Recombineering using RecTE from Pseudomonas syringae.
In this report, we describe the identification of functions that promote genomic recombination of linear DNA introduced into Pseudomonas cells by electroporation. The genes encoding these functions were identified in Pseudomonas syringae pv. syringae B728a based on similarity to the lambda Red Exo/Beta and RecET proteins encoded by the lambda and Rac bacteriophages of Escherichia coli. The ability of the pseudomonad-encoded proteins to promote recombination was tested in P. syringae pv. tomato DC3000 using a quantitative assay based on recombination frequency. The results show that the Pseudomonas RecT homolog is sufficient to promote recombination of single-stranded DNA oligonucleotides and that efficient recombination of double-stranded DNA requires the expression of both the RecT and RecE homologs. Additionally, we illustrate the utility of this recombineering system to make targeted gene disruptions in the P. syringae chromosome.
Swingle
Bryan
B
United States Department of Agriculture, Agricultural Research Service, 321 Plant Science Building, Tower Road, Ithaca, NY 14853, USA. Bryan.Swingle@ars.usda.gov
Bao
Zhongmeng
Z
Markel
Eric
E
Chambers
Alan
A
Cartinhour
Samuel
S
D010582:Bacteriophage lambda / Q000201:enzymology / Q000235:genetics; D004247:DNA / Q000235:genetics* / Q000378:metabolism*; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics; D004277:DNA, Single-Stranded / Q000235:genetics / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000235:genetics* / Q000378:metabolism*; D008969:Molecular Sequence Data; D044224:Pseudomonas syringae / Q000201:enzymology* / Q000235:genetics*; D011995:Recombination, Genetic; D017422:Sequence Analysis, DNA; D017385:Sequence Homology
D004269:DNA, Bacterial; D004277:DNA, Single-Stranded; D004268:DNA-Binding Proteins; D004247:DNA