<Emphasis Type="Italic">In vivo</Emphasis> fluorescence observation of parasporal inclusion formation in <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis>

A recombinant gene expressing a Cry1Ac-GFP fusion protein with a molecular mass of approximately 160 kD was constructed to investigate the expression of cry1Ac, the localization of its gene product Cry1Ac, and its role in crystal development in Bacillus thuringiensis. The cry1Ac-gfp fusion gene under the control of the cry1Ac promoter was cloned into the plasmid pHT304, and this construct was designated pHTcry1Ac-gfp. pHTcry1Ac-gfp was transformed into the crystal-negative strain, HD-73 cry⁻, and the resulting strain was named HD-73⁻(pHTcry1Ac-gfp). The gfp gene was then inserted into the large HD-73 endogenous plasmid pHT73 and fused with the 3′ terminal of the cry1Ac gene by homologous recombination, yielding HD-73Φ(cry1Ac-gfp)3534. Laser confocal microscopy and Western blot analyses showed for the first time that the Cry1Ac-GFP fusion proteins in both HD-73⁻(pHTcry1Ac-gfp) and HD-73Φ(cry1Ac-gfp)3534 were produced during asymmetric septum formation. Surprisingly, the Cry1Ac-GFP fusion protein showed polarity and was located near the septa in both strains. There was no significant difference between Cry1Ac-GFP and Cry1Ac in their toxicity to Plutella xylostella larvae.     

Characterization of the cry1Ac17 Gene from an Indigenous Strain of Bacillus thuringiensis subsp. kenyae

An indigenously isolated strain of Bacillus thuringiensis subsp. kenyae exhibited toxicity against lepidopteran as well as dipteran insects. The lepidopteran active cry1Ac protoxin gene coding sequence of 3.5 kb from this strain was cloned into vector pET28a(+). However, it could not be expressed in commonly used Escherichia coli expression hosts, BL21(DE3) and BL21(DE3)pLysS. This gene is classified as cry1Ac17 in the B. thuringiensis toxic nomenclature database. The coding sequence of this gene revealed that it contains about 3% codons, which are not efficiently translated by these expression hosts. Hence, this gene was expressed in a modified expression host, Epicurian coli BL21-Codonplus (DE3)-RIL. The expression of gene yielded a 130-kDa Cry1Ac17 protein. The protein was purified and its toxicity was tested against economically important insect pests, viz., Helicoverpa armigera and Spodoptera litura. LC₅₀ values obtained against these insects were 0.1 ng/cm³ and 1231 ng/cm², respectively. The higher toxicity of Cry1Ac17 protein, compared to other Cry1Ac proteins, toward these pests demonstrates the potential of this isolate as an important candidate in the integrated resistance management program in India.     

Increase in Insecticidal Toxicity by Fusion of the <Emphasis Type="Italic">cry1Ac</Emphasis> Gene from <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis> with the Neurotoxin Gene <Emphasis Type="Italic">hwtx-I</Emphasis>

A fusion gene was constructed by combining the cry1Ac gene of Bacillus thuringiensis strain 4.0718 with a neurotoxin gene, hwtx-1, which was synthesized chemically. In this process, an enterokinase recognition site sequence was inserted in frame between two genes, and the fusion gene, including the promoter and the terminator of the cry1Ac gene, was cloned into the shuttle vector pHT304 to obtain a new expression vector, pXL43. A 138-kDa fusion protein was mass-expressed in the recombinant strain XL002, which was generated by transforming pXL43 into B. thuringiensis acrystalliferous strain XBU001. Quantitative analysis indicated that the expressed protein accounted for 61.38% of total cellular proteins. Under atomic force microscopy, there were some bipyramidal crystals with a size of 1.0 × 2.0 μm. Bioassay showed that the fusion crystals from recombinant strain XL002 had a higher toxicity than the original Cry1Ac crystal protein against third-instar larvae of Plutella xylostella, with an LC₅₀ (after 48 h) value of 5.12 μg/mL. The study will enhance the toxicity of B. thuringiensis Cry toxins and set the groundwork for constructing fusion genes of the B. thuringiensis cry gene and other foreign toxin genes and recombinant strains with high toxicity. LiQiu Xia and XiaoShan Long contributed equally to this work.     

