A 28 kDa protein of the Bacillus thuringiensis serovar shandongiensis isolate 89-T-34-22 induces a human leukemic cell-specific cytotoxicity.

A 28 kDa protein that exhibits cytocidal activity specific for human leukemic T (MOLT-4) cells was purified from proteinase K-digested parasporal inclusion of a Bacillus thuringiensis serovar shandongiensis isolate. The N-terminal sequence of the protein was identical with that of the 32 kDa protein, regarded as a protoxin, of the inclusion proteins. The median effective concentration of this protein was 0.23 microg/ml against MOLT-4 cells and its specific activity was 7.9 times greater than that of the whole inclusion proteins. The 28 kDa protein induced necrosis-like cytotoxicity against MOLT-4 cells and the cytopathic effect with the passage of time was characterized by cell swelling, nuclear membrane isolation and chromatin condensation.

Similarity in moth-fly specific larvicidal activity between two serologically unrelated Bacillus thuringiensis strains.

Parasporal inclusions of a Bacillus thuringiensis isolate designated 92-KU-105-9 (H14/19) exhibited unusual larvicidal activity, specific for the moth-fly, Telmatoscopus albipunctatus (Diptera: Psychodidae), similar to that of a previously reported B. thuringiensis serovar leesis (H33) strain. The LC50 value of the purified inclusions was 4.92 micrograms ml-1 for the moth-fly larvae, while no mortality was shown in the mosquitoes Culex pipiens molestus and Anopheles stephensi, at protein concentrations up to 10 mg ml-1. Morphologically, the inclusion was a homogeneous globular body surrounded by an electron-dense, thick envelope. Multilamellar inner structure was evident between envelope membrane and inclusion matrix. SDS-PAGE revealed that the inclusions consist of five proteins with molecular masses of 72, 70, 68, 56 and 30 kDa. These proteins cross-reacted with the antibodies against inclusion proteins of the serovar leesis strain. High homologies existed in N-terminal amino acid sequences between the three major proteins (72, 70 and 68 kDa) and the two established protein classes, Cry4A and Cry10A.

Lectin activity of Bacillus thuringiensis parasporal inclusion proteins.

Parasporal inclusion proteins from a total of 151 Bacillus thuringiensis strains, consisting of 139 Japanese isolates and the type strains of 12 H serovars, were screened for haemagglutination (HA) activity against sheep erythrocytes. Of 58 B. thuringiensis strains with HA activity, nine strains exhibited high activity and the remaining 49 strains were moderately active. The strains with high HA activity were derived from phylloplanes and soils of five geographically different localities, and belonged to H serovars kurstaki and other undefined serotype(s). The HA activities in the four selected strains were generated only when alkali-solubilised parasporal inclusion proteins were proteolytically processed. Furthermore, the lectin activity of the four strains was strongly inhibited by preincubation with N-acetylgalactosamine. The lectin-producing B. thuringiensis strains were heterogeneous in other biological activities of parasporal inclusions: insecticidal activity and cytocidal action on human leukaemia T cells.

A novel class of mosquitocidal delta-endotoxin, Cry19B, encoded by a Bacillus thuringiensis serovar higo gene.

Partially digested HincII fragments of DNA from a mosquitocidal strain of Bacillus thuringiensis serovar higo were cloned into pBluescript II SK(+) and propagated in Escherichia coli. Recombinant cells were screened immunologically for the production of parasporal inclusion antigens. One E. coli clone harboring a recombinant plasmid exhibited larvicidal activity to Culex pipiens molestus, but not to Anopheles stephensi. Hybridization experiments revealed that the gene of the toxin protein is located on a 110 kb plasmid of B. thuringiensis serovar higo. Sequence analysis detected an open reading frame of 2046 nucleotides encoding a polypeptide of 682 amino acid residues with a predicted molecular weight of 78,467. The gene encoded five block regions commonly conserved in the insecticidal protein genes of B. thuringiensis. Amino acid sequence of the 78 kDa protein shared 49% identity and 56% similarity with that of the Cry19A protein from B. thuringiensis serovar jegathesan. A new class of delta-endotoxin protein, designated Cry19B, was established on the basis of this protein.

