Disruption of a Sugar Transporter Gene Cluster in a Hyperthermophilic Archaeon Using a Host-Marker System Based on Antibiotic Resistance

We have developed a gene disruption system in the hyperthermophilicarchaeon Thermococcus kodakaraensis using the antibiotic simvastatinand a fusion gene designed to overexpress the 3-hydroxy-3-methylglutarylcoenzyme A (HMG-CoA) reductase gene (hmg_Tk) with the glutamatedehydrogenase promoter. With this system, we disrupted the T.kodakaraensis amylopullulanase gene (apu_Tk) or a gene clusterwhich includes apu_Tk and genes encoding components of a putativesugar transporter. Disruption plasmids were introduced intowild-type T. kodakaraensis KOD1 cells, and transformants exhibitingresistance to 4 µM simvastatin were isolated. The transformantsexhibited growth in the presence of 20 µM simvastatin,and we observed a 30-fold increase in intracellular HMG-CoAreductase activity. The expected gene disruption via double-crossoverrecombination occurred at the target locus, but we also observedrecombination events at the hmg_Tk locus when the endogenoushmg_Tk gene was used. This could be avoided by using the correspondinggene from Pyrococcus furiosus (hmg_Pf) or by linearizing theplasmid prior to transformation. While both gene disruptionstrains displayed normal growth on amino acids or pyruvate,cells without the sugar transporter genes could not grow onmaltooligosaccharides or polysaccharides, indicating that thegene cluster encodes the only sugar transporter involved inthe uptake of these compounds. The ${Delta}$ apu_Tk strain could not growon pullulan and displayed only low levels of growth on amylose,suggesting that Apu_Tk is a major polysaccharide-degrading enzymein T. kodakaraensis.

INTRODUCTION

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Introduction

Hyperthermophiles are organisms that exhibit optimal growthat temperatures above 80°C (57). The organisms have attractedmuch attention from an evolutionary viewpoint as they occupythe deepest lineages within the phylogenies of both Archaeaand Bacteria based on rRNA sequences (56, 57). Hyperthermophilesare also focused upon as a source of (thermo)stable enzymesthat have the potential for application in a broad range oftechnologies (3, 21). There are now 14 complete genome sequencesand many more in progress, providing a wealth of primary structuraldata from which we can estimate the presence or absence of variousmetabolic and regulatory mechanisms. However, although biochemicaland structural analyses of hyperthermophile proteins are proceedingat a rapid pace, genetic studies to examine gene function invivo are still limited in number.

In contrast to the hyperthermophilic archaea, a wealth of genedisruption and shuttle vector systems has been developed forthe mesophilic archaea. In the halophilic archaea, proceduresallowing the uptake of bacteriophage (15), plasmid (14), andgenomic DNA (17) have been established with Halobacterium halobiumand Halobacterium (now Haloferax) volcanii. Stable shuttle vectorshave been developed (29, 37, 45), and homologous recombinationhas been demonstrated (34, 43). Selection methods include changesin phenotype from auxotrophy to prototrophy (17) and resistanceagainst a variety of antibiotics such as mevinolin (34, 37,45, 63), novobiocin (29), thiostrepton, and anisomycin (43).Genetic systems have also been developed for Haloarcula strains(16) and other Halobacterium strains including Halobacteriumsp. strain NRC-1 (8, 47, 62). In the methanogenic archaea, shuttlevectors and gene disruption systems have been developed in Methanococcusmaripaludis based on puromycin and the puromycin N-acetyltransferasegene (pac) (25, 48, 61) or neomycin and aminoglycoside phosphotransferasegenes (2). The puromycin-pac system has also been applied forgenetic manipulation in Methanococcus voltae (6, 7, 60), aswell as in various Methanosarcina species (19, 44). Genetictransformation has also been observed in the thermophilic methanogenMethanobacterium thermoautotrophicum (now Methanothermobacterthermautotrophicus) (64).

