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The complete nucleotide sequence of the plasmid pTX14-3 from Bacillus thuringiensissubsp. israelensishas been determined. The circular DNA molecule was 7649 bp and had a G + C content of 35.1%. Twenty-two open reading frames larger than 50 codons were identified. Ten of these open reading frames are suggested to be protein coding regions. The existence of the polypeptides encoded by the mob14-3and rep14-3genes were verified by maxi-cells analysis in Escherichia coli. Even though the rep14-3gene was expressed in E. colithe plasmid pTX14-3 was unable to replicate in this bacterium. The minimal region of the plasmid pTX14-3 required for replication in B. thuringiensiswas identified. Potential secondary structures upstream of the rep14-3gene indicated regulation by antisense RNA and transcription attenuation. Extensive sequence homology with the B. thuringiensissubsp. thuringiensisplasmid pG12 was found in the last part of the mob14-3gene, downstream of the rep14-3gene, and in the region containing the single-strand origin of replication (i.e., the minus origin) of pTX14-3. A sequence of 700 bp containing multiple direct repeats was found in an ORF encoding a glycine and proline rich protein of 35.9 kDa. 1.2 kbp upstream and 0.1 kbp downstream of this ORF was found a large direct repeat of 230 bp (87% identity). The region between this direct repeat was often spontaneously deleted from plasmid derivatives containing the entire pTX14-3.

pTX14-3 is a 7.5-kb cryptic plasmid isolated from a Bacillus thuringiensissubspecies israelensisstrain. Like many other small plasmids in gram-positive bacteria, pTX14-3 replicates via a single-stranded DNA intermediate. The nucleotide sequence of the replication region was determined and an open reading frame of 636 base pairs encoding a protein necessary for plasmid replication was identified by deletion analysis. No significant homology was found between this open reading frame and those encoding replication proteins identified on other plasmids isolated from gram-positive bacteria, nor could we find any homology to plus origins from other single-stranded DNA plasmids. Consequently, it seems that the replicon of pTX14-3 belongs to a new family of replicons in the group of single-stranded DNA plasmids. The sequence of the single-strand origin (i.e., the minus origin) responsible for the conversion of single-stranded plasmid DNA to double-stranded plasmid DNA was also determined. A partial homology between the minus origin of pTX14-3 and the Bacillus subtilisplasmid pBAA1 was identified. A previously identified locus that suppresses formation of high molecular weight multimers was also minimized and sequenced.