J. Biol. Chem.264:4740. 1988.)

18. I)onovan, W. P., ,J. NI. Gonzalez, M. P. Gilbert, and C.I)ankosik. 1988. Isolation and characterization of EG2158, anew strain of Bacillus thuria<gicnsi.s toxic to coleopteran larvae.and nucleotide sequence of the toxin gene. Mol. Gen. Genet.214:365-372.

19. Earp, D. J., and D. J. Ellar. 1987. Bacillus thuriagiaensis var.iu0ETisonai strain PG14: nucleotide sequence of a gene encoding

a 27 kDa crystal protein. Nucleic Acids Res. 15:3619.210. Eisenberg, D., E. Schwartz, M. Komaromy, and R. Wall. 1984.

Analysis of membrane and surface protein sequences with thehydrophobic moment plot. J. Mol. Biol. 179:125-142.

21. English, L. H., and L. C. Cantley. 1986. Delta endotoxin is apotent inhibitor of the (Na.K)-ATPase. J. Biol. Chem. 261:1170-1173.

2. Federici, B. A., P. Luthy, and J. E. lbarra. 1990. The paraspo-ral badv of Bacillus thiniagiCnsis subsp. israelcnsis: structure.pr-otein comilposition and toxicity. In H. de Barjac and S.Sutherland (ed.). Bacterial control of mosquitoes and black-flies: biochemistry. genetics and applications of Bacillus thiu-iagicasis and Bacillu isphacricus. in press. Rutgers UniversityP'ress. New Brunswick. N.J.

23. Fischhoff, D. A., K. S. Bowdisch, F. J. Perlak, P. G. Marrone,S. H. McCormick, J. G. Niedermeyer, D. A. Dean, K. Kusano-Kretzmer, E. .j. Mayer, D. E. Rochester, S. G. Rogers, andR. T. Fraley. 1987. Insect tolerant transgenic tomato plants.B3io/Technology 5:807-813.

M IC ROBio01- REV


24. Galjart, N. J., N. Sivasubramanian, and B. A. Federici. 1987.Plasmid location, cloning and sequence analysis of the geneencoding a 27.3-kilodalton cytolytic protein from Bacillusthuringiensis subsp. uournisoti (PG-14). Curr. Microbiol. 16:171-174.

25. Garduno, F., L. Thorne, A. M. Walfield, and T. J. Pollock.1988. Structural relatedness between mosquitocidal endotox-ins of Bacillus thuringiensis subsp. israciletsi.s. Appl. Environ.Microbiol. 54:277-279.

26. Garfield, J. L., and C. D. Stout. 1988. Crystallization andpreliminary X-ray diffraction studies of a toxic crystal proteinfrom a subspecies of Bacillus tluhurintgietnsis. J. Biol. Chem. 263:11800-11801.

27. Geiser, M., S. Schweitzer, and C. Grimm. 1986. The hypervari-able region in the genes coding for entomopathogenic crystalproteins of Bacillus tlhuirintgienlsis: nucleotide sequence of thekiurlidl gene of subsp. kiurstaiki HD1. Gene 48:109-118.

28. Gheysen, G., P. Dhaese, M. Van Montagu, and J. Schell. 1985.DNA flux across genetic barriers: the crown gall phenomenon,p. 11-47. In B. Hohn and E. S. Dennis (ed.), Genetic flux in

plants. Springer-Verlag, Vienna. Austria.29. Haider, M. Z., and D. J. Ellar. 1987. Characterization of the

toxicity and cytopathic specificity of a cloned Bacillus tlhuin-giensis crystal protein using insect cell culture. Mol. Micro-biol. 1:59-66.

30. Haider, M. Z., and D. J. Ellar. 1988. Nucleotide sequence of a

Bacillts thinigien.sis aizaiwai IC1 entomocidal crystal proteingene. Nucleic Acids Res. 16:10927.

31. Haider, M. Z., B. H. Knowles, and D. J. Ellar. 1986. Specificityof Bacillus tllrinigiensis var. colhncri insecticidal delta-endo-toxin by differential processing of the protoxin by larval gutproteases. Eur. J. Biochem. 156:531-540.

32. Harvey, W. R., M. Cioffi, J. A. T. Dow, and M. G. Wolfers-berger. 1983. Potassium ion transport ATPase in insect epithe-lium. J. Exp. Biol. 106:91-117.

