Cytolytic activity and immunological similarity of the Bacillus thuringiensis subsp. israelensis and Bacillus thuringiensis subsp. morrisoni isolate PG-14 toxins.

The parasporal bodies of the mosquitocidal isolates of Bacillus thuringiensis subsp. israelensis and B. thuringiensis subsp. morrisoni isolate PG-14 were compared with regard to their hemolytic and cytolytic activities and the immunological relatedness of the 28- and 65-kilodalton (kDa) proteins that occur in both subspecies. The alkali-solubilized parasporal bodies of B. thuringiensis subsp. israelensis caused 50% lysis of human erythrocytes at 1.14 micrograms/ml, whereas those of B. thuringiensis subsp. morrisoni caused similar lysis at 1.84 micrograms/ml. Preincubation of solubilized parasporal bodies with dioleolyl phosphatidylcholine significantly inhibited the hemolytic activity of both supspecies. In cytolytic assays against Aedes albopictus cells, the toxin concentrations causing 50% lysis for B. thuringiensis subsp. israelensis and B. thuringiensis subsp. morrisoni were 1.87 and 11.98 micrograms/ml, respectively. Polyclonal antibodies raised separately against the 25-kDa protein (a tryptic digest of the 28-kDa protein) or the 65-kDa protein of B. thuringiensis subsp. israelensis cross-reacted, respectively, with the 28- and the 65-kDa proteins of B. thuringiensis subsp. morrisoni. However, neither of these antibodies cross-reacted with the 135-kDa protein of either subspecies. These results indicate that the mosquitocidal and hemolytic properties of B. thuringiensis subsp. israelensis and B. thuringiensis subsp. morrisoni isolate PG-14 are probably due to the biologically related proteins that are present in the parasporal bodies of both subspecies. The lower hemolytic activity of the B. thuringiensis subsp. morrisoni may be due to the presence of lower levels of the 28-kDa protein in that subspecies.(ABSTRACT TRUNCATED AT 250 WORDS)     

Parasporal bodies of Bacillus thuringiensis subsp. morrisoni (PG-14) and Bacillus thuringiensis subsp. israelensis are similar in protein composition and toxicity

Abstract The mosquitocidal parasporal bodies of the PG-14 isolate of Bacillus thuringiensis ssp. morrisoni and B. thuringiensis ssp. israelensis were purified on sodium bromide gradients and compared using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) electron microscopy and bioassays against mosquito larvae. The parasporal bodies of both subspecies were spherical/ovoidal, approx. 0.7–1.2 μm in diameter, and contained major proteins of 28, 65, 126 and 135 kDa. In addition to these, the parasporal body of B. thuringiensis ssp. morrisoni contained at least one other major protein, of 144 kDa, which correlated with the presence of a quasi-bi-pyramidal inclusion not present in the B. thuringiensis ssp. israelensis parasporal body. The LC₅₀ for parasporal bodies of each subspecies was in the range of 3 ng/ml for fourth-instars of Aedes aegypti . These results indicate that B. thuringiensis Serotype 8a:8b, which is generally considered to produce proteins toxic to lepidopterous insects, is capable of producing a protein toxin complement similar to B. thuringiensis Serotype 14.     

Cytolytic Peptide Fragments of Cyt1Aa from Bacillus thuringiensis subsp. israelensis

Cyt1Aa is the major and most active component of the parasporal crystal of the Gram-positive soil entomopathogenic bacterium Bacillus thuringiensis subsp. israelensis. The Cyt1Aa protoxin exhibits some hemolytic and cytolytic activity. However, highly active 22–25 kDa toxins are obtained after proteolysis of Cyt1Aa from both the N- and the C-termini. As shown in this study, preliminary binding of the protoxin to polylamellary liposomes or partial denaturation of Cyt1Aa and further processing by several exogenous proteases yielded short 4.9–11.5 kDa cytolytic peptide fragments of Cyt1Aa. The shortest 51 amino acid peptide was obtained after pre-incubation of Cyt1Aa with SDS and proteolysis with proteinase K. This peptide was purified, identified as the Ile87–Asp137 fragment of Cyt1Aa and was shown to exhibit more than 30 % hemolysis of rabbit erythrocytes.     

Restriction analysis of plasmids of Bacillus thuringiensis subsp. israelensis H14

Restriction analysis of three small plasmids ( m _r; 3·65, 4·65 and 5·5 Md) of the mosquito pathogen Bacillus thuringiensis subsp. israelensis was performed. A restriction map of the 3–65 Md plasmid is presented. The stability of this plasmid, the availability of five single sites and the existence of a genetic transfer system make it ideal for development as a cloning vector.     

