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Title: Oligonucleotide recombination in gram negative bacteria

Author
item Swingle, Bryan
item Markel, Eric
item COSTANTINO, NINA - National Cancer Institute (NCI, NIH)
item BUBUNENKO, MIKHAIL - National Cancer Institute (NCI, NIH)
item Cartinhour, Samuel
item COURT, DONALD - National Cancer Institute (NCI, NIH)

Submitted to: Molecular Microbiology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 11/10/2009
Publication Date: 1/1/2010
Citation: Swingle, B.M., Markel, E.J., Costantino, N., Bubunenko, M., Cartinhour, S.W., Court, D. 2010. Oligonucleotide recombination in gram negative bacteria. Molecular Microbiology. 75(1):138-148.

Interpretive Summary: This report describes the initial identification and characterization of a novel form of DNA recombination. This finding is of fundamental importance because it appears to operate in bacteria as well as in other domains of life, including human cells. This finding is particularly important for biotechnology and biological engineering because it can be applied as a method to introduce specific changes directly in the genomes of microorganisms. In this report we document that bacterial genomic DNA can be changed to match the DNA sequence contained in a short single stranded DNA fragment. The short single stranded DNA fragments, which are used to direct the changes, are inexpensive and can be obtained from commercial sources. Additionally, these DNA fragments are made-to-order, thus can be designed to direct any change that the scientist requires. This finding also helps provide understanding of other mechanisms of DNA recombination, especially those mediated by phage (bacteria viruses). Many of these phages have functions that enhance the efficiency of recombination. We posit that our findings provide a contrast to the phage recombination functions and help show how the phage functions work.

Technical Abstract: 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.