Targeted gene disruption by homologous recombination in the hyperthermophilic archaeon Thermococcus kodakaraensis KOD1

Abstract

In contrast to the high accumulation in sequence data for hyperthermophilic archaea, methodology for genetically manipulating these strains is still at an early stage. This study aimed to develop a gene disruption system for the hyperthermophilic euryarchaeon Thermococcus kodakaraensis KOD1. Uracil-auxotrophic mutants with mutations in the orotidine-5'-monophosphate decarboxylase gene (pyrF) were isolated by positive selection using 5-fluoroorotic acid (5-FOA) and used as hosts for further transformation experiments. We then attempted targeted disruption of the trpE locus in the host strain by homologous recombination, as disruption of trpE was expected to result in tryptophan auxotrophy, an easily detectable phenotype. A disruption vector harboring the pyrF marker within trpE was constructed for double-crossover recombination. The host cells were transformed with the exogenous DNA using the CaCl(2) method, and several transformants could be selected based on genetic complementation. Genotypic and phenotypic analyses of a transformant revealed the unique occurrence of targeted disruption, as well as a phenotypic change of auxotrophy from uracil to tryptophan caused by integration of the wild-type pyrF into the host chromosome at trpE. As with the circular plasmid, gene disruption with linear DNA was also possible when the homologous regions were relatively long. Shortening these regions led to predominant recombination between the pyrF marker in the exogenous DNA and the mutated allele on the host chromosome. In contrast, we could not obtain trpE disruptants by insertional inactivation using a vector designed for single-crossover recombination. The gene targeting system developed in this study provides a long-needed tool in the research on hyperthermophilic archaea and will open the way to a systematic, genetic approach for the elucidation of unknown gene function in these organisms.

FIG. 1.

(A) Effect of 5-FOA on the growth of T. kodakaraensis KOD1. Cells were cultivated in ASW-AA liquid medium without 5-FOA (open circles) and with 0.75% 5-FOA (closed circles). (B) Uracil auxotrophy of T. kodakaraensis KU25. Cells were cultivated in ASW-AA liquid medium with or without addition of uracil (5 μg/ml). Symbols: open circles, KOD1 with Ura; open triangles, KOD1 without Ura; closed circles, KU25 with Ura; closed triangles, KU25 without Ura. Error bars represent standard deviations of three independent experiments.

FIG. 2.

Mutations in pyrF from T. kodakaraensis KU25 and KU27.

FIG. 3.

Schematic drawing of pUDT1 (A) and pUDT2 (B) for disruption of trpE in T. kodakaraensis. PDDC-R-PDDC-F (5′-TGGCTGCACTCCAGACCAAGGGCTACACCG-3′-5′-CCATCGGGGTCGAGCCTTCTGAGCTCCCCA-3′) were primers used in colony PCR analysis. The homologous regions in the circular DNAs and chromosome of T. kodakaraensis are shaded.

FIG. 4.

(A) Restriction map of the trp locus in T. kodakaraensis KOD1, KU25, and KW4. Restriction site abbreviations: Aa, Aat I; Ap, ApaI; E, Eco52 I. (B) Nucleotide sequence of the trpE locus from T. kodakaraensis KW4. Each four forms of letters denotes different regions as follows: underlined uppercase, trpE regions; uppercase, putative promoter region for pyrF; boldface uppercase, selectable marker pyrF; lowercase, pUC118-derived regions.

FIG. 5.

(A) Amplified DNA fragments after colony PCR analysis of T. kodakaraensis KOD1, KU25, and KW4 using PDDC-R-PDDC-F as primers. (B) Southern blot analysis using the trpE probe. Genomic DNAs of KOD1, KU25, and KW4 were digested with ApaI (lanes 1 to 3) and Eco52I (lanes 4 to 6) and hybridized with the trpE probe. (C) Southern blot analysis using the pyrF probe. Genomic DNAs of the three strains were digested with AatI (lanes 1 to 3) and ApaI (lanes 4 to 6) and hybridized with the pyrF probe.

FIG. 6.

Tryptophan auxotrophy of T. kodakaraensis KW4 in liquid medium (A) and on plate medium (B). (A) The cells were cultured in ASW-AAW⁻ liquid medium at 85°C. When needed, tryptophan and/or uracil was added in the medium at concentrations of 75 μg/ml and 5 μg/ml, respectively. Symbols: open circles, KW4 with Trp and Ura; open triangles, KW4 without Trp and with Ura; open squares, KW4 with Trp and without Ura; open diamonds, KW4 without Trp or Ura; closed circles, KU25 with Trp and Ura; closed triangles, KU25 without Trp and with Ura. Error bars represent standard deviations of three independent experiments. (B) Washed cells were spread on ASW-AAW⁻ plate medium containing uracil (10 μg/ml) at 85°C for 10 days. When needed, tryptophan (75 μg/ml) was added to the medium. (a) KW4 without Trp and with Ura; (b) KW4 with Trp and Ura; (c) KU25 without Trp and with Ura; (d) KU25 with Trp and Ura. The picture is composed of four different plates.