Isolation and Identification of novel toxins from a new mosquitocidal isolate from Malaysia, Bacillus thuringiensis subsp. jegathesan.

A new mosquitocidal Bacillus thuringiensis subsp., jegathesan, has recently been isolated from Malaysia. Parasporal crystal inclusions were purified from this strain and bioassayed against fourth-instar larvae of Culex quinquefasciatus, Aedes aegypti, Aedes togoi, Aedes albopictus, Anopheles maculatus, and Mansonia uniformis. The 50% lethal concentration of crystal inclusions for each species was 0.34, 8.08, 0.34, 17.59, 3.91, and 120 ng/ml, respectively. These values show that parasporal inclusions from this new subspecies have mosquitocidal toxicity comparable to that of inclusions isolated from B. thuringiensis subsp. israelensis. Solubilized and chymotrypsin-activated parasporal inclusions possessed low-level hemolytic activity. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that the crystals were composed of polypeptides of 77, 74, 72, 68, 55, 38, 35, 27, and 23 kDa. Analysis by Western blotting (immunoblotting) with polyclonal antisera raised against toxins purified from B. thuringiensis subsp. israelensis reveals that proteins in parasporal inclusions of subsp. jegathesan are distinct, because little cross-reactivity was shown. Analysis of the plasmid content of B. thuringiensis subsp. jegathesan indicates that the genes for toxin production may be located on 105- to 120-kb plasmids. Cry- clones that have been cured of these plasmids are nontoxic. Southern blot analysis of plasmid and chromosomal DNA from subsp. jegathesan showed little or low homology to the genes coding for CryIVA, CryIVB, and CryIVD from B. thuringiensis subsp. israelensis.     

Gene clusters located on two large plasmids determine spore crystal association (SCA) in Bacillus thuringiensis subsp. finitimus strain YBT-020

Crystals in Bacillus thuringiensis are usually formed in the mother cell compartment during sporulation and are separated from the spores after mother cell lysis. In a few strains, crystals are produced inside the exosporium and are associated with the spores after sporulation. This special phenotype, named 'spore crystal association' (SCA), typically occurs in B. thuringiensis subsp. finitimus. Our aim was to identify genes determining the SCA phenotype in B. thuringiensis subsp. finitimus strain YBT-020. Plasmid conjugation experiments indicated that the SCA phenotype in this strain was tightly linked with two large plasmids (pBMB26 and pBMB28). A shuttle bacterial artificial chromosome (BAC) library of strain YBT-020 was constructed. Six fragments from BAC clones were screened from this library and discovered to cover the full length of pBMB26; four others were found to cover pBMB28. Using fragment complementation testing, two fragments, each of approximately 35 kb and located on pBMB26 and pBMB28, were observed to recover the SCA phenotype in an acrystalliferous mutant, B. thuringiensis strain BMB171. Furthermore, deletion analysis indicated that the crystal protein gene cry26Aa from pBMB26, along with five genes from pBMB28, were indispensable to the SCA phenotype. Gene disruption and frame-shift mutation analyses revealed that two of the five genes from pBMB28, which showed low similarity to crystal proteins, determined the location of crystals inside the exosporium. Gene disruption revealed that the three remaining genes, similar to spore germination genes, contributed to the stability of the SCA phenotype in strain YBT-020. Our results thus identified the genes determining the SCA phenotype in B. thuringiensis subsp. finitimus.     

苏云金芽胞杆菌幕虫亚种晶胞粘连现象与晶体蛋白基因cry26Aa所在质粒有关

苏云金芽胞杆菌幕虫亚种T02菌株的伴胞晶体在芽胞外壁内侧形成,呈现晶胞粘连的现象。在此菌株中克隆了cry26 Aa和cry28 Aa两个基因,并对晶胞粘连现象与质粒的相关性做了系统研究。通过消除幕虫亚种T02菌株的质粒,得到了仅消除cry26 Aa所在质粒的菌株BMB1151和无质粒的菌株BMB1152。通过穿梭载体将cry26 Aa和cry28 Aa两个基因分别和同时转化无质粒突变株BMB1152并表达,形成的晶体与芽胞独立存在不能粘连,表明在幕虫亚种染色体背景下仅仅cry的表达不能形成晶胞粘连现象,从而推断晶胞粘连现象可能与幕虫亚种两个基因所在的质粒有关;进一步的研究发现将cry26 Aa在仅消除cry26 Aa所在质粒的突变株BMB1151中表达,形成的晶体与芽胞也分别独立存在不能粘连,从而进一步推断幕虫亚种晶胞粘连现象与cry26 Aa所在质粒有关。     

Properties of a 72-kilodalton mosquitocidal protein from Bacillus thuringiensis subsp. morrisoni PG-14 expressed in B. thuringiensis subsp. kurstaki by using the shuttle vector pHT3101.

