Conjugation-based genome engineering in Deinococcus radiodurans

Abstract

D. radiodurans has become an attractive microbial platform for the study of extremophile biology and industrial bioproduction. To improve the genomic manipulation and tractability of this species, the development of tools for whole genome engineering and design is necessary. Here, we report the development of a simple and robust conjugation-based transformation system from E. coli to D. radiodurans allowing for the introduction of stable, replicating plasmids expressing antibiotic resistance markers. Using this method with nonreplicating plasmids, we developed a protocol for creating sequential gene deletions in D. radiodurans by targeting re-striction-modification system genes. Importantly, we demonstrated a conjugation-based method for cloning the large (178 kb), high G+C content MP1 megaplasmid from D. radiodurans in E. coli. The conjugation-based tools described here will facilitate the development of D. radiodurans strains with synthetic genomes for biological studies and industrial applications. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=103 SRC="FIGDIR/small/464295v1_ufig1.gif" ALT="Figure 1"> View larger version (37K): org.highwire.dtl.DTLVardef@128437borg.highwire.dtl.DTLVardef@6669ddorg.highwire.dtl.DTLVardef@1b041eaorg.highwire.dtl.DTLVardef@b77f07_HPS_FORMAT_FIGEXP M_FIG C_FIG