Activity spectra of Bacillus thuringiensis delta-endotoxins against eight insect cell lines.

Eight continuous insect cell lines were tested for susceptibility to the delta-endotoxins of several lepidopteran-active strains and cloned-gene products of Bacillus thuringiensis. The assays were performed on cells suspended in agarose gel, which allowed the toxins activated at pH 10.5 to be applied directly in a high-pH buffer without causing solvent toxicity to the cells. The responses of the cell lines to the various toxins produced activity spectra that were used to identify functionally similar and dissimilar toxin proteins. IPRI-CF-1 and FPMI-MS-5, derived from neonate larvae of Choristoneura fumiferana and Manduca sexta, respectively, exhibited the greatest sensitivity to the toxins tested, whereas B. thuringiensis subsp. entomocidus had the broadest in vitro host range. Analysis of activity spectra led to the identification of the particular Cry protein that was responsible for the broad toxicity of this subspecies and demonstrated a distinct difference in toxin composition between two strains of subsp. sotto. The identical spectra observed for subsp. kurstaki HD-1 and NRD-12 is consistent with insect bioassay data obtained previously by other workers and supports the conclusion that there is virtually no difference in activity between these two strains. The in vitro assay system, referred to as the "lawn assay" and used to test B. thuringiensis activated toxins against insect cell lines, is particularly useful in mode-of-action studies and as a rapid, preliminary test for the presence of specific cytolytic proteins, rather than as a method for screening toxins of wild-type strains for insecticidal activity. The response of cells in vitro to B. thuringiensis toxins is often very different from that of the insect from which the cells were derived.

Activation and fragmentation of Bacillus thuringiensis delta-endotoxin by high concentrations of proteolytic enzymes.

Commercial enzymes and insect gut juice at various concentrations were used to digest Bacillus thuringiensis subsp. sotto Cry1Aa protoxin and examine the fragmentation pattern and effect on insecticidal activity. Trypsin at both high (5 mg/mL) and low (0.05 mg/mL) concentrations converted protoxin to toxin with no difference in insecticidal activity against Bombyx mori larvae. In both cases, the toxin protein had an apparent M(r) of 58.4 kDa (SDS-PAGE). Active toxin of identical M(r) was also produced with low concentrations of Pronase and subtilisin, but at high concentration, it was degraded into two protease-resistant fragments of apparent M(r) 31.8 and 29.6 kDa, and exhibited no insecticidal activity. Sequencing data established the primary cleavage site to be in domain II, the receptor-binding region of the toxin, in an exposed loop between two beta-sheet strands. Fragmentation was not observed, however, when the digests were analyzed by native protein techniques, but rather the toxin molecule appeared to be intact. The amount of activated toxin produced by Choristoneura fumiferana gut juice was markedly reduced when the gut-juice concentration was increased from 1 to 50% and correlated with a loss in insecticidal activity. However, no lower M(r) protease-resistant fragments were evident in the SDS-PAGE of these digests.

Functional effects of the bacterial insecticide Bacillus thuringiensis var. kurstaki on aquatic microbial communities.

Epilithic microbial communities were colonized on leaf disks and exposed to commercial preparations of Bacillus thuringiensis var. kurstaki (Btk) in aquatic microcosms. Responses in terms of microbial respiration, bacterial cell density, protozoan density, and microbial decomposition activity were measured. Test concentrations for treatments with Dipel 64AF and Dipel 8AF in microcosms were the expected environmental concentration (EEC) of 20 IU/ml, 100x the EEC, and 1000x the EEC. Bacterial cell density in the biofilm of leaf disks was significantly increased at concentrations as low as the EEC. There were no concomitant alterations in protozoan density. Microbial respiration was significantly increased, and decomposition activity was significantly decreased, but only at the artificially high concentration of 1000x the EEC. This effect was attributed to the spore-crystal component rather than formulation ingredients. Microbial decomposition of leaf material was also determined in outdoor stream channels treated at concentrations ranging from the EEC to 100x the EEC. Although there tended to be reduced decomposition activity in treated channels, there were no significant differences in mass loss of leaf material between treated and control channels. Various regression, classification, and ordination procedures were applied to the experimental data, and none indicated significant treatment effects. These results from laboratory and controlled field experiments indicate that contamination of watercourses with Btk is unlikely to result in significant adverse effects on microbial community function in terms of detrital decomposition.

Specificity of Activated CryIA Proteins from Bacillus thuringiensis subsp. kurstaki HD-1 for Defoliating Forest Lepidoptera.

The insecticidal activity of the CryIA(a), CryIA(b), and CryIA(c) toxins from Bacillus thuringiensis subsp. kurstaki HD-1 was determined in force-feeding experiments with larvae of Choristoneura fumiferana, C. occidentalis, C. pinus, Lymantria dispar, Orgyia leucostigma, Malacosoma disstria, and Actebia fennica. The toxins were obtained from cloned protoxin genes expressed in Escherichia coli. The protoxins were activated with gut juice from Bombyx mori larvae. Biological activity of the individual gene products as well as the native HD-1 toxin was assessed as the dose which prevented 50% of the insects from producing frass within 3 days (frass failure dose [FFD(50)]). The three toxins were about equally active against M. disstria. In the Choristoneura species, CryIA(a) and CryIA(b) were up to fivefold more toxic than CryIA(c). In the lymantriid species, CryIA(a) and CryIA(b) were up to 100-fold more toxic than CryIA(c). The toxicity of HD-1 was similar to that of the individual CryIA(a) or CryIA(b) toxins in all of these species. None of the CryIA toxins or HD-1 exhibited and toxicity towards A. fennica. Comparison of the observed FFD(50) of HD-1 with the FFD(50) expected on the basis of its crystal composition suggested a possible synergistic effect of the toxins in the two lymantriid species. Our results further illustrate the diversity of activity spectra of these highly related proteins and provide a data base for studies with forest insects to elucidate the molecular basis of toxin specificity.

An in vitro system for testing Bacillus thuringiensis toxins: the lawn assay.

A cell assay system was developed that allows Bacillus thuringiensis delta-endotoxins activated at high pH (10.5) to be tested in vitro without causing alkaline injury to target cells. The assay is carried out on a lawn of gel-suspended cells, requires only 1 microliter of sample per dose, and is quantitative, rapid, and sensitive. The threshold dose for toxicity of B. thuringiensis subsp. kurstaki HD-73 with IPRI-CF-1 cells was 24 pg protein. The assay is also very useful for identifying antibodies which inhibit toxicity and for detecting beta-exotoxin.