Evaluation of the cytogenetic damage induced by the organophosphorous insecticide acephate

The organophosphorous insecticide acephate was tested for its ability to induce in vitro cytogenetic effect in human peripheral lymphocytes by using the chromosomal aberrations (CAs), sister chromatid exchange (SCE) and micronuclei (MN) assay. The level of nuclear DNA damage of acephate was evaluated by using the comet assay. Concentrations of 12.5, 25, 50, 100 and 200 μg mL⁻¹ of acephate were used. All concentrations of acephate induced significant increase in the frequency of CAs and in the formation of MN dose dependently (r = 0.92 at 24 h, r = 0.95 at 48 h for CAs, r = 0.87 for MN). A significant increase was observed in induction of SCE at 50, 100 and 200 μg mL⁻¹ concentrations during 24 h treatment and at all concentrations (except 12.5 μg mL⁻¹) during 48 h treatment period in a dose-dependent manner (r = 0.84 at 24 h, r = 0.88 at 48 h). Acephate did not affect the replicative index and cytokinesis-block proliferation index (CBPI). However, it significantly decreased the mitotic index at all three highest concentrations (50, 100, 200 μg mL⁻¹) for 24 h treatment and at all concentrations (except 12.5 μg mL⁻¹) for 48 h treatment, dose-dependently (r = 0.94 at 24 h, r = 0.92 at 48 h). A significant increase in mean comet tail length was observed at 100 and 200 μg mL⁻¹ concentrations compared with negative control in a concentration-dependent manner (r = 0.94). The mean comet tail intensity was significantly increased at only 200 μg mL⁻¹ concentration. The present results indicate that acephate is a clastogenic, cytotoxic agent and it causes DNA damage at high concentrations in human lymphocytes in culture.     

Bioremoval of hexavalent chromium from water by a salt tolerant bacterium, Exiguobacterium sp. GS1

Pollution of terrestrial surfaces and aquatic systems by hexavalent chromium, Cr(VI), is a worldwide public health problem. A chromium resistant bacterial isolate identified as Exiguobacterium sp. GS1 by 16S rRNA gene sequencing displayed high rate of removal of Cr(VI) from water. Exiguobacterium sp. GS1 is 99% identical to Exiguobacterium acetylicum. The isolate significantly removed Cr(VI) at both high and low concentrations (1–200 μg mL⁻¹) within 12 h. The Michaelis–Menten K _m and V _max for Cr(VI) bioremoval were calculated to be 141.92 μg mL⁻¹ and 13.22 μg mL⁻¹ h⁻¹, respectively. Growth of Exiguobacterium sp. GS1 was indifferent at 1–75 μg mL⁻¹ Cr(VI) in 12 h. At initial concentration of 8,000 μg L⁻¹, Exiguobacterium sp. GS1 displayed rapid bioremoval of Cr(VI) with over 50% bioremoval in 3 h and 91% bioremoval in 8 h. Kinetic analysis of Cr(VI) bioremoval rate revealed zero-order in 8 h. Exiguobacterium sp. GS1 grew and significantly reduced Cr(VI) in cultures containing 1–9% salt indicating high salt tolerance. Similarly the isolate substantially reduced Cr(VI) over a wide range of temperature (18–45  °C) and initial pH (6.0–9.0). The T _opt and initial pH_opt were 35–40  °C and 7–8, respectively. Exiguobacterium sp. GS1 displayed a great potential for bioremediation of Cr(VI) in diverse complex environments.     

Efficient constitutive expression of chitinase in the mother cell of <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis> and its potential to enhance the toxicity of Cry1Ac protoxin

