Natural competence in the hyperthermophilic archaeon Pyrococcus furiosus facilitates genetic manipulation: construction of markerless deletions of genes encoding the two cytoplasmic hydrogenases

Abstract

In attempts to develop a method of introducing DNA into Pyrococcus furiosus, we discovered a variant within the wild-type population that is naturally and efficiently competent for DNA uptake. A pyrF gene deletion mutant was constructed in the genome, and the combined transformation and recombination frequencies of this strain allowed marker replacement by direct selection using linear DNA. We have demonstrated the use of this strain, designated COM1, for genetic manipulation. Using genetic selections and counterselections based on uracil biosynthesis, we generated single- and double-deletion mutants of the two gene clusters that encode the two cytoplasmic hydrogenases. The COM1 strain will provide the basis for the development of more sophisticated genetic tools allowing the study and metabolic engineering of this important hyperthermophile.

FIG. 1.

Strategy for obtaining a pyrF deletion and PCR analysis of the pyrF deletion in the COM1 strain. (A) A diagram of the pyrF genome region is shown with the pyrF deletion plasmid having 1-kb regions from up- and downstream of pyrF for homologous recombination and also containing the P_gdh-hmg cassette for selection of simvastatin resistance. Homologous recombination can occur at the upstream or downstream pyrF flanking regions, integrating the plasmid into the genome and generating a strain that is simvastatin resistant. Selection on 5-FOA selects for loss of the plasmid along with deletion of the pyrF gene. Bent arrows depict primers used for verification of pyrF deletion. (B) Gel depicting PCR products of the pyrF genome region in the COM1 strain compared to the wild type, amplified by primers outside the up- and downstream regions used for homologous recombination (see bent arrows in panel A).

FIG. 2.

COM1 is a uracil auxotroph. Growth curves of the COM1 strain (triangles) compared to the wild type (circles) in the presence (open symbols) and absence (closed symbols) of uracil. Culture growth was monitored by optical density at 660 nm. The slight increase in optical density for the COM1 strain cultured without uracil reflects a slight darkening of the medium due to incubation at 98°C. The lack of growth in the COM1 strain was verified by assaying protein concentrations for each time point. Each point represents an average of samples from two independent cultures, with error bars showing standard deviation.

FIG. 3.

Strategy for obtaining an SHI operon deletion and PCR analyses of the cytoplasmic hydrogenase operon deletions. (A) The SHI operon genome region is shown with the SHI operon deletion plasmid with 1-kb regions from up- and downstream of the operon for homologous recombination and also containing the P_gdh-pyrF cassette for selection of uracil prototrophy. Homologous recombination can occur at either the upstream or downstream SHI operon flanking regions, integrating the plasmid into the genome and generating a strain that is a uracil prototroph. PF0891, PF0892, PF0893, and PF0894 represent the genes coding for the SHI beta, gamma, delta, and alpha subunits, respectively. (B) Gel depicting PCR products of the SHI and SHII operon genome regions in the ΔSHI, ΔSHII, and ΔSHI ΔSHII strains compared to the COM1 strain, amplified by primers with at least one primer outside the homologous recombination regions.