Josette Chaufaux

Distinct mutations in PlcR explain why some strains of the Bacillus cereus group are nonhemolytic.

Bacillus thuringiensis, Bacillus cereus, and Bacillus anthracis are closely related species belonging to the Bacillus cereus group. B. thuringiensis and B. cereus generally produce extracellular proteins, including phospholipases and hemolysins. Transcription of the genes encoding these factors is controlled by the pleiotropic regulator PlcR. Disruption of plcR in B. cereus and B. thuringiensis drastically reduces the hemolytic, lecithinase, and cytotoxic properties of these organisms. B. anthracis does not produce these proteins due to a nonsense mutation in the plcR gene. We screened 400 B. thuringiensis and B. cereus strains for their hemolytic and lecithinase properties. Eight Hly- Lec- strains were selected and analyzed to determine whether this unusual phenotype was due to a mutation similar to that found in B. anthracis. Sequence analysis of the DNA region including the plcR and papR genes of these strains and genetic complementation of the strains with functional copies of plcR and papR indicated that different types of mutations were responsible for these phenotypes. We also found that the plcR genes of three B. anthracis strains belonging to different phylogenetic groups contained the same nonsense mutation, suggesting that this mutation is a distinctive trait of this species.

Chronic Exposure of the European Corn Borer (Lepidoptera: Crambidae) to Cry1Ab Bacillus thuringiensis Toxin

Abstract Transgenic corn expressing the insecticidal toxin from Bacillus thuringiensis Berliner is gaining support as an effective control technology for use against lepidopteran pests, particularly European corn borer, Ostrinia nubilalis Hübner (Lepidoptera: Crambidae). However, there is concern that widespread adoption of transgenic plants will rapidly lead to B. thuringiensis toxin resistance. Thus, long-term selection of O. nubilalis populations with the Cry1Ab B. thuringiensis toxin has been undertaken in several laboratories in the United States and in Europe. We present results from two independent selection experiments performed in laboratories at the University of Nebraska and at the Institut National de la Recherche Agronomique in France. Although the protocols and methods used by the two laboratories were different, the results were comparable. The highest level of resistance occurred at generation 7 (14-fold), generation 9 (13-fold), and generation 9 (32-fold) for three different strains. For each strain, the level of resistance fluctuated from generation to generation, although there were consistently significant decreases in toxin susceptibility across generations for all selected strains. These results suggest that low levels of resistance are common among widely distributed O. nubilalis populations.

Construction of new insecticidal Bacillus thuringiensis recombinant strains by using the sporulation non-dependent expression system of cryIIIA and a site specific recombination vector

Bacillus thuringiensis (Bt) delta-endotoxins are safe biological insecticidal proteins whose usefulness has long been recognized. The first commercialized Bt insecticidal formulations were composed of spore-crystal preparations derived from wild-type strains. These products generally have a limited insecticidal host range and several genetically modified strains have, therefore, been constructed using transformation procedures. However, addition of a new delta-endotoxin gene to strains already harboring other delta-endotoxin genes often resulted in broader-spectrum but less potent products because they produced significantly less of each of the crystal proteins. We report expression of the coding sequence of the sporulation specific cryIC gene from the non-sporulation-dependent cryIIIA promoter. Large amounts of CryIC accumulated in various Bt strains with different genetic backgrounds. Sporulation deficient Spo0A mutants, acrystalliferous derivatives and wild-type Bt strains expressing the engineered cryIII-cryIC gene were obtained. Introduction of the cryIII-cryIC gene whose product is highly active against Spodoptera littoralis into the Kto strain harboring the cryIA(c) gene active against Ostrinia nubilalis resulted in the construction of a new strain with increased potency and broader activity spectrum than the parent strain. Large amounts of each toxin were produced and the expression of the two genes seemed to be summed, presumably because the expression systems of the two genes are different. The plasmid shuttle vector used to introduce the cryIII-cryIC gene into the different Bt hosts utilizes the specific resolution site of transposon Tn4430 to enable construction of recombinant Bt strains that are free of foreign non-Bt DNA. This should facilitate the approval and acceptance for environmental release of the insecticidal recombinant products.

Esterase metabolism and reduced penetration are causes of resistance to deltamethrin in Spodoptera exigua HUB (Noctuidea; lepidoptera)

Abstract Using deltamethrin in toxicological experiments we have shown that a guatemalian strain of Spodoptera exigua has an LC 40 at least 100 times that of a sensitive one. Delayed penetration and cleavage of deltamethrin at the ester bond are two mechanisms responsible for this resistance. Degradation of deltamethrin is 17 times higher in the resistant strain compared to the sensitive one. This degradation is inhibited by DEF and paraoxon, indicating that esterases are likely involved in the metabolism of this insecticide. In addition it has been shown that the resistant strain has an enhanced esterase activity toward chromogenic substrates, such as naphthyl acetate or methylumbelliferyl acetate, the level depending on the substrate used. It is likely that activity toward chromogenic substrates and the hydrolysis of deltamethrin are related.