Properties of a 72-kilodalton mosquitocidal protein from Bacillus thuringiensis subsp. morrisoni PG-14 expressed in B. thuringiensis subsp. kurstaki by using the shuttle vector pHT3101.

The mosquitocidal properties of Bacillus thuringiensis subsp. israelensis and B. thuringiensis subsp. morrisoni PG-14 are attributable to protein inclusions grouped together within a parasporal body. In both of these strains, the mosquitocidal activity resides in proteins with molecular masses of 27, 72, 128, and 135 kDa. In an attempt to determine the toxicity of each protein, the shuttle vector pHT3101 was used to express the cryIVD gene (encoding the 72-kDa CryIVD protein) from B. thuringiensis subsp. morrisoni in an acrystalliferous mutant of B. thuringiensis subsp. kurstaki. With this system, parasporal inclusions of the 72-kDa protein were obtained that were comparable in size, shape, and toxicity to those produced by parental B. thuringiensis subsp. morrisoni. The inclusions were bar shaped, measured 500 by 300 by 150 nm, and were easily visible with phase-contrast microscopy by 16 h of cell growth. A 50% lethal concentration of 64 ng/ml for these inclusions was determined in bioassays against fourth instars of Culex quinquefasciatus, which was similar to the 50% lethal concentration of 55 ng/ml obtained for the 72-kDa inclusion from B. thuringiensis subsp. israelensis. In contrast, expression of the cryIVD gene in Escherichia coli was very low and only detectable by immunoblot analysis. These results demonstrate that the pHT3101-B. thuringiensis expression system can be used to express the CryIVD protein in quantities and with properties comparable to that obtained with the natural host. This system may prove useful for the expression of other B. thuringiensis proteins and, in particular, for reconstitution experiments with inclusions produced by the mosquitocidal subspecies of B. thuringiensis.     

Properties of a 72-kilodalton mosquitocidal protein from Bacillus thuringiensis subsp. morrisoni PG-14 expressed in B. thuringiensis subsp. kurstaki by using the shuttle vector pHT3101.

The mosquitocidal properties of Bacillus thuringiensis subsp. israelensis and B. thuringiensis subsp. morrisoni PG-14 are attributable to protein inclusions grouped together within a parasporal body. In both of these strains, the mosquitocidal activity resides in proteins with molecular masses of 27, 72, 128, and 135 kDa. In an attempt to determine the toxicity of each protein, the shuttle vector pHT3101 was used to express the cryIVD gene (encoding the 72-kDa CryIVD protein) from B. thuringiensis subsp. morrisoni in an acrystalliferous mutant of B. thuringiensis subsp. kurstaki. With this system, parasporal inclusions of the 72-kDa protein were obtained that were comparable in size, shape, and toxicity to those produced by parental B. thuringiensis subsp. morrisoni. The inclusions were bar shaped, measured 500 by 300 by 150 nm, and were easily visible with phase-contrast microscopy by 16 h of cell growth. A 50% lethal concentration of 64 ng/ml for these inclusions was determined in bioassays against fourth instars of Culex quinquefasciatus, which was similar to the 50% lethal concentration of 55 ng/ml obtained for the 72-kDa inclusion from B. thuringiensis subsp. israelensis. In contrast, expression of the cryIVD gene in Escherichia coli was very low and only detectable by immunoblot analysis. These results demonstrate that the pHT3101-B. thuringiensis expression system can be used to express the CryIVD protein in quantities and with properties comparable to that obtained with the natural host. This system may prove useful for the expression of other B. thuringiensis proteins and, in particular, for reconstitution experiments with inclusions produced by the mosquitocidal subspecies of B. thuringiensis.     

Delta endotoxin of Bacillus thuringiensis subsp. israelensis.