33. Hefford, M. A., R. Brousseau, G. Prefontaine, Z. Hanna, J. A.Condie, and P. C. K. Lau. 1987. Sequence of a lepidopterantoxin gene of Bacillus tlhlingiensis subsp. kiur.staiki NRD-12. J.Biotechnol. 6:307-322.

34. Heierson, A., R. Landen, A. Lovgren, G. Dalhammar, andH. G. Boman. 1987. Transformation of vegetative cells ofBacillus thluriitgiensis by plasmid DNA. J. Bacteriol. 169:1147-1152.

34 Heierson, A., R. Landen, A. Lovgren, G. Dalhammar, and H. G.Bowman. 1987. Transformation of vegetative cells of Bacillusthluiingienisis by plasmid DNA. J. Bacteriol. 169:1147-1152.

35. Herrnstadt, C., T. E. Gilroy, D. A. Sobieski, B. D. Bennet, andF. H. Gaertner. 1987. Nucleotide sequence and deduced aminoacid sequence of a coleopteran-active delta-endotoxin genefrom Bacillits thuringiensis subsp. stIa diego. Gene 57:37-46.

36. Herrnstadt, C., G. G. Soares, E. R. Wilcox, and D. L. Edwards.1986. A new strain of Bacillus tluriingiicgiensis with activityagainst coleopteran insects. Bio/Technology 4:305-308.

37. Hofmann, C., P. Luthy, R. Hutter, and V. Pliska. 1988. Bindingof the delta endotoxin from Bacillus thuringiensis to brush-border membrane vesicles of the cabbage butterfly (Pieri.sbrassicae). Eur. J. Biochem. 173:85-91.

38. Hofmann, C., H. Vanderbruggen, H. Hofte, J. Van Rie, S.Jansens, and H. Van Mellaert. 1988. Specificity of Bacillusthluinl£iigienisis 8-endotoxins is correlated with the presence ofhigh-affinity binding sites in the brush border membrane oftarget insect midguts. Proc. Natl. Acad. Sci. USA 85:7844-7848.

39. Hofte, H., H. de Greve, J. Seurinck, S. Jansens, j. Mahillon, C.Ampe, J. Vandekerckhove, H. Vanderbruggen, M. Van Mon-tagu, M. Zabeau, and M. Vaeck. 1986. Structural and func-tional analysis of a cloned delta endotoxin of Bacillus tlil/uinl-giensis Berliner 1715. Eur. J. Biochem. 161:273-280).

40. Hofte, H., J. Seurinck, A. Van Houtven, and M. Vaeck. 1987.Nucleotide sequence of a gene encoding an insecticidal proteinof Bacillus tliihuritngietisis var. tenebrionis toxic against Co-leoptera. Nucleic Acids Res. 15:7183.

41. Hofte, H., J. Van Rie, S. Jansens, A. Van Houtven, H. Vander-

bruggen, and M. Vaeck. 1988. Monoclonal antibody analysisand insecticidal spectrum of three types of lepidopteran-spe-cific insecticidal crystal proteins of Bacillus thuringicnsis.Appl. Environ. Microbiol. 54:2010-2017.

42. Hon&e, G., T. van der Saim, and B. Visser. 1988. Nucleotidesequence of crystal protein gene isolated from B. tituinigictIsissubspecies entomiocidius 60.5 coding for a toxin highly activeagainst Spotloptera species. Nucleic Acids Res. 16:6240.

43. Huber-Lukac, M., F. Jaquet, P. Luthy, R. Huetter, and D. G.Braun. 1986. Characterization of monoclonal antibodies to a

crystal protein of Bacillus tiuringiensis subsp. kur-staiki. Infect.Immun. 54:228-232.

44. Ibarra, J. E., and B. A. Federici. 1986. Isolation of a relativelynon-toxic 65-kilodalton protein inclusion from the parasporalbody of Bacillus tliuringiensis subsp. isrtailenisis. J. Bacteriol.165:527-533.

45. Jaquet, F., R. Hutter, and P. Luthy. 1987. Specificity ofBacillus thuringiensis delta-endotoxin. Appl. Environ. Micro-biol. 53:500-504.

