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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.
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).
The pXO2 ORF sequences detected in B. thuringiensis 33679 and B. thuringiensis AWO6 were distributed across the entire plasmid sequence, except in the 25.
The membrane containing B. thuringiensis AWO6 DNA was hybridized using a [α- 32P]dCTP-labeled probe prepared from a mixture of six PCR-amplified pXO2 ORF fragments (pXO2 ORFs 6, 10, 50, 63, 72, 81).
The two isolates with the most sequence conservation with pXO2 ORFs, B. thuringiensis isolates 33679 and AWO6, are known to contain large plasmids [ 12 19 ] . Four ORFs with high sequence similarity to B. thuringiensis AWO6 plasmid pAW63 were detected [ 22 ] , and a mixed pXO2 ORF probe hybridized with a PFGE fragment similar in size to pAW63.