Plasmid Transfer between the Bacillus thuringiensis Subspecies kurstaki and tenebrionis in Laboratory Culture and Soil and in Lepidopteran and Coleopteran Larvae

Abstract

Plasmid transfer between Bacillus thuringiensis subsp. kurstaki HD1 and B. thuringiensis subsp. tenebrionis donor strains and a streptomycin-resistant B. thuringiensis subsp. kurstaki recipient was studied under environmentally relevant laboratory conditions in vitro, in soil, and in insects. Plasmid transfer was detected in vitro at temperatures of 5 to 37°C, at pH 5.9 to 9.0, and at water activities of 0.965 to 0.995, and the highest transfer ratios (up to 10 ؊1 transconjugant/donor) were detected within 4 h. In contrast, no plasmid transfer was detected in nonsterile soil, and rapid formation of spores by the introduced strains probably contributed most to the lack of plasmid transfer observed. When a B. thuringiensis subsp. kurstaki strain was used as the donor strain, plasmid transfer was detected in killed susceptible lepidopteran insect (Lacanobia oleracea) larvae but not in the nonsusceptible coleopteran insect Phaedon chocleriae. When a B. thuringiensis subsp. tenerbrionis strain was used as the donor strain, no plasmid transfer was detected in either of these insects even when they were killed. These results show that in larger susceptible lepidopteran insects there is a greater opportunity for growth of B. thuringiensis strains, and this finding, combined with decreased competition due to a low initial background bacterial population, can provide suitable conditions for efficient plasmid transfer in the environment.Bacillus thuringiensis is a gram-positive, spore-forming bacterium that produces insecticidal crystal protein toxins during sporulation. B. thuringiensis was first found in diseased silkworms (Bombyx mori) in 1901 (11, 24) but has since been isolated from a range of environments, including insects, soil, dust from stored grain, and leaves from coniferous and deciduous trees (5,15,17,20). B. thuringiensis has been shown to contain a range of toxins and virulence determinants that may enhance its pathogenicity. However, the insecticidal crystal protein toxins or ␦-endotoxins are the primary determinants of pathogenicity. Generally, B. thuringiensis insecticidal protein toxin genes (cry) reside on large self-transmissible plasmids, and individual B. thuringiensis strains can harbor a diverse range of plasmids that can vary in number and in size from around 2 to 200 kb (4, 7-9, 16). Using plasmid curing, Gonzalez et al. (8,9) demonstrated that the cry genes are present on large plasmids which are more than 50 kb long and can be self-transmissible between strains by a conjugation-like mechanism. The cry genes are not randomly distributed and tend to be confined to relatively few plasmids (2, 3). For example, B. thuringiensis subsp. kurstaki HD1 contains 12 plasmids, but four of its cry genes (cry1Aa, cry1Ac, cry2A, and cry2B) reside on a single 110-MDa plasmid (6); the remaining cry gene (cry1Ab) occurs on an unstable 44-MDa plasmid (2, 3). Hence, plasmid behavior is of considerable significance for insecticidal activity in this bacterium.The transfer frequency of some of the large self-transmis...

Plasmid Transfer between Bacillus thuringiensis subsp. israelensis Strains in Laboratory Culture, River Water, and Dipteran Larvae

“…Plasmid pBC16 was introduced into B. thuringiensis subsp. israelensis IPS82 by electroporation by using a field strength of 8.75 kV/cm (400 ⍀, 25 F, 1.75 kV) and a Bio-Rad gene pulser (30). B. thuringiensis subsp.…”

“…This environment has proven to be conducive to gene exchange. Studies have found that plasmids can move between donor and recipient strains of B. thuringiensis in larvae of the lepidopteran insects Galleria mellonella, Spodoptera littoralis, and Lacanobia oleracea at levels similar to that found in broth culture (20,30) but not in the midgut environment of the coleopteran insect Phaedon cochleriae. So far, there have been no studies of transfer between B. thuringiensis strains in dipteran insects.…”

“…The effects of time, temperature, pH, and salt concentration on plasmid transfer were measured by using the standard broth mating procedure with appropriate modifications (30).…”

Role of Receptors in Bacillus thuringiensis Crystal Toxin Activity

“…Many cry genes are associated with transposable elements that may facilitate gene amplification, leading to the evolution of new toxins (29). In addition, most cry genes are found on plasmids, and horizontal transfer by conjugation may result in the creation of new strains with a novel complement of cry genes (166,167).…”

Characterization of a native Bacillus thuringiensis isolates from Malaysia that produces exosporium-enclosed parasporal inclusion

“…thuringiensis contains a set of self-replicating plasmids and they are transferred during conjugation (Thomas et al, 2000;Rolle at al., 2005). The set of plasmids carried by B. thuringiensis strains can vary in number and in size (4-150 MDa) (Fagundes et al, 2011).…”