Targeted Gene Disruption by Homologous Recombination in the Hyperthermophilic Archaeon Thermococcus kodakaraensis KOD1
Department of Synthetic Chemistry and Biological Chemistry, Graduate School of Engineering, Kyoto University, Yoshida-Honmachi, Sakyo-ku, Kyoto 606-8501$2:^{8|, 百拇医药
Accepted 2 October 2002$2:^{8|, 百拇医药
ABSTRACT$2:^{8|, 百拇医药
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 CaCl2 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.
INTRODUCTION/fu, 百拇医药
Among the living organisms, hyperthermophiles belonging to the third domain of life, Archaea, are attractive subjects of research from various standpoints. Many efforts to understand the strategies for adaptation to extremely high temperature environments have revealed numerous physiologically intriguing and unique properties of hyperthermophilic archaea (30, 36, 37). They have also attracted attention from an industrial point of view as potential resources for highly thermostable enzymes (3, 23, 40)./fu, 百拇医药
Along with the classical biochemistry and molecular cloning of structural genes, complete genome analyses in recent years have provided a new means of understanding the hyperthermophilic archaea. So far, entire genome sequences of 16 archaeal strains, including 10 hyperthermophiles, have been determined. This has revealed that genomes of hyperthermophilic archaea are rather small, and the small number of genes makes them ideal targets for studying the basic principles and evolution of life.
In spite of the many attractive aspects of hyperthermophilic archaea, progress of research on these organisms has been constantly hampered by the limitation of available tools for genetic manipulation. While genetic methodology has been developed for methanogenic archaea (19, 34) and extremely halophilic archaea (24, 34), comparable to those of their bacterial counterparts, manipulative strategies for the hyperthermophilic archaea are still at an early stage. With respect to the genus Sulfolobus in the kingdom Crenarchaeota, various genetic elements have been identified (44), and natural gene transfer via conjugation (26, 31) or virus infection (32) has been observed. Several cases of genetic manipulation have been reported with Sulfolobus by applying these exochromosomal genetic elements (1, 10, 11, 32, 35). In contrast, concerning hyperthermophiles of Euryarchaeota, genetic tools are further limited due to the small number of genetic elements identified and/or applicable in this kingdom. Although several autonomously replicating plasmids have been isolated from Pyrococcus and Thermococcus strains (6, 12, 41) and Archaeoglobus profundus (20), useful genetic tools have not been developed except for shuttle vectors based on pGT5 from Pyrococcus abyssi that could transform and be maintained in not only the closely related strain Pyrococcus furiosus but also Sulfolobus acidocaldarius (2, 4).
Moreover, it should be noted that there is no gene targeting system for hyperthermophilic archaea in the literature. The development of a system for the targeted disruption of genes would certainly be a major breakthrough in various fields of research on hyperthermophilic archaea, as although the archaeal genomes are relatively small, the function of approximately half of the genes cannot be predicted by primary structure comparisons. Along with biochemical analyses of individual gene products and/or structural genomics, analyses of phenotypic changes in knockout strains and genetic complementation techniques will no doubt contribute greatly to the identification of currently unknown gene functions.|ho, http://www.100md.com
Thermococcus kodakaraensis KOD1 (previously reported as Pyrococcus kodakaraensis KOD1) is a sulfur-reducing hyperthermophilic archaeon belonging to the Thermococcales in Euryarchaeota (22). Although the genome analyses of three Pyrococcus species, P. abyssi , P. furiosus (27; http://www.genome.utah.edu/sequence.html; http://www.ornl.gov/hgmis/publicat/99santa/157.html), and P.horikoshii (17; http://www.bio.nite.go.jp/ot3db_index.html),have already been completed among members of Thermococcales, the complete genome of a Thermococcus strain has not yet been determined. Recent culture-dependent and culture-independent studies have indicated that strains of the genus Thermococcus are more ubiquitous than Pyrococcus strains in various marine hydrothermal vent systems (15, 38). Therefore, Thermococcus strains, with their larger population, can be expected to play a major role in the ecology and metabolic activity of microbial consortia in extremely high temperature environments. In light of the importance of this genus, the genome project of T. kodakaraensis KOD1 is now in progress.
This study aimed to construct a gene disruption system for the hyperthermophilic archaeon T. kodakaraensis KOD1 for further functional analyses of genes identified by the genome project. For this purpose, we applied an orotidine-5'-monophosphate decarboxylase (OMPdecase) (PyrF)-deficient mutant of T. kodakaraensis KOD1, displaying uracil auxotrophy, as a host and the complementary pyrF gene as a selectable marker. 5-Fluoroorotic acid (5-FOA) was adopted to isolate the PyrF- mutants, as clones with resistance to this drug usually lack either PyrF or orotate phosphoribosyltransferase (OPRTase) (PyrE) activity. We here describe isolation of pyrF-deficient mutants of T. kodakaraensis KOD1 by 5-FOA positive selection (8, 9) and targeted disruption of trpE by homologous recombination under a selection based on complementation of uracil auxotrophy of the mutant.?ikv&, 百拇医药
MATERIALS AND METHODS?ikv&, 百拇医药
Strains and growth conditions. The strains and plasmids used in this study are listed in . T. kodakaraensis KOD1 and its derivatives were cultivated under strictly anaerobic conditions at 85°C in a rich growth medium (ASW-YT medium) or a synthetic medium containing amino acids (ASW-AA medium). ASW-YT medium was composed of a 1.25-fold dilution of artificial seawater (28) (0.8x ASW), yeast extract (5.0 g/liter), tryptone (5.0 g/liter), and elemental sulfur (2 g/liter) (pH 6.6). ASW-AA medium consisted of 0.8x ASW supplemented with a 5.0-ml/liter concentration of modified Wolfe's trace minerals [0.5 g of MnSO4 · 2H2O, 0.1 g of CoCl2, 0.1 g of ZnSO4, 0.01 g of CuSO4 · 5H2O, 0.01 g of AlK(SO4)2, 0.01 g of H3BO3, and 0.01 g of NaMoO4 · 2H2O per liter] (28), vitamin mixture (5.0 ml/liter) (28), 20 amino acids (250 mg of cysteine · HCl · H2O, 75 mg of alanine, 125 mg of arginine · HCl, 100 mg of asparagine · H2O, 50 mg of aspartic acid, 50 mg of glutamine, 200 mg of glutamic acid, 200 mg of glycine, 100 mg of histidine · HCl · H2O, 100 mg of isoleucine, 100 mg of leucine, 100 mg of lysine · HCl, 75 mg of methionine, 75 mg of phenylalanine, 125 mg of proline, 75 mg of serine, 100 mg of threonine, 75 mg of tryptophan, 100 mg of tyrosine, and 50 mg of valine per liter), and elemental sulfur (0.2 g/liter) (pH was adjusted to 6.9 with NaOH). When necessary, 5-FOA (Wako Pure Chemicals, Osaka, Japan) and uracil (Kohjin, Tokyo, Japan) were added to ASW-AA medium at the concentrations as described in the text. For investigation of tryptophan auxotrophy, tryptophan-deficient ASW-AA medium (ASW-AAW-) was used. To reduce the dissolved oxygen in the medium, 5.0% Na2S · 9H2O was added until the color of resazurin sodium salt (1.0 mg/liter) became clear. In the case of plate culture, 1.0% (wt/vol) Gelrite (Wako Pure Chemicals) was added to solidify the medium together with a 2.0-ml/liter concentration of polysulfide solution (10 g of Na2S · 9H2O and 3.0 g of elemental sulfur per 15 ml) instead of elemental sulfur and 5.0% Na2S · 9H2O solution. The cells inoculated on the plate medium were incubated at 85°C in anaerobic chambers (Tabai Espec, Osaka, Japan).
fig.ommitted\dzt, http://www.100md.com
Strains and plasmids used in this study, together with OMPdecase and OPRTase activities in T. kodakaraensis KOD1 and its mutants\dzt, http://www.100md.com
Escherichia coli strain DH5{alpha} , used for general DNA manipulation, was routinely cultivated at 37°C in Luria-Bertani medium (29) and supplemented with ampicillin (50 µg/ml) when needed.\dzt, http://www.100md.com
Mutagenesis with UV irradiation and isolation of 5-FOA-resistant mutants.\dzt, http://www.100md.com
T. kodakaraensis KOD1 was cultivated in 2.0 liters of ASW-AA liquid medium for 39 h, and the cells at the stationary phase were harvested by centrifugation (6,000 x g, 30 min). The following procedures were performed anaerobically in the anaerobic chambers. The cells were resuspended in 60 ml of ASW (3/100 volume), and a portion of the suspension (10 ml; 1010 cells) was poured into glass petri dishes. While being stirred, the suspension was subjected to UV irradiation at a distance of 20 cm from a 15-W germicidal lamp for appropriate periods of time (0, 30, 60, 90, and 120 s). Aliquots (200 µl) were spread on ASW-AA plate medium containing 0.75% 5-FOA for dominant selection of uracil-auxotrophic (Pyr-) mutants, together with uracil (10 µg/ml) to support growth of the resulting mutants. The cells were incubated at 85°C for 5 days. The number of viable cells was determined by inoculating portions of the cell suspension with appropriate dilution onto ASW-AA plate medium without 5-FOA and counting the colonies formed.
5-FOA-resistant colonies were isolated and cultivated in ASW-YT liquid medium. The grown cells were incubated for 2 days in ASW-AA liquid medium to avoid carryover of uracil and further subcultured in ASW-AA liquid medium with or without addition of uracil (5 µg/ml) to investigate the uracil auxotrophy of the isolates.4, http://www.100md.com
Enzyme assay.4, http://www.100md.com
Cell extracts of T. kodakaraensis KOD1 and its mutant strains were prepared as follows. Cells cultivated in ASW-YT liquid medium for 20 h were harvested by centrifugation (6,000 x g, 30 min) and lysed in 1/1,000 volume of 50 mM Tris-HCl (pH 7.5) containing 0.1% Triton X-100. After mixing with a vortex for 10 min, the supernatant after centrifugation (3,000 x g, 20 min) was used as the cell extract. The protein concentration was determined using a Bio-Rad (Hercules, Calif.) protein assay system with bovine serum albumin as a standard.4, http://www.100md.com
OMPdecase (PyrF) activity was measured by monitoring the decrease in absorbance at 285 nm derived from the conversion of orotidine-5'-monophosphate (OMP) to UMP ({varepsilon} 285 = 1,380 M-1 cm-1) (5). The assay mixture was composed of 100 mM Tris-HCl (pH 8.6), 1.5 mM MgCl2, 0.125 mM OMP, and the enzyme solution in a total volume of 1 ml. After preincubation of the assay mixture in a capped cuvette at 85°C for 5 min, the reaction was started by addition of the enzyme solution and was monitored at the same temperature for 10 min.
OPRTase (PyrE) activity was assayed by determining the decrease of orotic acid spectrophotometrically at 295 nm (5). When measuring enzyme samples from pyrF+ strains, successive decarboxylation of the reaction product, OMP, by the endogenous OMPdecase must be taken into account. Since the OMPdecase activity was higher than OPRTase activity in T. kodakaraensis, the OPRTase activity could accurately be determined with the {varepsilon} 295 of 3,670 M-1 cm-1, corresponding to the conversion of orotic acid to UMP via OMP. In case of PyrF- strains, we monitored the conversion of the starting substrate to OMP with a {varepsilon} 295 of 2,520 M-1 cm-1. The reaction was performed in 1-ml mixtures containing 100 mM Tris-HCl (pH 8.6), 1.5 mM MgCl2, 0.125 mM orotic acid, cell extract, and 1.6 mM 5-phosphoribosyl 1-pyrophosphate (5-PRPP). The assay mixture without 5-PRPP in a capped cuvette was preincubated at 85°C for 10 min, and the reaction was started by addition of 5-PRPP. The decrease in A295 was measured at the same temperature for 3 min.