46. Jarret, P. 1985. Potency factors in the delta-endotoxin ofBacillus thuringiensis var. ailzawai, and the significance ofplasmids in their control. J. Appl. Bacteriol. 58:437-448.

47. Klein, T. M., T. Gradziel, M. E. Fromm, and J. C. Sanford.1988. Factors influencing gene delivery into Zeci Inays cells byhigh-velocity microprojectiles. Bio/Technology 6:559-563.

48. Knowles, B. H., and D. J. Ellar. 1986. Characterization andpartial purification of a plasma membrane receptor for Bacillusthuringiensis var. kuri-staiki lepidopteran-specific 8-endotoxin.J. Cell Sci. 83:89-101.

49. Knowles, B. H., and D. J. Ellar. 1987. Colloid-osmotic lysis isa general feature of the mechanism of action of Bacillusthuringicnsis f-endotoxins with different insect specificities.Biochim. Biophys. Acta 924:509-518.

50. Knowles, B. H., and D. J. Ellar. 1988. Differential specificity oftwo insecticidal toxins from Bacillus tiulringicnsis var. aiiz-wati. Mol. Microbiol. 2:153-157.

51. Knowles, B. H., W. E. Thomas, and D. J. Ellar. 1984. Lectin-like binding of Bacillus thliin-i£gienisis var. kiur-sttaki lepi-dopteran-specific toxin is an initial step in insecticidal action.FEBS Lett. 168:197-202.

52. Kondo, S., N. Tamura, A. Kunitate, M. Hattori, A. Akashi, andI. Ohmori. 1987. Cloning and nucleotide sequencing of twoinsecticidal delta-endotoxin genes from Bacillus thuriensis.Agric. Biol. Chem. 51:455-463.

53. Krieg, A., A. Huger, G. Langenbruch, and W. Schnetter. 1983.Bacillus thuringiensis var. ten1ebr-ionIis: a new pathotype effec-tive against larvae of Coleoptera. J. Appl. Entomol. 96:500-508.

54. Kronstad, J. W., H. E. Schnepf, and H. R. Whiteley. 1983.Diversity of locations for the Bacillus tliihuingiensis crystalprotein gene. J. Bacteriol. 154:419-428.

55. Kronstad, J. W., and H. R. Whiteley. 1984. Inverted repeatsequences flank the Bacillus thuringiensis crystal protein gene.J. Bacteriol. 160:95-102.

56. Kronstad, J. W., and H. R. Whiteley. 1986. Three classes ofhomologous Bacillus thuringiensis crystal protein genes. Gene43:29-40.

57. Krywienzcyk, J. 1977. Antigenic composition of b-endotoxin asan aid in identification of Bacillus thuringiensis varieties.Publication IP-X-16. Insect Pathology Research Institute, Ca-nadian Forest Service, Sault Sainte Marie, Ontario, Canada.

58. Lecadet, M.-M., and H. de Barjac. 1981. Bacillus thuringiensisbeta-exotoxin, p. 293-321. In E. W. Davidson (ed.), Pathogen-esis of invertebrate microbial diseases. Allenheld, Osmun andCo., Totowa, N.J.

59. Lecadet, M.-M., and R. Dedonder. 1967. Enzymatic hydrolysisof the crystals of Bacillus thuringiensis by the proteases ofPieris brassicae. 1. Preparation and fractionation of the ly-sates. J. Invertebr. Pathol. 9:310-321.

60. Lecadet, M.-M., and D. Martouret. 1987. Host specificity ofthe Bacillus thuringiensis 8-endotoxin towar-d lepidopteranspecies: Spoudoptcra littoralis Bdv. and Picris brassicae L. J.lnvertebr. Pathol. 49:37-48.

VOL. 53, 1989


61. Lereclus, D., J. Ribier, A. Klier, G. Menou, and M.-M.Lecadet. 1984. A transposon-like structure related to the 6-endotoxin gene of Bacillus tlhuringiensis. EMBO J. 3:2561-2567.

62. Li, J., R. Henderson, J. Carroll, and D. Ellar. 1988. X-rayanalysis of the crystalline parasporal inclusion in Bacillusthuringiensis var. tecebr-ioniis. J. Mol. Biol. 199:543-544.

