Improved and versatile transformation system allowing multiple genetic manipulations of the hyperthermophilic archaeon Thermococcus kodakaraensis - PubMed (original) (raw)

Improved and versatile transformation system allowing multiple genetic manipulations of the hyperthermophilic archaeon Thermococcus kodakaraensis

Takaaki Sato et al. Appl Environ Microbiol. 2005 Jul.

Abstract

We have recently developed a gene disruption system for the hyperthermophilic archaeon Thermococcus kodakaraensis by utilizing a pyrF-deficient mutant, KU25, as a host strain and the pyrF gene as a selectable marker. To achieve multiple genetic manipulations for more advanced functional analyses of genes in vivo, it is necessary to establish multiple host-marker systems or to develop a system in which repeated utilization of one marker gene is possible. In this study, we first constructed a new host strain, KU216 (DeltapyrF), by specific and almost complete deletion of endogenous pyrF through homologous recombination. In this refined host, there is no need to consider unknown mutations caused by random mutagenesis, and unlike in the previous host, KU25, there is little, if any, possibility that unintended recombination between the marker gene and the chromosomal allele occurs. Furthermore, a new host-marker combination of a trpE deletant, KW128 (DeltapyrF DeltatrpE::pyrF), and the trpE gene was developed. This system made it possible to isolate transformants through a more simple selection procedure as well as to deduce the transformation efficiency, overcoming practical disadvantages of the first system. The effects of the transformation conditions were also investigated using this system. Finally, we have also established a system in which repeated utilization of the counterselectable pyrF marker is possible through its excision by pop-out recombination. Both endogenous and exogenous sequences could be applied as tandem repeats flanking the marker pyrF for pop-out recombination. A double deletion mutant, KUW1 (DeltapyrF DeltatrpE), constructed with the pop-out strategy, was demonstrated to be a useful host for the dual markers pyrF and trpE. Likewise, a triple deletion mutant, KUWH1 (DeltapyrF DeltatrpE DeltahisD), could also be constructed. The transformation systems developed here now provide the means for extensive genetic studies in this hyperthermophilic archaeon.

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Figures

FIG. 1.

FIG. 1.

Schematic diagram of targeted disruption of pyrF, trpE, and hisD in T. kodakaraensis KOD1, KU216, and KW128 using pUDPyrF, pUDTrpE, and pUDHisD, respectively. Relevant regions of the chromosome are illustrated for (from the top) strains KOD1, KU216, KW128, and KH3. The positions of primer sets used for analyses of targeted disruption of pyrF (CHDPYR-F/CHDPYR-R, closed arrowheads), trpE (CHDTRP-R/CHDTRP-F, open arrowheads), and hisD (CHDHID-R/CHDHID-F, closed arrows) are indicated. The gray, closed, open, and striped boldface bars indicate each region spanned by the pyrF, trpE, pyrF upstream, and trpE downstream probes used in Southern blot analyses, respectively. P_pyrF_ indicates the putative promoter region of the operon containing pyrF. Black stars indicate regions of target genes that were left intact in order to avoid disturbing nearby genes as described in Materials and Methods. Gene name abbreviations: Hypo, hypothetical gene; ino1, _myo_-inositol-1-phosphate synthase; PRb, predicted RNA-binding protein; tagD, cytidylyltransferase. Restriction site abbreviations: Ap, ApaI; Hc, HincII; Hd, HindIII.

FIG. 2.

FIG. 2.

PCR analyses of T. kodakaraensis strains KU216 (Δ_pyrF_), KW128 (Δ_pyrF_ Δ_trpE_::pyrF), and KH3 (Δ_pyrF_ Δ_trpE_::pyrF Δ_hisD_::trpE). (A) Amplification of pyrF and trpE loci in strains KOD1, KU216, and KW128 using CHDPYR-R/CHDPYR-F and CHDTRP-R/CHDTRP-F as primer sets, respectively. (B) Amplification of the hisD locus in T. kodakaraensis KOD1, KU216, KW128, and KH3 using CHDHID-R and CHDHID-F as primers. Primers used for these analyses are displayed in Fig. 1. M represents the DNA size marker, HindIII-digested λ DNA.

