A New Minimal Replicon ofBacillus anthracisPlasmid pXO1

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 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 ϳ10 3 CFU/g, while Bacillus cereus strains 569 and ATCC 10987 were transformed with efficiencies of 10 4 and 10 2 CFU/g, respectively. Around 95% of B. anthracis cells retained pMR after one round of sporulation and germination.

Identification of Three Noncontiguous Regions on Bacillus anthracis Plasmid pXO1 That Are Important for Its Maintenance

“…Phusion High-Fidelity DNA polymerase from New England BioLabs was used for the fragment PCR. Ready-To-Go PCR beads (GE Healthcare UK Limited) were used for routine DNA rearrangement analysis as described previously (1). All constructs were verified by DNA sequencing and/or restriction enzyme digestion.…”

“…B. anthracis was electroporated with unmethylated plasmid DNA isolated from E. coli SCS110 (lacking dam and dcm). Electroporation-competent B. anthracis cells were prepared and transformed as previously described (1).…”

“…This plasmid has some similarities to pXO1 (1). These plasmids encode the analogous proteins TubZ and RepX, which are reported to be required for replication of pBtoxis (TubZ) and pXO1 (RepX) (10,11).…”

“…Recently, we found that a pXO1 minireplicon (MR) plasmid including only open reading frames (ORFs) GBAA_pXO1_0020 to GBAA_pXO1_0023 is not stably maintained in B. anthracis, whereas the full-size parent pXO1 plasmid (having 181,677 bp and 217 ORFs) is extremely stable under the same growth conditions (1). (In all following text, ORF and gene names will refer to the B. anthracis Ames "Ancestor" strain plasmid pXO1, NCBI reference sequence NC_007322.2, and are abbreviated with retention of 3 digits, so, e.g., GBAA_pXO1_0020 is rendered as ba020 or ORF 20).…”

“…Two genetic tools developed for DNA manipulation in B. anthracis (the Cre-loxP [1] and Flp-FRT systems) were employed to delete pXO1 regions upstream and downstream of the minireplicon. The new Flp-FRT recombinase method was adapted from Saccharomyces cerevisiae (16) and used in B. anthracis in a manner analogous to that of the previously described Cre-loxP system (1). FRT sites and the Flp recombinase gene were cloned into newly constructed plasmids pSCF and pFPAS, respectively.…”