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A new minimal replicon of Bacillus anthracis plasmid 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 sigma(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 degrees C. The efficacy of transformation of plasmid-free B. anthracis Ames and Sterne strains by the original pMR was approximately 10(3) CFU/microg, while Bacillus cereus strains 569 and ATCC 10987 were transformed with efficiencies of 10(4) and 10(2) CFU/microg, respectively. Around 95% of B. anthracis cells retained pMR after one round of sporulation and germination.

SUBMITTER: Pomerantsev AP

PROVIDER: S-EPMC2725576 | biostudies-literature | 2009 Aug

REPOSITORIES: biostudies-literature

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A new minimal replicon of Bacillus anthracis plasmid pXO1.

Pomerantsev Andrei P AP Camp Andrew A Leppla Stephen H SH

Journal of bacteriology 20090605 16

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. an ...[more]

PMID: 19502400

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Project description:BackgroundCereulide, a depsipeptide structurally related to valinomycin, is responsible for the emetic type of gastrointestinal disease caused by Bacillus cereus. Recently, it has been shown that this toxin is produced by a nonribosomal peptide synthetase (NRPS), but its exact genetic organization and biochemical synthesis is unknown.ResultsThe complete sequence of the cereulide synthetase (ces) gene cluster, which encodes the enzymatic machinery required for the biosynthesis of cereulide, was dissected. The 24 kb ces gene cluster comprises 7 CDSs and includes, besides the typical NRPS genes like a phosphopantetheinyl transferase and two CDSs encoding enzyme modules for the activation and incorporation of monomers in the growing peptide chain, a CDS encoding a putative hydrolase in the upstream region and an ABC transporter in the downstream part. The enzyme modules responsible for incorporation of the hydroxyl acids showed an unusual structure while the modules responsible for the activation of the amino acids Ala and Val showed the typical domain organization of NRPS. The ces gene locus is flanked by genetic regions with high homology to virulence plasmids of B. cereus, Bacillus thuringiensis and Bacillus anthracis. PFGE and Southern hybridization showed that the ces genes are restricted to emetic B. cereus and indeed located on a 208 kb megaplasmid, which has high similarities to pXO1-like plasmids.ConclusionThe ces gene cluster that is located on a pXO1-like virulence plasmid represents, beside the insecticidal and the anthrax toxins, a third type of B. cereus group toxins encoded on megaplasmids. The ces genes are restricted to emetic toxin producers, but pXO1-like plasmids are also present in emetic-like strains. These data might indicate the presence of an ancient plasmid in B. cereus which has acquired different virulence genes over time. Due to the unusual structure of the hydroxyl acid incorporating enzyme modules of Ces, substantial biochemical efforts will be required to dissect the complete biochemical pathway of cereulide synthesis.

Dataset Information

A new minimal replicon of Bacillus anthracis plasmid pXO1.

Publications

A new minimal replicon of Bacillus anthracis plasmid pXO1.

Similar Datasets

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