|COSTANTINO, NINA - National Cancer Institute (NCI, NIH)|
|COURT, DONALD - National Cancer Institute (NCI, NIH)|
|BUBUNENKO, MIKHAIL - National Cancer Institute (NCI, NIH)|
Submitted to: Meeting Abstract
Publication Type: Abstract Only
Publication Acceptance Date: 10/6/2009
Publication Date: 10/11/2009
Citation: Swingle, B.M., Costantino, N., Court, D., Bubunenko, M.G., Cartinhour, S.W. 2009. Oligonucleotide recombination in bacteria. [abstract}. 17:28.
Technical Abstract: Today, there are more than 1,500 completed or draft bacterial genome sequences available for public access. To functionally analyze these genomes and to test the hypotheses that are generated from the sequence information we require new and generically useful tools. Recombineering (genetic engineering by recombination) offers a powerful strategy for site-specific mutagenesis of genomic loci and provides a method for rapid and precise functional genomic analysis. Here we present the results of experiments that were conducted to identify and characterize a novel mode of DNA recombination in bacteria. Using a series of genetic experiments we show that DNA oligonucleotides (oligos) introduced directly into bacteria by electroporation can recombine with the bacterial chromosome. We originally identified this phenomenon in Pseudomonas syringae and later found that Escherichia coli, Salmonella typhimurium and Shigella flexneri are also capable of oligo recombination. To more fully characterize this phenomenon, we tested the influence of oligo concentration, length and sequence on recombination frequency. The results of these experiments show that the concentration and length of the oligo have remarkable influences on the recombination frequency. Additionally, we found that the sequence of the oligo affects recombination in ways that are similar to lambda Red-mediated recombination, particularly with respect to the replication imposed strand bias and whether or not the oligos create mismatches that are efficiently repaired by the methyl directed mismatch repair (MMR) system.