Characterization of melanin produced by a wild-type strain of Bacillus cereus

Bacillus cereus 58 (Bc58)is a UV-resistant wild type strain that has an ability to produce a sorrel pigment induced by L-tyrosine.The Fourier-transform infrared (FT-IR)spectra and chemical tests of its pigment are similar to that of the standard melanin (Sigma).A bioassay shows that the LC50 of a Bacillus thuringiensis (Bt)formulation added with the melanin of Bc58 and exposed to UV for 5 h is 16.1 μg/ml,which is similar to that of the Bt formulation without UV treatment,however,it is almost double that of the Bt formulation exposed to UV without the melanin of Bc58.The result of SDS-PAGE indicates that the melanin of Bc58 can protect the insecticidal crystal proteins from degradation.This suggests that it is an excellent UV protective agent for the insecticidal crystal proteins of the Bt formulation.     

Characterization of melanin produced by a wild-type strain of Bacillus cereus

Bacillus cereus 58 (Bc58) is a UV-resistant wild type strain that has an ability to produce a sorrel pigment induced by L-tyrosine. The Fourier-transform infrared (FT-IR) spectra and chemical tests of its pigment are similar to that of the standard melanin (Sigma). A bioassay shows that the LC₅₀ of a Bacillus thuringiensis (Bt) formulation added with the melanin of Bc58 and exposed to UV for 5 h is 16.1 μg/ml, which is similar to that of the Bt formulation without UV treatment, however, it is almost double that of the Bt formulation exposed to UV without the melanin of Bc58. The result of SDS-PAGE indicates that the melanin of Bc58 can protect the insecticidal crystal proteins from degradation. This suggests that it is an excellent UV protective agent for the insecticidal crystal proteins of the Bt formulation. Translated from Microbiology, 2006, 33(1): 42–45 [译自: 微生物学通报]     

Isolation and characterisation of a novel bacteriocin produced by Bacillus thuringiensis strain B439

Bacillus thuringiensis strain B439 produces a bacteriocin-like inhibitory substance in its growth medium. This antimicrobial peptide, referred to as thuricin 439, acts as a bacteriocidal peptide and exhibits an apparent narrow range of inhibitory activity, essentially only affecting growth of Bacillus cereus and B. thuringiensis strains. It remains active over a relatively wide pH and temperature range, showing no loss of activity following heat treatments up to 80 degrees C. Purification of thuricin 439 was achieved using several chromatographic steps, which resulted in the identification of two peptides with inhibitory activity. These two peptides were shown to possess identical N-terminal sequences, but different molecular masses.     

A Bacillus thuringiensis host strain with high melanin production for preparation of light-stable biopesticides

The bacterium Bacillus thuringiensis produces a crystal protein with insecticidal properties; however, crystal proteins can be damaged by ultraviolet (UV) radiation. The aim of this study was to improve the stability of the insecticidal crystal protein (ICP) by constructing a mutant line that expresses high levels of the UV light-protecting pigment, melanin. BMB181, a B. thuringiensis mutant with high melanin production, was obtained after sub-culturing BMB171 for several generations at 42 °C. The melanin yield by BMB181 (without tyrosine supplementation) reached 8.55 mg/ml. The electroporation efficiency of BMB181 reached 10⁶ CFU/μg when a 6.7-kb foreign plasmid was used. Microscopic and SDS-PAGE analyses revealed that ICP (CryIAc10; GenBank: AAA73077.1), which is highly toxic to Lepidoptera, was synthesized efficiently by strain BMB181. The insecticidal properties of a recombinant line derived from strain BMB181, designated BMB32 (cry1Ac10/BMB181), was tested against the cotton bollworm, Helicoverpa armigera. After UV irradiation for 4 h, BMB32 had a half maximal inhibitory concentration value of 1.37 μg/ml, whereas the control line BMB31 (cry1Ac10/BMB171) had a median lethal dose value of 25.85 μg/ml. These results indicate that the B. thuringiensis mutant is a candidate for industrial scale production of light-stable insecticides.     