Previous studies revealed that chitinase could enhance the insecticidal activity of Bacillus thuringiensis and it has been used in combination with B. thuringiensis widely. However, the expression of B. thuringiensis chitinase is rather low and needs induction by chitin, which limits its field application. It would make sense to constitutively express the chitinase at a sufficiently high level to offer advantages in biological control of pests. In this study, a signal peptide-encoding sequence-deleted chitinase gene from B. thuringiensis strain 4.0718 under the control of dual overlapping promoters plus Shine–Dalgarno sequence and terminator sequence of cry1Ac3 gene was cloned into shuttle vector pHT315 and introduced into an acrystalliferous B. thuringiensis strain Cry⁻B. The recombinant plasmid was stably maintained over 240 generations in Cry⁻B. Chitinase was overexpressed within the sporangial mother cells in the form of spherical crystal-like inclusion bodies. The chitinase inclusions could be solubilized and exhibit chitinolytic activity in 30 mmol l⁻¹ Na₂CO₃–0.2% β-mercaptoethanol buffer at a wide range of alkaline pH values, and what’s more, the chitinase inclusions potentiated the insecticidal effect of Cry1Ac protoxin when used against larvae of Spodoptera exigua and Helicoverpa armigera.     

Production and activities of chitinases and hydrophobins from Lecanicillium lecanii

The production of chitinases and hydrophobins from Lecanicillium lecanii was influenced by the cultivation method and type of carbon source. Crude enzyme obtained from solid-substrate culture presented activities of exochitinases (32 and 51 kDa), endochitinases (26 kDa), β-N-acetylhexosaminidases (61, 80, 96 and 111 kDa). Additionally, submerged cultures produced exochitinases (32 and 45 kDa), endochitinases (10 and 26 kDa) and β-N-acetylhexosaminidases (61, 96 and 111 kDa). β-N-acetylhexosaminidases activity determined in solid-substrate culture with added chitin was ca. threefold (7.58 ± 0.57 U mg⁻¹) higher than submerged culture (2.73 + 0.57 U mg⁻¹). Similarly, hydrophobins displayed higher activities in solid-substrate culture (627.3 ± 2 μg protein mL⁻¹) than the submerged one (57.4 ± 4.7 μg protein mL⁻¹). Molecular weight of hydrophobins produced in solid-substrate culture was 7.6 kDa and they displayed surface activity on Teflon.     

Arsenic accumulation and thiol status in lichens exposed to As(V) in controlled conditions

Thalli of epiphytic lichen Hypogymnia physodes (L.) Nyl. and terricolous Cladonia furcata (Huds.) Schrad., collected from an area with background arsenic concentrations, were exposed to 0, 0.1, 1 and 10 μg mL⁻¹ arsenate (As(V)) solutions for 24 h. After exposure they were kept in the metabolically active state for 0, 24 and 48 h in a growth chamber. In the freeze dried samples glutathione (GSH), glutathione disulphide (GSSG), cysteine (Cys) and cystine were analysed and induction of phytochelatin (PC) synthesis measured by reversed-phase high-performance liquid chromatography in combination with fluorescence detection or UV spectrometry. Total arsenic content in thalli was measured by instrumental neutron activation analysis (INAA). In H. physodes, which contained higher amounts of arsenic compared to C. furcata, total glutathione content significantly decreased in samples exposed to 10 μg mL⁻¹ As(V), whereas in C. furcata a significant increase was observed. In both species PC synthesis was induced in thalli exposed to 10 μg mL⁻¹.     

An engineered Bacillus thuringiensis strain with insecticidal activity against Scarabaeidae (Anomala corpulenta) and Chrysomelidae (Leptinotarsa decemlineata and Colaphellus bowringi)

A genetically-engineered Bacillus thuringiensis (Bt) strain, 3A-HBF, with a broad insecticidal spectrum was constructed by introducing the recombinant plasmid pSTK-3A containing cry3Aa7 into the wild-type Bt strain HBF-1 containing the cry8Ca2 gene. The Cry3Aa7 protein produced by strain 3A-HBF was verified by SDS-PAGE and Western blotting. Flat rectangular crystals of Cry3Aa7 protein were observed besides spherical crystals (Cry8Ca2). The plasmid pSTK-3A was stable when strain 3A-HBF was grown in medium without antibiotics. The growth rate of 3A-HBF was not significantly different from that of the recipient strain, HBF-1. Strain 3A-HBF showed toxicity against two families of pests, Scarabaeidae and Chrysomelidae pests, which are susceptible to Cry8Ca (Anomala corpulenta) and Cry3Aa (Leptinotarsa decemlineata and Colaphellus bowringi). The 50% lethal concentrations of 3A-HBF against A. corpulenta, L. decemlineata and C. bowringi were 0.730 × 10⁸ c.f.u./g dry soil, 1.74 μg/ml and 1.15 μg/ml, respectively.     

