Gene knockout demonstrates that vip3A contributes to the pathogenesis of Bacillus thuringiensis toward Agrotis ipsilon and Spodoptera exigua.

Vip3A is an 89-kDa protein secreted by Bacillus thuringiensis during vegetative growth. To determine the importance of Vip3A for the insect pathogenicity of B. thuringiensis the vip3A gene was deleted from strain HD1, yielding strain HD1Deltavip3A. Compared with HD1, strain HD1Deltavip3A was one-fourth as toxic to Agrotis ipsilon larvae and less than one-tenth as toxic to Spodoptera exigua larvae. When streptomycin was included in the S. exigua diet the toxicity of HD1Deltavip3A was approximately half that of HD1. Addition of HD1 spores increased the toxicity of purified Cry1 protein more than 600-fold against S. exigua, whereas addition of HD1Deltavip3A spores increased toxicity of Cry1 protein approximately 10-fold. These results demonstrate that an important component of B. thuringiensis insecticidal activity against S. exigua is the synthesis of Vip3A protein by B. thuringiensis cells after ingestion of spores and crystal proteins by insect larvae.

Deletion of aprA and nprA genes for alkaline protease A and neutral protease A from bacillus thuringiensis: effect on insecticidal crystal proteins.

The aprA gene encoding alkaline protease A (AprA) was cloned from Bacillus thuringiensis subsp. kurstaki, and the cloned gene was used to construct aprA-deleted (aprA1) strains of B. thuringiensis. An aprA1 strain of B. thuringiensis that contained the wild-type gene for neutral protease A (nprA(+)) displayed levels of extracellular proteolytic activity that were similar to those of an aprA(+)nprA(+) strain. However, when EDTA was included in the protease assay to inhibit NprA activity the aprA1nprA(+) strain displayed only 2% of the extracellular proteolytic activity of the aprA(+)nprA(+) strain. A strain that was deleted for both aprA and nprA (aprA1nprA3 strain) failed to produce detectable levels of proteolytic activity either in the presence or absence of EDTA in the assay. Compared with the aprA(+)nprA(+) strain the aprA1nprA(+) strain yielded 10% more full-length Cry1Bb crystal protein and the aprA1nprA3 strain yielded 25% more full-length Cry1Bb protein. No significant differences were seen in the 50% lethal dose of Cry1Bb protein from aprA(+)nprA(+) and aprA1nprA3 strains against three species of lepidopteran insects. These results suggest that enhanced yield of certain crystal proteins can be obtained by deletion of the genes aprA and nprA which are the major extracellular proteases of B. thuringiensis.

Cloning of the nprA gene for neutral protease A of Bacillus thuringiensis and effect of in vivo deletion of nprA on insecticidal crystal protein.

The nprA gene, encoding Bacillus thuringiensis neutral protease A, was cloned by the use of gene-specific oligonucleotides. The size of neutral protease A deduced from the nprA sequence was 566 amino acids (60,982 Da). The cloned nprA gene was partially deleted in vitro, and the deleted allele, designated nprA3, was used to construct an nprA3 strain (neutral protease A-deficient strain) of B. thuringiensis. Growth and sporulation of the nprA3 strain were similar to those of an isogenic nprA+ strain, although the extracellular proteolytic activity of the nprA3 strain was significantly less than that of the nprA+ strain. The nprA3 strain produced insecticidal crystal proteins that were more stable than those of the isogenic nprA+ strain after solubilization in vitro, and sporulated cultures of the nprA3 strain contained higher concentrations of full-length insecticidal crystal proteins than did those of its isogenic counterpart. The absence of neutral protease A did not affect the insecticidal activity of a lepidopteran-specific crystal protein of B. thuringiensis. These results indicate that crystal protein stability and yield may be improved by deletion of specific proteases from B. thuringiensis.

Characterization of two genes encoding Bacillus thuringiensis insecticidal crystal proteins toxic to Coleoptera species.