From Bacillus thuringiensis subsp. israelensis, a proteinase-resistant protein was purified which exhibited toxicity to larval mosquitoes and cultured mosquito cells, lysed erythrocytes, and was lethal to mice. To extract the protein, a sporulating culture of B. thuringiensis subsp. israelensis was treated with alkali, neutralized, and incubated with trypsin and proteinase K. It was then purified by gel filtration and DEAE column chromatography. Up to 240 micrograms of toxic protein was purified from 1 g (wet weight) of culture pellet. Two closely related forms of toxic protein were obtained: the 25a and 25b proteins. The two forms comigrated near 25,000 daltons in a sodium dodecyl sulfate-polyacrylamide gel, were serologically related, and showed similar partial protease digestion profiles, but were distinguishable by DEAE chromatography and nondenaturing polyacrylamide gel electrophoresis. Protein sequencing data indicated the 25b protein lacked the two amino acids at the amino terminus of the 25a protein. A Western blot enzyme-linked immunosorbent assay of alkali-solubilized proteins that were not treated with proteases suggested the toxic 25a and 25b proteins were proteolytically derived from a larger molecule of about 28,000 daltons. Alkali-solubilized proteins from an acrystalliferous strain of B. thuringiensis subsp. israelensis and from B. thuringiensis subsp. kurstaki failed to cross-react with antibodies to the 25a protein.     

Optimization of medium composition for the production of mosquitocidal toxins from Bacillus thuringiensis subsp. israelensis

Optimization of chicken feather (CF) based culture medium for the production of Bacillus thuringiensis subsp. israelensis (Bti) biomass in combination with the agro industrial by-product (coconut cake, CC) and manganese chloride (MnCl2) has been evaluated. The biomass yield of Bti spore/crystal toxin was highest (12.06 g/L) from the test medium (CF+CC+MnCl2) compared to the reference medium (Luria Bertani, LB). Toxicity assay with Bti produced from the test medium against mosquito vectors (Culex quinquefasciatus, Anopheles stephensi and Aedes aegypti) was also satisfactory and results were comparable with bacteria produced from LB. The results suggest that Bti can be produced to the maximum extent possible as a potential mosquitocidal activity as suggested by the test medium (CF+CC+MnCl2).     

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.     

Minireplicon from pBtoxis of Bacillus thuringiensis subsp. israelensis

A 2.2-kb fragment containing a replicon from pBtoxis, the large plasmid that encodes the insecticidal endotoxins of Bacillus thuringiensis subsp. israelensis, was identified, cloned, and sequenced. This fragment contains cis elements, including iterons, found in replication origins of other large plasmids and suggests that pBtoxis replicates by a type A theta mechanism. Two genes, pBt156 and pBt157, encoding proteins of 54.4 kDa and 11.8 kDa, respectively, were present in an operon within this minireplicon, and each was shown by deletion analysis to be essential for replication. The deduced amino acid sequences of the 54.4-kDa and 11.8-kDa proteins showed no substantial homology with known replication (Rep) proteins. However, the 54.4-kDa protein contained a conserved FtsZ domain, and the 11.8 kDa protein contained a helix-turn-helix motif. As FtsZ proteins have known functions in bacterial cell division and the helix-turn-helix motif is present in Rep proteins, it is likely that these proteins function in plasmid replication and partitioning. The minireplicon had a copy number of two or three per chromosome equivalent in B. thuringiensis subsp. israelensis but did not replicate in B. cereus, B. megaterium, or B. subtilis. A plasmid constructed to synthesize large quantities of the Cry11A and Cyt1A endotoxins demonstrated that this minireplicon can be used to engineer vectors for cry and cyt gene expression.     

Genome Stability of Bacillus thuringiensis subsp. israelensis Isolates

Swedish soil isolates biochemically classified as Bacillus thuringiensis subsp. israelensis were further examined for genetic diversity by multilocus enzyme electrophoresis (MLEE), random amplified polymorphic DNA analysis (RAPD), pulse field gel electrophoresis (PFGE), and Southern blotting, and were compared with reference strains. All the tested strains belonging to the Bt. israelensis serotype H14 were found to be identical, as judged from the RAPD analysis. MLEE analysis gave a similar result; only one H14 strain was found to differ from the remaining H14 strains by one null allele. PFGE analysis confirmed a very close relationship between the H14 strains but revealed an SfiI restriction fragment of variable size. Southern blot analyses were carried out with probes for the chromosomally encoded flagellin gene(s) and the plasmid-encoded mosquitocidal toxins. All probes gave similar hybridization patterns in the H14 strains. The mosquito toxin probes hybridized only to the H14 strains, except for one probe hybridizing to strain 6:3, which was originally isolated from the same soil sample as strains 6:11 and 6:12. Because the RAPD, MLEE, and PFGE analyses showed that strain 6:3 appears to be unrelated to strains 6:11 and 6:12, the presence of a mosquito toxin sequence in strain 6:3 may suggest that gene transfer has occurred. Received: 8 July 1999 / Accepted: 9 August 1999     

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.     