Identity of hemolysins produced by Bacillus thuringiensis and Bacillus cereus

A hemolysin (Bt-hemolysin) produced by Bacillus thuringiensis var. kurstaki HD-1 producing crystalline toxin(s) was purified by successive treatments of ammonium sulfate (45-65%) and column chromatography using DEAE-cellulose, Sephadex G-75 and KB-002 (a hydroxyapatite column for fast protein liquid chromatography). A hemolysin (Bc-hemolysin) produced by B. cereus HG-6A was also purified by the same procedure. The purified Bt-hemolysin and Bc-hemolysin, both of which are thiol-activated hemolysins, were biologically, physicochemically and immunologically identical. These findings provide further evidence of the similarity of B. thuringiensis, which is being used as a biological insecticide, to B. cereus, a toxigenic organism of food poisoning.     

Characterization of mosquito larvicidal parasporal inclusions of a Bacillus thuringiensis serovar higo strain

The parasporal inclusion proteins of the type strain of Bacillus thuringiensis serovar higo (H44), that have moderate mosquitocidal activity, were characterized. The purified parasporal inclusions, spherical in shape, were examined for activity against the two mosquito species, Culex pipiens molestus and Anopheles stephensi and the moth-fly, Telmatoscopus albipunctatus . The LC₅₀ values of the inclusion for the two mosquitoes were 3·41 and 0·15 μg ml⁻¹, respectively. No mortality was shown for T. albipunctatus larvae by the inclusions at concentrations up to 1 mg ml⁻¹. Solubilized parasporal inclusions exhibited no haemolytic activity against sheep erythrocytes. Parasporal inclusions consisted of eight proteins with molecular masses of 98, 91, 71, 63, 59, 50, 44 and 27 kDa. Of these, the 50 and 44 kDa proteins were the major components. Analysis with immunoblotting revealed that, among several inclusion proteins of B. thuringiensis serovar israelensis, only two proteins of 130 kDa and 110 kDa reacted weakly with antibodies against higo proteins. N-terminal amino acid sequences of the 98, 91, and 71 kDa proteins showed 85–100% identity to those of the two established Cry protein classes, Cry4A and Cry10A.     

Thuricin: the bacteriocin produced by Bacillus thuringiensis

Bacillus thuringiensis serovar, thuringiensis (HD-2) demonstrated antibacterial activity against 48 of 56 strains of B. thuringiensis and against some other Gram-positive species but not against Gram-negative species. The antibacterial activity was not inducible by mitomycin C or by ultraviolet irradiation, and additional activity was not liberated from cells by sonication. Upon dilution of the antibacterial substance, zones of inhibition diminished without the appearance of plaques. Gel filtration chromatography indicated an Mr greater than 950,000 for the bacteriocin (thuricin) in its native form. The native thuricin was sedimented by ultracentrifugation, but electron microscopy of the pellet failed to reveal phage particles or phage components. Nondenaturing polyacrylamide gel electrophoresis (PAGE) of thuricin demonstrated the association of bacteriocin activity with a protein band which migrated only slightly into a 5% gel. Sodium dodecyl sulfate (SDS)-PAGE of partially purified thuricin revealed five major bands. Thuricin activity was substantially reduced by treatment with chymotrypsin, pronase, subtilisin, trypsin, and heat at 96 degrees C but not by treatment with lysozyme, phospholipase C, papain, peptidase, or organic solvents. It exhibited a bactericidal and bacteriolytic effect on a sensitive strain, B. thuringiensis serovar, canadensis (MF4). Partially purified preparations of thuricin had phospholipase A activity which was adsorbed by sensitive cells but not by cells which were insensitive to thuricin. Antibacterial activity was blocked by preincubation of thuricin with phospholipid. Loss of a 150-mDa plasmid was correlated with loss of thuricin production.     