Bacillus thuringiensis EG2838 and EG4961 are highly toxic to Colorado potato beetle larvae, and only strain EG4961 is toxic to southern corn rootworm larvae. To investigate the cause of the different insecticidal activities of EG2838 and EG4961, cryIII-type genes toxic to coleopterans were cloned from each strain. The cryIIIB gene, cloned as part of an 8.0-kb EcoRI fragment of EG2838 DNA, encoded a crystal protein (CryIIIB) of 74,237 Da. The cryIIIB2 gene, cloned as part of an 8.3-kb PstI-Asp718 fragment of EG4961 DNA, encoded a crystal protein (CryIIIB2) of 74,393 Da that was 94% identical to CryIIIB. Analysis of the transcriptional start sites showed that cryIIIB and cryIIIB2 were initiated from a conserved region located within 130 nucleotides upstream from the translation start sites of both genes. Although the CryIIIB and CryIIIB2 proteins were similar in sequence, they displayed distinct insecticidal activities: CryIIIB was one-third as toxic as CryIIIB2 to Colorado potato beetle larvae, and CryIIIB2, but not CryIIIB, was toxic to southern corn rootworm larvae. Genes encoding crystal proteins of approximately 32 and 31 kDa were located adjacent to the cryIIIB and cryIIIB2 genes, respectively. The 32- and 31-kDa crystal proteins failed to enhance the insecticidal activities of CryIIIB and CryIIIB2.

Two novel strains of Bacillus thuringiensis toxic to coleopterans.

Two novel strains of Bacillus thuringiensis were isolated from native habitats by the use of genes coding for proteins toxic to coleopterans (cryIII genes) as hybridization probes. Strain EG2838 (isolated by the use of the cryIIIA probe) contained a cryIIIA-hybridizing plasmid of approximately 100 MDa and synthesized crystal proteins of approximately 200 (doublet), 74, 70, 32, and 28 kDa. Strain EG4961 (isolated by the use of a cryIIIA-related probe) contained a cryIIIA-hybridizing plasmid of approximately 95 MDa and synthesized crystal proteins of 74, 70, and 30 kDa. Structural relationships among the crystal proteins of strains EG2838 and EG4961 were detected; antibodies to the CryIIIA protein toxic to coleopterans reacted with the 74- and 70-kDa proteins of EG2838 and EG4961, antibodies to the 32-kDa plus 28-kDa proteins of EG2838 reacted with the 30-kDa protein of EG4961, and antibodies to the 200-kDa proteins of EG2838 reacted with the 28-kDa protein of EG2838. Experiments with B. thuringiensis flagella antibody reagents demonstrated that EG2838 belongs to H serotype 9 (reference strain B. thuringiensis subsp. tolworthi) and that EG4961 belongs to H serotype 18 (reference strain B. thuringiensis subsp. kumamotoensis). A mixture of spores plus crystal proteins of either EG2838 or EG4961 was toxic to the larvae of Colorado potato beetle (Leptinotarsa decemlineata), and significantly, the EG4961 mixture was also toxic to the larvae of southern corn rootworm (Diabrotica undecimpunctata howardi). DNA restriction blot analysis suggested that strains EG2838 and EG4961 each contained a unique gene coding for a protein toxic to coleopterans.

Activation of a cryptic crystal protein gene of Bacillus thuringiensis subspecies kurstaki by gene fusion and determination of the crystal protein insecticidal specificity.

DNA hybridization with the insecticidal crystal protein gene cryllA (formerly cryBl) of Bacillus thuringiensis supspecies kurstaki has shown that subspecies kurstaki contains a cryllA-related sequence in addition to the cryllA gene (Donovan et al., 1988a). We have cloned the cryllA-related sequence and have determined that the sequence, which has been designated cryllB, is 89% identical to the cryllA gene. Recombinant B. thuringiensis cells harbouring the cloned cryllB gene produced very little CryllB protein. A high level of production of the CryllB protein was achieved by fusing the regulatory region of the crylllA crystal protein gene to the cryllB gene. The CryllB protein was found to be highly toxic to Lymantria dispar, Heliothis virescens and Trichoplusia ni, and was not toxic to Aedes aegypti.

Isolation and characterization of EG2158, a new strain of Bacillus thuringiensis toxic to coleopteran larvae, and nucleotide sequence of the toxin gene.

