Letters in Applied Microbiology 1998, 27, 62–66

Isolation of a non-insecticidal Bacillus thuringiensis strainbelonging to serotype H8a8b

H.-W. Park, J.-Y. Roh, Y.-H. Je, B.-R. Jin, H.-W. Oh 1, H.-Y. Park and S.-K. KangDepartment of Agricultural Biology, Seoul National University, Suwon, and 1Korea Research Institute of Bioscience& Biotechnology, Taejeon, Korea

1239/96: received and accepted 29 April 1998

H.-W. PARK, J.-Y. ROH, Y.-H. JE, B.-R. JIN, H.-W. OH, H.-Y. PARK AND S.-K. KANG. 1998. Bacillusthuringiensis strains non-toxic to Lepidoptera, Bombyx mori and Diptera, Culex pipiens pallenslarvae were isolated from Korean soil samples during an investigation of B. thuringiensisisolates highly toxic to insect pests. One of these isolates, NTB-88, produces parasporalinclusions about 138 kDa in size and is non-toxic to 19 insect species of three orders,Lepidoptera, Diptera and Coleoptera, even though it is highly susceptible to tryptic cleavage.Study of flagellar (H) antibodies of 33 B. thuringiensis strains revealed that NTB-88 hasan H antigen identical with that of subsp. morrisoni (serotype 8a8b). Comparisonof parasporal inclusion proteins and plasmid DNA patterns of strain NTB-88 with B.thuringiensis subsp. morrisoni HD-12 and B. thuringiensis subsp. morrisoni PG-14 showed thatthe isolate is a novel non-insecticidal B. thuringiensis strain belonging to serotype 8a8b.

INTRODUCTION

The most widely used microbial insecticides are those basedon Bacillus thuringiensis, a Gram-positive, spore-forming soilbacterium that synthesizes a proteinaceous parasporalinclusion during the sporulation phase. Many crystalline pro-teins have been characterized by their entomopathogenicactivity, being highly specific in their activity against severalinsect orders. For several crystal-producing B. thuringiensisstrains, however, no toxic activity has yet been demonstrated(Ohba and Aizawa 1986 ; Martin and Travers 1989 ; Meadowset al. 1992). Moreover, most B. thuringiensis strains recentlyreported as new serotypes seem to be non-toxic to insects(Rodriguez-Padilla et al. 1990 ; Pietrantonio and Gill 1992 ;Lee et al. 1994 ; Burtseva et al. 1995 ; Jingyuan et al. 1996).In addition, non-toxic strains of B. thuringiensis serotype H14,generally known to be highly toxic to mosquito larvae, havealso been reported (Ohba et al. 1988). There are no reports ofnon-insecticidal B. thuringiensis isolates belonging to serotypeH8a8b in which Lepidoptera-, Diptera- and Coleoptera-active strains have been reported. The present paper reportsa B. thuringiensis strain, NTB-88, possessing non-insecticidalparasporal inclusions and flagellar antigen H8a8b.

Correspondence to : Dr Seok-Kwon Kang, Department of AgriculturalBiology, College of Agriculture & Life Sciences, Seoul NationalUniversity, Suwon 441–744, Korea.

© 1998 The Society for Applied Microbiology

MATERIALS AND METHODS

Bacterial strains and culture media

Bacillus thuringiensis strain NTB-88 was isolated from soilsamples in Korea by the method of Ohba and Aizawa (1978).Bacillus thuringiensis subsp. morrisoni HD-12 and B.thuringiensis subsp. morrisoni PG-14, used as references, werekindly provided by Dr M. Ohba (Institute of BiologicalControl, Kyushu University, Japan). For obtaining crystalproteins and for extraction of plasmid DNA of B. thuringiensis,GYS and SPY media were used (Nickerson et al. 1974 ;Kronstad et al. 1983).

Preparation of H antisera and serological tests

H-antigens and the corresponding antisera were preparedfrom the type strains of serotypes H1 to H27 (Ohba andAizawa 1978). H-antigenic properties were analysed by tubeagglutination reactions according to the method described byOhba et al. (1981).