DNA manipulation and DNA sequencing.p*yg'}m, 百拇医药
General DNA manipulation was performed as described by Sambrook and Russel (29). Genomic DNA of T. kodakaraensis was isolated as described elsewhere (25). PCR was carried out using KOD Plus (Toyobo, Osaka, Japan) as DNA polymerase, and sequences of primers used for PCR are described in the text. When necessary, DNA fragments amplified by PCR were phosphorylated by T4 kinase (Toyobo). Restriction enzymes and modifying enzymes were purchased from Takara Shuzo (Kyoto, Japan) or Toyobo. DNA fragments after agarose gel electrophoresis were recovered and purified with GFX PCR DNA and a gel band purification kit (Amersham Pharmacia Biotech, Uppsala, Sweden). Plasmid DNA was isolated using Qiagen (Hilden, Germany) plasmid kits. DNA sequencing was performed using a BigDye terminator cycle sequencing kit and a model 3100 capillary sequencer (Applied Biosystems, Foster City, Calif.).p*yg'}m, 百拇医药
Construction of pUDT1 and pUDT2.p*yg'}m, 百拇医药
Two disruption vectors, pUDT1 and pUDT2, for homologous recombination in T. kodakaraensis with single- and double-crossover events, respectively, were constructed as follows. A DNA fragment (676 bp) containing Tk-pyrF was amplified from T. kodakaraensis KOD1 genomic DNA using the primers TK1-DUR-TK1-DUF (5'-GGGCATATGGAGGAGAGCAGGCTCATTCTGGCG-3'-5'-CTGAGGGGGTGTTTGACTTTCAA-3'; the underlined sequence indicates an NdeI site). The putative promoter region (130 bp) was amplified with the primers TK2-DPR-TK2-DPF (5'-GGGCTGCAGCCGCAACGCGCATTTTGCTCACCCGAAAA-3'-5'-GGGCATATGCATCACCTTTTTAACGGCCCTCTCCAAGAG-3'; the underlined sequences indicate PstI and NdeI sites, respectively). Both fragments were subcloned into pUC118 with the proper promoter-pyrF orientation. The resulting plasmid was designated pUD (3,944 bp). A truncated fragment of Tk-trpE (788 bp) was amplified using the primers TK3-DTR-TK3-DTF (5'-GGGGCATGCGGTGGCTTCGTTGGCTACGTCTCCTACG-3'-5'-GGGCTGCAGTTCGGGGCTCCGGTTAGTGTTCCCGCCG-3'; the underlined sequences indicate SphI and PstI sites, respectively), and then ligated with pUD at SphI and PstI sites, to obtain pUDT1 (4,732 bp) .
fig.ommitted8yx4*, http://www.100md.com
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.8yx4*, http://www.100md.com
For construction of pUDT2, a fragment (2,223 bp) containing Tk-trpE and the flanking regions was amplified using TK4-DT2R-TK4-DT2F (5'-GGGGTCGACCGGGTCTGGCGAGGGCAATGAGGGAC-3'-5'-GGGGAATTCGGTTATAGTGTTCGGAACGACCTTCACTC-3'; the underlined sequences indicate SalI and EcoRI sites, respectively) as primers, and subcloned into pUC118 at SalI and EcoRI sites. The plasmid obtained was named pUT4 (5,340 bp). pUD was digested with PvuII, followed by isolation of the fragment containing pyrF and its putative promoter region (1,104 bp). pUDT2 (6,012 bp) was obtained by insertion of the isolated fragment within pUT4 at blunted SacI sites in Tk-trpE .8yx4*, http://www.100md.com
Linear DNA fragments for homologous recombination in T. kodakaraensis, DT2-L1 (2,877 bp) and DT2-L2 (2,104 bp), were amplified from pUDT2 using the primer sets PDD-R-PDD-F (5'-CGGGTCTGGCGAGGGCAATGAGGGACAGCGCAGTCA-3'-5'-GGTTATAGTGTTCGGAACGACCTTCACT-3') and PDD-500R-PDD-500F (5'-GACCCTCGTCCTGGAGGTTGGCAATG-3'-5'-CGGCGTCCCCGGTGAGGGAGAAGTAA-3'), respectively. The amplified DNA fragments were purified after agarose gel electrophoresis.
Transformation of T. kodakaraensis.m;5[kg&, http://www.100md.com
The CaCl2 method for Methanococcus voltae PS (7) was modified for transformation of T. kodakaraensis. T. kodakaraensis KU25 was cultivated in ASW-YT liquid medium for 12 h, and the cells at the late exponential phase were harvested (17,000 x g, 5 min) from 3 ml of culture broth containing approximately 4 x 108 cells, and resuspended in 200 µl (1/15 volume) of transformation buffer (80 mM CaCl2 in 0.8x modified ASW that did not contain KH2PO4 to avoid precipitation between the calcium cation and the phosphate group) and kept on ice for 30 min. Then, 3 µg of DNA dissolved in Tris-EDTA buffer was added into the suspension, and the cells were incubated on ice for 1 h, which was followed by a heat shock at 85°C for 45 s and further incubation on ice for 10 min. As a control experiment, an equivalent volume of Tris-EDTA buffer was added to the cells instead of the DNA solution. The treated cells were cultured in 20 ml of ASW-AA liquid medium in the absence of uracil for two generations to select and concentrate Pyr+ transformants. The cells were then spread on ASW-AA plate medium without uracil and incubated for 5 to 8 days at 85°C. The resulting Pyr+ strains were analyzed by colony PCR and Southern hybridization using a DIG-DNA labeling and detection kit (Boehringer Mannheim, Mannheim, Germany).
RESULTSm!'d, 百拇医药
Isolation of uracil-auxotrophic mutants of T. kodakaraensis KOD1. We first set out to isolate uracil-auxotrophic mutants of T. kodakaraensis KOD1 using 5-FOA. The effects of 5-FOA on the growth of KOD1 were investigated in a synthetic medium with defined substrates (ASW-AA medium) and a nutrient-rich medium (ASW-YT medium). An important observation was that both media, in particular the ASW-AA medium, supported colony formation of T. kodakaraensis cells under anaerobic conditions when solidified with Gelrite. The plating efficiency of T. kodakaraensis was determined to be approximately 24%. 5-FOA has been reported to inhibit cell growth of various microorganisms, including the closely related P. abyssi (42), at concentrations from 0.05 to 0.1%. However, 5-FOA at this concentration range did not affect the cell growth of KOD1. When the 5-FOA concentration was increased in ASW-AA media, 0.75% was the threshold for complete inhibition of cell growth both in liquid medium and on plate medium. In contrast, 5-FOA showed little effects in the rich ASW-YT medium, even at high 5-FOA concentrations (~ 1.5%). Variable effects of 5-FOA in different media have also been observed in the case of yeasts grown on rich YPD medium (9). We therefore adopted ASW-AA plate medium with 0.75% 5-FOA for further selection of 5-FOA-resistant mutants of T. kodakaraensis KOD1.
fig.ommitted1'tv, http://www.100md.com
(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.1'tv, http://www.100md.com
The cells of the strain KOD1 were mutagenized by UV irradiation in an anaerobic chamber. The cell viability decreased with increase of the irradiation time; that is, viabilities of approximately 60, 40, 20, and 15% were observed after UV irradiation for 30, 60, 90, and 120 s, respectively. After this treatment, 5-FOA-resistant mutants were selected on ASW-AA plate media containing 0.75% 5-FOA and uracil (10 µg/ml). Independent experiments with different UV exposure times (30, 60, 90, or 120 s) gave several drug-resistant mutants with frequencies from 3 x 10-5 to 1 x 10-4. It should be noted that 5-FOA-resistant cells could also be isolated without UV irradiation (1 x 10-5 to 5 x 10-5 frequencies), suggesting the occurrence of spontaneous mutants during cell cultivation. Seventeen colonies, including seven colonies from the cultures without the UV irradiation, were randomly selected and their uracil auxotrophy was examined in ASW-AA liquid medium for three generations. Consequently, six strains were confirmed to be stable uracil-auxotrophic (Pyr-) mutants and designated as listed in , where KU18 and KU19 were possible spontaneous mutants.
Characterization of the mutants. In order to determine the deficient step in pyrimidine biosynthesis leading to uracil auxotrophy, OMPdecase (PyrF) and OPRTase (PyrE) activities in the cell extracts of the Pyr- mutants were assayed . The results indicated that the strains KU25 and KU27 were PyrF- mutants lacking OMPdecase activity, whereas the other four strains were PyrE- mutants with no OPRTase activity. The uracil-auxotrophic mutations in T. kodakaraensis examined here could be attributed to a biochemical defect in one of the two enzymes.+zcnn, 百拇医药
We subsequently performed genetic characterization of the two PyrF- mutant strains KU25 and KU27. The native pyrF gene from T. kodakaraensis KOD1 (Tk-pyrF) was identified in a phage clone of the genomic DNA library containing the gene encoding ribulose-1,5-bisphosphate carboxylase/oxygenase (13). Tk-pyrF encodes a protein of 213 amino acids, and the primary structure showed homology to the orthologues from P. abyssi (76% identity) and E. coli (24% identity). The corresponding genes in strains KU25 and KU27 were amplified by PCR, and the nucleotide sequences were determined. As shown in , a deletion of 96T was identified in pyrF from KU25, and the mutated gene corresponded to an ORF of only 99 bp due to the frame shift. In the case of KU27, there was a single nucleotide insertion (C) between 445C and 446G, which resulted in a mutant PyrF protein (176 amino acids) with an aberrant and shorter C-terminal region. These facts provided strong evidence that the strains KU25 and KU27 were actually pyrF-deficient mutants of T. kodakaraensis. We selected KU25 as a host strain for further transformation studies, because the pyrF gene was completely inactivated by the 1-bp deletion. shows the growth characteristic of KU25 compared to that of wild-type KOD1. In contrast to wild-type cells that could grow regardless of the addition of uracil, KU25 showed strict uracil auxotrophy in ASW-AA liquid medium. However, the mutant cells could grow on uracil-deficient ASW-AA plate medium even after preincubation of the cells under uracil starvation conditions to avoid carryover of uracil. Similar tendencies have been reported for the Pyr- mutants of the hyperthermophilic crenarchaeon S. solfataricus (21). It can be considered that the reason for this phenomenon is that Gelrite, used as a solidifier, might contain trace amounts of pyrimidine or related analogues, supporting the growth of the mutant cells.
fig.ommitted19)]]im, 百拇医药
Mutations in pyrF from T. kodakaraensis KU25 and KU27.19)]]im, 百拇医药
Disruption of trpE in T. kodakaraensis. We then performed targeted disruption of a gene in T. kodakaraensis KU25 by applying pyrF as a selectable marker. Tk-trpE, encoding the large subunit of anthranilate synthase in the tryptophan biosynthesis pathway (39), was chosen as the first target, because the disruption of this gene was expected to cause tryptophan auxotrophy, an easily detectable phenotype. Two gene disruption plasmids, namely, pUDT1 and pUDT2 , were constructed for homologous recombination through single- and double-crossover events, respectively. Both plasmids contained a marker cassette consisting of pyrF together with the putative promoter region. pUDT1 harbored an internal 788- bp fragment of trpE at an adjacent site to the marker cassette, and pUDT2 was constructed by inserting a 1,104-bp fragment of the marker cassette between the 5' region of trpE with trpD (1,020 bp) and the 3' region of trpE joined to trpG (753 bp).
