Bacillus thuringiensis serovar israelensis harbours, in addition to several circular plasmids, a small linear molecule of about 15 kb. Sequence analysis of this molecule, named pGIL01, showed the presence of at least 30 ORFs, five of which displayed similarity with proteins involved in phage systems: a B-type family DNA polymerase, a LexA-like repressor, two potential muramidases and a DNA-packaging protein (distantly related to the P9 protein of the tectiviral phage PRD1). Experimental evidence confirmed that pGIL01 indeed corresponds to the linear prophage of a temperate phage. This bacteriophage, named GIL01, produces small turbid plaques and is sensitive to organic solvents, which suggests the presence of lipid components in its capsid. Experiments using proteases and exonucleases also revealed that proteins are linked to the genomes of both pGIL01 prophage and GIL01 phage at their 59 extremities. Altogether, these features are reminiscent of those of phages found in the Tectiviridae family, and more specifically of those of PRD1, a broad-host-range phage of Gram-negative bacteria. Dot-blot hybridization, PFGE, PCR and RFLP analyses also showed the presence of pGIL01 variants in the Bacillus cereus group. INTRODUCTIONBacillus thuringiensis, the most widely used entomopathogenic bacterium, belongs to the Bacillus cereus sensu lato group. This cluster also includes B. cereus sensu stricto, an opportunist organism implicated in food poisoning (Granum & Lund, 1997), Bacillus anthracis, a human and animal pathogen, Bacillus mycoides and Bacillus pseudomycoides, characterized by their rhizoid growth (Nakamura, 1998), and the psychrotolerant Bacillus weihenstephanensis (Lechner et al., 1998). Despite their broad virulence, these bacteria are genetically closely related (their 16S RNA gene sequences share more than 99 % identity) and could be regarded as pertaining to a single species (Ash et al., 1991;Helgason et al., 2000).B. thuringiensis strains produce, during their sporulation, crystal toxins (delta-endotoxins) that are highly toxic to a number of insect larvae belonging to the orders Lepidoptera, Diptera and Coleoptera, but harmless to vertebrates. Classically, the numerous entomopathogenic B. thuringiensis strains, which have their own specific insecticidal activity, have been classified in different serotypes on the basis of their flagellar antigens. B. thuringiensis serovar (sv.) israelensis is active against dipteran species and is therefore one of the bioinsecticides of choice to control black flies and mosquitoes, both important vectors of human and animal diseases (for a recent review, see Glare & O'Callaghan, 2000).B. thuringiensis sv. israelensis strain H14 has been reported to contain at least eight DNA molecules, including three small (5?4, 6?7 and 7?6 kb) and four large (128, 145, 240 and 350 kb) circular plasmids, and one linear molecule (G. Jensen & L. Andrup, unpublished results). The pathogenicity of this strain only depends on the presence of the 128 kb plasmid which encodes the Cry and Cyt...

Among 48,901 samples of ready-to-eat food products at the Danish retail market, 0.5% had counts of Bacillus cereus-like bacteria above 10(4) cfu g(-1). The high counts were most frequently found in starchy, cooked products, but also in fresh cucumbers and tomatoes. Forty randomly selected strains had at least one gene or component involved in human diarrhoeal disease, while emetic toxin was related to only one B. cereus strain. A new observation was that 31 out of the 40 randomly selected B. cereus-like strains could be classified as Bacillus thuringiensis due to crystal production and/or content of cry genes. Thus, a large proportion of the B. cereus-like organisms present in food may belong to B. thuringiensis.

The aggregation phenotypes Agr؉ and Agr ؊ of Bacillus thuringiensis subsp. israelensis are correlated with a conjugation-like plasmid transfer and characterized by the formation of aggregates when the bacteria are socialized during exponential growth. We present evidence for the association of the Agr ؉ phenotype with the presence of the large (135-MDa) self-transmissible plasmid pXO16.

The potential of a bacterial toxin-antitoxin gene system for use in containment control in eukaryotes was explored. The Escherichia coli relE and relB genes were expressed in the yeast Saccharomyces cerevisiae. Expression of the relE gene was highly toxic to yeast cells. However, expression of the relB gene counteracted the effect of relE to some extent, suggesting that toxin-antitoxin interaction also occurs in S. cerevisiae. Thus, bacterial toxin-antitoxin gene systems also have potential applications in the control of cell proliferation in eukaryotic cells, especially in those industrial fermentation processes in which the escape of genetically modified cells would be considered highly risky.

The aggregation-mediated conjugation system in Bacillus thuringiensis subsp. israelensis encoded on the plasmid pXO16 is characterized by the formation of aggregates when Agr+ and Agr- cells are socialized in exponential growth. Using the aggregation phenotypes, we have identified potential recipients of the aggregation-plasmid pXO16 among Bacillus cereus, Bacillus subtilis, Bacillus megaterium, Bacillus sphaericus, and 24 subspecies of B. thuringiensis. We found 14 Agr- strains, i.e., potential recipients of the aggregation system encoded by plasmid pXO16. Five strains contained a conjugative apparatus of their own and were excluded from further examinations. To monitor the transfer of plasmid pXO16, we constructed a transposon insertion of the plasmid with Tn5401. The study of the plasmid transfer of pXO16::Tn5401 indicated the secretion of bacteriocins from both donor strain and recipient strains. Only one out of the nine strains examined was unable to receive the aggregation-plasmid pXO16 and express the aggregation phenotype and the conjugative abilities. It was found that the transfer of plasmid pXO16 to Bacillus thuringiensis subsp. israelensis Agr- strains was 100%. All recipients had acquired the aggregation-plasmid pXO16 and converted to the Agr+ phenotype.

No abstract

No abstract

The plasmid pHT73 containing cry1Ac and tagged with an erythromycin resistance gene was transferred from Bacillus thuringiensis subspecies kurstaki KT0 to several Bacillus cereus group strains by conjugation. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and phase contrast microscopy showed that the transconjugants containing plasmid pHT73 could express Cry1Ac toxin and produce bipyramidal crystalline inclusion bodies during sporulation. The study demonstrated that pHT73 could be transferred to B. thuringiensis subsp. kurstaki, several B. cereus strains and Bacillus mycoides. Under non-selective conditions, the stability of the pHT73 plasmid in the transconjugants was found to be 58.2-100% after 100 generations and 4-96% after 200 generations. The variations are mainly caused by the choice of receptor strain.