Tn5401 disruption of the spo0F gene, identified by direct chromosomal sequencing, results in CryIIIA overproduction in Bacillus thuringiensis.

The Bacillus thuringiensis spo0F gene was identified by chromosomal DNA sequencing of sporulation mutants derived from a B. thuringiensis transposon insertion library. A spo0F defect in B. thuringiensis, which was suppressed by multicopy hknA or kinA, resulted in the overproduction of the CryIIIA insecticidal crystal protein.     

Increase of the Bacillus thuringiensis Secreted Toxicity Against Lepidopteron Larvae by Homologous Expression of the vip3LB Gene During Sporulation Stage

The Vegetative insecticidal Vip3A proteins display a wide range of insecticidal spectrum against several agricultural insect pests. The fact that the expression of vip3 genes occurs only during the vegetative growth phase of Bacillus thuringiensis is a limiting factor in term of production level. Therefore, extending the synthesis of the Vip proteins to the sporulation phase is a good alternative to reach high levels of toxin synthesis. In this study, we have demonstrated that the maximal production of the secreted Vip3LB (also called Vip3Aa16) during the growth of the wild-type strain B. thuringiensis BUPM 95 is reached at the end of the vegetative growth phase, and that the protein remains relatively stable in the culture supernatant during the late sporulation stages. The vip3LB gene was cloned and expressed under the control of the sporulation dependant promoters BtI and BtII in B. thuringiensis BUPM 106 (Vip3(-)) and BUPM 95 (Vip3(+)) strains. The examination of the culture supernatants during the sporulation phase evidenced the synthesis of Vip3LB and its toxicity against the second-instars larvae of the Lepidopteron insect Spodoptera littoralis for the recombinant BUPM 106. Moreover, there was an increase of the Vip3LB synthesis level and an enhancement of the oral toxicity for the recombinant BUPM 95 resulting from the expression of the vip3LB gene during both the vegetative and sporulation phases and the relative stability of the Vip3LB protein.     

苏云金芽孢杆菌芽孢萌发相关基因gerM的克隆及功能研究

依照蜡状芽孢杆菌gerM基因的保守序列设计引物,从苏云金芽孢杆菌中扩增出640bp的DNA片段。以此为探针,从苏云金芽孢杆菌部分基因组酶切文库中成功地克隆到了一个4·5kb的DNA片段。序列分析表明,该片段包含一个完整的开放阅读框,其预测的编码产物与枯草芽孢杆菌GerM蛋白具有很高的同源性,将该基因命名为gerM。RT-PCR分析表明,gerM基因仅在芽孢形成的过程中表达。通过同源重组的策略构建了gerM基因的阻断突变株。研究表明,gerM基因的破坏影响苏云金芽孢杆菌芽孢萌发的速率和比例。     

Expression in Bacillus subtilis of the Bacillus thuringiensis cryIIIA toxin gene is not dependent on a sporulation-specific sigma factor and is increased in a spo0A mutant.

Expression of the Bacillus thuringiensis cryIIIA gene encoding a Coleoptera-specific toxin is weak during vegetative growth and is activated at the onset of the stationary phase. cryIIIA'-'lacZ fusions and primer extension analysis show that the regulation of cryIIIA expression is similar in Bacillus subtilis and in B. thuringiensis. Activation of cryIIIA expression was not altered in B. subtilis mutant strains deficient for the sigma H and sigma E sporulation-specific sigma factors or for minor sigma factors such as sigma B, sigma D, or sigma L. This result and the nucleotide sequence of the -35 and -10 regions of the cryIIIA promoter suggest that cryIIIA expression might be directed by the E sigma A form of RNA polymerase. Expression of the cryIIIA'-'lacZ fusion is shut off after t2 (2 h after time zero) of sporulation in the B. subtilis wild-type strain grown on nutrient broth sporulation medium. However, no decrease in cryIIIA-directed beta-galactosidase activity occurred in sigma H, kinA, or spo0A mutant strains. Moreover, beta-galactosidase activity was higher and remained elevated after t2 in the spo0A mutant strain. beta-Galactosidase activity was weak in abrB and spo0A abrB mutant strains, suggesting that AbrB is responsible for the higher level of cryIIIA expression observed in a spo0A mutant. However, both in spo0A and spo0A abrB mutant strains, beta-galactosidase activity remained elevated after t2, suggesting that even in the absence of AbrB, cryIIIA expression is controlled through modulation of the phosphorylated form of Spo0A. When the cryIIIA gene is expressed in a B. subtilis spo0A mutant strain or in the 168 wild-type strain, large amounts of toxins are produced and accumulate to form a flat rectangular crystal characteristic of the coleopteran-specific B. thuringiensis strains.     