A novel isolate of Bacillus thuringiensis serovar leesis that specifically exhibits larvicidal activity against the moth-fly, Telmatoscopus albipunctatus.

A soil isolate designated 88-KO-14-45, belonging to Bacillus thuringiensis serovar leesis (H33), exhibited larvicidal activity against the moth-fly, Telmatoscopus albipunctatus (Diptera: Psychodidae), but not for larvae of the culicine and aedine mosquitoes and Lepidoptera. Purified parasporal inclusions had an LC50 value of 5.78 micrograms/ml for the larval moth-fly, but gave no mortality against larvae of Culex pipiens molestus (Diptera: Culicidae) at protein concentrations up to 10 mg/ml. Electron microscopic observations revealed that the parasporal inclusions are homogeneous round-shaped bodies enclosed with thick, electron dense envelopes. Haemolytic activity against sheep erythrocytes was not detected in the solubilized inclusions. SDS-PAGE showed that the inclusions are composed of 72, 68, 56 and 30 kDa proteins. Immunologically, these proteins were unrelated to the inclusion proteins of B. thuringiensis serovar israelensis, while a 70 kDa protein of the strain 73-E-10-2 (B. thuringiensis serovar darmstadiensis) was seroactive to antibodies against proteins of 88-KO-14-45.

Cloning and characterization of a Bacillus thuringiensis serovar higo gene encoding a novel class of the delta-endotoxin protein, Cry27A, specifically active on the Anopheles mosquito.

A novel gene encoding a 98-kDa mosquitocidal delta-endotoxin protein, designated Cry27A, was cloned from a Bacillus thuringiensis serovar higo strain. The Cry27A protein contained the five sequence blocks of amino acids commonly conserved in most B. thuringiensis Cry proteins. Relatively high homologies, ranging from 43.0% to 84.4%, existed between the Cry27A protein and several established classes of mosquitocidal Cry proteins (Cry4A, Cry10A, Cry19A, Cry19B, and Cry20A) in the sequence of 51 N-terminal amino acids. The complete sequence of this protein, however, showed low levels (<40%) of amino acid identity to those of the known Cry proteins. Although the expression level of the cry27A gene was low in the transformants under the control of its own promoter, the use of the cyt1A promoter resulted in high-level expression of the gene, leading to the formation of inclusions. The expressed Cry27A protein showed larvicidal activity highly specific for Anopheles stephensi, but lacked the toxicity against Culex pipiens molestus and Aedes aegypti. The results suggest that the Cry27A protein is responsible for the Anopheles-preferential toxicity of the B. thuringiensis serovar higo strain.

Screening of the Bacillus thuringiensis Cry1Ac delta-endotoxin on the artificial phospholipid monolayer incorporated with brush border membrane vesicles of Plutella xylostella by optical biosensor technology.

The binding of Cry1Ac, an insecticidal protein of Bacillus thuringiensis, to a brush border membrane (BBM) isolated from midguts of the diamondback moth Plutella xylostella was examined by surface plasmon resonance (SPR)-based biosensor. BBM was mixed with 1,3-ditetradecylglycero-2-phosphocholine (PC14), a neutral charged artificial lipid, and was reconstructed to a monolayer on a hydrophobic chip for the biosensor. The binding of Cry1Ac to the reconstructed monolayer was analyzed by a two-state binding model, and it was shown that Cry1Ac bound to the monolayer in the first step with an affinity constant (K(1)) of 508 nM, followed by the second uni-molecular step with an equilibrium constant (K(2)) of 0.472. The overall affinity constant K(d) was determined to be 240 nM. The binding was markedly inhibited by N-acetyl-D-galactosamine (K(i)=8 mM). The monolayer was shown to retain a high affinity to Cry1Ac, providing an insect-free system for rapid and large-scale screening of B. thuringiensis insecticidal proteins by the SPR-based biosensor technology.

Bacillus thuringiensis soil populations naturally occurring in the Ryukyus, a subtropic region of Japan.