In the hyperthermophilic archaea, exchange and recombinationof chromosomal markers, as well as homologous recombinationof exogenous DNA, have been reported for Sulfolobus acidocaldarius(27, 28, 35, 50). A plasmid based on pNOB8 has been demonstratedto transform Sulfolobus solfataricus (22). An autonomously replicatingvector harboring a mutant hygromycin phosphotransferase genefor selection has also been developed for this species (12,13, 18). In the Euryarchaeota, a shuttle vector has been developedfor use in Pyrococcus abyssi (41). In terms of gene disruption,however, only two systems have been reported so far: one forThermococcus kodakaraensis (52, 53) from the Euryarchaeota andthe other for S. solfataricus from the Crenarchaeota (65). Bothsystems rely on homologous recombination. The former systemutilizes various host strains with amino acid/nucleotide auxotrophyand corresponding marker genes that complement the auxotrophy.The latter utilizes a lacS-deficient host strain and a modifiedbut active lacS marker gene with selection based on lactose-dependentgrowth. The two systems have proved to be powerful tools inexamining gene function in the respective strains (5, 30, 54,55) and can be expected to provide further genetic evidencethat will help in understanding the physiological roles of genesin these and closely related organisms.

In this study, we aimed to develop a gene disruption systemin hyperthermophiles using antibiotics and a marker gene thatwould confer resistance to transformant cells. This would relievethe necessity to prepare auxotrophic host cells and also allowselection of transformants in a nutrient-rich medium. Thus,the methodology should not only provide a convenient alternativefor gene disruption in T. kodakaraensis but also be helpfulin establishing gene disruption systems in other hyperthermophilicarchaea. We examined the possibilities of utilizing the mevinolinsystem established in the halophilic archaea. Mevinolin, alongwith its analog simvastatin, is a specific inhibitor of 3-hydroxy-3-methylglutarylcoenzyme A (HMG-CoA) reductase, an enzyme essential for archaealmembrane lipid biosynthesis (11, 36). HMG-CoA reductases havebeen extensively examined from a number of archaeal species(9, 10, 31). An overexpression construct of the HMG-CoA reductasegene can be expected to be applicable as a marker gene. Additionally,as the gene is originally present in the hyperthermophile, thereis no need for concern about the thermostability of the markergene product. As all archaeal strains are presumed to requirethe function of HMG-CoA reductase for lipid and membrane generation,the system described in this study has the potential for applicationin all hyperthermophilic archaea.

MATERIALS AND METHODS

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Materials and Methods

Strains, media, and plasmids. T. kodakaraensis KOD1 (4) and the mutant strains were cultivatedunder anaerobic conditions at 85°C in a nutrient-rich medium,ASW-YT (53), supplemented with various organic substrates orelemental sulfur when appropriate. ASW-YT medium was composedof 0.8x artificial seawater, 5.0 g liter^–1 of yeast extract,and 5.0 g liter^–1 of tryptone. Resazurin was added ata concentration of 0.8 mg liter^–1, and prior to inoculation,Na₂S was added to the medium until it became transparent. Inthe case of plate culture, instead of elemental sulfur and Na₂S9H₂O, 2 ml of a polysulfide solution (10 g of Na₂S 9H₂O and3 g of sulfur flowers in 15 ml of H₂O) per liter and Gelrite(10 g liter^–1) were added to solidify the medium. Allcomponents were purchased from Wako Pure Chemical Industries(Osaka, Japan) or Nacalai Tesque (Kyoto, Japan). When simvastatinwas added to the medium, simvastatin was dissolved in ethanol,and the amount of the solution added was adjusted so that theethanol concentration in the medium was constant at 0.1% (vol/vol).

DNA manipulation and sequence analysis. Escherichia coli strain DH5 ${alpha}$ and pUC18/pUC19 (Takara, Kyoto,Japan) were used for DNA manipulation and sequencing. E. colistrains were cultivated in Luria-Bertani medium at 37°Cwith ampicillin at a concentration of 100 µg ml^–1.Restriction and modification enzymes were purchased from Toyobo(Osaka, Japan) and Takara. Plasmid DNA was isolated with a plasmidmini kit from QIAGEN (Hilden, Germany). KOD Plus (Toyobo) wasused as a polymerase for PCR, and a GFX PCR DNA and gel bandpurification kit (GE Life Sciences, Little Chalfont, UnitedKingdom) was used to recover DNA fragments from agarose gelsafter electrophoresis. DNA sequencing was performed using aBigDye terminator cycle sequencing kit, version 3.1, and a model3100 capillary DNA sequencer (Applied Biosystems, Foster City,CA).

