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Applied and Environmental Microbiology, July 2006, p. 4899-4906, Vol. 72, No. 7
0099-2240/06/$08.00+0     doi:10.1128/AEM.00354-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Facile Recovery of Individual High-Molecular-Weight, Low-Copy-Number Natural Plasmids for Genomic Sequencing{dagger}

Laura E. Williams,1 Chris Detter,2 Kerrie Barry,2 Alla Lapidus,2 and Anne O. Summers1*

Department of Microbiology, University of Georgia, Athens, Georgia,1 U.S. Department of Energy, Joint Genome Institute, Walnut Creek, California2

Received 13 February 2006/ Accepted 3 May 2006

Sequencing of the large (>50 kb), low-copy-number (<5 per cell) plasmids that mediate horizontal gene transfer has been hindered by the difficulty and expense of isolating DNA from individual plasmids of this class. We report here that a kit method previously devised for purification of bacterial artificial chromosomes (BACs) can be adapted for effective preparation of individual plasmids up to 220 kb from wild gram-negative and gram-positive bacteria. Individual plasmid DNA recovered from less than 10 ml of Escherichia coli, Staphylococcus, and Corynebacterium cultures was of sufficient quantity and quality for construction of high-coverage libraries, as shown by sequencing five native plasmids ranging in size from 30 kb to 94 kb. We also report recommendations for vector screening to optimize plasmid sequence assembly, preliminary annotation of novel plasmid genomes, and insights on mobile genetic element biology derived from these sequences. Adaptation of this BAC method for large plasmid isolation removes one major technical hurdle to expanding our knowledge of the natural plasmid gene pool.

* Corresponding author. Mailing address: Department of Microbiology, University of Georgia, 527 Biological Sciences Building, Athens, GA 30602. Phone: (706) 542-2669. Fax: (706) 542-6140. E-mail: summers{at}uga.edu.

{dagger} Supplemental material for this article may be found at http://aem.asm.org.

Applied and Environmental Microbiology, July 2006, p. 4899-4906, Vol. 72, No. 7
0099-2240/06/$08.00+0     doi:10.1128/AEM.00354-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.



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