Morphological observation

Morphology was studied using both scanning and trans-mission electron microscopes according to the methoddescribed by Park et al. (1995).

NON-INSECTICIDAL BACILLUS THURINGIENSIS STRAIN 63

Solubilization and trypsinization of Bacillusthuringiensis crystal proteins

Solubilization of B. thuringiensis crystals was achieved byincubation in 50 mmol l−1 NaOH at 37 °C for 30 min (Pie-trantonio and Gill 1992). Solubilized crystal proteins ofB. thuringiensis were subjected to trypsin treatment by incu-bation at 37 °C for 2 h. Trypsin Type XIII (Sigma) frombovine pancreas was treated with 1-tosylamido-2-phenyl(ethyl) chloromethylketone (TPCK) and dissolved in doubledistilled water (500 mg ml−1). Solubilized crystal proteinaliquots (100 ml) were then subjected to trypsinization in a 1 : 5(v : v) trypsin to protein solution ratio. Gut juice treatment ofstrain NTB-88 was performed with gut juice of fifth instarlarvae of Bombyx mori.

SDS-PAGE

Samples were prepared from B. thuringiensis strains culturedin GYS at 30 °C for 5 d. When cell lysis had occurred, theinclusions were harvested and resuspended in distilled water.The crystals were analysed for protein components by SDS-PAGE using a 12·5% separating gel (Laemmli 1970). Gelswere stained with 0·1% Coomassie brilliant blue (Sigma).

Plasmid DNA isolation

The plasmid DNA of B. thuringiensis strains were extractedby the alkaline lysis method (Kronstad et al. 1983). PlasmidDNAs were run on a 0·7% agarose gel (FMC).

Bioassays

Activity of B. thuringiensis strain NTB-88 crystals was testedby feeding to second instar larvae of the purplish cochlidAustrapoda dentatus, the brown-tail moth Euproctis similis,the mulberry pyralid Glyphodes pyloalis, the oriental mothMonema flavescens, the tea cochlid Phrixolepia sericea and thewhite tiger moth Spilosoma subcarneum, third instar larvae ofthe wild silkworm Bombyx mandarina, the silkworm B. mori,the narrow-winged prominent Fentonia ocypete, the fall web-worm Hyphantria cunea, the oriental tobacco budwormHelicoverpa assulta, the beet armyworm Spodoptera exigua andthe tobacco cutworm S. litura (Dulmage et al. 1971), thirdinstar larvae of mosquitoes Culex pipiens pallens and C.tritaeniorhynchus (McLaughlin et al. 1984), and fifth instarlarvae of the mulberry longicorn beetle Aporiona germari(Donovan et al. 1992). In addition, adults of the soybeanbeetle Anomala rufocuprea, the Japanese beetle Popillia mutansand the rice weevil Sitophilus oryzae were tested. For Lep-idoptera and Diptera, mortality was recorded after 5 d whilefor Coleoptera, mortality was recorded after 7 d. All bioassayswere repeated on three different days.

© 1998 The Society for Applied Microbiology, Letters in Applied Microbiology 27, 62–66

RESULTS AND DISCUSSION

Serological studies indicated that the H antigenic structureof isolate NTB-88 isolated from soil samples in Korea wasidentical to that of subsp. morrisoni (H8a8b). The classi-fication of B. thuringiensis into serovars has been very usefulbut it has some limitations and has led to some confusionamong B. thuringiensis researchers (de Barjac and Frachon1990). There is no correlation at all between serotype classi-fication and spectrum of insecticidal activity. Several strainsproducing the same crystal proteins with the same spectrumof activity belong to different serotypes. In contrast, themorrisoni serotype (H8a8b) harbours strains that are activeagainst Lepidoptera (strain HD-12), Diptera (strain PG-14)and Coleoptera (B. thuringiensis subsp. tenebrionis) (Norris1964 ; Padua et al. 1984 ; Krieg et al. 1987).