T. kodakaraensis KU25 was transformed with 3 µg of pUDT1 or pUDT2 by the CaCl2 procedure, as described in Materials and Methods, where the cell viability after CaCl2 treatment was approximately 38%. Since the uracil auxotrophy of KU25 was not strict on the plate medium as mentioned above, Pyr+ transformants were first concentrated in a selective ASW-AA liquid medium not containing uracil. We could observe cell growth in all experiments when the cells were treated with pUDT1 or pUDT2, whereas no growth was seen in the control experiments. These results suggested that some recombination events took place within the cells, leading to uracil prototrophy. The concentrated cells were cultivated on ASW-AA plate medium without uracil, and colony PCR was performed using PDDC-R-PDDC-F primers to analyze the trpE locus in the isolates . The length of the amplified fragment from all 24 Pyr+ strains treated with pUDT1 was the same as that from the host strain KU25 (2.3 kbp) . This result indicated that disruption of trpE did not occur in this case. Spontaneous reversion of the pyrF mutation was unlikely, as control experiments without exogenous DNA did not lead to any prototrophs. We therefore examined the pyrF loci of three independent prototrophs. The lengths and nucleotide sequences of amplified fragments covering the pyrF region of each prototroph clarified that all three pyrF genes had reverted to the wild-type gene without integration of pUDT1 at this locus. Southern blot analysis using a probe spanning the pyrF region gave only a single band derived from the endogenous pyrF gene, which denies the possibility of nonspecific insertion of the pyrF marker in these chromosomes (data not shown). These results strongly suggested the occurrence of double-crossover recombination between the mutated pyrF on the chromosome and the intact gene in the plasmid. In contrast, when pUDT2 was added to the cells, a longer fragment (3.0 kbp), corresponding precisely to the length of a trpE::pyrF locus formed by double-crossover recombination , was amplified in 15 out of 24 Pyr+ strains . One of the positive isolates was selected and designated KW4 for further investigations.
fig.ommittedvaq'', 百拇医药
Transformation of T. kodakaraensis with various DNAsvaq'', 百拇医药
fig.ommittedvaq'', 百拇医药
(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.vaq'', 百拇医药
Genotype and phenotype of strain KW4. shows the longer fragment amplified from KW4 compared to the corresponding fragments from KOD1 and KU25 in the colony PCR analysis. The nucleotide sequence of the longer fragment from KW4 corresponded precisely to that of the disruption vector pUDT2. The orientation and the positioning of pyrF and the flanking regions derived from pUC118 were found as expected after a homologous double-crossover event at the trpE locus . In order to investigate whether unintended nonhomologous recombination events occurred in the chromosome of KW4 or not, we performed Southern blot analysis using trpE and pyrF probes. The regions spanned by these probes are displayed in . Only one signal was detected with the trpE probe against ApaI- and Eco52I-digested genomic DNA from KOD1, KU25, and KW4, but the signal against KW4 was longer . This agrees well with the results of PCR analysis mentioned above and further supports the occurrence of homologous recombination at the trpE locus. The pyrF probe gave identical signals derived from the endogenous pyrF locus in all three strains (7.3 and 11.0 kbp from Aat I- and ApaI-digested genomic DNA, respectively). In the case of KW4, one additional short band corresponding to pyrF within trpE could be detected . These results denied nonhomologous recombination and indicated that only targeted disruption of trpE had occurred in KW4.
fig.ommittedo-n\c^c, 百拇医药
(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.o-n\c^c, 百拇医药
To confirm the phenotype of KW4, we verified its growth properties in ASW-AAW- medium. While the host strain KU25 did not require tryptophan for growth, KW4 was a tryptophan-auxotrophic (Trp-) strain that could grow only in the presence of tryptophan. Growth of KW4 was no longer dependent on the addition of uracil, indicating that the Pyr- mutation in KU25 had been complemented in KW4, as expected. This tryptophan auxotrophy of KW4 was also clearly observed on the plate medium, as shown in . These results demonstrated that the disruption of trpE by the insertion of pyrF was responsible for the phenotypic change of auxotrophy from uracil to tryptophan.
fig.ommittedj)r$-, 百拇医药
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.j)r$-, 百拇医药
Effects of CaCl2 on transformation of T. kodakaraensis. In order to determine whether CaCl2 was essential for the transformation of T. kodakaraensis, KU25 cells were treated with transformation buffer without the CaCl2 supplemented in the experiments mentioned above (80 mM), and pUDT2 was added into the suspension. Contrary to our expectation, Pyr+ strains were obtained even in this case, and the longer trpE::pyrF locus was also detected by colony PCR analysis in 12 out of 24 Pyr+ isolates. This demonstrated that the high concentration of CaCl2 in the transformation buffer was not essential for DNA uptake and successive transformation of T. kodakaraensis.
Gene disruption by linear DNA and effect of the length of the homologous regions. We further investigated the possibility of gene disruption using linear DNA. Two linear DNA fragments, DT2-L1 (2,877 bp) and DT2-L2 (2,104 bp) were amplified by PCR using pUDT2 as a template. The former contained 1,020- and 753-bp regions homologous to the trp locus at both ends of the marker cassette, and the latter contained shorter homologous regions (two 500-bp regions) . After transformation with each linear DNA, we could obtain Pyr+ transformants in both cases. However, the trpE::pyrF locus generated by gene disruption of trpE was observed only in the experiments using DT2-L1. All 24 Pyr+ isolates using DT2-L2 harbored an intact trpE locus with the same length as that from the host strain. We then determined the nucleotide sequences of pyrF in several transformants that showed a Pyr+ phenotype without recombination at trpE. The results demonstrated the reversion of the deletion mutation in these isolates, as seen in the transformation with pUDT1, suggesting predominant recombination at the pyrF locus in these strains. These results indicated that gene disruption with linear DNA was possible when the homologous regions were relatively long, as in the case of DT2-L1. Shortening these regions, as in the case of DT2-L2, led to recombination at the pyrF locus. In any case, it should be noted that the ratio of trpE disruptants to Pyr+ isolates obtained with DT2-L1 (7 of 24) was lower than that obtained with the corresponding circular DNA, pUDT2 (15 of 24).
DISCUSSIONe|\, 百拇医药
In this study, we isolated uracil-auxotrophic (Pyr-) mutants of T. kodakaraensis KOD1 and achieved targeted disruption of the trpE locus in this archaeon by utilizing pyrF as a selectable marker. In spite of the fact that Pyr- mutants for several hyperthermophilic archaea have previously been described (14, 18, 21, 42), this is the first report on the development of a gene targeting system for a member of hyperthermophilic archaea in the literature.e|\, 百拇医药
5-FOA has been often applied for positive selection of uracil-auxotrophic mutants of various microorganisms. T. kodakaraensis KOD1 was also sensitive to this drug, but the sensitivity was lower than those of other microorganisms. 5-FOA-resistant mutants could be obtained by UV mutagenesis with frequencies from 3 x 10-5 to 1 x 10-4. Furthermore, spontaneous mutants could also be isolated with relatively high frequencies (1 x 10-5 to 5 x 10-5) compared to 1 x 10-6 to 3.5 x 10-6 for various Pyrococcus and Thermococcus strains (43). It has been reported that S. solfataricus exhibited high frequencies (10-5 to 10-4) for spontaneous Pyr- or ß-galactosidase mutants due to active transposable elements (21, 33). Although we are not aware of whether such transposable elements are present in the genome of T. kodakaraensis, genome analysis should clarify this in future studies.
Six out of the seventeen 5-FOA-resistant strains were stable uracil-auxotrophic mutants, which could be divided into two classes, PyrE- or PyrF- mutants. It is interesting that the two PyrF- mutants were obtained only under stronger UV mutagenesis conditions, whereas the four mutants obtained spontaneously and by low doses of UV were PyrE- strains. Similar tendencies have also been reported for UV-induced mutation of P. abyssi (42), spontaneous mutation of S. acidocaldarius (16), and transposon-mediated spontaneous mutation of S. solfataricus (21). These results suggested that the pyrE loci in hyperthermophilic archaea might be a mutational hotspot.6.vqqou, http://www.100md.com
The disruption of the target gene trpE was successfully performed by homologous recombination via a double-crossover event using the circular plasmid pUDT2. The internal region of trpE in the host strain KU25 could be replaced with the pyrF marker cassette, resulting in the phenotypic conversion from Pyr- to Trp-. As with the circular DNA, gene disruption with linear DNA (DT2-L1) was also possible, in which homologous regions of 1,020 and 753 bp were necessary for efficient double-crossover recombination at the targeted gene. We further found that recombination at pyrF became dominant when the homologous regions were 500 bp (DT2-L2). This may simply reflect the relative lengths of homologous DNA on the vector and chromosome. The host strain KU25 harbors an inactivated but nearly full-length pyrF gene of 641 bp on its chromosome, a possible target for recombination. As the disruption vector also contains an intact pyrF gene, it can be expected that longer homologous regions of the trp locus are necessary on the vector in order to obtain efficient recombination at the trp locus. Therefore, there still exists the possibility that gene disruption with much shorter homologous regions can occur in a host from which marker gene alleles on the chromosome have been completely removed. Additionally, intracellular digestion by exonucleases may also be a factor that affects the length of homologous DNA necessary for efficient targeted recombination. This may explain the lower ratio of trpE disruptants to Pyr+ isolates using DT2-L1 (7 of 24) compared with that using the corresponding circular DNA, pUDT2 (15 of 24).
In this study, we failed to detect the disruption of trpE with pUDT1, designed for single-crossover recombination, but instead observed reversion of the pyrF mutation in the host. This reversion can most likely be explained by double-crossover recombination at the pyrF locus, as in the case of the linear DT2-L2. This is supported by the facts that no spontaneous reversion could be observed in the control experiments without DNA and neither integration of pUDT1 at the pyrF locus nor nonspecific insertion of the pyrF marker in the chromosome could be detected by PCR and Southern blot analyses of the resulting prototrophs. The nonoccurrence of single-crossover recombination may be due to cleavage of the circular DNA by a host-controlled restriction system in T. kodakaraensis. Linearization of pUDT1 would prevent integration of the circular DNA into the chromosome. On the other hand, pUDT1 linearized by cleavage at regions other than pyrF would still retain the ability to recombine with the pyrF allele on the chromosome and explain the reversion observed in this case. It has been reported that methylation of an exogenous mobile intron with HaeIII methylase was effective for transformation of S. acidocaldarius, preventing its degradation within the archaeal cells (1). This methylation procedure has also been applied for transformation of P. furiosus and S. acidocaldarius using plasmid-based shuttle vectors (2, 4). Appropriate base modification of pUDT1 prior to transformation may lead to enhanced intracellular stability of the circular plasmid and allow the disruption of targeted genes by single-crossover recombination.
In this study, we initially adopted the CaCl2 procedure including heat shock at 85°C in order to chemically enhance the cellular competence of T. kodakaraensis. However, we subsequently found that the treatment of the cells with high concentrations of CaCl2 was not essential for transformation. Although we cannot rule out some contribution of the low concentrations of Ca2+ and Mg2+ (1.6 and 31 mM, respectively) in the 0.8x modified ASW, T. kodakaraensis seems to exhibit a natural competence to uptake exogenous DNA. A similar phenomenon has also been reported for the methanogenic archaeon M. voltae (7), whereas Sulfolobus strains have been generally transformed by electroporation (10, 11, 32, 35). Although the CaCl2 treatment has been successfully applied for the transformation of S. acidocaldarius and P. furiosus (2, 4), the effects of CaCl2 concentration on transformation efficiency have not been examined in detail. This natural competence of T. kodakaraensis may be a unique feature of this strain among the hyperthermophilic archaea and will certainly provide an advantage in future gene manipulation of this strain.
As described in Results, the Pyr- mutants of T. kodakaraensis could still grow on uracil-deficient plate medium, probably due to some pyrimidine-related compounds contaminating the medium. This fact made it difficult to evaluate the transformation efficiency, or in other words the number of transformants per microgram of DNA, for this archaeon. In contrast, the strain KW4, constructed in this study, showed strict tryptophan auxotrophy both in the liquid medium and on the plate medium, even when preincubation in tryptophan-deficient medium was omitted. The development of improved transformation systems for T. kodakaraensis is in progress by applying trpE disruptants and trpE as hosts and a selectable marker, respectively.*wm, 百拇医药
Homologous recombination is a powerful tool to analyze the function of any gene of interest. We now have the tools not only to disrupt genes but also to overexpress genes in the native host cell with the use of an appropriate promoter. We believe that the combination of complete genome information and a useful transformation system, enabling systematic gene disruption, will greatly contribute to advance the studies on the hyperthermophilic archaeon T. kodakaraensis. It is also of great interest whether our methodology can be applied in other Thermococcus species and the closely related Pyrococcus species.