Of 809 soil samples collected from the seven islands of the Ryukyus, Japan, 107 samples (13.2%) contained Bacillus thuringiensis. The frequency of B. thuringiensis among the B. cereus group was 1.1% (235/21842) on the average. The B. thuringiensis soil populations of the Ryukyus consisted of more than 22 H serogroups. The predominant H serotype was the H5ac/21 (serovar canadensis/colmeri), followed by the H3ad (serovar sumiyoshiensis) and H16 (serovar indiana). Geographically, most widely distributed H serogroups were the H16 and H10ac (serovar londrina); the former was recovered from five islands and the latter from three islands. Parasporal inclusions of the isolates were morphologically heterogeneous, roughly grouped into four categories: bipyramidal/cuboidal, spherical/ovoid, irregularly-pointed, and irregular-shaped. About 53% of the isolates formed spherical to ovoid parasporal inclusions. None of the isolates exhibited larvicidal activity against the silkworm, Bombyx mori. Only four isolates belonging to four different serotypes killed larvae of the mosquito, Aedes aegypti. These mosquito-specific isolates all produced spherical parasporal inclusions.

Larvicidal activity of Bacillus thuringiensis natural isolates; indigenous to Japan, against two nematoceran insect pests occurring in urban sewage environments.

A total of 1449 Bacillus thuringiensis strains, indigenous to Japan, were screened for larvicidal activity against two nematoceran insect pests, the mosquito, Culex pipiens molestus (Culicidae), and the moth-fly, Telmatoscopus albipunctatus (Psychodidae). Mosquito specific strains were abundant in H serotypes 3abc (serovar kurstaki), 3ade (fukuokaensis), 4ac (kenyae), 7 (aizawai), 11ac (kyushuensis) and 29 (amagiensis), while moth-fly specific strains were predominantly found in H serotype 17 (tohokuensis). Strains toxic to both insects were most frequently detected in H serotypes 10 (darmstadiensis) and 17/27. Seven selected B. thuringiensis strains were highly toxic to Culex and/or Telmatoscopus. There was a diversity in SDS-PAGE profiles of inclusion proteins of these strains.

Larval susceptibility of the diamondback moth, Plutella xylostella (Lepidoptera: Plutellidae), to Bacillus thuringiensis H serovars isolated in Japan.

A total of 1700 Japanese strains of Bacillus thuringiensis, belonging to at least 47 H serogroups, were examined for insecticidal activity against larvae of the diamondback moth, Plutella xylostella. The high-level toxicity was associated with 612 isolates (36.0%). Of these, 608 isolates (99.3%) fell into 13 H serogroups belonging to the low-numbered H serotypes, H1-H10. Conversely, most isolates belonging to the high-numbered serotypes (>H10) had little or no larvicidal activity; only one isolate of the serovar japonensis H23 was active. P xylostella larvae were susceptible to 89.8% of the serovar morrisoni H8a:8b strains and 85.7% of galleriae H5a:5b strains. High values of 60-80% were also obtained in six serovars (thuringiensis H1, alesti H3a:3c, kurstaki H3a:3b:3c, kenyae H4a:4c, aizawai H7, and tolworhi H9), while relatively low values of <60% in two other common serovars, sotto H4a:4b and darmstadiensis H10a:10b. Five selected isolates, belonging to H serovars other than kurstaki and aizawai, were 10-60 times less toxic than the reference strain HD-1 (serovar kurstaki). Parasporal inclusion proteins of these strains were immunologically unrelated to those of the strain HD-1 and the aizawai type strain.

Isolation of Bacillus thuringiensis from intertidal brackish sediments in mangroves.