Comparison of Bacillus thuringiensis subsp. israelensis CryIVA and CryIVB cloned toxins reveals synergism in vivo

When the gene for the mosquitocidal protein CryIVA was expressed in two strains of Bacillus thuringiensis (Bt) cured of their resident delta-endotoxin genes, the protein accumulated as large inclusions. The inclusions produced in the Bt subsp. kurstaki recipient strain were twice as soluble at alkaline pH as the inclusions produced in Bt subsp. israelensis. Solubilized protoxins were activated by treatment with mosquito gut extracts or trypsin for varying lengths of time and tested for in vitro cytotoxicity on cell lines of three genera of mosquito. CryIVA treated with any of the mosquito gut extracts for 6 h showed significant toxicity against Anopheles gambiae cells and slight activity on Culex quinquefasciatus cells. For CryIVB, the only significant cytotoxicity observed was against Aedes aegypti cells after treatment with Aedes gut extract. In in vivo bioassays, both CryIVA, purified from either of the Bt recipient strains, and CryIVB inclusions were similarly toxic to A. aegypti and A. gambiae larvae but CryIVA was 25-fold more toxic to C. quinquefasciatus. Synergism in vivo between the two toxins was revealed when results from assaying single toxins and mixtures were compared. Mixtures of CryIVA and CryIVB proved to be 5-fold more toxic to Culex than either toxin used singly and showed a reduced but similar synergism when tested against Aedes and Anopheles larvae. The synergism was not duplicated in vitro using cell lines from these three insects.     

Minireplicon from pBtoxis of Bacillus thuringiensis subsp. israelensis

A 2.2-kb fragment containing a replicon from pBtoxis, the large plasmid that encodes the insecticidal endotoxins of Bacillus thuringiensis subsp. israelensis, was identified, cloned, and sequenced. This fragment contains cis elements, including iterons, found in replication origins of other large plasmids and suggests that pBtoxis replicates by a type A theta mechanism. Two genes, pBt156 and pBt157, encoding proteins of 54.4 kDa and 11.8 kDa, respectively, were present in an operon within this minireplicon, and each was shown by deletion analysis to be essential for replication. The deduced amino acid sequences of the 54.4-kDa and 11.8-kDa proteins showed no substantial homology with known replication (Rep) proteins. However, the 54.4-kDa protein contained a conserved FtsZ domain, and the 11.8 kDa protein contained a helix-turn-helix motif. As FtsZ proteins have known functions in bacterial cell division and the helix-turn-helix motif is present in Rep proteins, it is likely that these proteins function in plasmid replication and partitioning. The minireplicon had a copy number of two or three per chromosome equivalent in B. thuringiensis subsp. israelensis but did not replicate in B. cereus, B. megaterium, or B. subtilis. A plasmid constructed to synthesize large quantities of the Cry11A and Cyt1A endotoxins demonstrated that this minireplicon can be used to engineer vectors for cry and cyt gene expression.     

苏云金芽胞杆菌以色列亚种20kDa蛋白质对CytA蛋白溶细胞作用的影响

为了证明苏云金芽胞杆菌以色列亚种２０ｋＤｅ蛋白质对ＣｙｔＡ蛋白溶细胞作用的影响, 根据２０ｋＤｅ蛋白质和ｃｙｔＡ蛋白基因的核苷酸序列,用ＡＭＰＬＩＦＹ程序设计了一套带有酶切位 点的引物,经ＰＣＲ扩增分别获得了２０ｋＤｅ蛋白质和ｃｙｔＡ蛋白基因。将其基因与表达载体 ｐＵＨＥ２４连接并转化到大肠杆菌ＸＬＩ和ＤＨＳ 分别获得含２０ｋＤａ蛋白质基因的克隆子 ＬＺ２９;含ｃｙｔＡ基因的克隆子ＬＺｃｙｔＡ和含有二者基因的重组子ＬＺ２０Ａ．在ＩＰＴＧ诱导下,测定 了不同克隆株基因表达产物对大肠杆菌细胞生长的影响。结果表明：ＬＺ２０的细胞生长不受影 响;ＬＺｃｙｔＡ的细胞被杀死;ＬＺ２０Ａ的细胞生长也不受影响。这表明２０ｋＤａ蛋白质基因与ｃｙｔＡ 蛋白基因重组后,２０ｋＤａ蛋白质基因表达产物可保护ＣｙｔＡ蛋白对大肠杆菌的溶细胞作用,而 巳这种作用并不因不同大肠杆菌受体而改变。     

Composition and Toxicity of the Inclusion of Bacillus thuringiensis subsp. israelensis