REFERENCEStff-, 百拇医药
Aagaard, C., J. Z. Dalgaard, and R. A. Garrett. 1995. Intercellular mobility and homing of an archaeal rDNA intron confers a selective advantage over intron- cells of Sulfolobus acidocaldarius. Proc. Natl. Acad. Sci. USA 92:12285-12289.tff-, 百拇医药
Aagaard, C., I. Leviev, R. N. Aravalli, P. Forterre, D. Prieur, and R. A. Garrett. 1996. General vectors for archaeal hyperthermophiles: strategies based on a mobile intron and a plasmid. FEMS Microbiol. Rev. 18:93-104.tff-, 百拇医药
Adams, M. W., and R. M. Kelly. 1998. Finding and using hyperthermophilic enzymes. Trends Biotechnol. 16:329-332.tff-, 百拇医药
Aravalli, R. N., and R. A. Garrett. 1997. Shuttle vectors for hyperthermophilic archaea. Extremophiles 1:183-191.tff-, 百拇医药
Beckwith, J. R., A. B. Pardee, R. Austrian, and F. Jacob. 1962. Coordination of the synthesis of the enzymes in the pyrimidine pathway of E. coli. J. Mol. Biol. 5:618-634.tff-, 百拇医药
Benbouzid-Rollet, N., P. López-García, L. Watrin, G. Erauso, D. Prieur, and P. Forterre. 1997. Isolation of new plasmids from hyperthermophilic Archaea of the order Thermococcales. Res. Microbiol. 148:767-775.
Bertani, G., and L. Baresi. 1987. Genetic transformation in the methanogen Methanococcus voltae PS. J. Bacteriol. 169:2730-2738.5&9u(u, 百拇医药
Boeke, J. D., F. LaCroute, and G. R. Fink. 1984. A positive selection for mutants lacking orotidine-5'-phosphate decarboxylase activity in yeast: 5-fluoro-orotic acid resistance. Mol. Gen. Genet. 197:345-346.5&9u(u, 百拇医药
Boeke, J. D., J. Trueheart, G. Natsoulis, and G. R. Fink. 1987. 5-Fluoroorotic acid as a selective agent in yeast molecular genetics. Methods Enzymol. 154:164-175.5&9u(u, 百拇医药
Cannio, R., P. Contursi, M. Rossi, and S. Bartolucci. 1998. An autonomously replicating transforming vector for Sulfolobus solfataricus. J. Bacteriol. 180:3237-3240.5&9u(u, 百拇医药
Elferink, M. G., C. Schleper, and W. Zillig. 1996. Transformation of the extremely thermoacidophilic archaeon Sulfolobus solfataricus via a self-spreading vector. FEMS Microbiol. Lett. 137:31-35.5&9u(u, 百拇医药
Erauso, G., S. Marsin, N. Benbouzid-Rollet, M. F. Baucher, T. Barbeyron, Y. Zivanovic, D. Prieur, and P. Forterre. 1996. Sequence of plasmid pGT5 from the archaeon Pyrococcus abyssi: evidence for rolling-circle replication in a hyperthermophile. J. Bacteriol. 178:3232-3237.
Ezaki, S., N. Maeda, T. Kishimoto, H. Atomi, and T. Imanaka. 1999. Presence of a structurally novel type ribulose-bisphosphate carboxylase/oxygenase in the hyperthermophilic archaeon, Pyrococcus kodakaraensis KOD1. J. Biol. Chem. 274:5078-5082.#4)[4, http://www.100md.com
Grogan, D. W., and R. P. Gunsalus. 1993. Sulfolobus acidocaldarius synthesizes UMP via a standard de novo pathway: results of biochemical-genetic study. J. Bacteriol. 175:1500-1507.#4)[4, http://www.100md.com
Holden, J. F., K. Takai, M. Summit, S. Bolton, J. Zyskowski, and J. A. Baross. 2001. Diversity among three novel groups of hyperthermophilic deep-sea Thermococcus species from three sites in the northeastern Pacific Ocean. FEMS Microbiol. Ecol. 36:51-60.#4)[4, http://www.100md.com
Jacobs, K. L., and D. W. Grogan. 1997. Rates of spontaneous mutation in an archaeon from geothermal environments. J. Bacteriol. 179:3298-3303.#4)[4, http://www.100md.com
Kawarabayasi, Y., M. Sawada, H. Horikawa, Y. Haikawa, Y. Hino, S. Yamamoto, M. Sekine, S. Baba, H. Kosugi, A. Hosoyama, Y. Nagai, M. Sakai, K. Ogura, R. Otsuka, H. Nakazawa, M. Takamiya, Y. Ohfuku, T. Funahashi, T. Tanaka, Y. Kudoh, J. Yamazaki, N. Kushida, A. Oguchi, K. Aoki, and H. Kikuchi. 1998. Complete sequence and gene organization of the genome of a hyper-thermophilic archaebacterium, Pyrococcus horikoshii OT3. DNA Res. 5:55-76.
Kondo, S., A. Yamagishi, and T. Oshima. 1991. Positive selection for uracil auxotrophs of the sulfur-dependent thermophilic archaebacterium Sulfolobus acidocaldarius by use of 5-fluoroorotic acid. J. Bacteriol. 173:7698-7700.\fhs'x, http://www.100md.com
Lange, M., and B. K. Ahring. 2001. A comprehensive study into the molecular methodology and molecular biology of methanogenic Archaea. FEMS Microbiol. Rev. 25:553-571.\fhs'x, http://www.100md.com
López-García, P., P. Forterre, J. van der Oost, and G. Erauso. 2000. Plasmid pGS5 from the hyperthermophilic archaeon Archaeoglobus profundus is negatively supercoiled. J. Bacteriol. 182:4998-5000.\fhs'x, http://www.100md.com
Martusewitsch, E., C. W. Sensen, and C. Schleper. 2000. High spontaneous mutation rate in the hyperthermophilic archaeon Sulfolobus solfataricus is mediated by transposable elements. J. Bacteriol. 182:2574-2581.\fhs'x, http://www.100md.com
Morikawa, M., Y. Izawa, N. Rashid, T. Hoaki, and T. Imanaka. 1994. Purification and characterization of a thermostable thiol protease from a newly isolated hyperthermophilic Pyrococcus sp. Appl. Environ. Microbiol. 60:4559-4566.
Niehaus, F., C. Bertoldo, M. Kähler, and G. Antranikian. 1999. Extremophiles as a source of novel enzymes for industrial application. Appl. Microbiol. Biotechnol. 51:711-729.9, 百拇医药
Peck, R. F., S. Dassarma, and M. P. Krebs. 2000. Homologous gene knockout in the archaeon Halobacterium salinarum with ura3 as a counterselectable marker. Mol. Microbiol. 35:667-676.9, 百拇医药
Ramakrishnan, V., and M. W. W. Adams. 1995. Preparation of genomic DNA from sulfur-dependent hyperthermophilic archaea, p. 95-96. In F. T. Robb and A. R. Place (ed.), Archea: a laboratory manual—thermophiles. Cold Spring Harbor Press, Cold Spring Harbor, N.Y.9, 百拇医药
Reilly, M. S., and D. W. Grogan. 2001. Characterization of intragenic recombination in a hyperthermophilic archaeon via conjugational DNA exchange. J. Bacteriol. 183:2943-2946.9, 百拇医药
Robb, F. T., D. L. Maeder, J. R. Brown, J. DiRuggiero, M. D. Stump, R. K. Yeh, R. B. Weiss, and D. M. Dunn. 2001. Genomic sequence of hyperthermophile, Pyrococcus furiosus: implications for physiology and enzymology. Methods Enzymol. 330:134-157.
Robb, F. T., and A. R. Place. 1995. Media for Thermophiles, p. 167-168. In F. T. Robb and A. R. Place (ed.), Archea: a laboratory manual—thermophiles. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.501], http://www.100md.com
Sambrook, J., and D. Russel. 2001. Molecular cloning: a laboratory manual, 3rd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.501], http://www.100md.com
Schäfer, G., M. Engelhard, and V. Müller. 1999. Bioenergetics of the archaea. Microbiol. Mol. Biol. Rev. 63:570-620.501], http://www.100md.com
Schleper, C., I. Holz, D. Janekovic, J. Murphy, and W. Zillig. 1995. A multicopy plasmid of the extremely thermophilic archaeon Sulfolobus effects its transfer to recipients by mating. J. Bacteriol. 177:4417-4426.501], http://www.100md.com
Schleper, C., K. Kubo, and W. Zillig. 1992. The particle SSV1 from the extremely thermophilic archaeon Sulfolobus is a virus: demonstration of infectivity and of transfection with viral DNA. Proc. Natl. Acad. Sci. USA 89:7645-7649.501], http://www.100md.com
Schleper, C., R. Röder, T. Singer, and W. Zillig. 1994. An insertion element of the extremely thermophilic archaeon Sulfolobus solfataricus transposes into the endogenous ß-galactosidase gene. Mol. Gen. Genet. 243:91-96.
Sowers, K. R., and H. J. Schreier. 1999. Gene transfer systems for the Archaea. Trends Microbiol. 7:212-219.|n?@', 百拇医药
Stedman, K. M., C. Schleper, E. Rumpf, and W. Zillig. 1999. Genetic requirements for the function of the archaeal virus SSV1 in Sulfolobus solfataricus: construction and testing of viral shuttle vectors. Genetics 152:1397-1405.|n?@', 百拇医药
Stetter, K. O. 1996. Hyperthermophilic prokaryotes. FEMS Microbiol. Rev. 18:149-158.|n?@', 百拇医药
Stetter, K. O. 1999. Extremophiles and their adaptation to hot environments. FEBS Lett. 452:22-25.|n?@', 百拇医药
Takai, K., T. Komatsu, F. Inagaki, and K. Horikoshi. 2001. Distribution of archaea in a black smoker chimney structure. Appl. Environ. Microbiol. 67:3618-3629.|n?@', 百拇医药
Tang, X. F., S. Ezaki, H. Atomi, and T. Imanaka. 2001. Anthranilate synthase without an LLES motif from a hyperthermophilic archaeon is inhibited by tryptophan. Biochem. Biophys. Res. Commun. 281:858-865.|n?@', 百拇医药
Vieille, C., and G. J. Zeikus. 2001. Hyperthermophilic enzymes: sources, uses, and molecular mechanisms for thermostability. Microbiol. Mol. Biol. Rev. 65:1-43.
Ward, D. E., I. M. Revet, R. Nandakumar, J. H. Tuttle, W. M. de Vos, J. van der Oost, and J. DiRuggiero. 2002. Characterization of plasmid pRT1 from Pyrococcus sp. strain JT1. J. Bacteriol. 184:2561-2566.m'vd7.j, http://www.100md.com
Watrin, L., S. Lucas, C. Purcarea, C. Legrain, and D. Prieur. 1999. Isolation and characterization of pyrimidine auxotrophs, and molecular cloning of the pyrE gene from the hyperthermophilic archaeon Pyrococcus abyssi. Mol. Gen. Genet. 262:378-381.m'vd7.j, http://www.100md.com
Watrin, L., and D. Prieur. 1996. UV and ethyl methanesulfonate effects in hyperthermophilic archaea and isolation of auxotrophic mutants of Pyrococcus strains. Curr. Microbiol. 33:377-382.m'vd7.j, http://www.100md.com
Zillig, W., H. P. Arnold, I. Holz, D. Prangishvili, A. Schweier, K. Stedman, Q. She, H. Phan, R. Garrett, and J. K. Kristjansson. 1998. Genetic elements in the extremely thermophilic archaeon Sulfolobus. Extremophiles 2:131-140.(Takaaki Sato Toshiaki Fukui Haruyuki Atomi and Tadayuki Imanaka)
Accepted 2 October 2002$2:^{8|, 百拇医药
ABSTRACT$2:^{8|, 百拇医药
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 CaCl2 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.