63. Luithy, P., and H. R. Ebersold. 1981. Bacillus tliirlgwiniiesisdelta-endotoxin: histopathology and molecular mode of action,p. 235-267. In E. W. Davidson (ed.). Pathogenesis of inverte-brate microbial diseases. Allenheld, Osmun and Co. Totowa,N.J.

64. Mahillon, J., J. Seurinck, L. V. Rompuy, J. Delcour, and M.Zabeau. 1985. Nucleotide sequence and structural organizationof an insertion sequence element (1S231) from Bacillus thur-igielcisis strain Berliner 1715. EMBO J. 4:3895-3899.

64a.Masson, L., P. Marcotte, G. Prefontaine, and R. Brousseau.1989. Nucleotide sequence of a gene cloned from Bacillustlhuringinsis subspecies entotnocidus coding for an insecti-cidal protein toxic for Bouuhbv mlori. Nucleic Acids Res. 17:446.

65. McCabe, D. E., W. F. Swain, B. J. Martinell, and P. Christou.1988. Stable transformation of soybean (Glycine max.) byparticle acceleration. Bio/Technology 6:923-926.

66. McGaughey, W. H. 1985. Insect resistance to the biologicalinsecticide Bacillus thlrin-igietusis. Science 299:193-195.

67. McGaughey, W. H., and D. E. Johnson. 1987. Toxicity ofdifferent serotypes and toxins of Bacillus thuliug-iicgi.sis toresistant and susceptible Indianmeal moths (Lepidoptera:Pyralidae). J. Econ. Entomol. 80:1122-1126.

68. McLean, K. M., and H. R. Whiteley. 1987. Expression inEschcrichia coli of a cloned crystal protein gene of Bacillusthuringiensis subsp. israeletisis. J. Bacteriol. 169:1017-1023.

69. McPherson, S. A., F. J. Perlack, R. L. Fuchs, P. G. Marrone,P. B. Lavrik, and D. A. Fischhoff. 1988. Characterization of thecoleopteran-specific protein gene of Bacillus thiuringicnsis var.tcicbn-iounis. Bio/Technology 6:61-66.

70. Middlebrook, J. L., and R. B. Dorland. 1984. Bacterial toxins:cellular mechanisms of action. Microbiol. Rev. 48:199-221.

71. Nagamatsu, Y., Y. Itai, C. Hatanaka, G. Funatsu, and K.Hayashi. 1984. A toxic fragment from the entomocidal crystalprotein of Bacillus thuringicnsis. Agric. Biol. Chem. 48:611-619.

72. Obukowicz, M. G., F. J. Perlack, K. Kusano-Kretzmer, E. J.Meyer, and L. S. Watrud. 1986. Integration of the deltaendotoxin gene of Bacillus thurnienigisis into the chromosomeof root colonizing strains of pseudomonads using Tn5. Gene45:327-331.

73. Oeda, K., K. Oshie, M. Shimizu, K. Nakamura, H. Yamamoto,1. Nakayama, and H. Ohkawa. 1987. Nucleotide sequence ofthe insecticidal protein gene of Bacillus thuringiensis strainaiztawai IPL7 and its high level expression in Escherichia coli.Gene 53:113-119.

74. Pao-intara, M., C. Angsuthanasombat, and S. Panyim. 1988.The mosquito larvicidal activity of 130 kDa delta-endotoxin ofBacillus thuringiensis var. israelensis resides in the 72 kDaamino-terminal fragment. Biochem. Biophys. Res. Commun.153:294-300.

75. Pfannenstiel, M. A., G. A. Couche, E. J. Ross, and K. W.Nickerson. 1986. Immunological relationships among proteinsmaking up the Bacillus tluringiensis subsp. israelensis crystal-line toxin. AppI. Environ. Microbiol. 52:644-649.

76. Sacchi, V. F., P. Parenti, G. M. Hanozet, B. Giordana, P.Luthy, and M. G. Wolfersberger. 1986. Bacillus thliuni-igiengisistoxin inhibits K+-gradient-dependent amino acid transportacross the brush-border membrane of Pietis blra.ssicace midgutcells. FEBS Lett. 204:213-218.

77. Sanchis, V., D. Lereclus, G. Menou, J. Chaufaux, and M.-M.Lecadet. 1988. Multiplicity of delta-endotoxin genes with dif-ferent insecticidal specificities in Bacillus tlhul-iingietisis aizava,i7.29. Mol. Microbiol. 2:393-404.

