This Article
Right arrow Full Text
Right arrow Full Text (PDF)
Right arrow An erratum has been published
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowReprints and Permissions
Right arrow Copyright Information
Right arrow Books from ASM Press
Right arrow MicrobeWorld
Google Scholar
Right arrow Articles by Pomerantsev, A. P.
Right arrow Articles by Leppla, S. H.
PubMed
Right arrow PubMed Citation
Right arrow Articles by Pomerantsev, A. P.
Right arrow Articles by Leppla, S. H.

 Previous Article  |  Next Article 

Journal of Bacteriology, August 2009, p. 5134-5146, Vol. 191, No. 16
0021-9193/09/$08.00+0     doi:10.1128/JB.00422-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

A New Minimal Replicon of Bacillus anthracis Plasmid pXO1 {triangledown}

Andrei P. Pomerantsev, Andrew Camp, and Stephen H. Leppla*

Bacterial Toxins and Therapeutics Section, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland 20892-3202

Received 27 March 2009/ Accepted 28 May 2009

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°C. The efficacy of transformation of plasmid-free B. anthracis Ames and Sterne strains by the original pMR was ~103 CFU/µg, while Bacillus cereus strains 569 and ATCC 10987 were transformed with efficiencies of 104 and 102 CFU/µg, respectively. Around 95% of B. anthracis cells retained pMR after one round of sporulation and germination.

* Corresponding author. Mailing address: Bacterial Toxins and Therapeutics Section, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892-3202. Phone: (301) 594-2865. Fax: (301) 480-0326. E-mail: sleppla{at}niaid.nih.gov

{triangledown} Published ahead of print on 5 June 2009.

Journal of Bacteriology, August 2009, p. 5134-5146, Vol. 191, No. 16
0021-9193/09/$08.00+0     doi:10.1128/JB.00422-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.





Copyright © 2009 by the American Society for Microbiology.   For an alternate route to Journals.ASM.org, visit: http://intl-journals.asm.org | More Info»