INTRODUCTION/fu, 百拇医药
Among the living organisms, hyperthermophiles belonging to the third domain of life, Archaea, are attractive subjects of research from various standpoints. Many efforts to understand the strategies for adaptation to extremely high temperature environments have revealed numerous physiologically intriguing and unique properties of hyperthermophilic archaea (30, 36, 37). They have also attracted attention from an industrial point of view as potential resources for highly thermostable enzymes (3, 23, 40)./fu, 百拇医药
Along with the classical biochemistry and molecular cloning of structural genes, complete genome analyses in recent years have provided a new means of understanding the hyperthermophilic archaea. So far, entire genome sequences of 16 archaeal strains, including 10 hyperthermophiles, have been determined. This has revealed that genomes of hyperthermophilic archaea are rather small, and the small number of genes makes them ideal targets for studying the basic principles and evolution of life.
In spite of the many attractive aspects of hyperthermophilic archaea, progress of research on these organisms has been constantly hampered by the limitation of available tools for genetic manipulation. While genetic methodology has been developed for methanogenic archaea (19, 34) and extremely halophilic archaea (24, 34), comparable to those of their bacterial counterparts, manipulative strategies for the hyperthermophilic archaea are still at an early stage. With respect to the genus Sulfolobus in the kingdom Crenarchaeota, various genetic elements have been identified (44), and natural gene transfer via conjugation (26, 31) or virus infection (32) has been observed. Several cases of genetic manipulation have been reported with Sulfolobus by applying these exochromosomal genetic elements (1, 10, 11, 32, 35). In contrast, concerning hyperthermophiles of Euryarchaeota, genetic tools are further limited due to the small number of genetic elements identified and/or applicable in this kingdom. Although several autonomously replicating plasmids have been isolated from Pyrococcus and Thermococcus strains (6, 12, 41) and Archaeoglobus profundus (20), useful genetic tools have not been developed except for shuttle vectors based on pGT5 from Pyrococcus abyssi that could transform and be maintained in not only the closely related strain Pyrococcus furiosus but also Sulfolobus acidocaldarius (2, 4).
Moreover, it should be noted that there is no gene targeting system for hyperthermophilic archaea in the literature. The development of a system for the targeted disruption of genes would certainly be a major breakthrough in various fields of research on hyperthermophilic archaea, as although the archaeal genomes are relatively small, the function of approximately half of the genes cannot be predicted by primary structure comparisons. Along with biochemical analyses of individual gene products and/or structural genomics, analyses of phenotypic changes in knockout strains and genetic complementation techniques will no doubt contribute greatly to the identification of currently unknown gene functions.|ho, http://www.100md.com
Thermococcus kodakaraensis KOD1 (previously reported as Pyrococcus kodakaraensis KOD1) is a sulfur-reducing hyperthermophilic archaeon belonging to the Thermococcales in Euryarchaeota (22). Although the genome analyses of three Pyrococcus species, P. abyssi , P. furiosus (27; http://www.genome.utah.edu/sequence.html; http://www.ornl.gov/hgmis/publicat/99santa/157.html), and P.horikoshii (17; http://www.bio.nite.go.jp/ot3db_index.html),have already been completed among members of Thermococcales, the complete genome of a Thermococcus strain has not yet been determined. Recent culture-dependent and culture-independent studies have indicated that strains of the genus Thermococcus are more ubiquitous than Pyrococcus strains in various marine hydrothermal vent systems (15, 38). Therefore, Thermococcus strains, with their larger population, can be expected to play a major role in the ecology and metabolic activity of microbial consortia in extremely high temperature environments. In light of the importance of this genus, the genome project of T. kodakaraensis KOD1 is now in progress.
This study aimed to construct a gene disruption system for the hyperthermophilic archaeon T. kodakaraensis KOD1 for further functional analyses of genes identified by the genome project. For this purpose, we applied an orotidine-5'-monophosphate decarboxylase (OMPdecase) (PyrF)-deficient mutant of T. kodakaraensis KOD1, displaying uracil auxotrophy, as a host and the complementary pyrF gene as a selectable marker. 5-Fluoroorotic acid (5-FOA) was adopted to isolate the PyrF- mutants, as clones with resistance to this drug usually lack either PyrF or orotate phosphoribosyltransferase (OPRTase) (PyrE) activity. We here describe isolation of pyrF-deficient mutants of T. kodakaraensis KOD1 by 5-FOA positive selection (8, 9) and targeted disruption of trpE by homologous recombination under a selection based on complementation of uracil auxotrophy of the mutant.?ikv&, 百拇医药
MATERIALS AND METHODS?ikv&, 百拇医药
Strains and growth conditions. The strains and plasmids used in this study are listed in . T. kodakaraensis KOD1 and its derivatives were cultivated under strictly anaerobic conditions at 85°C in a rich growth medium (ASW-YT medium) or a synthetic medium containing amino acids (ASW-AA medium). ASW-YT medium was composed of a 1.25-fold dilution of artificial seawater (28) (0.8x ASW), yeast extract (5.0 g/liter), tryptone (5.0 g/liter), and elemental sulfur (2 g/liter) (pH 6.6). ASW-AA medium consisted of 0.8x ASW supplemented with a 5.0-ml/liter concentration of modified Wolfe's trace minerals [0.5 g of MnSO4 · 2H2O, 0.1 g of CoCl2, 0.1 g of ZnSO4, 0.01 g of CuSO4 · 5H2O, 0.01 g of AlK(SO4)2, 0.01 g of H3BO3, and 0.01 g of NaMoO4 · 2H2O per liter] (28), vitamin mixture (5.0 ml/liter) (28), 20 amino acids (250 mg of cysteine · HCl · H2O, 75 mg of alanine, 125 mg of arginine · HCl, 100 mg of asparagine · H2O, 50 mg of aspartic acid, 50 mg of glutamine, 200 mg of glutamic acid, 200 mg of glycine, 100 mg of histidine · HCl · H2O, 100 mg of isoleucine, 100 mg of leucine, 100 mg of lysine · HCl, 75 mg of methionine, 75 mg of phenylalanine, 125 mg of proline, 75 mg of serine, 100 mg of threonine, 75 mg of tryptophan, 100 mg of tyrosine, and 50 mg of valine per liter), and elemental sulfur (0.2 g/liter) (pH was adjusted to 6.9 with NaOH). When necessary, 5-FOA (Wako Pure Chemicals, Osaka, Japan) and uracil (Kohjin, Tokyo, Japan) were added to ASW-AA medium at the concentrations as described in the text. For investigation of tryptophan auxotrophy, tryptophan-deficient ASW-AA medium (ASW-AAW-) was used. To reduce the dissolved oxygen in the medium, 5.0% Na2S · 9H2O was added until the color of resazurin sodium salt (1.0 mg/liter) became clear. In the case of plate culture, 1.0% (wt/vol) Gelrite (Wako Pure Chemicals) was added to solidify the medium together with a 2.0-ml/liter concentration of polysulfide solution (10 g of Na2S · 9H2O and 3.0 g of elemental sulfur per 15 ml) instead of elemental sulfur and 5.0% Na2S · 9H2O solution. The cells inoculated on the plate medium were incubated at 85°C in anaerobic chambers (Tabai Espec, Osaka, Japan).
fig.ommitted\dzt, http://www.100md.com
Strains and plasmids used in this study, together with OMPdecase and OPRTase activities in T. kodakaraensis KOD1 and its mutants\dzt, http://www.100md.com
Escherichia coli strain DH5{alpha} , used for general DNA manipulation, was routinely cultivated at 37°C in Luria-Bertani medium (29) and supplemented with ampicillin (50 µg/ml) when needed.\dzt, http://www.100md.com
Mutagenesis with UV irradiation and isolation of 5-FOA-resistant mutants.\dzt, http://www.100md.com
T. kodakaraensis KOD1 was cultivated in 2.0 liters of ASW-AA liquid medium for 39 h, and the cells at the stationary phase were harvested by centrifugation (6,000 x g, 30 min). The following procedures were performed anaerobically in the anaerobic chambers. The cells were resuspended in 60 ml of ASW (3/100 volume), and a portion of the suspension (10 ml; 1010 cells) was poured into glass petri dishes. While being stirred, the suspension was subjected to UV irradiation at a distance of 20 cm from a 15-W germicidal lamp for appropriate periods of time (0, 30, 60, 90, and 120 s). Aliquots (200 µl) were spread on ASW-AA plate medium containing 0.75% 5-FOA for dominant selection of uracil-auxotrophic (Pyr-) mutants, together with uracil (10 µg/ml) to support growth of the resulting mutants. The cells were incubated at 85°C for 5 days. The number of viable cells was determined by inoculating portions of the cell suspension with appropriate dilution onto ASW-AA plate medium without 5-FOA and counting the colonies formed.
5-FOA-resistant colonies were isolated and cultivated in ASW-YT liquid medium. The grown cells were incubated for 2 days in ASW-AA liquid medium to avoid carryover of uracil and further subcultured in ASW-AA liquid medium with or without addition of uracil (5 µg/ml) to investigate the uracil auxotrophy of the isolates.4, http://www.100md.com
Enzyme assay.4, http://www.100md.com
Cell extracts of T. kodakaraensis KOD1 and its mutant strains were prepared as follows. Cells cultivated in ASW-YT liquid medium for 20 h were harvested by centrifugation (6,000 x g, 30 min) and lysed in 1/1,000 volume of 50 mM Tris-HCl (pH 7.5) containing 0.1% Triton X-100. After mixing with a vortex for 10 min, the supernatant after centrifugation (3,000 x g, 20 min) was used as the cell extract. The protein concentration was determined using a Bio-Rad (Hercules, Calif.) protein assay system with bovine serum albumin as a standard.4, http://www.100md.com
OMPdecase (PyrF) activity was measured by monitoring the decrease in absorbance at 285 nm derived from the conversion of orotidine-5'-monophosphate (OMP) to UMP ({varepsilon} 285 = 1,380 M-1 cm-1) (5). The assay mixture was composed of 100 mM Tris-HCl (pH 8.6), 1.5 mM MgCl2, 0.125 mM OMP, and the enzyme solution in a total volume of 1 ml. After preincubation of the assay mixture in a capped cuvette at 85°C for 5 min, the reaction was started by addition of the enzyme solution and was monitored at the same temperature for 10 min.
OPRTase (PyrE) activity was assayed by determining the decrease of orotic acid spectrophotometrically at 295 nm (5). When measuring enzyme samples from pyrF+ strains, successive decarboxylation of the reaction product, OMP, by the endogenous OMPdecase must be taken into account. Since the OMPdecase activity was higher than OPRTase activity in T. kodakaraensis, the OPRTase activity could accurately be determined with the {varepsilon} 295 of 3,670 M-1 cm-1, corresponding to the conversion of orotic acid to UMP via OMP. In case of PyrF- strains, we monitored the conversion of the starting substrate to OMP with a {varepsilon} 295 of 2,520 M-1 cm-1. The reaction was performed in 1-ml mixtures containing 100 mM Tris-HCl (pH 8.6), 1.5 mM MgCl2, 0.125 mM orotic acid, cell extract, and 1.6 mM 5-phosphoribosyl 1-pyrophosphate (5-PRPP). The assay mixture without 5-PRPP in a capped cuvette was preincubated at 85°C for 10 min, and the reaction was started by addition of 5-PRPP. The decrease in A295 was measured at the same temperature for 3 min.