Construction of the gene disruption vectors. Two disruption vectors, pUDapu and pUDmal, were constructedfor the targeted disruption of the T. kodakaraensis amylopullulanasegene apu (apu_Tk) and the sugar transporter gene cluster includingapu_Tk, respectively, via double-crossover homologous recombination.Overexpression cassettes for the HMG-CoA reductase gene fromT. kodakaraensis (hmg_Tk) were constructed by replacing the nativepromoter with a putative promoter region (–554 to –4)of the glutamate dehydrogenase gene (49). The region –3to –1 was replaced by 5'-CAT-3' in order to incorporatean NdeI site for fusion of the promoter to the coding regionof hmg_Tk. Cassettes were designed so that one had SmaI sitesat both ends, while another had an XbaI site upstream of thepromoter and a BamHI site downstream of hmg_Tk. The two cassetteswere inserted into pUC18 and sequenced. For construction ofthe apu_Tk disruption plasmid, a DNA fragment including apu_Tkalong with its flanking regions (about 1,000 bp) was amplifiedfrom the genomic DNA of T. kodakaraensis KOD1 with the primerset APU-F1 and APU-TRANS-R1 (5'-AATTCAGAACGGCAAGCTCTACGTAACAGACGGCA-3'and 5'-GCGTCGTAGATGTCCTCGGGCCTTATGCCGAAGAT-3', respectively)and inserted into pUC18 at the HincII site. An inverse PCR wasthen carried out to amplify the flanking regions and pUC18,thereby removing the coding region of gene. The primers usedwere APU-R2 and APU-F2 (5'-CTTATCACCTCACTCTTTAAGGCCTCCAACAGTGA-3'and 5'-AGAGGGTGGCGGAATCTGCGGCCCGGCGTTCCTCG-3', respectively).The DNA fragment was ligated with the hmg_Tk overexpression cassetteexcised with SmaI and designated pUDapu. For disruption of thesugar transporter gene cluster, DNA fragments of the 5' and3' flanking regions (about 1,000-bp) of the gene cluster wereamplified with the primer pair TRANS-F1 and TRANS-R2 (5'-AGTTCTCAAATCGGACCTTCCGCCGATGGAAAAGT-3'and 5'-TGTTTATCACCTAGTTATCTCGTTGCATTTGAGTA-3', respectively)and the pair TRANS-F2 and APU-TRANS-R1 (TRANS-F2, 5'-TCCCCAGGATCCGGCGGTGGTGAAGAGGGTGGCGG-3').The 5' flanking region was inserted into pUC19 at the HincIIsite, followed by insertion of the overexpression cassette inthe XbaI and BamHI sites. The 3' flanking region was then insertedin the BamHI and SmaI sites, resulting in the plasmid pUDmal.

Transformation of T. kodakaraensis. Transformation procedures were performed as described previously(52, 53), but the host strain used in this study was the wild-typeT. kodakaraensis KOD1. After transformation, cells were cultivatedin ASW-YT liquid medium supplemented with 0.2% (wt/vol) elementalsulfur (ASW-YT-S⁰) in the presence of 4 µM simvastatinat 85°C for 12 h. The cells were further grown in ASW-YT-S⁰liquid medium with 8 µM simvastatin at 85°C and spreadon ASW-YT (polysulfide) plate medium containing 4 µM simvastatinand incubated at 85°C. Genomic DNA was isolated from thetransformants and analyzed by PCR and Southern blot analysis.