Intertidal brackish sediments in mangroves were examined for isolation of Bacillus thuringiensis strains with novel toxicity spectra. A total of 18 B. thuringiensis isolates were recovered from eight sediment samples (36.4%) out of 22 samples tested. The frequency of B. thuringiensis was 1.3% among the colonies of Bacillus cereus/B. thuringiensis group. While five isolates were allocated to the four H serogroups, the majority of the isolates were serologically untypable or untestable. Two isolates belonging to the serovar israelensis/tochigiensis (H14/19) exhibited strong toxicities against larvae of the mosquito, Culex pipiens molestus, and mammalian cells (sheep erythrocyte and two human cancer cell lines) in vitro. The other 16 isolates showed no toxicity against the mosquito and mammalian cells. None of the isolates showed larvicidal activity against the diamondback moth, Plutella xylostella. Strong lectin activities against sheep erythrocytes were associated with two serologically untestable isolates and an H3 isolate.

Red swamp crawfish (Procambarus clarkii): an alternative experimental host in the study of white spot syndrome virus.

The pathogenicity of white spot syndrome virus (WSSV) for the red swamp crawfish (Procambarus clarkii) was investigated after infection by intramuscular (i.m.) injection and oral route. The cumulative mortality of crawfish injected i.m. with WSSV reached 100% in 5 days. After oral feeding WSSV-infected kuruma shrimp (Penaeus japonicus) muscle tissues to the crawfish the cumulative mortality of this host reached 100% in 11 days. On reinfection trials, all the crawfish fed WSSV-infected crawfish muscle tissues died in 9 days. All the shrimp injected with a filtrate of infected crawfish heart tissues died in 12 days with typical signs of white spot syndrome (WSS). Electron microscopy clearly demonstrated that WSSV propagated in the cells of the crawfish midgut. This study showed that the red swamp crawfish can be used as alternative experimental host in the study of WSSV.

Isolation and characterization of a novel Bacillus thuringiensis strain expressing a novel crystal protein with cytocidal activity against human cancer cells.

AIMS: To characterize a novel, unusual, Bacillus thuringiensis strain, to clone its Cry gene and determine the spectrum of action of the encoded Cry protein. METHODS AND RESULTS: The B. thuringiensis strain, referred to as M15, was isolated from dead two-spotted spider mites (Tetranychus urticae Koch; Arthropoda: Arachnida: Tetranychidae). It is an autoagglutination-positive strain and is therefore non-serotypeable. A sporulated culture produces a roughly spherical parasporal inclusion body, the crystal, tightly coupled to the spore. Although the crystal appears to be composed of at least two major polypeptides of 86 and 79 kDa as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, Southern hybridization indicates that the corresponding crystal protein gene is likely present in only one copy. The crystal protein gene was cloned and, based on nucleotide sequence homology with an orthologous cry31Aa1 gene, assigned the name cry31Aa2. Although initially isolated from spider mites, B. thuringiensis M15 is non-toxic to spider mites and it does not produce the wide spectrum beta-exotoxin. Assays on mammalian cells, however, reveal that Cry31Aa2, when cleaved with trypsin, is cytocidal to some human cancer cells but not to normal human cells. No cytocidal activity was induced after protease treatment of Cry31Aa2 with either chymotrypsin or proteinase K. Trypsin, chymotrypsin and proteinase K cleavage sites were determined. CONCLUSIONS: The B. thuringiensis strain M15 exhibits specific cytocidal activities against some human cancer cells. SIGNIFICANCE AND IMPACT OF THE STUDY: This study raises questions as to the actual role of this bacterial strain and its crystal protein in the environment. It may be possible to further develop the Cry31Aa2 protein to target specific human cancer cells.

Larvicidal toxicity of Japanese Bacillus thuringiensis against the mosquito Anopheles stephensi.

Japanese isolates of Bacillus thuringiensis were screened for larvicidal activity against the mosquito Anopheles stephensi, the urban malaria vector of the Indian subcontinent. Among more than 30 strains identified, larvicidal activity causing > 80% mortality in 72 h was demonstrated for 41/1449 (2.8%) isolates. The majority of strains and isolates (97.2%) exhibited little or no larvicidal activity. Anopheles-active strains belonged to more than 12 H serotypes, especially H3ade (serovar fukuokaensis) and H44 (serovar higo). SDS-PAGE profiles of inclusion proteins showed 4 distinct types among 6 active strains examined. The most active Japanese isolates were H20 strain 89-T-34-14 (LC50 4.4 micrograms/ml) and H44 serovar higo strain 74-E-45-24 (LC50 7.6 micrograms/ml), respectively, 13-fold and 23-fold less active than the international standard H14 serovar israelensis (LC50 0.33 microgram/ml).