DNA manipulation and DNA sequencing.p*yg'}m, 百拇医药
General DNA manipulation was performed as described by Sambrook and Russel (29). Genomic DNA of T. kodakaraensis was isolated as described elsewhere (25). PCR was carried out using KOD Plus (Toyobo, Osaka, Japan) as DNA polymerase, and sequences of primers used for PCR are described in the text. When necessary, DNA fragments amplified by PCR were phosphorylated by T4 kinase (Toyobo). Restriction enzymes and modifying enzymes were purchased from Takara Shuzo (Kyoto, Japan) or Toyobo. DNA fragments after agarose gel electrophoresis were recovered and purified with GFX PCR DNA and a gel band purification kit (Amersham Pharmacia Biotech, Uppsala, Sweden). Plasmid DNA was isolated using Qiagen (Hilden, Germany) plasmid kits. DNA sequencing was performed using a BigDye terminator cycle sequencing kit and a model 3100 capillary sequencer (Applied Biosystems, Foster City, Calif.).p*yg'}m, 百拇医药
Construction of pUDT1 and pUDT2.p*yg'}m, 百拇医药
Two disruption vectors, pUDT1 and pUDT2, for homologous recombination in T. kodakaraensis with single- and double-crossover events, respectively, were constructed as follows. A DNA fragment (676 bp) containing Tk-pyrF was amplified from T. kodakaraensis KOD1 genomic DNA using the primers TK1-DUR-TK1-DUF (5'-GGGCATATGGAGGAGAGCAGGCTCATTCTGGCG-3'-5'-CTGAGGGGGTGTTTGACTTTCAA-3'; the underlined sequence indicates an NdeI site). The putative promoter region (130 bp) was amplified with the primers TK2-DPR-TK2-DPF (5'-GGGCTGCAGCCGCAACGCGCATTTTGCTCACCCGAAAA-3'-5'-GGGCATATGCATCACCTTTTTAACGGCCCTCTCCAAGAG-3'; the underlined sequences indicate PstI and NdeI sites, respectively). Both fragments were subcloned into pUC118 with the proper promoter-pyrF orientation. The resulting plasmid was designated pUD (3,944 bp). A truncated fragment of Tk-trpE (788 bp) was amplified using the primers TK3-DTR-TK3-DTF (5'-GGGGCATGCGGTGGCTTCGTTGGCTACGTCTCCTACG-3'-5'-GGGCTGCAGTTCGGGGCTCCGGTTAGTGTTCCCGCCG-3'; the underlined sequences indicate SphI and PstI sites, respectively), and then ligated with pUD at SphI and PstI sites, to obtain pUDT1 (4,732 bp) .
fig.ommitted8yx4*, http://www.100md.com
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.8yx4*, http://www.100md.com
For construction of pUDT2, a fragment (2,223 bp) containing Tk-trpE and the flanking regions was amplified using TK4-DT2R-TK4-DT2F (5'-GGGGTCGACCGGGTCTGGCGAGGGCAATGAGGGAC-3'-5'-GGGGAATTCGGTTATAGTGTTCGGAACGACCTTCACTC-3'; the underlined sequences indicate SalI and EcoRI sites, respectively) as primers, and subcloned into pUC118 at SalI and EcoRI sites. The plasmid obtained was named pUT4 (5,340 bp). pUD was digested with PvuII, followed by isolation of the fragment containing pyrF and its putative promoter region (1,104 bp). pUDT2 (6,012 bp) was obtained by insertion of the isolated fragment within pUT4 at blunted SacI sites in Tk-trpE .8yx4*, http://www.100md.com
Linear DNA fragments for homologous recombination in T. kodakaraensis, DT2-L1 (2,877 bp) and DT2-L2 (2,104 bp), were amplified from pUDT2 using the primer sets PDD-R-PDD-F (5'-CGGGTCTGGCGAGGGCAATGAGGGACAGCGCAGTCA-3'-5'-GGTTATAGTGTTCGGAACGACCTTCACT-3') and PDD-500R-PDD-500F (5'-GACCCTCGTCCTGGAGGTTGGCAATG-3'-5'-CGGCGTCCCCGGTGAGGGAGAAGTAA-3'), respectively. The amplified DNA fragments were purified after agarose gel electrophoresis.
Transformation of T. kodakaraensis.m;5[kg&, http://www.100md.com
The CaCl2 method for Methanococcus voltae PS (7) was modified for transformation of T. kodakaraensis. T. kodakaraensis KU25 was cultivated in ASW-YT liquid medium for 12 h, and the cells at the late exponential phase were harvested (17,000 x g, 5 min) from 3 ml of culture broth containing approximately 4 x 108 cells, and resuspended in 200 µl (1/15 volume) of transformation buffer (80 mM CaCl2 in 0.8x modified ASW that did not contain KH2PO4 to avoid precipitation between the calcium cation and the phosphate group) and kept on ice for 30 min. Then, 3 µg of DNA dissolved in Tris-EDTA buffer was added into the suspension, and the cells were incubated on ice for 1 h, which was followed by a heat shock at 85°C for 45 s and further incubation on ice for 10 min. As a control experiment, an equivalent volume of Tris-EDTA buffer was added to the cells instead of the DNA solution. The treated cells were cultured in 20 ml of ASW-AA liquid medium in the absence of uracil for two generations to select and concentrate Pyr+ transformants. The cells were then spread on ASW-AA plate medium without uracil and incubated for 5 to 8 days at 85°C. The resulting Pyr+ strains were analyzed by colony PCR and Southern hybridization using a DIG-DNA labeling and detection kit (Boehringer Mannheim, Mannheim, Germany).
RESULTSm!'d, 百拇医药
Isolation of uracil-auxotrophic mutants of T. kodakaraensis KOD1. We first set out to isolate uracil-auxotrophic mutants of T. kodakaraensis KOD1 using 5-FOA. The effects of 5-FOA on the growth of KOD1 were investigated in a synthetic medium with defined substrates (ASW-AA medium) and a nutrient-rich medium (ASW-YT medium). An important observation was that both media, in particular the ASW-AA medium, supported colony formation of T. kodakaraensis cells under anaerobic conditions when solidified with Gelrite. The plating efficiency of T. kodakaraensis was determined to be approximately 24%. 5-FOA has been reported to inhibit cell growth of various microorganisms, including the closely related P. abyssi (42), at concentrations from 0.05 to 0.1%. However, 5-FOA at this concentration range did not affect the cell growth of KOD1. When the 5-FOA concentration was increased in ASW-AA media, 0.75% was the threshold for complete inhibition of cell growth both in liquid medium and on plate medium. In contrast, 5-FOA showed little effects in the rich ASW-YT medium, even at high 5-FOA concentrations (~ 1.5%). Variable effects of 5-FOA in different media have also been observed in the case of yeasts grown on rich YPD medium (9). We therefore adopted ASW-AA plate medium with 0.75% 5-FOA for further selection of 5-FOA-resistant mutants of T. kodakaraensis KOD1.
fig.ommitted1'tv, http://www.100md.com
(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.1'tv, http://www.100md.com
The cells of the strain KOD1 were mutagenized by UV irradiation in an anaerobic chamber. The cell viability decreased with increase of the irradiation time; that is, viabilities of approximately 60, 40, 20, and 15% were observed after UV irradiation for 30, 60, 90, and 120 s, respectively. After this treatment, 5-FOA-resistant mutants were selected on ASW-AA plate media containing 0.75% 5-FOA and uracil (10 µg/ml). Independent experiments with different UV exposure times (30, 60, 90, or 120 s) gave several drug-resistant mutants with frequencies from 3 x 10-5 to 1 x 10-4. It should be noted that 5-FOA-resistant cells could also be isolated without UV irradiation (1 x 10-5 to 5 x 10-5 frequencies), suggesting the occurrence of spontaneous mutants during cell cultivation. Seventeen colonies, including seven colonies from the cultures without the UV irradiation, were randomly selected and their uracil auxotrophy was examined in ASW-AA liquid medium for three generations. Consequently, six strains were confirmed to be stable uracil-auxotrophic (Pyr-) mutants and designated as listed in , where KU18 and KU19 were possible spontaneous mutants.
Characterization of the mutants. In order to determine the deficient step in pyrimidine biosynthesis leading to uracil auxotrophy, OMPdecase (PyrF) and OPRTase (PyrE) activities in the cell extracts of the Pyr- mutants were assayed . The results indicated that the strains KU25 and KU27 were PyrF- mutants lacking OMPdecase activity, whereas the other four strains were PyrE- mutants with no OPRTase activity. The uracil-auxotrophic mutations in T. kodakaraensis examined here could be attributed to a biochemical defect in one of the two enzymes.+zcnn, 百拇医药
We subsequently performed genetic characterization of the two PyrF- mutant strains KU25 and KU27. The native pyrF gene from T. kodakaraensis KOD1 (Tk-pyrF) was identified in a phage clone of the genomic DNA library containing the gene encoding ribulose-1,5-bisphosphate carboxylase/oxygenase (13). Tk-pyrF encodes a protein of 213 amino acids, and the primary structure showed homology to the orthologues from P. abyssi (76% identity) and E. coli (24% identity). The corresponding genes in strains KU25 and KU27 were amplified by PCR, and the nucleotide sequences were determined. As shown in , a deletion of 96T was identified in pyrF from KU25, and the mutated gene corresponded to an ORF of only 99 bp due to the frame shift. In the case of KU27, there was a single nucleotide insertion (C) between 445C and 446G, which resulted in a mutant PyrF protein (176 amino acids) with an aberrant and shorter C-terminal region. These facts provided strong evidence that the strains KU25 and KU27 were actually pyrF-deficient mutants of T. kodakaraensis. We selected KU25 as a host strain for further transformation studies, because the pyrF gene was completely inactivated by the 1-bp deletion. shows the growth characteristic of KU25 compared to that of wild-type KOD1. In contrast to wild-type cells that could grow regardless of the addition of uracil, KU25 showed strict uracil auxotrophy in ASW-AA liquid medium. However, the mutant cells could grow on uracil-deficient ASW-AA plate medium even after preincubation of the cells under uracil starvation conditions to avoid carryover of uracil. Similar tendencies have been reported for the Pyr- mutants of the hyperthermophilic crenarchaeon S. solfataricus (21). It can be considered that the reason for this phenomenon is that Gelrite, used as a solidifier, might contain trace amounts of pyrimidine or related analogues, supporting the growth of the mutant cells.
fig.ommitted19)]]im, 百拇医药
Mutations in pyrF from T. kodakaraensis KU25 and KU27.19)]]im, 百拇医药
Disruption of trpE in T. kodakaraensis. We then performed targeted disruption of a gene in T. kodakaraensis KU25 by applying pyrF as a selectable marker. Tk-trpE, encoding the large subunit of anthranilate synthase in the tryptophan biosynthesis pathway (39), was chosen as the first target, because the disruption of this gene was expected to cause tryptophan auxotrophy, an easily detectable phenotype. Two gene disruption plasmids, namely, pUDT1 and pUDT2 , were constructed for homologous recombination through single- and double-crossover events, respectively. Both plasmids contained a marker cassette consisting of pyrF together with the putative promoter region. pUDT1 harbored an internal 788- bp fragment of trpE at an adjacent site to the marker cassette, and pUDT2 was constructed by inserting a 1,104-bp fragment of the marker cassette between the 5' region of trpE with trpD (1,020 bp) and the 3' region of trpE joined to trpG (753 bp).
