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 from Bacillus 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 pAMbeta1, pIP501, and pSM19035, indicating that the pAW63 replicon belongs to the pAMbeta1 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 pAMbeta1 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 the Enterococcus 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 repC and 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.

FIG. 1

Map of the replication region of pAW63. Relevant restriction sites and ORFs larger than 100 codons are shown, and potential ribosome binding sites (RB) are marked. The locations of the origin of replication, two inverted repeats (IR), and a region containing multiple iterons are indicated. Three derivatives of the cloned fragment pAW002 and their replication capacities in B. subtilis are shown.

FIG. 2

Origin of the cloned replicon. (A) Agarose gel electrophoresis of plasmids isolated from strains AW06 (pAW63), AW05 (pHT73), wild-type strain HD73 (pAW63 and pHT73), and AW43 (cured of all large plasmids). (B) Autoradiograph of a Southern blot of the gel shown in panel A in which the 3.3-kb HindIII fragment from pAW002 was used as a probe.

FIG. 3

Alignment of the origin of replication of plasmid pAMβ1 and the regions downstream of the rep genes from plasmids pAW63 and p43. The starting point of leading-strand synthesis of pAMβ1 is indicated as a triangle, and termination of its lagging-strand synthesis maps a further 16 bp downstream (14). The stop codons of the rep genes are underlined.

FIG. 4

Region of pAW63 containing multiple iterons. (A) The repeated sequences are underlined, and arrows show the orientations. Mismatches from the consensus sequence are indicated with triangles. Sequences highlighted in boldface type represent regions of high A+T content (83 to 87%). The putative initiation codon and ribosome binding site (RB) of rep63B are also shown.

FIG. 5

Strains harboring plasmids with similarity to the pAW63 replicon. (A) Agarose gel electrophoresis of a total plasmid preparation from B. thuringiensis subsp. kurstaki and B. thuringiensis subsp. alesti strains with regions displaying similarity to the 5.8-kb HindIII fragment from the pAW63 replicon, restricted with HindIII. (B) Autoradiograph of a Southern blot of the gel shown in panel A in which pAW105 was used as a probe. The strain numbers (Tables 1 and 2) are indicated above each lane.