Journal
APPLIED AND ENVIRONMENTAL MICROBIOLOGY
Volume 78, Issue 13, Pages 4669-4676Publisher
AMER SOC MICROBIOLOGY
DOI: 10.1128/AEM.00936-12
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Funding
- Bio-Energy Science Center [DE-PS02-06ER64304]
- Office of Biological and Environmental Research [FG02-08ER64690]
- Chemical Sciences, Geosciences and Biosciences Division, Office of Basic Energy Sciences, Office of Science, U.S. Department of Energy [DE-FG05-95ER20175]
- predoctoral Graduate Training In Genetics grant [NIH 5T32GM007103-30]
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We recently reported the isolation of a mutant of Pyrococcus furiosus, COM1, that is naturally and efficiently competent for DNA uptake. While we do not know the exact nature of this mutation, the combined transformation and recombination frequencies of this strain allow marker replacement by direct selection using linear DNA. In testing the limits of its recombination efficiency, we discovered that marker replacement was possible with as few as 40 nucleotides of flanking homology to the target region. We utilized this ability to design a strategy for selection of constructed deletions using PCR products with subsequent excision, or pop-out, of the selected marker. We used this method to construct a markerless deletion of the trpAB locus in the GLW101 (COM1 Delta pyrF) background to generate a strain (JFW02) that is a tight tryptophan auxotroph, providing a genetic background with two auxotrophic markers for further strain construction. The utility of trpAB as a selectable marker was demonstrated using prototrophic selection of plasmids and genomic DNA containing the wild-type trpAB alleles. A deletion of radB was also constructed that, surprisingly, had no obvious effect on either recombination or transformation, suggesting that its gene product is not involved in the COM1 phenotype. Attempts to construct a radA deletion mutation were unsuccessful, suggesting that this may be an essential gene. The ease and speed of this procedure will facilitate the construction of strains with multiple genetic changes and allow the construction of mutants with deletions of virtually any nonessential gene.
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