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Total genomic DNA from B. thuringiensis strain AWO6 hybridized with 23 pXO2 ORF fragments, including all ORFs tested in the region between ORF 5 and ORF 38 (Table 3).
Pulsed field gel electrophoresis was used to separate plasmid and chromosomal DNA in B. thuringiensis AWO6, and a Southern hybridization blot using a mixed pool of pXO2-derived probes (ORFs 6, 10, 50, 63, 72, 81) was performed to determine if any of the ORFs were present on the pAW63 plasmid (Figure).
A dot-blot hybridization assay was performed using DNA from B. thuringiensis strain AWO6 as probe against PCR-amplified pXO2 ORF DNA applied to a nylon membrane.
3 kb pXO2 cap gene-containing region did not hybridize with B. thuringiensis strain AWO6 DNA.
B. thuringiensis AWO6 is a strain containing a 70 kb plasmid designated pAW63 [ 12 21 ] . This strain was derived from B. thuringiensis HD73 by curing of its crystal toxin bearing plasmid, pHT73 [ 12 21 ] . The pAW63 plasmid contains a replication complex that is classified as a member of the pAMB-1 family of theta replicating plasmids that are present in a broad range of Gram positive species [ 22 ] . Plasmid pXO2 also appears to be a pAMB1-like theta replicating plasmid [ 23 ] and elements surrounding the replication complex are present in both pXO2 and pAW63 (see pXO2 ORFs 35, 37, 38, 39 in Tables 2and 3).