Overexpression of Bacillus thuringiensis HknA, a histidine protein kinase homology, bypasses early Spo mutations that result in CryIIIA overproduction.

The Bacillus thuringiensis CryIIIA insecticidal crystal protein (ICP) is a vegetatively expressed protein that is toxic to coleopteran insect larvae. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of the asporogenous B. thuringiensis subsp. morrisoni strain EG1351, which harbors the native cryIIIA-encoding 88-MDa plasmid, showed a 2.5-fold overproduction of the CryIIIA protein compared with that of an isogenic wild-type strain. Further studies showed that neither CryIIIA protein synthesis nor CryIIIA protein processing was affected in strain EG1351 during vegetative growth. In an attempt to characterize the EG1351 mutation by complementation of function, the hknA gene was identified and cloned from a B. thuringiensis cosmid library. Primer extension analysis of hknA mRNA in wild-type B. thuringiensis demonstrated that the hknA gene is transcribed during vegetative growth from a sigma A-like promoter. Multiple copies of either the hknA gene or the Bacillus subtilis kinA (spoIIJ) gene were shown to bypass the sporulation defect in strain EG1351 as well as a spo0F mutation in B. thuringiensis EG1634. Additional studies showed that the hknA gene was not defective in strain EG1351. The results of this study suggest that hknA encodes a novel histidine protein kinase involved in B. thuringiensis sporulation. We also propose that the CryIIIA-overproducing phenotype of strain EG1351 is most likely due to a defect in the phosphorylation of Spo0A and confirm that CryIIIA production is not dependent on sporulation.     

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.     

Microcalorimetric investigation on the growth model and the protein yield of Bacillus thuringiensis

A novel microcalorimetric technique based on the bacterial heat output was applied to evaluate the special growth model, the protein expression and the generation time of Bacillus thuringiensis for the first time. The thermogenic curves of the aerobic metabolism of B. thuringiensis strains YBT-833, YBT-1520 and YBT-833-2-1 were determined by using an LKB-2277 BioActivity Monitor. The analysis of the thermogenic curves indicated both the mutant strain and the wild-type strains followed the same linear growth model during sporulation. The metabolism heat output revealed heat output was correlated to the yield of the insecticidal crystal proteins (ICPs) very well, the more protein product, and the less heat output. Based on the data acquired, we proposed that this method could be a useful tool in monitoring the fermentation of B. thuringiensis.     

A novel strain of Bacillus thuringiensis (NCIMB 40152) active against coleopteran insects

Abstract: A novel Bacillus thuringiensis strain, NCIMB 40152, was isolated from dead Tenebrio molitor L. larvae. The isolate is endowed with specific insecticide activity against the Colorado potato beetle ( Leptinotarsa decemlineata (Say), Coleoptera, Crysomelidae), but has no effect against lepidopteran or dipteran insects.
During the sporulation process it produces diamond-shaped crystals and small inclusions. Rare flat, square crystals are alsò produced. The strain can be differentiated from the other reported B. thuringiensis strains active against Coleoptera on the basis of physiological and biochemical tests, flagellar serotype and toxin electrophoretic patterns.
NCIMB 40152 toxin is easily extractable from spore/crystal preparations in neutral distilled water without detergents or reducing agents.     

Proteomic analysis of Bacillus thuringiensis at different growth phases by using an automated online two-dimensional liquid chromatography-tandem mass spectrometry strategy

The proteome of a new Bacillus thuringiensis subsp. kurstaki strain, 4.0718, from the middle vegetative (T(1)), early sporulation (T(2)), and late sporulation (T(3)) phases was analyzed using an integrated liquid chromatography (LC)-based protein identification system. The system comprised two-dimensional (2D) LC coupled with nanoscale electrospray ionization (ESI) tandem mass spectrometry (MS/MS) on a high-resolution hybrid mass spectrometer with an automated data analysis system. After deletion of redundant proteins from the different batches and B. thuringiensis subspecies, 918, 703, and 778 proteins were identified in the respective three phases. Their molecular masses ranged from 4.6 Da to 477.4 Da, and their isoelectric points ranged from 4.01 to 11.84. Function clustering revealed that most of the proteins in the three phases were functional metabolic proteins, followed by proteins participating in cell processes. Small molecular and macromolecular metabolic proteins were further classified according to the Kyoto Encyclopedia of Genes and Genome and BioCyc metabolic pathway database. Three protoxins (Cry2Aa, Cry1Aa, and Cry1Ac) as well as a series of potential intracellular active factors were detected. Many significant proteins related to spore and crystal formation, including sporulation proteins, help proteins, chaperones, and so on, were identified. The expression patterns of two identified proteins, CotJc and glutamine synthetase, were validated by Western blot analysis, which further confirmed the MS results. This study is the first to use shotgun technology to research the proteome of B. thuringiensis. Valuable experimental data are provided regarding the methodology of analyzing the B. thuringiensis proteome (which can be used to produce insecticidal crystal proteins) and have been added to the related protein database.     