T. kodakaraensis KU25 was transformed with 3 µg of pUDT1 or pUDT2 by the CaCl2 procedure, as described in Materials and Methods, where the cell viability after CaCl2 treatment was approximately 38%. Since the uracil auxotrophy of KU25 was not strict on the plate medium as mentioned above, Pyr+ transformants were first concentrated in a selective ASW-AA liquid medium not containing uracil. We could observe cell growth in all experiments when the cells were treated with pUDT1 or pUDT2, whereas no growth was seen in the control experiments. These results suggested that some recombination events took place within the cells, leading to uracil prototrophy. The concentrated cells were cultivated on ASW-AA plate medium without uracil, and colony PCR was performed using PDDC-R-PDDC-F primers to analyze the trpE locus in the isolates . The length of the amplified fragment from all 24 Pyr+ strains treated with pUDT1 was the same as that from the host strain KU25 (2.3 kbp) . This result indicated that disruption of trpE did not occur in this case. Spontaneous reversion of the pyrF mutation was unlikely, as control experiments without exogenous DNA did not lead to any prototrophs. We therefore examined the pyrF loci of three independent prototrophs. The lengths and nucleotide sequences of amplified fragments covering the pyrF region of each prototroph clarified that all three pyrF genes had reverted to the wild-type gene without integration of pUDT1 at this locus. Southern blot analysis using a probe spanning the pyrF region gave only a single band derived from the endogenous pyrF gene, which denies the possibility of nonspecific insertion of the pyrF marker in these chromosomes (data not shown). These results strongly suggested the occurrence of double-crossover recombination between the mutated pyrF on the chromosome and the intact gene in the plasmid. In contrast, when pUDT2 was added to the cells, a longer fragment (3.0 kbp), corresponding precisely to the length of a trpE::pyrF locus formed by double-crossover recombination , was amplified in 15 out of 24 Pyr+ strains . One of the positive isolates was selected and designated KW4 for further investigations.
fig.ommittedvaq'', 百拇医药
Transformation of T. kodakaraensis with various DNAsvaq'', 百拇医药
fig.ommittedvaq'', 百拇医药
(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.vaq'', 百拇医药
Genotype and phenotype of strain KW4. shows the longer fragment amplified from KW4 compared to the corresponding fragments from KOD1 and KU25 in the colony PCR analysis. The nucleotide sequence of the longer fragment from KW4 corresponded precisely to that of the disruption vector pUDT2. The orientation and the positioning of pyrF and the flanking regions derived from pUC118 were found as expected after a homologous double-crossover event at the trpE locus . In order to investigate whether unintended nonhomologous recombination events occurred in the chromosome of KW4 or not, we performed Southern blot analysis using trpE and pyrF probes. The regions spanned by these probes are displayed in . Only one signal was detected with the trpE probe against ApaI- and Eco52I-digested genomic DNA from KOD1, KU25, and KW4, but the signal against KW4 was longer . This agrees well with the results of PCR analysis mentioned above and further supports the occurrence of homologous recombination at the trpE locus. The pyrF probe gave identical signals derived from the endogenous pyrF locus in all three strains (7.3 and 11.0 kbp from Aat I- and ApaI-digested genomic DNA, respectively). In the case of KW4, one additional short band corresponding to pyrF within trpE could be detected . These results denied nonhomologous recombination and indicated that only targeted disruption of trpE had occurred in KW4.
fig.ommittedo-n\c^c, 百拇医药
(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.o-n\c^c, 百拇医药
To confirm the phenotype of KW4, we verified its growth properties in ASW-AAW- medium. While the host strain KU25 did not require tryptophan for growth, KW4 was a tryptophan-auxotrophic (Trp-) strain that could grow only in the presence of tryptophan. Growth of KW4 was no longer dependent on the addition of uracil, indicating that the Pyr- mutation in KU25 had been complemented in KW4, as expected. This tryptophan auxotrophy of KW4 was also clearly observed on the plate medium, as shown in . These results demonstrated that the disruption of trpE by the insertion of pyrF was responsible for the phenotypic change of auxotrophy from uracil to tryptophan.
fig.ommittedj)r$-, 百拇医药
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.j)r$-, 百拇医药
Effects of CaCl2 on transformation of T. kodakaraensis. In order to determine whether CaCl2 was essential for the transformation of T. kodakaraensis, KU25 cells were treated with transformation buffer without the CaCl2 supplemented in the experiments mentioned above (80 mM), and pUDT2 was added into the suspension. Contrary to our expectation, Pyr+ strains were obtained even in this case, and the longer trpE::pyrF locus was also detected by colony PCR analysis in 12 out of 24 Pyr+ isolates. This demonstrated that the high concentration of CaCl2 in the transformation buffer was not essential for DNA uptake and successive transformation of T. kodakaraensis.
Gene disruption by linear DNA and effect of the length of the homologous regions. We further investigated the possibility of gene disruption using linear DNA. Two linear DNA fragments, DT2-L1 (2,877 bp) and DT2-L2 (2,104 bp) were amplified by PCR using pUDT2 as a template. The former contained 1,020- and 753-bp regions homologous to the trp locus at both ends of the marker cassette, and the latter contained shorter homologous regions (two 500-bp regions) . After transformation with each linear DNA, we could obtain Pyr+ transformants in both cases. However, the trpE::pyrF locus generated by gene disruption of trpE was observed only in the experiments using DT2-L1. All 24 Pyr+ isolates using DT2-L2 harbored an intact trpE locus with the same length as that from the host strain. We then determined the nucleotide sequences of pyrF in several transformants that showed a Pyr+ phenotype without recombination at trpE. The results demonstrated the reversion of the deletion mutation in these isolates, as seen in the transformation with pUDT1, suggesting predominant recombination at the pyrF locus in these strains. These results indicated that gene disruption with linear DNA was possible when the homologous regions were relatively long, as in the case of DT2-L1. Shortening these regions, as in the case of DT2-L2, led to recombination at the pyrF locus. In any case, it should be noted that the ratio of trpE disruptants to Pyr+ isolates obtained with DT2-L1 (7 of 24) was lower than that obtained with the corresponding circular DNA, pUDT2 (15 of 24).
DISCUSSIONe|\, 百拇医药
In this study, we isolated uracil-auxotrophic (Pyr-) mutants of T. kodakaraensis KOD1 and achieved targeted disruption of the trpE locus in this archaeon by utilizing pyrF as a selectable marker. In spite of the fact that Pyr- mutants for several hyperthermophilic archaea have previously been described (14, 18, 21, 42), this is the first report on the development of a gene targeting system for a member of hyperthermophilic archaea in the literature.e|\, 百拇医药
5-FOA has been often applied for positive selection of uracil-auxotrophic mutants of various microorganisms. T. kodakaraensis KOD1 was also sensitive to this drug, but the sensitivity was lower than those of other microorganisms. 5-FOA-resistant mutants could be obtained by UV mutagenesis with frequencies from 3 x 10-5 to 1 x 10-4. Furthermore, spontaneous mutants could also be isolated with relatively high frequencies (1 x 10-5 to 5 x 10-5) compared to 1 x 10-6 to 3.5 x 10-6 for various Pyrococcus and Thermococcus strains (43). It has been reported that S. solfataricus exhibited high frequencies (10-5 to 10-4) for spontaneous Pyr- or ß-galactosidase mutants due to active transposable elements (21, 33). Although we are not aware of whether such transposable elements are present in the genome of T. kodakaraensis, genome analysis should clarify this in future studies.
Six out of the seventeen 5-FOA-resistant strains were stable uracil-auxotrophic mutants, which could be divided into two classes, PyrE- or PyrF- mutants. It is interesting that the two PyrF- mutants were obtained only under stronger UV mutagenesis conditions, whereas the four mutants obtained spontaneously and by low doses of UV were PyrE- strains. Similar tendencies have also been reported for UV-induced mutation of P. abyssi (42), spontaneous mutation of S. acidocaldarius (16), and transposon-mediated spontaneous mutation of S. solfataricus (21). These results suggested that the pyrE loci in hyperthermophilic archaea might be a mutational hotspot.6.vqqou, http://www.100md.com
The disruption of the target gene trpE was successfully performed by homologous recombination via a double-crossover event using the circular plasmid pUDT2. The internal region of trpE in the host strain KU25 could be replaced with the pyrF marker cassette, resulting in the phenotypic conversion from Pyr- to Trp-. As with the circular DNA, gene disruption with linear DNA (DT2-L1) was also possible, in which homologous regions of 1,020 and 753 bp were necessary for efficient double-crossover recombination at the targeted gene. We further found that recombination at pyrF became dominant when the homologous regions were 500 bp (DT2-L2). This may simply reflect the relative lengths of homologous DNA on the vector and chromosome. The host strain KU25 harbors an inactivated but nearly full-length pyrF gene of 641 bp on its chromosome, a possible target for recombination. As the disruption vector also contains an intact pyrF gene, it can be expected that longer homologous regions of the trp locus are necessary on the vector in order to obtain efficient recombination at the trp locus. Therefore, there still exists the possibility that gene disruption with much shorter homologous regions can occur in a host from which marker gene alleles on the chromosome have been completely removed. Additionally, intracellular digestion by exonucleases may also be a factor that affects the length of homologous DNA necessary for efficient targeted recombination. This may explain the lower ratio of trpE disruptants to Pyr+ isolates using DT2-L1 (7 of 24) compared with that using the corresponding circular DNA, pUDT2 (15 of 24).
In this study, we failed to detect the disruption of trpE with pUDT1, designed for single-crossover recombination, but instead observed reversion of the pyrF mutation in the host. This reversion can most likely be explained by double-crossover recombination at the pyrF locus, as in the case of the linear DT2-L2. This is supported by the facts that no spontaneous reversion could be observed in the control experiments without DNA and neither integration of pUDT1 at the pyrF locus nor nonspecific insertion of the pyrF marker in the chromosome could be detected by PCR and Southern blot analyses of the resulting prototrophs. The nonoccurrence of single-crossover recombination may be due to cleavage of the circular DNA by a host-controlled restriction system in T. kodakaraensis. Linearization of pUDT1 would prevent integration of the circular DNA into the chromosome. On the other hand, pUDT1 linearized by cleavage at regions other than pyrF would still retain the ability to recombine with the pyrF allele on the chromosome and explain the reversion observed in this case. It has been reported that methylation of an exogenous mobile intron with HaeIII methylase was effective for transformation of S. acidocaldarius, preventing its degradation within the archaeal cells (1). This methylation procedure has also been applied for transformation of P. furiosus and S. acidocaldarius using plasmid-based shuttle vectors (2, 4). Appropriate base modification of pUDT1 prior to transformation may lead to enhanced intracellular stability of the circular plasmid and allow the disruption of targeted genes by single-crossover recombination.
In this study, we initially adopted the CaCl2 procedure including heat shock at 85°C in order to chemically enhance the cellular competence of T. kodakaraensis. However, we subsequently found that the treatment of the cells with high concentrations of CaCl2 was not essential for transformation. Although we cannot rule out some contribution of the low concentrations of Ca2+ and Mg2+ (1.6 and 31 mM, respectively) in the 0.8x modified ASW, T. kodakaraensis seems to exhibit a natural competence to uptake exogenous DNA. A similar phenomenon has also been reported for the methanogenic archaeon M. voltae (7), whereas Sulfolobus strains have been generally transformed by electroporation (10, 11, 32, 35). Although the CaCl2 treatment has been successfully applied for the transformation of S. acidocaldarius and P. furiosus (2, 4), the effects of CaCl2 concentration on transformation efficiency have not been examined in detail. This natural competence of T. kodakaraensis may be a unique feature of this strain among the hyperthermophilic archaea and will certainly provide an advantage in future gene manipulation of this strain.
As described in Results, the Pyr- mutants of T. kodakaraensis could still grow on uracil-deficient plate medium, probably due to some pyrimidine-related compounds contaminating the medium. This fact made it difficult to evaluate the transformation efficiency, or in other words the number of transformants per microgram of DNA, for this archaeon. In contrast, the strain KW4, constructed in this study, showed strict tryptophan auxotrophy both in the liquid medium and on the plate medium, even when preincubation in tryptophan-deficient medium was omitted. The development of improved transformation systems for T. kodakaraensis is in progress by applying trpE disruptants and trpE as hosts and a selectable marker, respectively.*wm, 百拇医药
Homologous recombination is a powerful tool to analyze the function of any gene of interest. We now have the tools not only to disrupt genes but also to overexpress genes in the native host cell with the use of an appropriate promoter. We believe that the combination of complete genome information and a useful transformation system, enabling systematic gene disruption, will greatly contribute to advance the studies on the hyperthermophilic archaeon T. kodakaraensis. It is also of great interest whether our methodology can be applied in other Thermococcus species and the closely related Pyrococcus species.