A novel strain of Bacillus thuringiensis was isolated from soybean grain dust from Kansas and found to be toxic to larvae of Leptinotarsa decemlineata (Colorado potato beetle). The strain (EG2158) synthesized two parasporal crystals: a rhomboid crystal composed of a 73116 dalton protein of approximately 30 kDa. Plasmid transfer and gene cloning experiments demonstrated that the 73 kDa protein was encoded on an 88 MDa plasmid and that the protein was toxic to the larvae of Colorado potato beetle (CPB). The sequence of the 73 kDa protein, as deduced from the sequence of its gene (cryC), was found to have regions of similarity with several B. thuringiensis crystal proteins: the lepidopteran-toxic P1 proteins of var. kurstaki and berliner, the lepidopteran- and dipteran-toxic P2 (or CRYB1) protein of var. kurstaki, and the dipteran-toxic 130 kDa protein of var. israelensis. While B. megaterium cells harboring the cryC gene from EG2158 synthesized significant amounts of the 73 kDa CRYC protein, Escherichia coli cells did not. The cryC-containing B. megaterium cells produced rhomboid crystals that were toxic to CPB larvae.

Molecular characterization of a gene encoding a 72-kilodalton mosquito-toxic crystal protein from Bacillus thuringiensis subsp. israelensis.

A gene encoding a 72,357-dalton (Da) crystal protein of Bacillus thuringiensis var. israelensis was isolated from a native 75-MDa plasmid by the use of a gene-specific oligonucleotide probe. Bacillus megaterium cells harboring the cloned gene (cryD) produced significant amounts of the 72-kDa protein (CryD), and the cells were highly toxic to mosquito larvae. In contrast, cryD-containing Escherichia coli cells did not produce detectable levels of the 72-kDa CryD protein. The sequence of the CryD protein, as deduced from the sequence of the cryD gene, was found to contain regions of homology with two previously described B. thuringiensis crystal proteins: a 73-kDa coleopteran-toxic protein and a 66-kDa lepidopteran- and dipteran-toxic protein of B. thuringiensis subsp. kurstaki. A second gene encoding the B. thuringiensis subsp. israelensis 28-kDa crystal protein was located approximately 1.5 kilobases upstream from and in the opposite orientation to the cryD gene.

Gene encoding a morphogenic protein required in the assembly of the outer coat of the Bacillus subtilis endospore.

Endospores of Bacillus subtilis are encased in a two-layer protein shell known as the coat, which consists of a lammellar-like inner layer and an electron-dense outer layer. We report the cloning of the structural gene (designated cotE) for an alkali-soluble coat protein of 24 kD and show that the cotE gene product is a morphogenic protein required in the assembly of the outer coat. The nucleotide sequence of cotE reveals an open reading frame capable of encoding a 181-residue-long polypeptide of 21 kD. A cotE mutant was created by replacing the chromosomal gene, which was located at 145 degrees on the chromosome, with an in vitro constructed, deletion-mutated gene. The resulting cotE mutant formed normal-looking (optically refractile) spores that were heat resistant but were sensitive to lysozyme and somewhat impaired in germination. Ultrastructural analysis indicated that the mutant spores lacked the electron-dense outer layer of the coat but retained a normal-looking inner coat. The mutant spores were pleiotropically deficient in several coat proteins, including the product of cotE and the products of previously cloned cot genes A-C. Based on experiments in which expression of the cotA and cotC genes was found to be unimpaired in cotE mutant cells, we infer that the cotE gene product is involved in the assembly of the products of cotA-cotC, and certain other proteins into the electron-dense outer layer of the coat.

Amino acid sequence and entomocidal activity of the P2 crystal protein. An insect toxin from Bacillus thuringiensis var. kurstaki.