Bacillus thuringiensis strain NTB-88 produces typicalbipyramidal parasporal inclusions (Fig. 1). There were nosignificant differences in the shapes or sizes of vegetativecells, spores and parasporal inclusions of isolate NTB-88 andstrain HD-12. Interestingly, however, NTB-88 was not toxicto a range of insects including 13 lepidopteran, two dipteranand four coleopteran species, even at the highest dosage testedof 1×107 crystal forming units ml−1.

The parasporal inclusion proteins of isolate NTB-88 wereanalysed and compared with those of B. thuringiensis subsp.morrisoni HD-12 and B. thuringiensis subsp. morrisoni PG-14 (Fig. 2). SDS-PAGE analysis of the solubilized fractionshowed that the isolate has a major crystal protein with anestimated molecular mass of about 138 kDa in a 12·5% poly-acrylamide gel (Fig. 2a). Western blot analyses were per-formed with crystals of strains HD-12 and PG-14, usingantisera of NTB-88 crystal proteins. There was no significantpositive reaction between any crystal components and NTB-88 antisera (data not shown).

There have been several reports that non-toxic crystals ofB. thuringiensis have low solubility even at high pH. Crystalsof two non-toxic isolates from Japan that contain a majorprotein of 60 kDa and several minor proteins are insoluble in0·1 mol l−1 bicarbonate buffer, pH 10·2, after incubation at27 °C for 2 h (Ohba et al. 1987). These crystals dissolve onthe addition of silkworm gut juice but the solubility is muchless than that of crystals of other B. thuringiensis strains. Thecrystals of B. thuringiensis subsp. shandongiensis (H22), alsoknown to be non-insecticidal, are not soluble at pH 12 whilecrystals from most B. thuringiensis strains dissolve above pH9·5 (Pietrantonio and Gill 1992). Furthermore, Due et al.(1994) have reported insoluble non-toxic B. thuringiensisstrains with bipyramidal crystals containing about 130 kDaprotein. The toxicity of crystal proteins is dependent on thesolubility and interaction of structurally related protoxinswithin an inclusion, and probably play an important rolein determining ease of solubility under alkaline conditions.

64 H.-W. PARK ET AL.

Fig. 1 Electron micrographs of the isolateBacillus thuringiensis NTB-88. (a), Scanningelectron micrograph of the spore–crystalcomplex after autolysis. (b), Transmissionelectron micrograph of sporulating cellscontaining a parasporal crystal enclosedwithin the exosporium. The spore andcrystal are represented as S and C,respectively. Bar, 1·0 mm

Fig. 2 SDS-PAGE analysis of crystalproteins of Bacillus thuringiensisstandards and isolate NTB-88. (a), non-solubilized (lanes 2, 5 and 8), solubilized(lanes 3, 6 and 9) and trypsin-digested(lanes 4, 7 and 10) crystal proteins ofthe isolate NTB-88 (lanes 2, 3 and 4) ; B.thuringiensis subsp. morrisoni HD-12(lanes 5, 6 and 7) and B. thuringiensissubsp. morrisoni PG-14 (lanes 8, 9 and 10).(b), Crystal proteins of the isolate NTB-88 treated with Bombyx mori gut juiceaccording to the time interval, 1 min (lane3), 5 min (lane 4), 10 min (lane 5), 15min (lane 6) and 60 min (lane 7).Untreated crystals (lane 2) and B. morigut juice (lane 8) were also loaded ascontrols. Molecular masses are given to theleft in kDa

Comparison of the ability of strain NTB-88 crystals toundergo trypsinization showed that these crystals are fairlysusceptible to typtic cleavage, as are those of strains HD-12and PG-14. When the solubilized proteins were treated withtrypsin, the 138 kDa proteins of isolate NTB-88 underwenttrypsinization and produced a trypsin resistant fragment ofabout 70 kDa, which is slightly higher than the similar proteinfor strain HD-12. In addition, crystal proteins of isolateNTB-88, absolutely non-toxic to 19 insect species, treatedwith Bombyx mori gut juice underwent rapid proteolysis (Fig.2b). Crystals were also treated with gut juice from S. exigualarvae, and the same result was obtained (data not shown).These results suggest that the ‘non-soluble therefore non-toxic’ theory should be considered only for certain isolates ofB. thuringiensis.