Specificity-determining regions of a lepidopteran-specific insecticidal protein produced by Bacillus thuringiensis

The lepidopteran-specific, insecticidal crystal proteins of Bacillus thuringiensis vary in toxicity to different species of lepidopteran larvae. We report studies of CryIA(a) and CryIA(c), two related proteins that have different degrees of toxicity to Heliothis virescens yet very similar degrees of toxicity to Manduca sexta. The amino acid differences between these proteins are located primarily between residues 280 and 722. We have constructed a series of chimeric proteins and determined their toxicities to both insects. The most significant findings arise from the replacement of three segments of the cryIA(c) gene with homologous portions of the cryIA(a) gene: codons 332-428, 429-447, and 448-722. Each of these segments contributed substantially and largely additively toward efficacy for H. virescens. However, replacement of the 429-447 segment of cryIA(c) gene with the cryIA(a) sequence resulted in a 27-50-fold reduction in toxicity toward M. sexta whereas the reduction in toxicity to H. virescens was only 3-4-fold. Subdivision of the 429-447 segment and replacements involving residues within this segment reduced toxicity to M. sexta by 5- to more than 2000-fold whereas toxicity to H. virescens was only reduced 3-10-fold. These observations indicate that: 1) different but overlapping regions of the cryIA(c) gene determine specificity to each of the two test insects; 2) some of the examined gene segments interact in determining specificity; and 3) different sequences in the cryIA(a) and cryIA(c) genes are required for maximal toxicity to M. sexta.     

Characterization of a Bacillus thuringiensis strain with a broad spectrum of activity against lepidopteran insects

The insect pathogen Bacillus thuringiensis is suitable for use in biological control, and certain strains have been developed as commercial bioinsecticides. The molecular and biological characterization of a Bacillus thuringiensis subsp. aizawai strain, named HU4‐2, revealed its potential as a bioinsecticide. The strain was found to contain eight different cry genes: cry1Ab, cry1Ad, cry1C, cry1D, cry1F, cry2, cry9Ea1, and a novel cry1I‐type gene. Purified parasporal crystals from strain HU4‐2 comprised three major proteins of 130–145 kDa, which were tested for their insecticidal potency to four species of Lepidoptera (Helicoverpa armigera, Spodoptera exigua, S. littoralis, and S. frugiperda) and three species of mosquito (Culex pipiens pipiens, Aedes aegypti, and Anopheles stephensi). The crystal proteins were highly toxic against all the species of Lepidoptera tested, moderately toxic against two of the mosquito species (C. pipiens and Ae. aegypti), but no toxicity was observed against a third species of mosquito (An. stephensi) at the concentrations used in our study. The LC₅₀ values of the HU4‐2 Bt strain against H. armigera larvae (5.11 µg/ml) was similar to that of HD‐1 Bt strain (2.35 µg/ml), the active ingredient of the commercial product Dipel®. Additionally, the LC₅₀ values of the HU4‐2 Bt strain against S. littoralis, S. frugiperda, and S. exigua (2.64, 2.22, and 3.38 µg/ml, respectively) were also similar to that of the Bt strain isolated from the commercial product Xentari® for the same three species of Spodoptera (1.94, 1.34, and 2.19 µg/ml, respectively). Since Xentari® is significantly more toxic to Spodoptera spp. than Dipel® and, reciprocally, Dipel® is significantly more toxic against H. armigera than Xentari®, we discuss the potential of the HU4‐2 strain to control all these important lepidopteran pests.     

Thuricin 7: a novel bacteriocin produced by Bacillus thuringiensis BMG1.7, a new strain isolated from soil

AIMS: Detection and identification of new antagonistic activities towards Bacillus cereus and relatives. METHODS AND RESULTS: Twenty Bacillus thuringiensis strains were screened for their capacity to express bacteriocin-like agents. Strain BMG1.7, isolated from soil, showed an antagonistic activity called thuricin 7. Thuricin 7 was active against several species of the genus Bacillus, including three of the four known B. thuringiensis/B. cereus bacteriocin producers, as well as against Streptococcus pyogenes and Listeria monocytogenes strains. Antimicrobial activity was lost after treatment with proteinase K. The active protein had an apparent molecular weight of 11.6 kDa, and was secreted at the end of the exponential growth phase. Thuricin 7 retained 55% of the activity after incubation at 98 degrees C for 30 min. The mode of action of thuricin 7 was shown to be bactericidal and bacteriolytic. CONCLUSION: Thuricin 7 is a novel bacteriocin produced by a newly isolated Bacillus thuringiensis strain BMG1.7. SIGNIFICANCE AND IMPACT OF THE STUDY: The characteristics of thuricin 7 indicate that it is a new bacteriocin which may have interesting biotechnological applications due to its relatively large activity spectrum.     