The gene encoding the 66-kDa entomocidal protein (P2 protein or mosquito factor) from Bacillus thuringiensis var. kurstaki has been isolated by the use of a 62-mer oligonucleotide probe that encoded 21 amino acids of the P2 protein NH2 terminus. The DNA sequence of the gene, designated cryBI, was unique from the published sequences of other B. thuringiensis genes. However, the amino acid sequence of the P2 protein, as deduced from the DNA sequence of the cryBI gene, was found to contain a sequence of 100 amino acids having 37% homology to a group of B. thuringiensis entomocidal proteins, the P1 proteins. Late stationary phase Bacillus megaterium cells harboring the cloned B. thuringiensis cryBI gene contained large aggregates of the P2 protein, and the cells were highly toxic to both lepidopteran and dipteran larvae. In contrast, Escherichia coli cells harboring the cloned cryBI gene contained very low levels of the P2 protein. DNA blot hybridization experiments showed that certain B. thuringiensis strains contained at least one cryBI-related DNA sequence in addition to the cryBI gene itself.

Polynucleotide phosphorylase and ribonuclease II are required for cell viability and mRNA turnover in Escherichia coli K-12.

The isolation of a temperature-sensitive allele of RNase II (rnb) by in vitro mutagenesis has permitted the demonstration that RNase II and polynucleotide phosphorylase (PNPase) are required for cell viability and mRNA turnover in Escherichia coli. Double-mutant strains carrying the pnp-7 and rnb-500 alleles (PNPase deficient and RNase II thermolabile) ceased growing in Luria broth within 30 min after shift to the nonpermissive temperature. Cessation of growth was accompanied by an accumulation of mRNA fragments 100-1500 nucleotides long. In contrast, single-mutant and wild-type control strains grew normally at the nonpermissive temperature and did not accumulate mRNA. No significant changes in rRNA patterns were observed in any of the strains.

Purification and characterization of orotidine-5'-phosphate decarboxylase from Escherichia coli K-12.

Using blue Sepharose affinity chromatography, we purified orotidine-5'-phosphate decarboxylase over 600-fold, to near homogeneity, from strains of Escherichia coli harboring the cloned pyrF gene on the multicopy plasmid pDK26. The purified enzyme has a subunit molecular weight of 27,000 but appears to be catalytically active as a dimer. In contrast to yeast enzymes, orotidine-5'-phosphate decarboxylase from E. coli is unstable at pH 6.0. The specific activity and Km values were 220 U/mg and 6 microM, respectively.

Cloning and physical analysis of the pyrF gene (coding for orotidine-5'-phosphate decarboxylase) from Escherichia coli K-12.

The structural gene (pyrF) for orotidine-5'-phosphate decarboxylase (OMPase, EC 4.1.1.23) of Escherichia coli K-12 has been cloned as part of two PvuII fragments (1.2 and 0.9 kb) to form the recombinant plasmid pDK26. Extracts of E. coli [pDK26] had 80-fold higher levels of OMPase activity than wild-type strains without the plasmid. Maxicell analysis showed that pDK26 encoded two proteins of Mr 27 000 [pyrF(OMPase)] and 15 000 (Z) in addition to the ampicillin-resistance determinant. The approximate initiation site and direction of transcription of the pyrF gene have been determined. Extracts of strains that were deficient in polynucleotide phosphorylase (PNPase) had higher levels of OMPase activity than isogenic PNPase+ strains when one or two copies of the pyrF gene were present per cell either in the chromosome or on a low copy number plasmid. However, no significant difference in OMPase activity was seen in PNPase- strains that contained the pyrF gene cloned in a multicopy plasmid. Southern hybridization experiments showed that the yeast gene for OMPase (URA3) and the E. coli pyrF gene had less than 70% DNA sequence homology.

Amplification of ribonuclease II (rnb) activity in Escherichia coli K-12.

A 7.1 kb HindIII-XhoI fragment of E. coli DNA which contains the structural gene for ribonuclease II (rnb) has been cloned in the recombinant plasmid pDK24. At least two constitutively expressed genes are encoded on the fragment as shown by maxicell analysis. On denaturing polyacrylamide gels RNase II appears as a single 72,000 dalton species. The approximate site of transcription initiation of the rnb gene has been mapped. Although derivatives of E. coli harboring pDK24 contained 10-fold more RNase II activity that wild type strains without the plasmid, the degradation rate of mRNA was similar in all strains tested. Strains deficient in both RNase II and polynucleotide phosphorylase appear inviable.