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.     

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.     

Construction of Bacillus thuringiensis wild-type S76 and Cry- derivatives expressing a green fluorescent protein: two potential marker organisms to study bacteria-plant interactions

Collectively, the species Bacillus thuringiensis, Bacillus cereus, and Bacillus anthracis represent microorganisms of high economic, medical, and biodefense importance. Although the genetic correlation and pathogenic characteristics have been extensively dissected, the ecological properties of these three species in their natural environments remain poorly understood. Thus, a tractable marker for detecting these bacteria under specific environmental and physiological conditions is a valuable tool. With this purpose, a plasmid (pAD43-25) carrying a functional gfp gene sequence (gfpmut3A) was introduced into the wild-type strain Bacillus thuringiensis subsp. kurstaki S76, which bears approximately 11 plasmids, allowing constitutive synthesis of green fluorescent protein (GFP) during vegetative growth (strain S76GFP+). Additionally, this vector was transferred to a plasmid-cured (Cry-) B. thuringiensis host. Bright green cells were detected by fluorescence microscopy in both recombinants by 2 h after inoculation in liquid medium and could be seen throughout the remaining cultivation time until complete sporulation was accomplished. For strain S76GFP+ protein profile and plasmid DNA analyses indicate, respectively, that this recombinant maintained Cry proteins expression and resident plasmid outline. Thus, in addition to the potential of strain S76GFP+ as a marker organism in bacteria-plant interaction studies, the production and stability of active GFPmut3a make this unique expression system a useful experimental model to study adaptive changes of host-plasmid as well as plasmid-plasmid relationships in a population of cells stressed by the production of a recombinant protein.     

Characterization of csh203::Tn917lac, a mutation in Bacillus subtilis that makes the sporulation sigma factor sigma-H essential for normal vegetative growth.

spo0H encodes a sigma factor, sigma-H, of RNA polymerase that is required for sporulation in Bacillus subtilis. Null mutations in spo0H block the initiation of sporulation but have no obvious effect on vegetative growth. We have characterized an insertion mutation, csh203::Tn917lac, that makes spo0H essential for normal growth. In otherwise wild-type cells, the csh203::Tn917lac insertion mutation has no obvious effect on cell growth, viability, or sporulation. However, in combination with a mutation in spo0H, the csh203 mutation causes a defect in vegetative growth. The csh203::Tn917lac insertion mutation was found to be located within orf23, the first gene of the rpoD (sigma-A) operon. The transposon insertion separates the major vegetative promoters P1 and P2 from the coding regions of two essential genes, dnaG (encoding DNA primase) and rpoD (encoding the major sigma factor, sigma-A) and leaves these genes under the control of minor promoters, including P4, a promoter controlled by sigma-H. The chs203 insertion mutation caused a 2- to 10-fold increase in expression of promoters recognized by RNA polymerase containing sigma-H. The increased expression of genes controlled by sigma-H in the csh203 single mutant, as well as the growth defect of the csh203 spo0H double mutant, was due to effects on rpoD and not to a defect in orf23 or dnaG.     

Vegetative expression of the delta-endotoxin genes of Bacillus thuringiensis subsp. kurstaki in Bacillus subtilis.

Bacillus thuringiensis subsp. kurstaki total DNA was digested with BglII and cloned into the BamHI site of plasmid pUC9 in Escherichia coli. A recombinant plasmid, pHBHE, expressed a protein of 135,000 daltons that was toxic to caterpillars. A HincII-SmaI double digest of pHBHE was then ligated to BglII-cut plasmid pBD64 and introduced into Bacillus subtilis by transformation. The transformants were identified by colony hybridization and confirmed by Southern blot hybridization. A 135,000-dalton protein which bound to an antibody specific for the crystal protein of B. thuringiensis was detected from the B. subtilis clones containing the toxin gene insert in either orientation. A toxin gene insert cloned into a PvuII site distal from the two drug resistance genes of the pBD64 vector also expressed a 135,000-dalton protein. These results suggest that the toxin gene is transcribed from its own promoter. Western blotting of proteins expressed at various stages of growth revealed that the crystal protein expression in B. subtilis begins early in the vegetative phase, while in B. thuringiensis it is concomitant with the onset of sporulation. The cloned genes when transferred to a nonsporulating strain of B. subtilis also expressed a 135,000-dalton protein. These results suggest that toxin gene expression in B. subtilis is independent of sporulation. Another toxin gene encoding a 130,000- to 135,000-dalton protein was cloned in E. coli from a library of B. thuringiensis genes established in lambda 1059. This gene was then subcloned in B. subtilis. The cell extracts from both clones were toxic to caterpillars. Electron microscope studies revealed the presence of an irregular crystal inclusion in E. coli and a well-formed bipyramidal crystal in B. subtilis clones similar to the crystals found in B. thuringiensis.     

