Replication Mechanism and Sequence Analysis of the Replicon of pAW63, a Conjugative Plasmid from Bacillus thuringiensis

Abstract

A 5.8-kb fragment of the large conjugative plasmid pAW63 fromBacillus thuringiensis subsp. kurstaki HD73 containing all the information for autonomous replication was cloned and sequenced. By deletion analysis, the pAW63 replicon was reduced to a 4.1-kb fragment harboring four open reading frames (ORFs). Rep63A (513 amino acids [aa]), encoded by the largest ORF, displayed strong similarity (40% identity) to the replication proteins from plasmids pAMβ1, pIP501, and pSM19035, indicating that the pAW63 replicon belongs to the pAMβ1 family of gram-positive theta-replicating plasmids. This was confirmed by the facts that no single-stranded DNA replication intermediates could be detected and that replication was found to be dependent on host-gene-encoded DNA polymerase I. An 85-bp region downstream of Rep63A was also shown to have strong similarity to the origins of replication of pAMβ1 and pIP501, and it is suggested that this region contains the bona fide pAW63 ori. The protein encoded by the second large ORF, Rep63B (308 aa), was shown to display similarity to RepB (34% identity over 281 aa) and PrgP (32% identity over 310 aa), involved in copy control of theEnterococcus faecalis plasmids pAD1 and pCF10, respectively. No significant similarity to known proteins or DNA sequences could be detected for the two smallest ORFs. However, the location, size, hydrophilicity, and orientation of ORF6 (107 codons) were analogous to those features of the putative genes repCand prgO, which encode stability functions on plasmids pAD1 and pCF10, respectively. The cloned replicon of plasmid pAW63 was stably maintained in Bacillus subtilis and B. thuringiensis and displayed incompatibility with the native pAW63. Hybridization experiments using the cloned replicon as a probe showed that pAW63 has similarity to large plasmids from other B. thuringiensis subsp. kurstaki strains and to a strain of B. thuringiensis subsp. alesti.

Germination and conjugation of Bacillus thuringiensis subsp. israelensis in the intestine of gnotobiotic rats

“…Previous studies have shown similarity between genes from B. thuringiensis and other bacteria, e.g. enterococci (Wilcks et al 1999;Van der Auwera et al 2005) and Clostridium sp. (Barloy et al 1998), indicating that genes have been shared between B. thuringiensis and indigenous intestinal species.…”

Elaboration of an electroporation protocol for large plasmids and wild-type strains ofBacillus thuringiensis

“…The size of plasmid DNA badly affects electroporation transformation efficiency (Ohse et al 1995;Turgeon et al 2006). Bacillus thuringiensis plasmids consist of two groups: (i) the small plasmids (<15 kb), which perform rolling-circle replication (RC) using a single-stranded intermediate (Andrup et al 2003), and (ii) the large plasmids that normally follow a theta replicating mode with low copy number (Wilcks et al 1999;Huang et al 2006). Large theta replicating plasmids always gave transformation efficiencies lower than those obtained with the small RCR plasmids (Turgeon et al 2006).…”

Genomics of the group of organisms

“…Based on similarity to other replication proteins identified from plasmids in the B. cereus group and other Gram-positive organisms, the origin of replication was readily identified in pXO2 [101,102]. While well characterized in B. thuringiensis plasmid pAW63, the pXO2 origin of replication was not functionally characterized until recently [80].…”