The mosquitocidal properties of Bacillus thuringiensis subsp. israelensis and B. thuringiensis subsp. morrisoni PG-14 are attributable to protein inclusions grouped together within a parasporal body. In both of these strains, the mosquitocidal activity resides in proteins with molecular masses of 27, 72, 128, and 135 kDa. In an attempt to determine the toxicity of each protein, the shuttle vector pHT3101 was used to express the cryIVD gene (encoding the 72-kDa CryIVD protein) from B. thuringiensis subsp. morrisoni in an acrystalliferous mutant of B. thuringiensis subsp. kurstaki. With this system, parasporal inclusions of the 72-kDa protein were obtained that were comparable in size, shape, and toxicity to those produced by parental B. thuringiensis subsp. morrisoni. The inclusions were bar shaped, measured 500 by 300 by 150 nm, and were easily visible with phase-contrast microscopy by 16 h of cell growth. A 50% lethal concentration of 64 ng/ml for these inclusions was determined in bioassays against fourth instars of Culex quinquefasciatus, which was similar to the 50% lethal concentration of 55 ng/ml obtained for the 72-kDa inclusion from B. thuringiensis subsp. israelensis. In contrast, expression of the cryIVD gene in Escherichia coli was very low and only detectable by immunoblot analysis. These results demonstrate that the pHT3101-B. thuringiensis expression system can be used to express the CryIVD protein in quantities and with properties comparable to that obtained with the natural host. This system may prove useful for the expression of other B. thuringiensis proteins and, in particular, for reconstitution experiments with inclusions produced by the mosquitocidal subspecies of B. thuringiensis.     

Properties of a 72-kilodalton mosquitocidal protein from Bacillus thuringiensis subsp. morrisoni PG-14 expressed in B. thuringiensis subsp. kurstaki by using the shuttle vector pHT3101.

The mosquitocidal properties of Bacillus thuringiensis subsp. israelensis and B. thuringiensis subsp. morrisoni PG-14 are attributable to protein inclusions grouped together within a parasporal body. In both of these strains, the mosquitocidal activity resides in proteins with molecular masses of 27, 72, 128, and 135 kDa. In an attempt to determine the toxicity of each protein, the shuttle vector pHT3101 was used to express the cryIVD gene (encoding the 72-kDa CryIVD protein) from B. thuringiensis subsp. morrisoni in an acrystalliferous mutant of B. thuringiensis subsp. kurstaki. With this system, parasporal inclusions of the 72-kDa protein were obtained that were comparable in size, shape, and toxicity to those produced by parental B. thuringiensis subsp. morrisoni. The inclusions were bar shaped, measured 500 by 300 by 150 nm, and were easily visible with phase-contrast microscopy by 16 h of cell growth. A 50% lethal concentration of 64 ng/ml for these inclusions was determined in bioassays against fourth instars of Culex quinquefasciatus, which was similar to the 50% lethal concentration of 55 ng/ml obtained for the 72-kDa inclusion from B. thuringiensis subsp. israelensis. In contrast, expression of the cryIVD gene in Escherichia coli was very low and only detectable by immunoblot analysis. These results demonstrate that the pHT3101-B. thuringiensis expression system can be used to express the CryIVD protein in quantities and with properties comparable to that obtained with the natural host. This system may prove useful for the expression of other B. thuringiensis proteins and, in particular, for reconstitution experiments with inclusions produced by the mosquitocidal subspecies of B. thuringiensis.     

Identification of self-transmissible plasmids in four Bacillus thuringiensis subspecies.

