The pore-forming domain of Bacillus thuringiensis insecticidal Cry toxins is formed of seven amphipathic α-helices. Because pore formation is thought to involve conformational changes within this domain, the possible role of its interhelical loops in this crucial step was investigated with Cry9Ca double mutants, which all share the previously characterized R164A mutation, using a combination of homology modeling, bioassays and electrophysiological measurements. The mutations either introduced, neutralized or reversed an electrical charge carried by a single residue of one of the domain I loops. The ability of the 28 Cry9Ca double mutants to depolarize the apical membrane of freshly isolated Manduca sexta larval midguts was tested in the presence of either midgut juice or a cocktail of protease inhibitors because these conditions had been shown earlier to greatly enhance pore formation by Cry9Ca and its R164A single-site mutant. Most mutants retained toxicity toward neonate larvae and a pore-forming ability in the electrophysiological assay, which were comparable to those of their parental toxin. In contrast, mutants F130D, L186D and V189D were very poorly toxic and practically inactive in vitro. On the other hand, mutant E129A depolarized the midgut membrane efficiently despite a considerably reduced toxicity, and mutant Q192E displayed a reduced depolarizing ability while conserving a near wild-type toxicity. These results suggest that the conditions found in the insect midgut, including high ionic strength, contribute to minimizing the influence of surface charges on the ability of Cry9Ca and probably other B. thuringiensis toxins to form pores within their target membrane.

The toxicity of Bacillus thuringiensis is temperature sensitive. Incubation of Caenorhabditis elegans with nematicidal B. thuringiensis strains at 16, 20, and 25 degrees C shows that toxicity decreases as temperature declines. At 16 degrees C, toxicity is completely lost, while it is maximal at 25 degrees C. Toxicity is pH sensitive and is significantly reduced when nematodes are incubated with the weak bases NH4Cl, chloroquine, acridine orange, methyl red, and neutral red. Based on these results, we proposed the hypothesis that the nematicidal factor is effectively internalized into the intestinal cells, a sharp deviation from the insecticidal B. thuringiensis toxins acting at the level of the brush border membrane. Although the absence of purified toxin prevents a more definitive elucidation of the mode of action, the results of this third and final part of this series of publications convincingly indicate that nematicidal B. thuringiensis do not hold the same promise as a biological control agent as the insecticidal B. thuringiensis strains.

Light-microscopic observations of the toxic action of Bacillus thuringiensis spore/crystals reveals that, in Caenorhabditis elegans, the intestine is destroyed in two stages over a period of 24 h. The anterior ring of four cells is the first and foremost target. Observations indicate that the intestine is the only tissue being destroyed. Screening of fourteen additional rhabditid nematode species against three nematicidal B. thuringiensis strains active against C. elegans, resulted in only one additional sensitive nematode species, and indicates a high species specificity of the nematicidal factor. However, in contrast to insect-specific B. thuringiensis toxins, the nematicidal toxin exhibits low developmental stage specificity against C. elegans; all developmental stages, including adult nematodes are sensitive. Moreover, sensitivity increases as development proceeds. Using ethyl methyl sulfonate induced mutagenesis two mutants of C. elegans have been recovered, exhibiting reduced sensitivity of up to 50 % against one of the nematicidal strains. Moreover, one of the mutants exhibited cross-resistance to a second nematicidal B. thuringiensis strain against which it was not screened. Preliminary data indicate that the reduced sensitivity in the mutants is not due to reduced pharyngeal pumping activity.

TransrrUssion electron rrUcroscopy is used to describe the intoxication in CaenorhabdiLis elegans, feeding on toxic spore/crystals of Bacillus thuringiensis. The toxin acts directly against the intestine, fLrst by affecting the anteriormost ring of four intestinal cells. Over a period of 12 hours, these cells lose much of their volume, the microvilli regress slowly, several cell organelles undergo dramatic change and are ultimately destroyed. No ruprure of the apical intestinal cell membrane is observed. Non-intestinal tissues seem unaffected. This srudy indicates considerable ultrastrucrural differences in the mode of action bet\veen the nematicidal toxin and the insecticidal crystal toxins from B. thun'ngiensis.

During screening of Bacillus thuringiensis isolates for nematicidal activity it was observed that spores of B. thuringiensis germinated in the intestine of bacteriophagous nematodes in the presence of antibiotics. This phenomenon was studied more closely by scanning electron microscopy. The nematodes were fed with bacterial spore-crystal mixtures in axenic culture medium supplemented with tetracyclin and chloramphenicol. Germination of spores was rare but was more frequently observed in Panagrellus redivivus than in other nematode species investigated. Germination of spores in the nematode intestine resulted in the colonization of the entire nematode within 24 hr. Crude nematode tissue preparations supported germination and subsequent growth of B. thuringiensis spores and vegetative cells. The mechanism for the loss of antibiotic activity in the nematode intestine is unknown. Since B. thuringiensis requires a nutrient-rich environment for reproduction, e.g., a cadaver, bacteriophagous nematodes may serve as suitable hosts for B. thuringiensis.

The nematicidal activity of the spore-crystal mixtures of three Bacillus thuringiensis isolates against hatched juveniles and adults of Caenorhabditis elegans was investigated. Toxicity was determined by adding 50-mu l aliquots of the spore-crystal mixtures to microtitre plate wells containing 50-mu l aqueous suspensions of 200-400 hatched juveniles and adults of C. elegans. Nematode mortality was observed from 8 hours incubation onwards; after 24 hours incubation no more significant increases in nematode mortality occurred. Nematode mortality varied from about 50 to 60 % when the nematicidal activity was tested in distilled water and was usually somewhat higher (but less than 10 %) when tested in axenic medium. Toxicity varied between the three isolates. Concentrations of at least 10(8) particles/ml were necessary to cause a nematode mortality higher than 30 %. Nematicidal activity was only observed when spore-crystal mixtures from at least 2-day-old cultures, consisting of about 50 % of vegetative cells, often containing a spore, and for about 50 % of a mixture of spores and crystals, were used. Heating to 75 degrees C and higher for 24 hours and autoclaving at 120 degrees C for 20 min destroyed the nematicidal activity of all three isolates. Differences in stability of the nematicidal activity were observed between the three isolates. In two isolates the nematicidal activity did not decline after storage at 28 degrees C for 15 days; in the third isolate the nematicidal activity declined after storage at 28 degrees C for 7 days. Multiple freezing at -20 degrees C or -70 degrees C and thawing had no effect on the nematicidal activity of two isolates but decreased the nematicidal activity of the third isolate. pH changes resulted in differences in stability of the nematicidal activity between the three isolates. These results may indicate the presence of different toxins.

Eleven cellulase genes from Gram-positive bacteria were cloned in a Lactobacillus plantarum silage inoculum. Eight of these genes were expressed as active enzymes from their original promotors and translation signals. Where tested, the enzymes produced by transformed L.plantarum had the same temperature and pH optimum as enzymes produced in the original host, or in transformed Escherichia coli. Using chloramphenicol acetyltransferase as a cell-internal marker enzyme, it could be demonstrated that at least endoglucanase D from Clostridium thermocellum was actively secreted by transformed L. plantarum. In growing L. plantarum cultures, most of the enzymes were irreversibly inactivated when the pH decreased below 4.5. If the transformed strains were to be applied as an inoculum in silage, this pH inactivation might be useful in preventing overdigestion of the crop fibre.