78. Schnepf, H. E., and H. R. Whiteley. 1985. Delineation of atoxin-encoding segment of a Bacillls thlluningiensis crystal

protein gene. J. Biol. Chem. 260:6273-6280.79. Schnepf, H. E., H. C. Wong, and H. R. Whiteley. 1985. The

amino acid sequence of a crystal protein from Bacillus thur-inigiecisis deduced from the DNA base sequence. J. Biol.Chem. 260:6264-6272.

80. Schnepf, H. E., H. C. Wong, and H. R. Whiteley. 1987.Expression of a cloned Bacillus thilringiensis crystal proteingene in E.scherichia coli. J. Bacteriol. 169:4110-4118.

81. Sekar, V., D. V. Thompson, M. J. Maroney, R. G. Bookland,and M. J. Adang. 1987. Molecular cloning and characterizationof the insecticidal crystal protein gene of Bacillus tlhuringicnsisvar. tenebriounis. Proc. Natl. Acad. Sci. USA 84:7036-7040.

82. Sen, K., G. Honda, N. Koyama, M. Nishida, A. Neki, H. Sakai,M. Himeno, and T. Komano. 1988. Cloning and nucleotidesequences of the two 130 kDa insecticidal protein genes ofBacillus thuringuiensis var. isralcensis. Agric. Biol. Chem. 52:873-878.

83. Shibano, Y., A. Yamagata, T. Amachi, and M. Takanami. 1986.Complete structure of an insecticidal crystal protein gene fromBacillus thuringicnsis, p. 307-32t). Ill A. T. Ganesan and J. A.Hoch (ed.), Bacillus molecular genetics and biotechnologyapplications. Academic Press. Inc., Orlando. Fla.

84. Shibano, Y., A. Yamagata, N. Nakamura, T. lizuka, H. Sugi-saki, and M. Takanami. 1985. Nucleotide sequence coding forthe insecticidal fragment of the Bacillus tlhiinigiensis crystalprotein gene. Gene 34:243-251.

85. Shimizu, M., K. Oshie, K. Nakamura, Y. Takada, K. Oeda, andH. Ohkawa. 1988. Cloning and expression in Escherichia coliof the 135-kDa insecticidal protein gene from Bacillus tiluin-gicuisis subsp. aizlaa(li IPL7. Agric. Biol. Chem. 52:1565-1573.

86. Shivakumar, A. G., G. .J. Gundling, T. A. Benson, D. Casuto,M. F. Miller, and B. B. Spear. 1986. Vegetative expression ofthe 6-endotoxin genes of Bacillus thuringiensis subsp. kuirstuikiin Bacillus siubtilis. J. B3acteriol. 166:194-204.

87. Thomas, W. E., and D. J. Ellar. 1983. Bacillus thuringicesisvar. isracluelesis crystal 8-endotoxin: effects on insect andmammalian cells in vitro and in vivo. J. Cell Sci. 60:181-197.

88. Thorne, L., F. Garduno, T. Thompson, D. Decker, M. Zounes,M. Wild, A. M. Walfield, and T. Pollock. 1986. Structuralsimilarity between the Lepidoptera- and Diptera-specific insec-ticidal endotoxin genes of Bacillus tdurinieng sis subsp. kur-sttuki and isr-aeleusis. J. Bacteriol. 166:801-811.

89. Tungpradubkul, S., C. Settasatien, and S. Panyim. 1988. Thecomplete nucleotide sequence of a 130 kDa mosquito-larvicidaldelta-endotoxin gene of Bacillus tlhluringietisis var. israieleisis.Nucleic Acids Res. 16:1637-1638.

90. Vaeck, M., A. Reynaerts, H. Hofte, S. Jansens, M. De Beukel-eer, C. Dean, M. Zabeau, M. Van Montagu, and J. Leemans.1987. Transgenic plants protected from insect attack. Nature(London) 328:33-37.

91. Waalwijck, C., A. M. Dullemans, M. E. S. van Workum, and B.Visser. 1985. Molecular cloning and the nucleotide sequence ofthe Mr 28 000 crystal protein gene of Bacillus tldiuuit,giengsiissubsp. israelctisis. Nucleic Acids Res. 13:8206-8217.