The transfer of plasmids by mating from four Bacillus thuringiensis subspecies to Bacillus anthracis and Bacillus cereus recipients was monitored by selecting transcipients which acquired plasmid pBC16 (Tcr). Transcipients also inherited a specific large plasmid from each B. thuringiensis donor at a high frequency along with a random array of smaller plasmids. The large plasmids (ca. 50 to 120 megadaltons), pXO13, pXO14, pXO15, and pXO16, originating from B. thuringiensis subsp. morrisoni, B. thuringiensis subsp. toumanoffi, B. thuringiensis subsp. alesti, and B. thuringiensis subsp. israelensis, respectively, were demonstrated to be responsible for plasmid mobilization. Transcipients containing any of the above plasmids had donor capability, while B. thuringiensis strains cured of each of them were not fertile, indicating that the plasmids confer conjugation functions. Confirmation that pXO13, pXO14, and pXO16 were self-transmissible was obtained by the isolation of fertile B. anthracis and B. cereus transcipients that contained only pBC16 and one of these plasmids. pXO14 was efficient in mobilizing the toxin and capsule plasmids, pXO1 and pXO2, respectively, from B. anthracis transcipients to plasmid-cured B. anthracis or B. cereus recipients. DNA-DNA hybridization experiments suggested that DNA homology exists among pXO13, pXO14, and the B. thuringiensis subsp. thuringiensis conjugative plasmids pXO11 and pXO12. Matings performed between strains which each contained the same conjugative plasmid demonstrated reduced efficiency of pBC16 transfer. However, in many instances when donor and recipient strains contained different conjugative plasmids, the efficiency of pBC16 transfer appeared to be enhanced.     

A 54-kilodalton protein encoded by pBtoxis is required for parasporal body structural integrity in Bacillus thuringiensis subsp. israelensis

Strains of Bacillus thuringiensis such as B. thuringiensis subsp. israelensis (ONR-60A) and B. thuringiensis subsp. morrisoni (PG-14) pathogenic for mosquito larvae produce a complex parasporal body consisting of several protein endotoxins synthesized during sporulation that form an aggregate of crystalline inclusions bound together by a multilamellar fibrous matrix. Most studies of these strains focus on the molecular biology of the endotoxins, and although it is known that parasporal body structural integrity is important to achieving high toxicity, virtually nothing is known about the matrix that binds the toxin inclusions together. In the present study, we undertook a proteomic analysis of this matrix to identify proteins that potentially mediate assembly and stability of the parasporal body. In addition to fragments of their known major toxins, namely, Cry4Aa, Cry4Ba, Cry11Aa, and Cyt1Aa, we identified peptides with 100% identity to regions of Bt152, a protein coded for by pBtoxis of B. thuringiensis subsp. israelensis, the plasmid that encodes all endotoxins of this subspecies. As it is known that the Bt152 gene is expressed in B. thuringiensis subsp. israelensis, we disrupted its function and showed that inactivation destabilized the parasporal body matrix and, concomitantly, inclusion aggregation. Using fluorescence microscopy, we further demonstrate that Bt152 localizes to the parasporal body in both strains, is absent in other structural or soluble components of the cell, including the endospore and cytoplasm, and in ligand blots binds to purified multilamellar fibrous matrix. Together, the data show that Bt152 is essential for stability of the parasporal body of these strains.     

Transfer of chromosomal genes and plasmids in Bacillus thuringiensis.

A low frequency of chromosomal gene transfer from Bacillus thuringiensis to Bacillus cereus was detected by cell mating, with a tryptophan marker being the most frequently transferred gene among four that were tested. The process was resistant to DNase and was not mediated by cell filtrates. Among several B. thuringiensis subspecies tested, transfer was best with a derivative of B. thuringiensis subsp. kurstaki HD1, which lost several plasmids. All of the B. cereus recombinants contained at least one plasmid from the donor B. thuringiensis; frequently, it was a plasmid that encoded a protoxin gene. In matings with B. thuringiensis subsp. kurstaki HD1, a 29-megadalton plasmid that contained a ca. 2.5-kilobase region of homology with the chromosome was always transferred. No detectable transfer of chromosomal genes was found in B. thuringiensis subsp. kurstaki HD1 strains lacking this plasmid, suggesting that there may be chromosome mobilization.     