The multisegmented ovoidal inclusion of Bacillus thuringiensis subsp. israelensis was found to be composed of two structurally and biochemically distinct components. Electron microscopy of the inclusion revealed it to be composed mainly of osmiophobic or lightly stained segments crystallized in a lattice showing a repeat of approximately 4.3 nm. These light segments of the inclusions were shared by osmiophylic darkly stained segments with a crystal lattice repeat of approximately 7.8 nm. The lightly stained segments were soluble at pH 9.2 in sodium dodecyl sulfate-dithiothreitol-Tris-hydrochloride. The extracts of lightly stained segments were lytic to mammalian erythrocytes, and the precipitate obtained by lowering the pH to 5.2 was toxic to the larvae of Aedes egypti. The dark inclusion segments remaining, besides being much less toxic to larvae, were nonlytic to erythrocytes and were soluble at pH 10.5 in sodium dodecyl sulfate-dithiothreitol-Tris-hydrochloride. The light segment was composed of two major polypeptide doublets with molecular weights of 145,000 and 135,000, and 27,000 and 26,500, and the dark segments were composed of a single major polypeptide with a molecular-weight of 70,000. Hence, the inclusion of B. thuringiensis subsp. israelensis is more complex than previously reported, and we conclude that the toxin may be the polypeptide with a molecular weight of 27,000 and 26,500.     

Fate of Bacillus thuringiensis subsp. israelensis under Simulated Field Conditions

The fate of Bacillus thuringiensis subsp. israelensis in a natural aquatic habitat was studied in a model system by using laboratory-simulated field waters and a mutant of the bacterium resistant to three antibiotics. Contact with mud of a sporal culture of the mutant resulted in an immediate disappearance of the larvicidal activity but had no influence on viability. The cessation of toxicity was caused by bacterial adsorption on soil particles, since 99.8% of the bacteria was found in the mud fraction within 45 min, with concurrent disappearance from the supernatant. When the mud was stirred, the bacteria could be redetected. The viability count of the mud suspension remained practically constant for at least 22 days, indicating that the spores were still fully viable but were incapable of germinating and multiplying in the mud under our experimental conditions. Approximately 8% of the colony forming ability of the bacteria could be separated from the mud by vigorous mixing followed by immediate filtration. The filtrated spores retained their toxicity, killing 90% of the larval populations even after 22 days incubation in the soil. The inactivation of the toxic activity of B. thuringiensis subsp. israelensis in the mud was therefore a reversible process and was probably due to masking of the bacteria, thus making the bacteria and their toxin inaccessible to the larvae. In the simulated field waters without mud, we observed only a very slow inhibition of the larvicidal activity. In contrast to the activity in the mud suspension, this activity could not be restored.     

Purification of the mosquitocidal and cytolytic proteins of Bacillus thuringiensis subsp. israelensis

Two proteins from parasporal crystals of Bacillus thuringiensis subsp. israelensis were purified to electrophoretic homogeneity by gel filtration and anion-exchange chromatography. The larger of the two proteins (molecular weight, 68,000) was not cytolytic, whereas the smaller protein (molecular weight, 28,000) was highly cytolytic when assayed against rat erythrocytes. When these proteins were assayed against larvae of the yellow fever mosquito, Aedes aegypti, the larger protein was at least 100-fold more toxic than the smaller protein. Although proteolytic activity was not detected in solubilized crystals nor in purified protein preparations, the toxin (molecular weight, 68,000) was readily degraded to smaller, nontoxic molecules, even when maintained at 4 degrees C. Mixtures of the two purified proteins were significantly more toxic to mosquito larvae than was either protein alone. Thus, it is likely that both the mosquitocidal and the cytolytic protein play roles in the overall insecticidal action of the parasporal crystal produced by this bacterium.     

Novel fermentation media for production of Bacillus thuringiensis subsp. israelensis

The production of Bacillus thuringiensis subsp. israelensis (deBarjac) (Bti) as a biopesticide is not cost-effective using existing fermentation technology. In this study, we explored the use of several less expensive alternative culture media (potato, common sugar, and Bengal gram) for the growth and production of Bti. Growth was obtained in all tested media and was comparable to that obtained in conventional medium (Luria-Bertani). Toxicity assays showed that the toxin produced from the novel growth media were effective in killing larvae of Culex quinquefasciatus, Anopheles stephensi, and Aedes aegypti and toxicity was comparable to that produced from Luria-Bertani medium. These observations suggest that potato can be used as a cheap source of culture medium for the production of Bti toxin in mosquito control programs.