Characterization and sequence analysis of a small plasmid from Bacillus thuringiensis var. kurstaki strain HD1-DIPEL

The complete nucleotide sequence of a small (2.055 kb) plasmid pHD2 from Bacillus thuringiensis var. kurstaki strain HD1-DIPEL was obtained. The sequence encoded two open reading frames (ORFs) which corresponded to polypeptides of Mr 26,447 and 9122. Comparison of the sequence with those obtained for other plasmids from Gram-positive organisms suggested that pHD2 may belong to the extensive and highly interrelated family of plasmids exhibiting replication via a ssDNA intermediate: a putative nick site was proposed on the basis of sequence homology and one ORF exhibited distant homology with the site-specific topoisomerases encoded by the pT181 family of staphylococcal plasmids, while the other ORF exhibited considerable similarity to a small polypeptide (RepA) encoded by plasmid pLS1. Constructs consisting of pHD2, pBR322, and the chloramphenicol resistance gene from pC194 were capable of stable maintenance in B. thuringiensis var. israelensis, but were subject to apparently specific deletions in the heterologous host. The same constructs could not be established in Bacillus subtilis.