REFERENCEStff-, 百拇医药
Aagaard, C., J. Z. Dalgaard, and R. A. Garrett. 1995. Intercellular mobility and homing of an archaeal rDNA intron confers a selective advantage over intron- cells of Sulfolobus acidocaldarius. Proc. Natl. Acad. Sci. USA 92:12285-12289.tff-, 百拇医药
Aagaard, C., I. Leviev, R. N. Aravalli, P. Forterre, D. Prieur, and R. A. Garrett. 1996. General vectors for archaeal hyperthermophiles: strategies based on a mobile intron and a plasmid. FEMS Microbiol. Rev. 18:93-104.tff-, 百拇医药
Adams, M. W., and R. M. Kelly. 1998. Finding and using hyperthermophilic enzymes. Trends Biotechnol. 16:329-332.tff-, 百拇医药
Aravalli, R. N., and R. A. Garrett. 1997. Shuttle vectors for hyperthermophilic archaea. Extremophiles 1:183-191.tff-, 百拇医药
Beckwith, J. R., A. B. Pardee, R. Austrian, and F. Jacob. 1962. Coordination of the synthesis of the enzymes in the pyrimidine pathway of E. coli. J. Mol. Biol. 5:618-634.tff-, 百拇医药
Benbouzid-Rollet, N., P. López-García, L. Watrin, G. Erauso, D. Prieur, and P. Forterre. 1997. Isolation of new plasmids from hyperthermophilic Archaea of the order Thermococcales. Res. Microbiol. 148:767-775.
Bertani, G., and L. Baresi. 1987. Genetic transformation in the methanogen Methanococcus voltae PS. J. Bacteriol. 169:2730-2738.5&9u(u, 百拇医药
Boeke, J. D., F. LaCroute, and G. R. Fink. 1984. A positive selection for mutants lacking orotidine-5'-phosphate decarboxylase activity in yeast: 5-fluoro-orotic acid resistance. Mol. Gen. Genet. 197:345-346.5&9u(u, 百拇医药
Boeke, J. D., J. Trueheart, G. Natsoulis, and G. R. Fink. 1987. 5-Fluoroorotic acid as a selective agent in yeast molecular genetics. Methods Enzymol. 154:164-175.5&9u(u, 百拇医药
Cannio, R., P. Contursi, M. Rossi, and S. Bartolucci. 1998. An autonomously replicating transforming vector for Sulfolobus solfataricus. J. Bacteriol. 180:3237-3240.5&9u(u, 百拇医药
Elferink, M. G., C. Schleper, and W. Zillig. 1996. Transformation of the extremely thermoacidophilic archaeon Sulfolobus solfataricus via a self-spreading vector. FEMS Microbiol. Lett. 137:31-35.5&9u(u, 百拇医药
Erauso, G., S. Marsin, N. Benbouzid-Rollet, M. F. Baucher, T. Barbeyron, Y. Zivanovic, D. Prieur, and P. Forterre. 1996. Sequence of plasmid pGT5 from the archaeon Pyrococcus abyssi: evidence for rolling-circle replication in a hyperthermophile. J. Bacteriol. 178:3232-3237.
Ezaki, S., N. Maeda, T. Kishimoto, H. Atomi, and T. Imanaka. 1999. Presence of a structurally novel type ribulose-bisphosphate carboxylase/oxygenase in the hyperthermophilic archaeon, Pyrococcus kodakaraensis KOD1. J. Biol. Chem. 274:5078-5082.#4)[4, http://www.100md.com
Grogan, D. W., and R. P. Gunsalus. 1993. Sulfolobus acidocaldarius synthesizes UMP via a standard de novo pathway: results of biochemical-genetic study. J. Bacteriol. 175:1500-1507.#4)[4, http://www.100md.com
Holden, J. F., K. Takai, M. Summit, S. Bolton, J. Zyskowski, and J. A. Baross. 2001. Diversity among three novel groups of hyperthermophilic deep-sea Thermococcus species from three sites in the northeastern Pacific Ocean. FEMS Microbiol. Ecol. 36:51-60.#4)[4, http://www.100md.com
Jacobs, K. L., and D. W. Grogan. 1997. Rates of spontaneous mutation in an archaeon from geothermal environments. J. Bacteriol. 179:3298-3303.#4)[4, http://www.100md.com
Kawarabayasi, Y., M. Sawada, H. Horikawa, Y. Haikawa, Y. Hino, S. Yamamoto, M. Sekine, S. Baba, H. Kosugi, A. Hosoyama, Y. Nagai, M. Sakai, K. Ogura, R. Otsuka, H. Nakazawa, M. Takamiya, Y. Ohfuku, T. Funahashi, T. Tanaka, Y. Kudoh, J. Yamazaki, N. Kushida, A. Oguchi, K. Aoki, and H. Kikuchi. 1998. Complete sequence and gene organization of the genome of a hyper-thermophilic archaebacterium, Pyrococcus horikoshii OT3. DNA Res. 5:55-76.
Kondo, S., A. Yamagishi, and T. Oshima. 1991. Positive selection for uracil auxotrophs of the sulfur-dependent thermophilic archaebacterium Sulfolobus acidocaldarius by use of 5-fluoroorotic acid. J. Bacteriol. 173:7698-7700.\fhs'x, http://www.100md.com
Lange, M., and B. K. Ahring. 2001. A comprehensive study into the molecular methodology and molecular biology of methanogenic Archaea. FEMS Microbiol. Rev. 25:553-571.\fhs'x, http://www.100md.com
López-García, P., P. Forterre, J. van der Oost, and G. Erauso. 2000. Plasmid pGS5 from the hyperthermophilic archaeon Archaeoglobus profundus is negatively supercoiled. J. Bacteriol. 182:4998-5000.\fhs'x, http://www.100md.com
Martusewitsch, E., C. W. Sensen, and C. Schleper. 2000. High spontaneous mutation rate in the hyperthermophilic archaeon Sulfolobus solfataricus is mediated by transposable elements. J. Bacteriol. 182:2574-2581.\fhs'x, http://www.100md.com
Morikawa, M., Y. Izawa, N. Rashid, T. Hoaki, and T. Imanaka. 1994. Purification and characterization of a thermostable thiol protease from a newly isolated hyperthermophilic Pyrococcus sp. Appl. Environ. Microbiol. 60:4559-4566.
Niehaus, F., C. Bertoldo, M. Kähler, and G. Antranikian. 1999. Extremophiles as a source of novel enzymes for industrial application. Appl. Microbiol. Biotechnol. 51:711-729.9, 百拇医药
Peck, R. F., S. Dassarma, and M. P. Krebs. 2000. Homologous gene knockout in the archaeon Halobacterium salinarum with ura3 as a counterselectable marker. Mol. Microbiol. 35:667-676.9, 百拇医药
Ramakrishnan, V., and M. W. W. Adams. 1995. Preparation of genomic DNA from sulfur-dependent hyperthermophilic archaea, p. 95-96. In F. T. Robb and A. R. Place (ed.), Archea: a laboratory manual—thermophiles. Cold Spring Harbor Press, Cold Spring Harbor, N.Y.9, 百拇医药
Reilly, M. S., and D. W. Grogan. 2001. Characterization of intragenic recombination in a hyperthermophilic archaeon via conjugational DNA exchange. J. Bacteriol. 183:2943-2946.9, 百拇医药
Robb, F. T., D. L. Maeder, J. R. Brown, J. DiRuggiero, M. D. Stump, R. K. Yeh, R. B. Weiss, and D. M. Dunn. 2001. Genomic sequence of hyperthermophile, Pyrococcus furiosus: implications for physiology and enzymology. Methods Enzymol. 330:134-157.
Robb, F. T., and A. R. Place. 1995. Media for Thermophiles, p. 167-168. In F. T. Robb and A. R. Place (ed.), Archea: a laboratory manual—thermophiles. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.501], http://www.100md.com
Sambrook, J., and D. Russel. 2001. Molecular cloning: a laboratory manual, 3rd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.501], http://www.100md.com
Schäfer, G., M. Engelhard, and V. Müller. 1999. Bioenergetics of the archaea. Microbiol. Mol. Biol. Rev. 63:570-620.501], http://www.100md.com
Schleper, C., I. Holz, D. Janekovic, J. Murphy, and W. Zillig. 1995. A multicopy plasmid of the extremely thermophilic archaeon Sulfolobus effects its transfer to recipients by mating. J. Bacteriol. 177:4417-4426.501], http://www.100md.com
Schleper, C., K. Kubo, and W. Zillig. 1992. The particle SSV1 from the extremely thermophilic archaeon Sulfolobus is a virus: demonstration of infectivity and of transfection with viral DNA. Proc. Natl. Acad. Sci. USA 89:7645-7649.501], http://www.100md.com
Schleper, C., R. Röder, T. Singer, and W. Zillig. 1994. An insertion element of the extremely thermophilic archaeon Sulfolobus solfataricus transposes into the endogenous ß-galactosidase gene. Mol. Gen. Genet. 243:91-96.
Sowers, K. R., and H. J. Schreier. 1999. Gene transfer systems for the Archaea. Trends Microbiol. 7:212-219.|n?@', 百拇医药
Stedman, K. M., C. Schleper, E. Rumpf, and W. Zillig. 1999. Genetic requirements for the function of the archaeal virus SSV1 in Sulfolobus solfataricus: construction and testing of viral shuttle vectors. Genetics 152:1397-1405.|n?@', 百拇医药
Stetter, K. O. 1996. Hyperthermophilic prokaryotes. FEMS Microbiol. Rev. 18:149-158.|n?@', 百拇医药
Stetter, K. O. 1999. Extremophiles and their adaptation to hot environments. FEBS Lett. 452:22-25.|n?@', 百拇医药
Takai, K., T. Komatsu, F. Inagaki, and K. Horikoshi. 2001. Distribution of archaea in a black smoker chimney structure. Appl. Environ. Microbiol. 67:3618-3629.|n?@', 百拇医药
Tang, X. F., S. Ezaki, H. Atomi, and T. Imanaka. 2001. Anthranilate synthase without an LLES motif from a hyperthermophilic archaeon is inhibited by tryptophan. Biochem. Biophys. Res. Commun. 281:858-865.|n?@', 百拇医药
Vieille, C., and G. J. Zeikus. 2001. Hyperthermophilic enzymes: sources, uses, and molecular mechanisms for thermostability. Microbiol. Mol. Biol. Rev. 65:1-43.
Ward, D. E., I. M. Revet, R. Nandakumar, J. H. Tuttle, W. M. de Vos, J. van der Oost, and J. DiRuggiero. 2002. Characterization of plasmid pRT1 from Pyrococcus sp. strain JT1. J. Bacteriol. 184:2561-2566.m'vd7.j, http://www.100md.com
Watrin, L., S. Lucas, C. Purcarea, C. Legrain, and D. Prieur. 1999. Isolation and characterization of pyrimidine auxotrophs, and molecular cloning of the pyrE gene from the hyperthermophilic archaeon Pyrococcus abyssi. Mol. Gen. Genet. 262:378-381.m'vd7.j, http://www.100md.com
Watrin, L., and D. Prieur. 1996. UV and ethyl methanesulfonate effects in hyperthermophilic archaea and isolation of auxotrophic mutants of Pyrococcus strains. Curr. Microbiol. 33:377-382.m'vd7.j, http://www.100md.com
Zillig, W., H. P. Arnold, I. Holz, D. Prangishvili, A. Schweier, K. Stedman, Q. She, H. Phan, R. Garrett, and J. K. Kristjansson. 1998. Genetic elements in the extremely thermophilic archaeon Sulfolobus. Extremophiles 2:131-140.(Takaaki Sato Toshiaki Fukui Haruyuki Atomi and Tadayuki Imanaka)