92. Wabiko, H., K. C. Raymond, and L. A. Bulla, Jr. 1986.Bacillus tdlirin-i,giensis entomocidal protoxin gene sequence andgene product analysis. DNA 5:305-314.

93. Ward, E. S., and D. J. Ellar. 1986. Bacillus thurlini*gieisis var.isracleletisis delta-endotoxin: nucleotide sequence and charac-terization of the transcripts in Bacillums thauringiensis and Esch-ericlia col. J. Mol. Biol. 191:1-11.

94. Ward, E. S., D. J. Ellar, and C. N. Chilcott. 1988. Single aminoacid changes in the Bacillhus thuringicnsis var. israelensis6-endotoxin affect the toxicity and expression of the protein. J.Mol. Biol. 202:527-535.

95. Ward, E. S., A. R. Ridley, D. J. Ellar, and J. A. Todd. 1986.Bacillus thluuringiensis var. israelenusis deltcl-endotoxin: cloningand expression of the toxin in sporogenic and asporogenicstrains of Bacilluus slibtili.s. J. Mol. Biol. 191:13-22.

96. Ward, S., and D. Ellar. 1987. Nucleotide sequence of aBacillus thiurinugienisis var. isi-aclelusis gene encoding a 130 kDadelta-endotoxin. Nucleic Acids Res. 15:7195.

MICROBIOL. REV.


97. Whiteley, H. R., and H. E. Schnepf. 1986. The molecularbiology of parasporal crystal body formation in Bacillus tlhur-intgienisis. Annu. Rev. Microbiol. 40:549-576.

98. Widner, W. R., and H. R. Whiteley. 1989. Two highly relatedinsecticidal crystal proteins of Bacillus thluiijigienisis subsp.kiur-st(uki possess different host range specificities. J. Bacteriol.171:965-974.

99. Wit, D. P., H. Carlson, and J. C. Hodgdon. 1986. Cytotoxicityof Bacillus thlriingietasis 6-endotoxin to cultured CF-1 cellsdoes not correlate with in i1ivo activity towards spruce bud-worm larvae, p. 3-6. In R. A. Samson. J. M. Vlak, and P.Peters (ed.), Fundamental and applied aspects of invertebratepathology. Society for Invertebrate Pathology. San Diego,Calif.

100. Wong, H. C., and S. Chang. 1985. Identification of a positiveretroregulator that functions in Escherichia coli and Bacillusslubtilis, p. 104-109. In J. A. Hoch and P. Setlow (ed.),Molecular biology of microbial differentiation. American Soci-ety for Microbiology, Washington, D.C.

101. Wong, H. C., H. E. Schnepf, and H. R. Whiteley. 1983.Transcriptional and translational start sites for the Bacillus

thiuringiensis crystal protein gene. J. Biol. Chem. 258:1960-1967.

102. Wu, D., and F. N. Chang. 1985. Synergism in mosquitocidalactivity of 26 and 65 kDa proteins from Bacillits thluiritugieusissubsp. israleinsis crystal. FEBS Lett. 190:232-236.

103. Yamamoto, T. 1983. Identification of entomocidal toxins ofBacillus tiliiiigiensis by high performance liquid chromatog-raphy. J. Gen. Microbiol. 129:2595-2603.

104. Yamamoto, T., J. A. Garcia, and H. T. Dulmage. 1983.Immunological properties of the entomocidal proteins of Bai-cilhls thUuringiensis and its insecticidal activity. J. lnvertebr.Pathol. 41:122-130.

105. Yamamoto, T., and R. E. McLaughlin. 1981. Isolation of a

protein from the parasporal crystal of Bacillius thlingiiensisvar. kiur.statki toxic to the mosquito larva, Aedes taeCiOrhyn-(lulls. Biochem. Biophys. Res. Commun. 103:414-421.

106. Yamamoto, T., 1. A. Watkinson, L. Kim, M. V. Sage, R.Stratton, N. Akanda, Y. Li, D.-P. Ma, and B. A. Roe. 1988.Nucleotide sequence of the gene coding for a 130-kDa mosqui-tocidal protein of Bacillius tlhuirinigiensis israelensis. Gene 66:107-120.

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Insecticidal Crystal Proteins of Bacilllus thuringiensis