Southern blot analysis. A digoxigenin-DNA labeling and detection kit (Roche Diagnostics,Basel, Switzerland) was used according to the manufacturer'sinstructions. The probes within the coding regions of hmg_Tkand apu_Tk were amplified, respectively, with the primer pairHMG-F and HMG-R (5'-TGAGAACATCGGGCACTACTCAATAGATCCCAACC-3' and5'-ACCAACGAGGTTCTTGCGGTAGTTCACCTCGGCTA-3', respectively) andthe pair APU-F and APU-R (5'-CTCAACGACAAGACCCTTGAAATCCTAGCGGAGAA-3'and 5'-GGCTCATCTTATCTTTGTTTTCCATGAGGGCCTTT-3', respectively).The probe within the coding region of the malE gene of T. kodakaraensis(malE_Tk) was amplified with the primers MalE-F and MalE-R (5'-CACTTCCCCGACCGAGACCACTACTACCTCACCCA-3'and 5'-CTGCTGGGTGTTGTAGTCGGCAGTCGGGGCCATGT-3', respectively).The probe within the hmg gene from P. furiosus (hmg_Pf) was amplifiedwith the primers PfHMG-F and PfHMG-R (5'-AAAGCACATTGGCCACTACTCAATTGATCCAAACG-3'and 5'-ACCCACTAAGTTCTTTAGGTAGTTTACTTCAGCGA-3', respectively),and the probe corresponding to the promoter region of the glutamatedehydrogenase gene was amplified with the primers GDHp-F andGDHp-R (5'-ATATCCCACCTCCGATTCCGTTGGTATTTAATCGG-3' and 5'-TACCACCTCATTTCGGTAATCTGCGAGGTTAACTT-3',respectively). Genomic DNA from the wild-type and gene disruptionmutant strains was digested with PvuII.

Growth properties of T. kodakaraensis and mutant strains. T. kodakaraensis KOD1 and the mutant strains were grown in ASW-YT-S⁰medium at 85°C for 12 h and inoculated into 15 ml of ASW-YT-S⁰or ASW-YT medium supplemented with 0.5% (wt/vol) sodium pyruvate,a 0.5% (wt/vol) concentration of a specific maltooligosaccharide(3, 4, 5, 6, or 7 glucose units), 0.5% (wt/vol) amylose (polysaccharideconsisting of glucose connected solely by ${alpha}$ -1,4-glycosidic bonds),or 0.5% (wt/vol) pullulan (polysaccharide consisting of maltotrioseunits connected by ${alpha}$ -1,6-glycosidic bonds). Cell densities (opticaldensity at 660 nm) were measured at appropriate intervals witha UV spectrometer mini photo 518R (Taitec, Koshigaya, Japan).In order to estimate resistance toward simvastatin, cells werecultivated in 15 ml of ASW-YT-S⁰ supplemented with 1, 5, 10,or 20 µM simvastatin.

Measurements of HMG-CoA reductase activity. Activity measurements were performed at 60°C in a finalvolume of 1 ml containing cell extracts, 200 µM NADPH,and 0.5 mM HMG-CoA (Sigma, St. Louis, MO) in 50 mM potassiumphosphate buffer (pH 7.0). The consumption of NADPH was monitoredat 340 nm by a UV-visible light spectrophotometer (UV-1600PC;Shimadzu, Kyoto, Japan). Cell extracts were prepared as follows.T. kodakaraensis and the disruptants were cultivated in ASW-YT-S⁰medium at 85°C for approximately 8 h. Cells were collectedand sonicated on ice, and the supernatant after centrifugation(20,000 x g for 30 min at 4°C) was used as the cell extract.Protein concentrations were determined with a protein assaykit (Bio-Rad, Hercules, CA) using bovine serum albumin as astandard.

RESULTS

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Results

Effect of various concentrations of simvastatin on the growth of T. kodakaraensis. As isopentenyl diphosphate is the major precursor for archaeallipid membranes, we supposed that inhibition of HMG-CoA reductasewould have severe effects on the growth of T. kodakaraensis(Fig. 1). Our main concerns were whether the uptake and inhibitoryeffects of simvastatin were sufficient to allow use of the antibioticat realistic concentrations and whether the compound was stableenough at temperatures of >80°C to inhibit growth forseveral days, which is necessary for the formation of colonies.

