Developing genetic tools for the hyperthermophilic archaeon, Pyrococcus furiosus
Farkas, Joel Andrew
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Pyrococcus furiosus is a hyperthermophilic marine archeon that has been extensively studied for more than 25 years. We previously identified an auxotrophic mutant, GLW101 (COM1 ΔpyrF), which is naturally and efficiently competent for DNA uptake. Replicating shuttle vectors have been constructed based on the chromosomal origin of replication (oriC). In the process of identifying the minimum replication origin required for autonomous plasmid replication in P. furiosus, we discovered that several previously predicted features were not essential for stable autonomous plasmid replication. A minimum region required to promote plasmid DNA replication was identified, and plasmids based on this sequence readily transformed P. furiosus GLW101. The plasmids replicated autonomously, existed in a single copy, were structurally unchanged after transformation and were stable without selection for more than 100 generations. We found that the combined transformation and recombination frequencies of the GLW101 strain allow marker replacement using linear DNA. We discovered that marker replacement was possible with as few as 40 nucleotides of flanking homology to the target region and adapted 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 background to generate a strain (JFW02), which is a tight tryptophan auxotroph, providing a doubly auxotrophic strain. 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 but had no obvious effect on either recombination or transformation, suggesting that it 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 deletion of virtually any nonessential gene. These advances will facilitate a number of research venues including hydrogen production, natural competence and CRISPR function. The development of multiply marked strains and a reporter gene system are remaining challenges to the advancement of the genetics effort.