Bacillus thuringiensisδ-Endotoxin Proteins Show a Correlation in Toxicity and Short Circuit Current Inhibition Against Helicoverpa zea

Pesticidal activity of Bacillus thuringiensisδ-endotoxins, Cry1Aa, Cry1Ab, Cry1Ac, and Cry2A, was determined by using the force-feeding bioassay method to 4^th instar larvae of Helicoverpa zea. H. zea was susceptible to Bt toxins in the order Cry1Ac > Cry1Ab > Cry1Aa > Cry2A with 63.60, 89.04, 159.65, and 375.78 ng/larvae respectively. The abilities of selected Bacillus thuringiensis toxins to inhibit short circuit current (I_SC) in midgut epithelia of H. zea were also investigated by voltage clamp assay. The voltage-clamp studies were conducted on isolated midguts, measuring the inhibition of short circuit current (I_SC) by activated toxin. A Cry1Aa toxin dilution of 33.3 and 500 ng/ml resulted in inhibition of I_SC of −2.29 μA/min (lag time 15 min) and −4.48 μA/min (lag time, 2 min) respectively. The Cry1Ab dilution of 25 ng/ml inhibited I_SC to −1.39 μA/min, a lag time of 14 min, and 333.3 ng/ml dilution resulted in decay of I_SC−2.49 μA/min, lag time 1 min respectively. The Cry1Ac lower dilution 16.7 ng/ml inhibited I_SC to −1.39 μA/min, lag time 4 min, and a high dilution 333.3 ng/ml decay I_SC to −2.44 μA/min, lag time 1 min. The inhibition of I_SC (−1.10 μA/min, lag time 25) at lower dilution (33.3 ng/ml) and high dilution (500 ng/ml), decay (−2.38 μA/min, lag time 5 min), showed a correlation between toxin concentration and inhibitory response with Cry2A toxin. The lag time decreased with increasing concentration of toxin applied, which is additional evidence of dose response besides direct correlation of toxicity assays and I_SC. Received: 7 April 2000 / Accepted: 2 May 2000     

Insecticide-tolerant and plant growth promoting <Emphasis Type="Italic">Bradyrhizobium</Emphasis> sp. (<Emphasis Type="Italic">vigna</Emphasis>) improves the growth and yield of greengram [<Emphasis Type="Italic">Vigna radiata</Emphasis> (L.) Wilczek] in insecticide-stressed soils

This study was designed to identify rhizobial strains specific to greengram expressing higher tolerance against insecticides, fipronil and pyriproxyfen, and synthesizing plant growth regulators even amid insecticide-stress. Of the 50 bradyrhizobial isolates, the Bradyrhizobium sp. strain MRM6 showed tolerance up to 1,600 μg mL⁻¹ against each of fipronil and pyriproxyfen. The tolerant Bradyrhizobium sp. (vigna) produced plant growth promoting substances in substantial amounts, both in the presence and absence of insecticides. The strain MRM6 was further used to investigate its impact on greengram grown in soils treated with 200 (the recommended dose), 400 and 600 μg kg⁻¹ soil of fipronil and 1,300 (the recommended dose), 2,600 and 3,900 μg kg⁻¹ soil of pyriproxyfen. Fipronil at 600 μg kg⁻¹ soils and pyriproxyfen at 3,900 μg kg⁻¹ soils had greatest toxic effects and decreased plant biomass, symbiotic efficiency, nutrient uptake and seed yield of greengram plants. The Bradyrhizobium sp. (vigna) inoculant when used with fipronil and pyriproxyfen significantly increased the measured parameters compared to the plants grown in soils treated solely with the same concentration of each insecticide. This study inferred that the Bradyrhizobium sp. (vigna) strain MRM6 may be exploited as bio-inoculant to increase the productivity of greengram exposed to insecticide-stressed soils.     