Characterization of a mosquitocidal Bacillus thuringiensis serovar sotto strain isolated from Okinawa, Japan

AIMS: To characterize the mosquitocidal activity of parasporal inclusions of the Bacillus thuringiensis serovar sotto strain 96-OK-85-24, for comparison with two well-characterized mosquitocidal strains. METHODS AND RESULTS: The strain 96-OK-85-24 significantly differed from the existing mosquitocidal B. thuringiensis strains in: (1) lacking the larvicidal activity against Culex pipiens molestus and haemolytic activity, and (2) SDS-PAGE profiles, immunological properties and N-terminal amino acid sequences of parasporal inclusion proteins. CONCLUSIONS: It is clear from the results that the strain 96-OK-85-24 synthesizes a novel mosquitocidal Cry protein with a unique toxicity spectrum. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first report of the occurrence of a mosquitocidal B. thuringiensis strain with an unusual toxicity spectrum, lacking the activity against the culicine mosquito.     

Characterization of a Bacillus thuringiensis strain which is toxic to the housefly Musca domestica

Abstract A Bacillus thuringiensis isolate has been discovered which is toxic to the common housefly ( Musca domestica ) as well as other Diptera and Lepidoptera . Crystal δ-endotoxins purified from this isolate killed 50% of Musca larvae at a concentration of 10.2 μg/ml, and β-exotoxin was not detected. Sodium dodecyl polyacrylamide gel electrophoresis of the purified crystals revealed three protein species which were related to CryIA(b), CryIB and CryIIA toxins on the basis of immunoreactivity and amino-terminal sequence determination. Southern blot and DNA restriction analyses suggested that the strain has sequences related to one cry IA(b), one cry IIA, and two cry IIB genes.     

Characterization of a Bacillus thuringiensis strain collection isolated from diverse Costa Rican natural ecosystems

Costa Rican natural ecosystems are among the most diverse in the world. For this reason, we isolated strains of the entomopathogenic bacteria Bacillus thuringiensis (Bt) to determine their diversity, distribution and abundance. A total of 146 Bt strains were obtained from environmental samples collected from diverse natural ecosystems and life zones of Costa Rica. We recovered Bt strains from 71%, 63%, 61% and 54% of soil samples, fresh leaves, other substrates and leaf litter respectively. Bt was isolated in 65% of the samples collected in the humid tropical forest in national parks (Braulio Carrillo, Gandoca Manzanillo, Sierpe, Hitoy Cerere, and Cahuita), and in 59% of the samples collected in the dry tropical forest (Parque Nacional Marino las Baulas, Palo Verde and Santa Rosa). In the very humid tropical forest (Tortuguero) Bt was isolated in 75% of the samples and in the very humid tropical forest transition perhumid (Carara) it was found in 69% of the samples. The strains exhibit a diverse number, size and morphology of parasporal inclusion bodies: irregular (47%), oval (20%), bipyramidal (3%), bipyramidal and cubic (1%), bipyramidal, oval and irregular (5%) and bipyramidal, oval and cubic crystals (2%). Strains isolated from Braulio Carrillo, Tortuguero and Cahuita, presented predominantly irregular crystals. On the other hand, more than 60% of the isolates from Térraba-Sierpe and Hitoy-Cerere had medium oval crystals. Strains from Gandoca-Manzanillo, Palo Verde and Carara presented mainly combinations of oval and irregular crystals. Nevertheless, the greatest diversity in crystal morphology was observed in those from Santa Rosa, Llanos del Rio Medio Queso and Parque Marino las Baulas. Protein analyses of the crystal-spore preparations showed delta-endotoxin with diverse electrophoretic patterns, with molecular weights in the range of 20 to 160 kDa. Fifty six percent of the strains amplified with the cry2 primer, 54% with vip3, 20% with cry1, 9% with cry3-cry7 and 8% with cry8. The cry11 and cyt genes were found in 8% and 7% of the strains, respectively. When analyzed with specific primers for the cryl subfamily, 13 different genetic profiles were obtained. In addition, twenty-four strains did not amplify with any of the primers used, suggesting they contain novel cry genes. The diversity of Bt genes found in this collection indicates it could have great potential for the control of different species of insect pests. The toxicological characterization of the strains by bioassays against important insect pests will provide useful information about their potential use for the formulation of biological insecticides and their respective cry and vip genes for the transformation of crops to confer resistance to insects.     