Pathogenicity of intrathoracically administrated Bacillus thuringiensis spores in Blatta orientalis

The ability of Bacillus thuringiensis to produce septicaemia in Periplaneta americana and Blatta orientalis has been investigated. Spores and crystals from several wild-type strains as well as spores of a B. thuringiensis crystal-deficient mutant, were first orally administrated at high doses, and no significant mortality was recorded. Intrathoracic injection of spore suspensions in P. americana revealed that this species is not very susceptible to B. thuringiensis spores. B. orientalis, by contrast, was found to be very susceptible to B. thuringiensis, with a LD(50) of about 35,000 spores, that is similar to that reported on Lepidoptera challenged with parenterally injected B. thuringiensis.     

Larvicidal activity of Bacillus thuringiensis subsp. israelensis in the dipteran Haematobia irritans.

A strain of Bacillus thuringiensis subsp. israelensis was found to be larvicidal to horn flies, Haematobia irritans (L. [Diptera:Muscidae]). The toxic activity was particulate, appeared during sporulation, and could be prevented by the addition of streptomycin before sporulation. Density gradient centrifugation in Renografin was used to separate endospores, crystals, and low-density particulate matter (fraction 3) from sporulated preparations. Larvicidal activity was restricted to purified crystals and fraction 3, indicating that delta-endotoxin of B. thuringiensis subsp. israelensis was active against horn fly larvae. Purified crystals produced mortality during larval feeding stages, but not pupal stages. Fraction 3 produced significant mortality during both larval and pupal stages. The mortality data indicated the presence of at least two dipteran-active toxins.     

Larvicidal activity of Bacillus thuringiensis subsp. israelensis in the dipteran Haematobia irritans

A strain of Bacillus thuringiensis subsp. israelensis was found to be larvicidal to horn flies, Haematobia irritans (L. [Diptera:Muscidae]). The toxic activity was particulate, appeared during sporulation, and could be prevented by the addition of streptomycin before sporulation. Density gradient centrifugation in Renografin was used to separate endospores, crystals, and low-density particulate matter (fraction 3) from sporulated preparations. Larvicidal activity was restricted to purified crystals and fraction 3, indicating that delta-endotoxin of B. thuringiensis subsp. israelensis was active against horn fly larvae. Purified crystals produced mortality during larval feeding stages, but not pupal stages. Fraction 3 produced significant mortality during both larval and pupal stages. The mortality data indicated the presence of at least two dipteran-active toxins.     

苏云金芽孢杆菌gerA操纵子在芽孢萌发中的作用

芽孢萌发的营养诱导剂通过与特异的萌发受体结合激活下游的萌发过程,从而使芽孢经过一系列的遗传变化及生化反应恢复营养生长.从苏云金芽孢杆菌(Bacillus thuringiensis)中克隆到一个与枯草芽孢杆菌(Bacillus subtilis)gerA操纵子和蜡状芽孢杆菌(Bacillus cereus)gerR操纵子同源的gerA操纵子.苏云金芽孢杆菌gerA操纵子含有3个开放读码框:gerAA、gerAC和gerAB,该操纵子在产孢起始3个小时后开始转录.gerA的破坏阻断了L-丙氨酸诱导的芽孢萌发并且延迟了肌苷诱导的萌发.在L-丙氨酸诱导芽孢萌发的过程中D-环丝氨酸能够提高芽孢的萌发率.     

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.     

A method for the determination of bacterial spore DNA content based on isotopic labelling, spore germination and diphenylamine assay; ploidy of spores of several Bacillus species.

A reliable method for measuring the spore DNA content, based on radioactive DNA labelling, spore germination in absence of DNA replication and diphenylamine assay, was developed. The accuracy of the method, within 10-15%, is adequate for determining the number of chromosomes per spore, provided that the genome size is known. B subtilis spores were shown to be invariably monogenomic, while those of larger bacilli Bacillus megaterium, Bacillus cereus and Bacillus thuringiensis, often, if not invariably, contain two genomes. Attempts to modify the spore DNA content of B subtilis by altering the richness of the sporulation medium, the sporulation conditions (liquid or solid medium), or by mutation, were apparently unsuccessful. An increase of spore size with medium richness, not accompanied by an increase in DNA content, was observed. The implication of the apparently species-specific spore ploidy and the influence of the sporulation conditions on spore size and shape are discussed.     

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.     

Effect of Sm(3+) ion on growth of Bacillus thuringiensis by microcalorimetry

By using an LKB-2277 Bioactivity Monitor, cycle-flow method, the thermogenic curves of aerobic growth for Bacillus thuringiensis cry II strain at 28 degrees C have been obtained. The metabolic thermogenic curves of B. thuringiensis cry II contained two distinct patterns: the first reflects the changes during the bacterial growth phase and the second corresponds to the sporulation phase. From these thermogenic curves in the absence and presence of Sm(3+) ions, the thermokinetic parameters such as the growth rate constants k, the interval time tau(I), the maximum power P (max 1) and heat-output Q(log) for log phase, the maximum power P (max 2) and heatoutput Q(stat) for stationary phase, the heat-output Q(spor) for sporulation phase and total heat effects QT are calculated. Sm(3+) ion has promoting action on the growth of B. thuringiensis cry II in its lower concentration range; on the other hand, this ion has inhibitory action on the sporulation of B. thuringiensis in its higher concentration range. We also found that the effects of Sm(3+) ion on B. thuringiensis during the sporulation phase were far greater than that during the bacterial phase. It is concluded that the application of B. thuringiensis for controlling insecticides is not affected by the presence of the rare-earth elements in the environmental ecosystem.