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FIG. 1. The mevalonate pathway for isoprenoid lipid biosynthesis in Archaea. The reaction catalyzed by HMG-CoA reductase is boxed with dotted lines. It should be noted that the two reactions converting mevalonate phosphate to isopentenyl diphosphate are distinct from the reactions in the classical mevalonate pathway (26).

We examined the growth of T. kodakaraensis KOD1 in the presenceof various concentrations of simvastatin in the nutrient-richmedium ASW-YT-S⁰. Simvastatin was dissolved in ethanol, andthe amount of ethanol added to the medium was constant at 0.1%(vol/vol). In ASW-YT-S⁰ medium, T. kodakaraensis KOD1 cellsreach the stationary phase within 24 h. No effect on growthwas observed with the addition of ethanol alone. In the presenceof 1 or 2 µM simvastatin, growth was observed only after24 h, while 48 h was necessary for growth with 3 µM simvastatin.At concentrations of 4 or 5 µM simvastatin, growth wasnot observed for at least 5 days, indicating that these concentrationswould be suitable for selecting transformants with resistanceagainst simvastatin. We also confirmed that these concentrationswere sufficient to prevent colony formation of T. kodakaraensisKOD1 on nutrient-rich plate medium.

A cassette for the overexpression of the HMG-CoA reductase gene. As simvastatin is a competitive inhibitor of HMG-CoA reductase,we expected that overexpression of its gene from T. kodakaraensis(hmg_Tk) would reduce the inhibitory effects of simvastatin oncell growth. Previous studies have indicated that the enzymeglutamate dehydrogenase is abundant in T. kodakaraensis cellsgrown in various media (49), suggesting that the gene (gdh_Tk)is under the control of a strong promoter. We therefore utilizeda 551-bp intergenic region between the coding regions of gdh_Tk(TK1431) and the adjacent gene TK1432 and fused the region upstreamof hmg_Tk (see Materials and Methods). This overexpression cassette(P_gdh-hmg) was used as the marker gene for construction of disruptionplasmids (Fig. 2A).

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FIG. 2. Disruption of the apu_Tk and mal_Tk loci of T. kodakaraensis. (A) Design of the hmg_Tk overexpression cassette using the 5' upstream flanking region of gdh_Tk. (B) Gene organization of the putative maltooligosaccharide transporter of T. kodakaraensis. TK1774* represents the correct sequence of the apu_Tk gene (see text). Black arrows indicate the gene(s) disrupted in this study. (C) The two plasmids constructed for the disruption of the apu_Tk and mal_Tk loci via double-crossover recombination. (D) PCR analyses of the apu_Tk and mal_Tk loci confirming gene disruption. Primers were designed in the 5' and 3' flanking regions of the gene(s) to be disrupted. DNA size markers were run in lane M, and their sizes (bp) are indicated to the left of the gels. The results of PCR with wild-type T. kodakaraensis KOD1 and five individual transformants are indicated in lane W and lanes 1 to 5, respectively. The arrowheads to the right of the gels indicate the amplified fragments expected before and after recombination. The decreases in lengths of the amplified fragments reflect the differences in length between apu_Tk ( $~$ 3,500 bp) and P_gdh-hmg ( $~$ 2,000 bp) and between mal_Tk ( $~$ 7,000 bp) and P_gdh-hmg. Nonspecific amplifications of DNA fragments observed for the wild-type mal_Tk locus were due to the prolonged reaction time necessary to amplify the entire locus.

Design and construction of the gene disruption plasmids. The genes disrupted in this study were a putative amylopullulanasegene (apu_Tk, or TK1774) and a gene cluster including apu_Tk andthree additional genes encoding the components of a sugar transporter(TK1771 to TK1773) of T. kodakaraensis (Fig. 2B) (24). In thelatter stages of this study, we discovered an error in the originalgenome sequence of TK1774 (an excess A at position 1,581,978of the genome). The correct sequence leads to a protein witha change and elongation in sequence in the C-terminal regionfrom residue Asn1070 (see Discussion). In this report we willrefer to the corrected apu_Tk gene as TK1774* and, for simplicity,to the four-gene cluster (TK1771 to TK1774*) as mal_Tk.

