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 strains of Bacillus thuringiensis subsp. israelensis was studied under a range of environmentally relevant laboratory conditions in vitro, in river water, and in mosquito larvae. Mobilization of pBC16 was detected in vitro at a range of temperatures, pH values, and available water conditions, and the maximum transfer ratio was 10 ؊3 transconjugant per recipient under optimal conditions. Transfer of conjugative plasmid pXO16ϻTn5401 was also detected under this range of conditions. However, a maximum transfer ratio of 1.0 transconjugant per recipient was attained, and every recipient became a transconjugant. In river water, transfer of pBC16 was not detected, probably as a result of the low transfer frequency for this plasmid and the formation of spores by the introduced donor and recipient strains. In contrast, transfer of plasmid pXO16ϻTn5401 was detected in water, but at a lower transfer ratio (ca. 10 ؊2 transconjugant per donor). The number of transconjugants increased over the first 7 days, probably as a result of new transfer events between cells, since growth of both donor and recipient cells in water was not detected. Mobilization of pBC16 was not detected in killed mosquito larvae, but transfer of plasmid pXO16::Tn5401 was evident, with a maximum rate of 10 ؊3 transconjugant per donor. The reduced transfer rate in insects compared to broth cultures may be accounted for by competition from the background bacterial population present in the mosquito gut and diet or by the maintenance of a large population of B. thuringiensis spores in the insects.

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Ultraviolet (u.v.) laser irradiation has been used to inactivate Bacillus subtilis spores deposited on to planar aluminium-and polyethylene-coated packaging surfaces. Kill kinetics were found to be diphasic, with an initial rapid inactivation phase followed by tailing. Although no de®nitive evidence was obtained, it is thought that spores located within packaging crevices/pores were primarily responsible for the observed tailing. Surviving spores were also found on the unexposed underside of cards and, to a lesser extent, within clumps. The log count reduction in B. subtilis was dependent on spore loading and total u.v. dose. In comparison, packaging surface composition,¯uence (2±18 Jm À2 ) and frequency (40±150 Hz) had only a negligible effect. By irradiating boards carrying 10 6 spores, with a dose of 11Á5 J cm À2 , a log count reduction >5 was obtained. The mode of spore inactivation was primarily through DNA disruption. This was con®rmed by the high sensitivity of spores lacking protective, small, acid-soluble proteins, in addition to the high frequency of auxotrophic and asporogenous mutations found amongst survivors.

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