Figure 3 shows the typical plasmid pattern of strain NTB-88 compared with those of control strains HD-12 and PG-14.This pattern showed only three plasmids, and some plasmid

© 1998 The Society for Applied Microbiology, Letters in Applied Microbiology 27, 62–66

comigration was observed with plasmids of both controlstrains. Strain NTB-88 had a plasmid of about 150 Mdal insize known to carry the crystal protein genes in strains HD-12 and PG-14 (Kronstad et al. 1983 ; Padua and Federici1990). However, the plasmid pattern of strain NTB-88 wastotally different from those of the control strains.

In conclusion, the characterization of the isolate NTB-88, which produces about 138 kDa crystal proteins highlysusceptible to tryptic cleavage and shows no activity against19 insect species, has demonstrated its uniqueness among B.thuringiensis strains that belong to serotype H8a8b.

ACKNOWLEDGEMENTS

This work received financial support from the ResearchCenter for New Bio-Materials in Agriculture, Seoul NationalUniversity and from the Ministry of Agriculture, Forestryand Fisheries (Grant No. 295000-0), Korea.

NON-INSECTICIDAL BACILLUS THURINGIENSIS STRAIN 65

Fig. 3 Plasmid patterns of the isolate NTB-88 (lane 1), Bacillusthuringiensis subsp. morrisoni HD-12 (lane 2) and B. thuringiensissubsp. morrisoni PG-14 (lane 3). M indicates Lambda DNAdigested with HindIII

REFERENCES

Burtseva, L.I., Burlak, V.A., Kalmikova, G.V., de Barjac, H. andLecadet, M.M. (1995) Bacillus thuringiensis novosibirsk (serotypeH24a24c), a new subspecies from the west Siberian plain. Journalof Invertebrate Pathology 66, 92–93.

de Barjac, H. and Frachon, E. (1990) Classification of Bacillusthuringiensis strains. Entomophaga 35, 233–240.

Donovan, W.P., Rupar, M.J., Slaney, A.C., Malvar, T., Gawron-Burke, M.C. and Johnson, T.B. (1992) Characterization of twogenes encoding Bacillus thuringiensis insecticidal crystal proteinstoxic to Coleoptera species. Applied and Environmental Micro-biology 58, 3921–3927.

Du, C., Martin, P.A.W. and Nickerson, K.W. (1994) Comparisonof desulfide contents and solubility of alkaline pH of insecticidaland noninsecticidal Bacillus thuringiensis protein crystals. Appliedand Environmental Microbiology 60, 3847–3853.

Dulmage, H.T., Boening, O.P., Rehnborg, C.S. and Hansen, G.D.(1971) A proposed standardized bioassay for formulations ofBacillus thuringiensis based on the International Unit. Journal ofInvertebrate Pathology 18, 240–245.

Jingyuan, D., Ling, Y., Bo, W., Xixia, L., Ziniu, Y. and Lecadet,M.M. (1996) Bacillus thuringiensis subspecies huazhongensis, ser-otype H40, isolated from soils in the People’s Republic of China.Letters in Applied Microbiology 22, 42–45.

Krieg, A., Schnetter, W., Huger, A.M. and Langenbruch, G.A.

© 1998 The Society for Applied Microbiology, Letters in Applied Microbiology 27, 62–66

(1987) Bacillus thuringiensis subsp. tenebrionis, strain B1256-82 : athird pathotype within the H-serotype 8a8b. Systematic andApplied Microbiology 9, 138–141.

Kronstad, J.W., Schnepf, H.E. and Whiteley, H.R. (1983) Diversityof locations for Bacillus thuringiensis crystal protein genes. Journalof Bacteriology 154, 419–428.

Laemmli, U.K. (1970) Cleavage of structural proteins during theassembly of the head of bacteriophage T4. Nature 227, 680–685.