苏云金芽胞杆菌cry26Aa和cry28Aa基因共表达可在芽胞外壁内侧形成伴胞晶体

苏云金芽胞杆菌幕虫亚种的伴胞晶体在预芽胞外壁内侧形成,呈现晶体芽胞粘连的现象。根据已发表的cry26Aa1和cry28Aa1基因序列设计引物,从苏云金芽胞杆菌幕虫亚种T02中扩增得到cry26Aa和cry28Aa基因,通过穿梭载体将这两个基因分别和同时转化到苏云金芽胞杆菌无晶体突变株BMB171后,透射电镜下可在芽胞外壁内侧和外侧同时观察到伴胞晶体,而单独表达时可在芽胞外壁外侧观察到伴胞晶体。结果表明,伴胞晶体在芽胞外壁内侧表达不单独依赖于启动子的时空调控,可能还受到晶体蛋白相互作用的影响。     

Charactererization of novel non-toxic Bacillus thuringiensis isoloates from Korea

J.Y. ROH, H.W. PARK, B.R. JIN, H.S. KIM, Y.M. YU AND S.K. KANG. 1996. Four Bacillus thuringiensis isolates from soil samples produced parasporal inclusions which were non-toxic to insects. The isolates were named B. thuringiensis NTB-1, NTB-2, NTB-3 and NTB-4. The parasporal inclusions were shown to be ovoid by phase contrast and scanning electron microscopy. The serotypes of the four isolates were determined by agglutination using 33 antisera; NTB-1 and NTB-4 seemed to be subsp. isruelensis ,and NTB-2 seemed to be subsp. pondzcheriensis . NTB-3 did not react with the 33 antisera. However, comparison of parasporal protein and plasmid DNA patterns of the four isolates with those of 15 known non-toxic B. thuringiensis strains demonstrated that the four isolates are novel.     

类似S-层蛋白的苏云金芽胞杆菌伴胞晶体蛋白基因的克隆

芽胞杆菌CTC菌被鉴定为苏云金芽胞杆菌,鞭毛血清型H2,幕虫亚种;产生卵圆形伴胞晶体,伴胞晶体蛋白为100kD;测定了该蛋白 N－末端序列,该序列与炭疽芽胞杆菌的细胞表面S－层蛋白具92－93％相似性,根据Southern杂交制作了该晶体蛋白基因ctc所在位置的限制性酶切图谱,分别克隆了该基因5’和3’端所在2．9kb XbaI片段和3．1kb Cla I DNA片段,彼此间具0．6kb重叠,通过拼接获得含完整ctc基因的克隆,含该基因的大肠杆菌与表达S－层蛋白的大肠杆菌具相似生长特征,初步表明CTC菌株的伴胞晶体由细胞表面S－层蛋白组成,苏云金芽胞杆菌区别于蜡状芽胞杆菌和炭疽芽胞杆菌的唯一标准是能形成伴胞晶体,由于S－层是细胞表面的结构成分,本文对CTC菌株鉴定为苏云金芽胞杆菌以及伴胞晶体作为苏云金芽胞杆菌鉴别的唯一标准提出了质疑。     

Characterization of mosquitocidal activity of Bacillus thuringiensis subsp. fukuokaensis crystal proteins.

The mosquitocidal crystals of Bacillus thuringiensis subsp. fukuokaensis were isolated and bioassayed against fourth-instar larvae of two mosquito species. The 50% lethal concentration values of the crystals to Aedes aegypti and Culex quinquefasciatus were 4.1 and 2.9 micrograms/ml, respectively. In addition, the solubilized crystals had hemolytic activity; 50 micrograms/ml was the lowest detectable level. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis revealed that the crystals consisted of polypeptides of 90, 86, 82, 72, 50, 48, 37, and 27 kDa. When the solubilized inclusion was treated with C. quinquefasciatus midgut brush border membrane vesicles or Manduca sexta gut juice, only one major protein was detected. This protein retained mosquitocidal activity but had no detectable hemolytic activity. Immunological analysis of this subspecies and the subspecies israelensis, kyushuensis and darmstadiensis by using polyclonal antisera raised against the whole-crystal protein of B. thuringiensis subsp. fukuokaensis revealed that the proteins in subsp. fukuokaensis are distinct from proteins in the other subspecies because little cross-reaction was observed. Analysis of the plasmid pattern showed that the crystal protein genes are located on a plasmid of 130 MDa. Analysis of plasmid and chromosomal DNA from subsp. fukuokaensis showed little homology to the 72-kDa toxin gene (PG-14) of B. thuringiensis subsp. morrisoni. However, some of the proteins of B. thuringiensis subsp. fukuokaensis are homologous to other B. thuringiensis toxins because N-terminal amino acid analysis revealed that the 90-kDa protein is encoded by a cryIV gene type.     