Purification and characterization of a lipopeptide produced by Bacillus thuringiensis CMB26

AIMS: To isolate an antagonist for use in the biological control of phytopathogenic fungi including Colletotrichum gloeosporioides, then to purify and characterize the biocontrol agent produced by the antagonist. METHODS AND RESULTS: Bacteria that exhibited antifungal activity against the causative agent pepper anthracnose were isolated from soil, with Bacillus thuringiensis CMB26 showing the strongest activity. A lipopeptide produced by B. thuringiensis CMB26 was precipitated by adjusting the pH 2 with 3 n HCl and extracted using chloroform/methanol (2:1, v/v) and reversed-phase HPLC. The molecular weight was estimated as 1447 Da by MALDI-TOF mass spectrometry. Scanning electron and optical microscopies showed that the lipopeptide has activity against Escherichia coli O157:ac88, larvae of the cabbage white butterfly (Pieris rapae crucivora) and phytopathogenic fungi. The lipopeptide had cyclic structure and the amino acid composition was L-Glu, D-Orn, L-Tyr, D-allo-Thr, D-Ala, D-Val, L-Pro, and L-Ile in a molar ratio of 3:1:2:1:1:2:1:1. The purified lipopeptide showed the same amino acid composition as fengycin, but differed slightly in fatty acid composition, in which the double bond was at carbons 13-14 (m/z 303, 316) and there was no methyl group. CONCLUSION: A lipopeptide was purified and characterized from B. thuringiensis CMB26 and found to be similar to the lipopeptide fengycin. This lipopeptide can function as a biocontrol agent, and exhibits fungicidal, bactericidal, and insecticidal activity. SIGNIFICANCE AND IMPACT OF THE STUDY: Compared with surfactin and iturin, the lipopeptide from B. thuringiensis CMB26 showed stronger antifungal activity against phytopathogenic fungi. This lipopeptide is a candidate for the biocontrol of pathogens in agriculture.     

Identification of a second cytotoxic protein produced by Bacillus thuringiensis A1470

Bacillus thuringiensis A1470 produces multiple proteins with similar molecular masses (~30 kDa) with cytotoxicity against human cell lines. One that was previously identified, parasporin-4, is a β-pore-forming toxin. The N-terminal sequence of a second cytotoxic protein was identical to a partial sequence of parasporin-2 produced by B. thuringiensis A1547. PCR was performed on total plasmid DNA from A1470 by using primers for parasporin-2 to amplify a gene which was then cloned. The cloned gene differed from A1547 parasporin-2 by 8 bp and the predicted protein differed by four amino acids. The gene was expressed in Escherichia coli, and the cytotoxic activities of the recombinant protein against four human cell lines (MOLT-4, Jurkat, HeLa, and HepG2) were similar to those of A1547 parasporin-2. We then confirmed that the A1470 strain simultaneously produces parasporin-2 and parasporin-4.     

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.     

