A New Minimal Replicon of Bacillus anthracis Plasmid pXO1

Abstract

ABSTRACT An 8,883-bp mini-pXO1 plasmid containing a replicon from Bacillus anthracis pXO1 (181.6 kb) was identified by making large deletions in the original plasmid using a newly developed Cre- loxP system. Portions of the truncated mini-pXO1 were cloned into an Escherichia coli vector unable to replicate in B. anthracis . A 5.95-kb region encompassing three putative genes was identified as the minimal pXO1 fragment required for replication of the resulting recombinant shuttle plasmid (named pMR) in B. anthracis . Deletion analysis showed that the only genes essential for replication were the pXO1-14 and pXO1-16 genes, which are transcribed in opposite directions and encode predicted proteins of 66.5 and 67.1 kDa, respectively. The ORF14 protein contains a helix-turn-helix motif, while the ORF16 upstream region contains attributes of a theta-replicating plasmid origin of replication (Ori), namely, an exclusively A+T-containing segment, five 9-bp direct repeats, an inverted repeat, and a σ A -dependent promoter for the putative replication initiator Rep protein (ORF16). Spontaneous mutations generated in the ORF14, ORF16, and Ori regions of pMR during PCR amplification produced a temperature-sensitive plasmid that is unable to replicate in B. anthracis at 37°C. The efficacy of transformation of plasmid-free B. anthracis Ames and Sterne strains by the original pMR was ∼10 3 CFU/μg, while Bacillus cereus strains 569 and ATCC 10987 were transformed with efficiencies of 10 4 and 10 2 CFU/μg, respectively. Around 95% of B. anthracis cells retained pMR after one round of sporulation and germination.