Characterization of a new strain of Bacillus thuringiensis, a producer of an endotoxin against coleoptera

A new strain of Bacillus thuringiensis 2-7 was found to belong to the serotype H8. Cells of this strain contained irregular and flat crystalline inclusions and two large plasmids. The gene responsible for crystal formation is most likely located on the large plasmid greater than 105 MDa in size. Comparison of the cry gene of B. thuringiensis 2-7 and the cryIIIA gene of B. thuringiensis subsp. tenebrionis showed that their nucleotide sequences are identical.     

High-level cryIVD and cytA gene expression in Bacillus thuringiensis does not require the 20-kilodalton protein, and the coexpressed gene products are synergistic in their toxicity to mosquitoes.

Interactions among the 20-kDa protein gene and the cytA and cryIVD genes located in a 9.4-kb HindIII fragment were studied. A series of plasmids containing a combination of these different genes was constructed by using the Escherichia coli/Bacillus thuringiensis shuttle vector pHT3101. The plasmids were then used to transform an acrystalliferous strain, cryB, derived from B. thuringiensis subsp. kurstaki. The results from sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblot analyses suggest that although the 20-kDa protein is required for the efficient CytA protein production in E. coli, it is not required in B. thuringiensis. With or without the truncated 20-kDa protein gene, the CtyA and/or CryIVD proteins are produced and form parasporal inclusions in B. thuringiensis cells. However, more-efficient expression is obtained when a second protein, probably acting as a chaperonin, is present. In addition, the time course studies show that the CytA and CryIVD proteins are coordinately produced. Both the crude B. thuringiensis culture and purified inclusions from each recombinant B. thuringiensis strain are toxic to Culex quinquefasciatus larvae. The parasporal inclusions formed in B. thuringiensis cells are mosquitocidal, with CytA synergizing CryIVD toxicity.     

Characterization of mosquitocidal activity of Bacillus thuringiensis subsp. fukuokaensis crystal proteins

The mosquitocidal crystals of Bacillus thuringiensis subsp. fukuokaensis were isolated and bioassayed against fourth-instar larvae of two mosquito species. The 50% lethal concentration values of the crystals to Aedes aegypti and Culex quinquefasciatus were 4.1 and 2.9 micrograms/ml, respectively. In addition, the solubilized crystals had hemolytic activity; 50 micrograms/ml was the lowest detectable level. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis revealed that the crystals consisted of polypeptides of 90, 86, 82, 72, 50, 48, 37, and 27 kDa. When the solubilized inclusion was treated with C. quinquefasciatus midgut brush border membrane vesicles or Manduca sexta gut juice, only one major protein was detected. This protein retained mosquitocidal activity but had no detectable hemolytic activity. Immunological analysis of this subspecies and the subspecies israelensis, kyushuensis and darmstadiensis by using polyclonal antisera raised against the whole-crystal protein of B. thuringiensis subsp. fukuokaensis revealed that the proteins in subsp. fukuokaensis are distinct from proteins in the other subspecies because little cross-reaction was observed. Analysis of the plasmid pattern showed that the crystal protein genes are located on a plasmid of 130 MDa. Analysis of plasmid and chromosomal DNA from subsp. fukuokaensis showed little homology to the 72-kDa toxin gene (PG-14) of B. thuringiensis subsp. morrisoni. However, some of the proteins of B. thuringiensis subsp. fukuokaensis are homologous to other B. thuringiensis toxins because N-terminal amino acid analysis revealed that the 90-kDa protein is encoded by a cryIV gene type.     