Recovery of Bacillus thuringiensis from marine sediments of Japan.

Marine sediments from a Japanese bay were examined for the occurrence of Bacillus thuringiensis. Of 1313 colonies belonging to the Bacillus cereus/B. thuringiensis group, 22 (1.7%) were allocated to B. thuringiensis. Marine isolates of B. thuringiensis consisted of heterogeneous multiple H serogroups; 10 isolates were assigned to the eight serovars (kurstaki, sumiyoshiensis, sotto, aizawai, darmstadiensis, thompsoni, neoleonensis, and higo); two motile isolates failed to react with the reference antisera; and the others were serologically untestable. Insecticidal activities were associated with two kurstaki isolates (toxic to both Lepidoptera and Diptera) and a higo isolate (Diptera-specific). None of the parasporal inclusion proteins of the 22 isolates exhibited in vitro cytotoxic activity against two vertebrate cells, sheep erythrocytes and HeLa cells. All B. thuringiensis isolates had no halophilism, although seawater-based medium supported their growth, sporulation, and formation of parasporal inclusions.

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.

Bacillus thuringiensis serovar higo (flagellar serotype 44), a new serogroup with a larvicidal activity preferential for the anopheline mosquito.

Eight strains of Bacillus thuringiensis, isolated in Japan, formed spherical parasporal inclusions and exhibited low to moderate larvicidal activities for two mosquito species, Anopheles stephensi and Culex pipiens molestus, but not for another dipteran, Telmatoscopus albipunctatus, or two lepidopterans, Bombyx mori and Hyphantria cunea. The anopheline toxicity (LC50 = 6.3 micrograms ml-1) was > 10 times greater than the activity on the Culex mosquito. These strains were assigned to a previously undescribed flagellar (H) antigenic group. On the basis of the representative strain, 92-KU-137-4, a serogroup with H antigen 44, Bacillus thuringiensis serovar higo was established as new.

Bacillus thuringiensis serovar shandongiensis strain 89-T-34-22 produces multiple cytotoxic proteins with similar molecular masses against human cancer cells.

AIMS: To prove that Bacillus thuringiensis serovar shandongiensis strain 89-T-34-22 produces several novel cytotoxic proteins against human leukaemic T cells. METHODS AND RESULTS: Parasporal inclusion protein was solubilized and processed by proteinase K and was separated by anion-exchange chromatography. Cytopathic effects of each fraction against MOLT-4 and Jurkat cells were monitored. CONCLUSIONS: Existence of at least two novel cytotoxic proteins was suggested and N-terminal sequences of the newly identified proteins were determined to be QSTTDVIREY and X (Y or I) (P or I) NLANELA (X indicates uncertain amino acids). Molecular masses of the two proteins were approx. 27-28 kDa. SIGNIFICANCE AND IMPACT OF THE STUDY: In this study, we demonstrated that the strain 89-T-34-22 produces at least two novel cytotoxic proteins with similar molecular masses against human cancer cells. This is the first strain of B. thuringiensis which produces multiple cytotoxic proteins against human cancer cells.

Specificity of lectin activity of Bacillus thuringiensis parasporal inclusion proteins.

Sheep erythrocyte-agglutinating parasporal inclusion proteins from four Bacillus thuringiensis strains (FITC-20, FITC-73, FITC-76 and IBC-1456) were examined for lectin activity against erythrocytes from four mammalian (rabbit, horse, cow and guinea pig) and an avian (chicken) species. Of the five erythrocyte species, only rabbit cells were agglutinated with the protein of the human cancer cell-killing strain IBC-1456. No haemagglutination (HA) activities were shown in other protein-erythrocyte combinations. The lectin activity of the strain IBC-1456 against rabbit cells was strongly inhibited by preincubation with D-galactose. Overall results revealed that the B. thuringiensis lectins have a preference for sheep erythrocytes.

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 microgram.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.