|BAO, ZHONGMENG - Cornell University|
|CHAMBERS, ALAN - Cornell University|
Submitted to: Applied and Environmental Microbiology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 6/1/2010
Publication Date: 8/1/2010
Citation: Swingle, B.M., Bao, Z., Markel, E.J., Chambers, A., Cartinhour, S.W. 2010. Recombineering using RecTE from Pseudomonas syringae. Applied and Environmental Microbiology. 76(15):4960-4968.
Interpretive Summary: This report describes a new and important advance for biotechnology and genetic engineering in bacteria. Here, we describe the identification of two enzymes that work together to enhance the exchange of genetic information in bacteria. These functions were identified in the plant pathogenic bacterium Pseudomonas syringae pv. syringae B728a, which causes bacterial brown spot of bean. The genetic exchange that we observed was between two DNA molecules. One of the DNA molecules was a short DNA fragment that was introduced into the bacteria using standard methods and the second was the bacterial chromosome. This type of genetic exchange is extremely useful because the short DNA fragments can be modified to contain specific changes and using the enzymes that we discovered, these changes can then be incorporated into the bacterial chromosome. This procedure enables efficient engineering of specific sites in the organism's DNA, which can be used to study the function of specific regions of DNA or to alter the capabilities of the organism for industrial purposes.
Technical Abstract: In this report we describe the identification of functions that promote recombination of DNA introduced into Pseudomonas cells by electroporation. The genes encoding these functions were identified in Pseudomonas syringae pv. syringae B728a based on similarity to other recombinases encoded by E. coli bacteriophage. The genes identified in the P. syringae genomic sequence potentially encode proteins similar to RecE and RecT of the Rac prophage found in many strains of E. coli K12. The Rac recE and recT genes encode a 3 prime to 5 prime exonuclease and a single stranded DNA (ssDNA) annealing protein respectively. The ability of the pseudomonad RecET functions to promote recombination was tested in P. syringae pv. tomato DC3000 (DC3000) using a quantitative assay of recombination frequency that we developed. The results show that the pseudomonad RecT is sufficient to promote recombination of ssDNA oligonucleotides and that efficient recombination of double stranded DNA requires the expression of both RecE and RecT. These results are consistent with the functions proposed for RecE and RecT based on protein similarity. Additionally we characterized the oligo concentration and length requirements for RecT mediated oligo recombination in order to optimize the conditions to achieve the highest possible levels of recombination.