Complete genome sequence of Bacillus thuringiensis serovar finitimus strain YBT-020

Bacillus thuringiensis is a gram-positive, spore-forming bacterium that forms parasporal crystals at the onset of the sporulation phase of its growth. Here, we report the complete genome sequence of B. thuringiensis serovar finitimus strain YBT-020, whose parasporal crystals consist of Cry26Aa and Cry28Aa crystal proteins and are located between the exosporium and the spore coat and remain adhering to the spore after sporulation.     

Regulation of protoxin synthesis in Bacillus thuringiensis.

A derivative of Bacillus thuringiensis subsp. kurstaki (HD-1) formed parasporal inclusions at 25 degrees C, but not at 32 degrees C. This strain differed from the parent only in the loss of a 110-megadalton (Md) plasmid, but plasmid and chromosomal copies of protoxin genes were present in both strains. On the basis of temperature shift experiments, the sensitive period appeared to be during midexponential growth, long before the time of protoxin synthesis at 3 to 4 h after the end of exponential growth. The conditional phenotype could be transferred by cell mating to naturally acrystalliferous Bacillus cereus. In all such cases, a 29-Md protoxin -encoding plasmid was transferred, but this plasmid alone was barely sufficient for protoxin synthesis. Protoxin production increased to detectable levels, but well below those of the parental donor strain, by simultaneous transfer of a 44-Md protoxin -encoding plasmid. Transfer of a 5-Md plasmid with the two larger protoxin -coding plasmids resulted in a protoxin synthesis level approaching that of the donor strain. A role for some of the cryptic plasmids of kurstaki in parasporal body formation was implied. In contrast, a closely related B. thuringiensis strain, HD73 , produced crystals at both 25 and 32 degrees C even when the capacity was transferred on a 50-Md plasmid to B. cereus. The amount of protoxin produced in these B. cereus transcipients , however, was somewhat less than that produced in the parental strain HD73 , implying that catabolic differences, gene dosage, or the presence of a chromosomal gene (or a combination of these) may be necessary for maximum production. A regulatory component of the 29-Md plasmid appeared to be trans-acting and dominant since B. cereus transcipients containing the 29-Md plasmid from kurstaki and the 50-Md plasmid from HD73 produced more protoxin at 25 degrees C than at 30 degrees C. Similar results were obtained when protoxin synthetic capacity was transferred from B. thuringiensis subsp. israelensis to the conditional B. thuringiensis subsp. kurstaki strain.     

Transfer of chromosomal genes and plasmids in Bacillus thuringiensis

A low frequency of chromosomal gene transfer from Bacillus thuringiensis to Bacillus cereus was detected by cell mating, with a tryptophan marker being the most frequently transferred gene among four that were tested. The process was resistant to DNase and was not mediated by cell filtrates. Among several B. thuringiensis subspecies tested, transfer was best with a derivative of B. thuringiensis subsp. kurstaki HD1, which lost several plasmids. All of the B. cereus recombinants contained at least one plasmid from the donor B. thuringiensis; frequently, it was a plasmid that encoded a protoxin gene. In matings with B. thuringiensis subsp. kurstaki HD1, a 29-megadalton plasmid that contained a ca. 2.5-kilobase region of homology with the chromosome was always transferred. No detectable transfer of chromosomal genes was found in B. thuringiensis subsp. kurstaki HD1 strains lacking this plasmid, suggesting that there may be chromosome mobilization.     

Fine structure of the Baccilus thuringiensis spore.

The thin-sectioned spore of Bacillus thuringiensis resembles that of Bacillus cereus in fine structure. Planar inclusions occur between the exosporium and spore coat and are structured differently from the parasporal crystal outside the exosporium.     

Isolation of a non-insecticidal Bacillus thuringiensis strain belonging to serotype H8a8b

Bacillus thuringiensis strains non-toxic to Lepidoptera, Bombyx mori and Diptera, Culex pipiens pallens larvae were isolated from Korean soil samples during an investigation of B. thuringiensis isolates highly toxic to insect pests. One of these isolates, NTB-88, produces parasporal inclusions 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 has an H antigen identical with that of subsp. morrisoni (serotype 8a8b). Comparison of 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 that the isolate is a novel non-insecticidal B. thuringiensis strain belonging to serotype 8a8b.