Characterization study of the sporulation kinetics of Bacillus thuringiensis

A wild-type and an rDNA strain of Bacillus thuringiensis were cultured in a net-draft-tube modified 20-L airlift bioreactor. A comparison of the sporulation patterns suggests that the early sporulation strain has a lower final spore count. Results from off-gas analysis suggests that the CO(2) profile could be an alternative indication to spore counts for the examination of fermentation performance or even the mortality in bioassay of the cultivation product. The difference in mortality tests exhibited by the microorganism was attributed to different patterns of sporulation as well as different levels of gene control inside the cell itself. The sporulation kinetics of B. thuringiensis was simulated by a simple modified Hill equation, where the initial glucose concentration could affect the timing of the onset of sporulation. The equation matches well with the experimental sporulation data for B. thuringiensis in both wild-type and rDNA strains.     

Expression of the mel gene from Pseudomonas maltophilia in Bacillus thuringiensis

AIMS: The objective of this work was to express a novel mel gene, responsible for melanin formation, in Bacillus thuringiensis. METHODS AND RESULTS: A novel mel gene from Pseudomonas maltophilia was sub-cloned into B. thuringiensis using a shuttle vector plasmid and electroporation. Results revealed that the mel gene was expressed under the control of the CryIIIA promoter in B. thuringiensis and conferred u.v. protection on the recipient strain. CONCLUSIONS: The novel mel gene from Ps. maltophilia expressed in B. thuringiensis conferred u.v. protection on the recipient strain. SIGNIFICANCE AND IMPACT OF THE STUDY: Products containing B. thuringiensis for pest control are sensitive to u.v. degradation. As melanin has the ability to act as a u.v. absorber, a recombinant B. thuringiensis strain producing melanin provides a new stability for B. thuringiensis preparations.     

A 20-kilodalton protein preserves cell viability and promotes CytA crystal formation during sporulation in Bacillus thuringiensis.

The effect of a 20-kDa protein on cell viability and CytA crystal production in its natural host, Bacillus thuringiensis, was studied by expressing the cytA gene in the absence or presence of this protein. In the absence of the 20-kDa protein, B. thuringiensis cells either were killed during sporulation (strain cryB) or produced very small CytA crystals (strain 4Q7). Expression of cytA in the presence of the 20-kDa protein, however, preserved cell viability, especially in strain cryB, and in both strains yielded bipyramidal crystals of the CytA protein that were larger than those of wild-type B. thuringiensis. These results suggest that the 20-kDa protein promotes crystal formation, perhaps by chaperoning CytA molecules during synthesis and crystallization, concomitantly preventing the CytA protein from interacting lethally with the bacterial host cell.     

Specificity and efficacy of purified Bacillus thuringiensis proteins against agronomically important insects

The host range and relative efficacy of three purified Bacillus thuringiensis insect control proteins were determined against 17 different agronomically important insects representing five orders and one species of mite. The three B. thuringiensis proteins were single gene products from B. thuringiensis ssp. kurstaki HD-1 (CryIA(b)) and HD-73 (CryIA(c)), both lepidopteran-specific proteins, and B. thuringiensis ssp. tenebrionis (CryIIIA), a coleopteran-specific protein. Seven insects showed sensitivity to both B. thuringiensis ssp. kurstaki proteins, whereas only 1 of the 18 insects was sensitive to B. thuringiensis ssp. tenebrionis protein. The level of B. thuringiensis ssp. kurstaki protein required for 50% mortality (LC50) varied by 2000-fold for these 7 insects. A larval growth inhibition assay was developed to determine the amount of B. thuringiensis ssp. kurstaki protein required to inhibit larval growth by 50% (EC50). This extremely sensitive assay enabled detection of B. thuringiensis ssp. kurstaki HD-73 levels as low as 1 ng/ml.