Oligonucleotide recombination in Gram-negative bacteria

Authors

  • Bryan Swingle,

    Corresponding author
    1. United States Department of Agriculture-Agricultural Research Service, Ithaca, NY 14853, USA.
    2. Department of Plant Pathology and Plant-Microbe Biology, Cornell University, Ithaca, NY 14853, USA.
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  • Eric Markel,

    1. United States Department of Agriculture-Agricultural Research Service, Ithaca, NY 14853, USA.
    2. Department of Plant Pathology and Plant-Microbe Biology, Cornell University, Ithaca, NY 14853, USA.
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  • Nina Costantino,

    1. Gene Regulation and Chromosome Biology Laboratory, Center for Cancer Research, and
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  • Mikhail G. Bubunenko,

    1. Basic Research Program, SAIC-Frederick, Inc., National Cancer Institute at Frederick, Frederick, MD 21702, USA.
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  • Samuel Cartinhour,

    1. United States Department of Agriculture-Agricultural Research Service, Ithaca, NY 14853, USA.
    2. Department of Plant Pathology and Plant-Microbe Biology, Cornell University, Ithaca, NY 14853, USA.
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  • Donald L. Court

    1. Gene Regulation and Chromosome Biology Laboratory, Center for Cancer Research, and
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*E-mail Bryan.Swingle@ars.usda.gov; Tel. (+1) 607 255 6733; Fax (+1) 607 255 4471.

Summary

This report describes several key aspects of a novel form of RecA-independent homologous recombination. We found that synthetic single-stranded DNA oligonucleotides (oligos) introduced into bacteria by transformation can site-specifically recombine with bacterial chromosomes in the absence of any additional phage-encoded functions. Oligo recombination was tested in four genera of Gram-negative bacteria and in all cases evidence for recombination was apparent. The experiments presented here were designed with an eye towards learning to use oligo recombination in order to bootstrap identification and development of phage-encoded recombination systems for recombineering in a wide range of bacteria. The results show that oligo concentration and sequence have the greatest influence on recombination frequency, while oligo length was less important. Apart from the utility of oligo recombination, these findings also provide insights regarding the details of recombination mediated by phage-encoded functions. Establishing that oligos can recombine with bacterial genomes provides a link to similar observations of oligo recombination in archaea and eukaryotes suggesting the possibility that this process is evolutionary conserved.

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