Lee, H.H., Lee, J.A., Lee, K.Y. et al. (1994) New serovars of Bacillusthuringiensis : B. thuringiensis ser. coreanensis (Serotype H25), B.thuringiensis ser. leesis (Serotype H33), and B. thuringiensis ser.konkukian (Serotype H34). Journal of Invertebrate Pathology 63,217–219.

Martin, P.A.W. and Travers, R.S. (1989) Worldwide abundanceand distribution of Bacillus thuringiensis isolates. Applied andEnvironmental Microbiology 55, 2437–2442.

McLaughlin, R.E., Dulmage, H.T., Alls, R. et al. (1984) US stan-dard bioassay for the potency assessment of Bacillus thuringiensisserotype H-14 against mosquito larvae. Bulletin of the Ento-mological Society of America 30, 26–29.

Meadows, M.P., Ellis, D.J., Butt, J., Jarrett, P. and Burges, H.D.(1992) Distribution, frequency, and diversity of Bacillusthuringiensis in an animal feed mill. Applied and EnvironmentalMicrobiology 58, 1344–1350.

Nickerson, K.W., St. Julian, G. and Bulla Jr., L.A. (1974) Physi-ology of spore forming bacteria associated with insects : radio-respirometric survey of carbohydrate metabolism in the 12serotypes of Bacillus thuringiensis. Applied Microbiology 28, 129–132.

Norris, J.R. (1964) The classification of Bacillus thuringiensis. Journalof Applied Bacteriology 27, 439–447.

Ohba, M. and Aizawa, K. (1978) Serological identification of Bacillusthuringiensis and related bacteria isolated in Japan. Journal ofInvertebrate Pathology 32, 303–309.

Ohba, M. and Aizawa, K. (1986) Distribution of Bacillus thuringiensisin soils of Japan. Journal of Invertebrate Pathology 17, 277–282.

Ohba, M., Ono, K., Aizawa, K. and Iwanami, S. (1981) Two newsubspecies of Bacillus thuringiensis isolated in Japan : Bacillusthuringiensis subsp. kumamotoensis (Serotype 18) and Bacillusthuringiensis subsp. tochigiensis (Serotype 19). Journal of Invert-ebrate Pathology 38, 184–190.

Ohba, M., Yu, Y.M. and Aizawa, K. (1987) Non-toxic isolates ofBacillus thuringiensis producing parasporal inclusions withunusual protein components. Letters in Applied Microbiology 11,85–89.

Ohba, M., Yu, Y.M. and Aizawa, K. (1988) Occurrence of non-insecticidal Bacillus thuringiensis flagellar serotype 14 in the soilof Japan. Systematic and Applied Microbiology 11, 85–89.

Padua, L.E. and Federici, B.A. (1990) Development of mutantsof the mosquitocidal bacterium Bacillus thuringiensis subspeciesmorrisoni (PG-14) toxic to lepidopterous or dipterous insects.FEMS Microbiology Letters 66, 257–262.

Padua, L.E. Ohba, M. and Aizawa, K. (1984) Isolation of a Bacillusthuringiensis strain (serotype 8a:8b) highly and selectively toxicagainst mosquito larvae. Journal of Invertebrate Pathology 44, 12–17.

66 H.-W. PARK ET AL.

Park, H.W., Kim, H.S., Lee, D.W., Yu, Y.M., Jin, B.R. and KangS.K. (1995) Expression and synergistic effect of three types ofcrystal protein genes in Bacillus thuringiensis. Biochemical andBiophysical Research Communications 214, 602–607.

Pietrantonio, P.V. and Gill, S.S. (1992) The parasporal inclusionsof Bacillus thuringiensis subsp. shandongiensis : characterization andscreening for insecticidal activity. Journal of Invertebrate Path-ology 59, 295–302.

© 1998 The Society for Applied Microbiology, Letters in Applied Microbiology 27, 62–66

Rodriguez-Padilla, C., Galan-Wong, L., de Barjac, H., Roman-Calderon, E., Tamez-Guerra, R. and Dulmage, H. (1990) Bacillusthuringiensis subspecies neoleonensis serotype H-24, a new sub-species which produces a triangular crystal. Journal of InvertebratePathology 56, 280–282.