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 beta-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 beta-exotoxin I. Strains producing significant amounts of beta-exotoxin I were more frequently isolated from invertebrate samples than from dust, water, soil, or plant samples.

␤-Exotoxin I is a nonspecific insecticidal metabolite secreted by some Bacillus thuringiensis strains. Several studies of B. thuringiensis strains that have lost the capacity to produce ␤-exotoxin I have suggested that there is a strong correlation between high levels of ␤-exotoxin I production and the ability to synthesize crystal proteins. In this study, we showed that a mutant strain, B. thuringiensis 407-1(Cry ؊ )(Pig ؉ ), with no crystal gene, produced considerable amounts of ␤-exotoxin I together with a soluble brown melanin pigment. Therefore, ␤-exotoxin I production can take place after a strain has lost the plasmids bearing the cry genes, which suggests that these curable plasmids probably contain determinants involved in the regulation of ␤-exotoxin I production. Using a mini-Tn10 transposon, we constructed a library of strain 407-1(Cry ؊ )(Pig ؉ ) mutants. We screened for nonpigmented mutants with impaired ␤-exotoxin I production and identified a genetic locus harboring two genes (berA and berB) essential for ␤-exotoxin I production. The deduced amino acid sequence of the berA gene displayed significant similarity to the ATP-binding domains of the DRI (drug resistance and immunity) family of ATP-binding cassette (ABC) proteins involved in drug resistance and immunity to bacteriocins and lantibiotics. The berB gene encodes a protein with six putative transmembrane helices, which probably constitutes the integral membrane component of the transporter. The demonstration that berAB is required for ␤-exotoxin I production and/or resistance in B. thuringiensis adds an adenine nucleotide analog to the wide range of substrates of the superfamily of ABC proteins. We suggest that berAB confers ␤-exotoxin I immunity in B. thuringiensis, through active efflux of the molecule.

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 g of ␤-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 g/ml. However, these amounts of ␤-exotoxin I were below the threshold for detectable toxicity against this insect species. Secretion of large amounts of ␤-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 ␤-exotoxin I.Bacillus thuringiensis is a sporogenic soil bacterium which forms characteristic crystalline inclusions composed of insecticidal crystal (Cry) proteins that are highly specific for the larvae of several insect pests (27). Because of this, B. thuringiensis has been widely used in biological pest control (19, 22a). Many isolates also produce an assortment of various other virulence factors that are secreted into the culture medium (9). These factors include the vegetative insecticidal proteins Vip1, Vip2, and Vip3, which do not display any sequence homology with each other or with any known protein (10,29,30), the Cry1I toxin (17), and ␤-exotoxin I (2, 5, 15, 23), a nonproteinaceous toxin. Unlike the Vip and Cry toxins, ␤-exotoxin I is not specific and thus may have detrimental effects on nontarget organisms (16,22); it is particularly active against dipteran species, but it is also active against coleopteran, lepidopteran, and some nematode species (12). The mechanism of action of ␤-exotoxin I is not fully understood. However, this toxin is an adenine nucleotide analogue (11) that has been found to interfere with RNA polymerase (1, 28). Thus, it has been proposed that this molecule inhibits the synthesis of RNA by competing with ATP for binding sites, thereby affecting insect molting and pupation and causing teratological effects at sublethal doses (4, 16). ␤-Exotoxin I displays some toxicity to mammalian cells (1, 22) and has been banned from public use based on World Health Organization advice (31). However, unless a bioassay or high-performance liquid chromatography (HPLC) analysis (6,13,14) is performed, it is impossible to predict whether a strain produces ␤-exotoxin I. Previous studies have shown that ␤-exotoxin I production is often linked to the presence of plasmids bearing cry genes (cry plasmids); several experiments have shown that the ability to secrete ␤-exotoxin I and the ability to produce crystals were transferred together to Bacillus cereus and B. thuringiensis recipient strains by conjugation (21, 24). Conversely, strains that had lost the capacity to synthesize crystal toxins following loss of cry plasmids also were unable to secrete ␤-exotoxin I, although the parental str...

␤-Exotoxin I is an insecticidal nucleotide analogue secreted by various Bacillus thuringiensis strains. In this report, we describe the characterization and transcriptional analysis of a gene cluster, designated sigW-ecfXecfY, that is essential for ␤-exotoxin I production in B. thuringiensis subsp. thuringiensis strain 407-1. In this strain, the disruption of the sigW cluster resulted in nontoxic culture supernatants. sigW encodes a protein of 177 residues that is 97 and 94% identical to two putative RNA polymerase extracytoplasmic-function-type sigma factors from Bacillus anthracis strain Ames and Bacillus cereus strain ATCC 14579, respectively. It is also 50, 30, and 26% identical to SigW from Clostridium perfringens and SigW and SigX from Bacillus subtilis, respectively. EcfX, encoded by the gene following sigW, significantly repressed the expression of sigW when both genes were overtranscribed, suggesting that it could be the anti-sigma factor of SigW. Following the loss of its curable cry plasmid, strain 407 became unable to synthesize crystal toxins, in contrast to the mutant strain 407-1(Cry ؊ )(Pig ؉ ), which overproduced this molecule in the absence of this plasmid. Transcriptional analysis of sigW indicated that this gene was expressed during the stationary phase and only in the 407-1(Cry ؊ )(Pig ؉ ) mutant. This suggests that in the wild type-407(Cry ؉ ) strain, ␤-exotoxin I was produced from determinants located on a cry gene-bearing plasmid and that sigW is able to induce ␤-exotoxin I production in B. thuringiensis in the absence of cry gene-bearing plasmids. Although the signal responsible for this activation is unknown, these results indicate that ␤-exotoxin I production in B. thuringiensis can be restored or induced via an alternative pathway that requires sigW expression.

Laboratory observations revealed that late-instar larvae of the eastern spruce budworm (Choristoneura fumiferana (Clemens)) (Lepidoptera: Tortricidae) spend most of their time spinning, wandering, and resting; less than 10% is spent feeding. Larvae feed in a discontinuous pattern of short feeding bouts separated by much longer intervals of nonfeeding activity. Over a 2 h observation period, feeding bouts averaged 2.2 min and were separated by 17.4 min intervals for 4th-instar larvae as compared to 3.3 min bouts separated by 33.4 min intervals for 5th-instar larvae. The duration of a feeding bout was positively correlated with the duration of the subsequent interval, not with the duration of preceding intervals, suggesting that feeding-bout frequency is governed primarily by post-ingestion processes. It is postulated that short feeding bouts followed by long intervals limit the window for ingesting an efficacious dose of aerially applied insecticides such as Bacillus thuringiensis.