Identification of carotenoids with high antioxidant capacity produced by extremophile microorganisms

In this study, the carotenoids produced by the extremophile microorganisms Halococcus morrhuae, Halobacterium salinarium and Thermus filiformis were separated and identified by high-performance liquid chromatography connected to a diode array detector and a tandem mass spectrometer. The in vitro scavenging capacity of the carotenoid extracts against radical and non-radical species was evaluated. In halophilic microorganisms, the following carotenoids were identified: bacterioruberin, bisanhydrobacterioruberin, trisanhydrobacterioruberin and their derivatives. In the thermophilic bacterium, the carotenoids all-trans-zeaxanthin, zeaxanthin monoglucoside, thermozeaxanthins and thermobiszeaxanthins were identified. The antioxidant capacities of the carotenoid extracts of H. morrhuae (trolox equivalent antioxidant capacity = 5.07 and IC₅₀ = 0.85 μg mL⁻¹) and H. salinarium (trolox equivalent antioxidant capacity = 5.28 and IC₅₀ = 0.84 μg mL⁻¹) were similar and higher than those of the bacterium T. filiformis (trolox equivalent antioxidant capacity = 2.87 and IC₅₀ = 2.41 μg mL⁻¹). This difference is related to the presence of acyclic carotenoids with both large numbers of conjugated double bounds and of hydroxyl groups in the major carotenoid of the halophilic microorganisms.     

Drift-ice and under-ice water communities in the Gulf of Bothnia (Baltic Sea)

The algal, protozoan and metazoan communities within different drift-ice types (newly formed, pancake and rafted ice) and in under-ice water were studied in the Gulf of Bothnia in March 2006. In ice, diatoms together with unidentified flagellates dominated the algal biomass (226 ± 154 μg ww l⁻¹) and rotifers the metazoan and protozoan biomass (32 ± 25 μg ww l⁻¹). The under-ice water communities were dominated by flagellates and ciliates, which resulted in lower biomasses (97 ± 25 and 21 ± 14 μg ww l⁻¹, respectively). The under-ice water and newly formed ice separated from all other samples to their own cluster in hierarchical cluster analysis. The most important discriminating factors, according to discriminant analysis, were chlorophyll-a, phosphate and silicate. The under-ice water/newly formed ice cluster was characterized by high nutrient and low chlorophyll-a values, while the opposite held true for the ice cluster. Increasing trends in chlorophyll-a concentration and biomass were observed with increasing ice thickness. Within the thick ice columns (>40 cm), the highest chlorophyll-a concentrations (6.6–22.2 μg l⁻¹) were in the bottom layers indicating photoacclimation of the sympagic community. The ice algal biomass showed additional peaks in the centric diatom-dominated surface layers coinciding with the highest photosynthetic efficiencies [0.019–0.032 μg C (μg Chl-a ⁻¹ h⁻¹) (μE m⁻² s⁻¹)⁻¹] and maximum photosynthetic capacities [0.43-1.29 μg C (μg Chl-a ⁻¹ h⁻¹)]. Rafting and snow-ice formation, determined from thin sections and stable oxygen isotopic composition, strongly influenced the physical, chemical and biological properties of the ice. Snow-ice formation provided the surface layers with nutrients and possibly habitable space, which seemed to have favored centric diatoms in our study.     

Improving Toxicity of <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis> Strain Contains the <Emphasis Type="Italic">cry8Ca</Emphasis> Gene Specific to <Emphasis Type="Italic">Anomala corpulenta</Emphasis> Larvae

The cry8C-type gene designated cry8Ca2, which was cloned and sequenced from a Bacillus thuringiensis isolate HBF-1 in China, consisted of an open reading frame of 3483 bp encoding a protein of 1160 amino-acid residues. Sequence analysis showed that the Cry8Ca2 protoxin of 130.5 kDa had 99.9% sequence homology with the previously reported Cry8Ca1 protein, with one mismatch between the two amino-acid sequences. When the Cry8Ca2 toxin was expressed in a crystal-negative strain of B. thuringiensis (HD-73⁻), elliptical crystals were produced. Cell extracts from this recombinant strain showed insecticidal activity against Anomala corpulenta larva. Mutant cry8Ca2 genes, produced by polymerase chain reaction amplification with Taq DNA polymerase, were used to develop recombinant B. thuringiensis strains. Mutants producing higher levels of insecticidal activity were identified by bioassay. Thirty-five mutants forming crystals were characterized, and two of them showed significantly increased insecticidal activity against A. corpulenta larva. The 50% lethality concentrations (LC₅₀) of the two mutants were 0.2334 × 10⁸ and 0.2591 × 10⁸ colony-forming units g⁻¹, considerably lower than the LC₅₀ of the wild-type strain HBF-1 (0.9583 × 10⁸ CFU g⁻¹) and that of B. thuringiensis serovar japonensis strain Buibui (1.0752 × 10⁸ CFU g⁻¹).     