FIG. 3.

FIG. 3.

Southern blot analyses of T. kodakaraensis strains KU216 (Δ_pyrF_), KW128 (Δ_pyrF_ Δ_trpE_::pyrF), and KH3 (Δ_pyrF_ Δ_trpE_::pyrF Δ_hisD_::trpE). (A) The pyrF upstream probe was used against genomic DNAs of KOD1 and KU216 digested with HincII. (B) The pyrF probe was used against genomic DNAs of KOD1, KU216, KW128, and KUW1 digested with ApaI. (C) The trpE downstream probe was used against genomic DNAs of KOD1, KU216, KW128, and KUW1 digested with ApaI. (D) The trpE probe was used against genomic DNAs of KOD1, KW128, and KH3 digested with HindIII. The bars on the left side of each panel indicate the mobility of fragments in the DNA size marker, HindIII-digested λ DNA. Regions spanned by probes used for these analyses are displayed in Fig. 1.

FIG. 4.

FIG. 4.

Schematic diagram of sequential disruption of trpE and hisD through excision of the pyrF marker by pop-out recombination. (A) Construction of strains KUW1 (Δ_pyrF_ Δ_trpE_) and KUWH1 (Δ_pyrF_ Δ_trpE_ Δ_hisD_) using type I pop-out vectors harboring tandem repeats of the endogenous 3′ region of the target gene flanking pyrF on both sides. (B) Construction of strains KUWc1 and KUWcHc1 using type II pop-out vectors harboring tandem repeats of the exogenous 2μ′ region flanking pyrF on both sides. The regions shaded in gray indicate the tandem repeat regions in each strategy. Open arrowheads and closed arrows indicate primer sets CHDTRP-R/CHDTRP-F and CHDHID-R/CHDHID-F for analyses of targeted disruption of trpE and hisD, respectively. Restriction site abbreviation: Ap, ApaI. All genes adjacent to the target genes are the same as those mentioned in the legend of Fig. 1.

FIG. 4.

FIG. 4.

Schematic diagram of sequential disruption of trpE and hisD through excision of the pyrF marker by pop-out recombination. (A) Construction of strains KUW1 (Δ_pyrF_ Δ_trpE_) and KUWH1 (Δ_pyrF_ Δ_trpE_ Δ_hisD_) using type I pop-out vectors harboring tandem repeats of the endogenous 3′ region of the target gene flanking pyrF on both sides. (B) Construction of strains KUWc1 and KUWcHc1 using type II pop-out vectors harboring tandem repeats of the exogenous 2μ′ region flanking pyrF on both sides. The regions shaded in gray indicate the tandem repeat regions in each strategy. Open arrowheads and closed arrows indicate primer sets CHDTRP-R/CHDTRP-F and CHDHID-R/CHDHID-F for analyses of targeted disruption of trpE and hisD, respectively. Restriction site abbreviation: Ap, ApaI. All genes adjacent to the target genes are the same as those mentioned in the legend of Fig. 1.

FIG. 5.

FIG. 5.

PCR analyses of _pyrF_-trpE double deletion mutants and _pyrF_-_trpE_-hisD triple deletion mutants of T. kodakaraensis constructed by repeated utilization of the pyrF marker using pop-out strategy. (A) Amplification of the trpE locus in strains KU216, KuW1, KUW1, KuWc1, and KUWc1 using CHDTRP-R/CHDTRP-F as a primer set. (B) Amplification of pyrF, trpE, and hisD loci in strains KU216, KUW1, and KUWH1 using CHDPYR-R/CHDPYR-F, CHDTRP-R/CHDTRP-F, and CHDHID-R/CHDHID-F as primer sets, respectively. (C) Amplification of pyrF, trpE, and hisD loci in strains KU216, KUWc1, and KUWcHc1 using CHDPYR-R/CHDPYR-F, CHDTRP-R/CHDTRP-F, and CHDHID-R/CHDHID-F as primer sets, respectively. Primer sets used for these analyses were displayed in Fig. 1 and 4. M represents the DNA size marker, HindIII-digested λ DNA.

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