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 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...

Thuringiensin: A Thermostable Secondary Metabolite from Bacillus thuringiensis with Insecticidal Activity against a Wide Range of Insects

“…However, it is known to be an ATP analog [11] that interferes with RNA polymerase [23,24]. Thu inhibits the synthesis of RNA by competing with ATP on binding sites, affecting insect molting and pupation, and causing teratological effects at sublethal doses [25,26,27]. The disease symptoms caused by Thu in insects are different from those caused by insecticidal cry protein, and are only observed during insect molting and pupation.…”

An Extracytoplasmic-Function Sigma Factor Is Involved in a Pathway Controlling β-Exotoxin I Production in Bacillus thuringiensis subsp. thuringiensis Strain 407-1

“…In the case of B. thuringiensis, which is generally regarded as an insect pathogen, its ability to produce high levels of ␤-exotoxin I during stationary phase can make a significant contribution towards determining the insecticidal effects of individual strains. This could explain why genetic determinants involved in ␤-exotoxin I production are present on the same plasmids as certain cry genes (12). These plasmid-borne elements are more likely to be regulatory genes, whereas the genes responsible for ␤-exotoxin I biosynthesis are probably chromosomal.…”

“…To date, most studies on ␤-exotoxin I have focused on its mode of action and toxicology (10,36,47), and few studies have been done to unravel the regulatory mechanisms and genetic determinants involved in ␤-exotoxin I production. However, ␤-exotoxin I production has often been linked to the presence of plasmids of various sizes bearing cry genes (12). 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 (32).…”

Bacillus cereus produces several nonproteinaceous insecticidal exotoxins

“…For A. grandis and S. littoralis, the bioassays were carried out as described earlier (Espinasse et al, 2002). For A. fabae, 10 young adults were placed in a glass cylinder closed on one side with Saran Wlm and on the other side by two ParaWlm membranes.…”

“…The best known of these exotoxins is -exotoxin I, a heat-stable, water-soluble, and low molecular weight compound (701 Da). About 50% of the B. thuringiensis strains secrete detectable amounts of this toxin during the stationary growth phase (Espinasse et al, 2002). -Exotoxin I is highly toxic by oral route to a wide range of insect species.…”