Genetic variation of <Emphasis Type="Italic">Fusarium oxysporum</Emphasis> isolates forming fumonisin B<Subscript>1</Subscript> and moniliformin

Thirty single-spore isolates of a toxigenic fungus, Fusarium oxysporum, were isolated from asparagus spears and identified by species-specific polymerase chain reaction (PCR) and translation elongation factor 1-α (TEF) sequence analysis. In the examined sets of F. oxysporum isolates, the DNA sequences of mating type genes (MAT) were identified. The distribution of MAT idiomorph may suggest that MAT1-2 is a predominant mating type in the F. oxysporum population. F. oxysporum is mainly recognised as a producer of moniliformin—the highly toxic secondary metabolite. Moniliformin content was determined by high-performance liquid chromatography (HPLC) analysis in the range 0.05–1,007.47 μg g⁻¹ (mean 115.93 μg g⁻¹) but, also, fumonisin B₁ was detected, in the concentration range 0.01–0.91 μg g⁻¹ (mean 0.19 μg g⁻¹). There was no association between mating types and the mycotoxins biosynthesis level. Additionally, a significant intra-species genetic diversity was revealed and molecular markers associated with toxins biosynthesis were identified.     

Characterization of Two Novel <Emphasis Type="Italic">cry8</Emphasis> Genes from <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis> Strain BT185

Two novel cry8-type genes, cry8Ea1 and cry8Fa1, obtained from a Holotrichia parallela–specific Bacillus thuringiensis strain, BT185, were characterized. Findings showed that cry8Ea1 and cry8Fa1 encoded polypeptides of 1164 and 1174 amino acid residues, respectively. The deduced amino acid sequences of both Cry8Ea1 and Cry8Fa1 polypeptides are the most similar to that of Cry8Ba1. Eight conserved blocks (blocks 1–8) exist in Cry8Ea1 and Cry8Fa1 polypeptides compared with known Cry proteins. Cry8Ea1 and the Cry8Fa1 toxins could form spheric crystals when they were expressed in the acrystalliferous mutant strain HD73⁻. The spores and crystals from the recombinant strain containing cry8Ea1 were toxic to Holotrichia parallela, with an LC₅₀ of 0.0875 × 10⁸ colony-forming units (CFU)/g. However, Cry8Fa1 expressed in the recombinant strain was not toxic to H. parallela, Anomala corpulenta, or H. oblita.     

Identification of cry-Type Genes on 20-kb DNA Associated with Cry1 Crystal Proteins from Bacillus thuringiensis

Heterologous DNA fragments (20-kb) associated with Cry1 crystal proteins (protoxins) from a soil-isolated Bacillus thuringiensis strain 4.0718 were isolated and analyzed. RFLP patterns of the PCR products showed that the 20-kb DNA fragments harbored cry1Aa, cry1Ac, cry2Aa, and cry2Ab genes. Furthermore, a 4.2-kb DNA fragment, which contained the promoter, the coding region, and the terminator of cry1Ac gene, was cloned from the 20-kb DNAs by PCR, and then the cry1Ac gene was expressed in an acrystalliferous B. thuringiensis strain 4Q7 by using E. coli-B. thuringiensis shuttle vector pHT3101. SDS-PAGE and microscopy studies revealed that the recombinant could express 130-kDa Cry1Ac protoxin and produce bipyramidal crystals during sporulation. Bioassay results proved that crystal-spore mixture from the recombinant was toxic to Plutella xylostella. This was the first report of cry-type genes present on 20-kb DNA associated with Cry1 protoxins of B. thuringiensis.