Characterization of a Novel Strain of Bacillus thuringiensis

Bacillus thuringiensis is a well-known species of entomopathogenic bacteria that is widely used as a biopesticide against many insect pests. Insecticidal proteins, coded for by genes located in plasmids, form typical parasporal, crystalline inclusions during sporulation. In this report, an unusual strain of B. thuringiensis subserovar oyamensis (LBIT-113), isolated from living larvae of Anopheles pseudopunctipennis in Mexico, was characterized by its ultrastructure, the protein composition of its parasporal crystal, plasmid pattern, and toxicological properties against several insect and noninsect targets. The parasporal crystal is enclosed within the spore's outermost envelope (exosporium), as determined by transmission electron microscopy, and exhibits a square, flat shape. Its main components are two proteins with sizes of 88 and 54 kDa. Despite some crystal morphology resemblance, both proteins are immunologically unrelated to the Cry IIIA protein, as shown by immunoblot analysis, when probed with antisera raised against the 88-kDa protein and the Cry IIIA protein. Partial N-terminal sequence of the 88-kDa protein revealed a unique amino acid arrangement among the Cry proteins. Solubilization of the crystal proteins was achieved at 3.3 M NaBr, and its digestion with trypsin showed only one ca. 60-kDa peptide, as observed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The patterns of three plasmids of strain LBIT-113 were considerably different from those of B. thuringiensis subspp. kurstaki, tenebrionis, and israelensis. Parasporal crystals showed no toxicity to larvae of four species of caterpillar, three species of mosquito, two species of beetle, one species of cricket, one species of ant, one species of aphid, one species of nematode, one species of ostracod, one species of ameba, and one species of rotifer.     

Bacteriocin-like inhibitor substances produced by Mexican strains of Bacillus thuringiensis

Bacteriocins are antimicrobial peptides synthesized and secreted by bacteria and could potentially be used as natural food preservatives. Here, we report the production of bacteriocin-like inhibitor substances (Bt-BLIS) by five Mexican strains of Bacillus thuringiensis. Bacillus thuringiensis subsp. morrisoni (LBIT 269), B. thuringiensis subsp. kurstaki (LBIT 287), B. thuringiensis subsp kenyae (LBIT 404), B. thuringiensis subsp. entomocidus (LBIT 420) and B. thuringiensis subsp. tolworthi (LBIT 524) produced proteinaceous Bt-BLIS with high levels of activity against Bacillus cereus and other gram-positive bacteria. Although none was active against the gram-negative bacteria, Escherichia coli, Shigella species and Pseudomonas aeruginosa, the five Bt-BLIS demonstrated antimicrobial activity against Vibrio cholerae, the etiologic agent of cholera. Biochemical and biophysical studies demonstrated that the five Bt-BLIS could be categorized into two groups, those produced by LBIT 269 and 287 (Group A) and LBIT 404, 420, 524 (Group B), based on relative time of peptide synthesis, distinctive bacterial target specificity and stability in a wide range of temperatures and pH. Because of their stability and bactericidal activities against B. cereus and V. cholerae agents of emetic, diarrheal and lethal syndromes in humans, these Bt-BLIS could potentially be used as biodegradable preservatives in the food industry.     

Isolation, purification and physicochemical characterization of water-soluble Bacillus thuringiensis melanin

Melanins are widely used in medicine, pharmacology, cosmetics and other fields. Although several technologies for the purification of water-insoluble dioxyphenylalanine (DOPA) melanins have been described, a source of water-soluble melanin is highly desirable. Here we describe an effective procedure for the isolation and purification of water-soluble melanin using the culture medium of Bacillus thuringiensis subsp. galleriae strain K1. Water-soluble melanin from this organism has an isoelectric point (pI=3.0-3.2) and was purified optimally by adsorbtion using the IA-1r resin and elution as a concentrated solution. The purified melanin obtained exhibited a similar infra-red absorbtion spectrum to synthetic melanin and contained quinolic and phenolic structures and an amino acid content of around 20% after acid hydrolysis. The molecular weight of the purified melanin determined by SDS-PAGE was 4 kDa and the electromagnetic spin resonance spectrum of the purified microbial melanin was a slightly asymmetric singlet without hyperfine structure with about 7 Gauss width of the line between points of the maximum incline and g=2.006. The concentration of paramagnetic centers in melanin is 0.21x10(18) spin/g. The results obtained provide a rapid, simple and inexpensive method for the large scale purification of water soluble melanin that may have widespread applications.