Occurrence and linkage between secreted insecticidal toxins in natural isolates of Bacillus thuringiensis.

Little is known about the occurrence and linkage between secreted insecticidal virulence factors in natural populations of Bacillus thuringiensis (Bt). We carried out a survey of 392 Bt strains isolated from various samples originating from 31 countries. The toxicity profile of the culture supernatants of these strains was determined individually against Anthonomus grandis (Coleoptera) and Spodoptera littoralis (Lepidoptera). We analyzed β-exotoxin I production and searched for the genes encoding Vip1–2, Vip3, and Cry1I toxins in 125 of these strains. Our results showed that these insecticidal toxins were widespread in Bt but that their distribution was nonrandom, with significant linkage observed between vip3 and cry1I and between vip1–2 and β-exotoxin I. Strains producing significant amounts of β-exotoxin I were more frequently isolated from invertebrate samples than from dust, water, soil, or plant samples.

Correspondence of High Levels of Beta-Exotoxin I and the Presence of cry1B in Bacillus thuringiensis

ABSTRACT Examination of 640 natural isolates of Bacillus thuringiensis showed that the 58 strains (9%) whose supernatants were toxic to Anthonomus grandis (Coleoptera: Curculionidae) produced between 10 and 175 μg of β-exotoxin I per ml. We also found that 55 (46%) of a sample of 118 strains whose culture supernatants were not toxic to A. grandis nevertheless produced between 2 and 5 μg/ml. However, these amounts of β-exotoxin I were below the threshold for detectable toxicity against this insect species. Secretion of large amounts of β-exotoxin I was strongly associated with the presence of cry1B and vip2 genes in the 640 natural B. thuringiensis isolates studied. We concluded that strains carrying cry1B and vip2 genes also possess, on the same plasmid, genetic determinants necessary to promote high levels of production of β-exotoxin I.

Did the introduction of maize into Europe provide enemy-free space to Ostrinia nubilalis? Parasitism differences between two sibling species of the genus Ostrinia.

We examined whether maize offers enemy‐free space (EFS) to its pest Ostrinia nubilalis, and may thereby have contributed to its divergence from the sibling species, Ostrinia scapulalis, feeding mainly on mugwort, when introduced into Europe five centuries ago. We collected Ostrinia larvae on maize (70 populations, 8425 individuals) and mugwort (10 populations, 1184 individuals) and recorded parasitism using both traditional (counting emerging parasitoids) and molecular methods (detection by specific polymerase chain reaction). The main parasitoid was Macrocentrus cingulum (Braconidae). On mugwort, parasitism was twice that on maize, and parasitoid‐related mortality was 8 times higher. This suggests that maize affords substantial EFS to Ostrinia feeding on it. The lower Mortality:Infestation ratio in maize suggests that O. nubilalis’ immune response might be stronger than that of O. scapulalis. If so, adapting to maize and diverging from O. scapulalis would decrease the impact of parasitism on O. nubilalis at both ecological and evolutionary levels.

A comparison and analysis of the toxicity and receptor binding properties of Bacillus thuringiensis CryIC ∂‐endotoxin on Spodoptera littoralis and Bombyx mori

The binding of l‐[35S]methionine in vivo labelled CryIC toxin to its receptor in brush border membrane vesicle (BBMVs) prepared from Spodoptera littoralis and Bombyx mori was studied. Both insect species were highly susceptible to the CryIC toxin in bioassays, B. mori being 7‐fold more sensitive to CryIC than S. littoralis (LC50s of 10 ng/cm2 and 70 ng/cm2, respectively). Competition and direct binding experiments revealed saturable high‐affinity binding sites on BBMVs from both insects which had similar binding characteristics for the CryIC toxin (K d = 10 nM, B max = 8 to 9 pmol/mg BBMVs and IC50 = 37 nM for both insect species). Thus a specific receptor for the CryIC toxin is present in both insect species and the 7‐fold greater potency of CryIC towards B. mori is not due to qualitative or quantitative differences in binding affinity or receptor site concentration. Dissociation experiments also indicated that the binding of [35S]CryIC to B. mori BBMVs is partially reversible.

The cryic gene from Bacillus thuringiensis provides protection against Spodoptera littoralis in young transgenic plants

Abstract A 3′-end truncated crystal protein gene derived from Bacillus thuringiensis subsp. aizawai 7.29, encoding the toxic fragment of the insecticidal CRYIC protein, was placed under the control of the CaMV 35S promoter with a duplicated enhancer. Its expression in tobacco conferred significant insecticidal activity towards the important pest Spodoptera littoralis . Expression of the CRYIC toxin is at its maximum level at the early stages of plant development then decreases as the plants become older.