Amylopullulanases, or type II pullulanases, exhibit both ${alpha}$ -amylaseand pullulanase activities and can therefore cleave both ${alpha}$ -1,4-and ${alpha}$ -1,6-glucosidic bonds (20). There are a number of otherhomologs on the genome (24), some with putative signal sequencesfor secretion, expected to harbor the ability to degrade ${alpha}$ -linkedpolysaccharides. In particular, the TK1884 protein has beenexperimentally confirmed to exhibit ${alpha}$ -amylase activity (59).On the other hand, in contrast to the two sugar transporterspresent in Pyrococcus furiosus (Mal-I, PF1739 to PF1744, andMal-II, PF1933 to PF1938) (33), only one putative gene clusteris found on the T. kodakaraensis genome (TK1771 to TK1775) (24).Based on primary structure similarity, the transporter fromT. kodakaraensis corresponds to Mal-II, suggesting that it isspecific to maltooligosaccharides with three or more glucoseunits. By disrupting apu_Tk, we expected to gain insight intothe actual degree of influence apu_Tk has, among the multipleamylase homologs of T. kodakaraensis, on the degradation ofvarious extracellular polysaccharides. Growth characteristicsof the mal_Tk disruptant were expected to clarify the presenceor absence of other sugar transporters as well as to provideinformation on the substrate specificity of the Mal_Tk transporterin vivo.

Similar to the design of gene disruption plasmids in a previouslydescribed system using pyrF or trpE as selectable markers (52),P_gdh-hmg was inserted between the 5' and 3' flanking regions( $~$ 1,000 bp) of the target gene(s) (Fig. 2C). The plasmids pUDapuand pUDmal were used to transform wild-type T. kodakaraensisKOD1, and transformants were selected based on their resistancetoward simvastatin.

Isolation of the gene disruption strains ${Delta}$ apu_Tk and ${Delta}$ mal_Tk. After transformation, cells were grown in ASW-YT-S⁰ liquid mediumin the presence of 4 µM simvastatin. Growth was observedwith cells transformed with pUDapu and pUDmal but not for cellstreated without plasmid. Cells were further inoculated in thesame liquid medium with 8 µM simvastatin and then spreadon plate medium with 4 µM simvastatin. Five colonies wereselected for each gene disruption and grown in ASW-YT-S⁰ medium.We examined the apu_Tk and mal_Tk loci by PCR (Fig. 2D). As expected,we observed shorter amplified fragments from the transformantsthan from the wild-type strain, corresponding to the decreasein length brought about by the replacement of apu_Tk and mal_Tkby P_gdh-hmg.

Extent of simvastatin resistance of the transformants. As the transformants harbored P_gdh-hmg on their genomes, weexamined their resistance against various concentrations ofsimvastatin (Fig. 3). One ${Delta}$ apu_Tk and one ${Delta}$ mal_Tk transformant weregrown in the presence of 1, 5, 10, and 20 µM simvastatin,and their growth characteristics were compared with those observedin medium without simvastatin. Although the wild-type straincould not grow at all with 5 µM simvastatin, specificgrowth rates and cell yields of the transformants were stillcomparable to those in the absence of simvastatin. The degreeof inhibition became prominent at higher concentrations, butwe found that concentrations over 20 µM were necessaryto completely inhibit growth of the transformants. We furtherexamined the levels of HMG-CoA reductase activity in the wild-typeand transformant cells. Specific activity in the cell extractsof wild-type cells was approximately 25 nmol min^–1 mg^–1.In contrast, the level observed in the extracts of the ${Delta}$ apu_Tkstrain was 760 nmol min^–1 mg^–1, indicating an increasein activity of over 30-fold. The resistance against simvastatinand the increase in HMG-CoA reductase activity in the transformantsare consistent with the presumption that simvastatin inhibitsgrowth of T. kodakaraensis KOD1 by specifically inhibiting theactivity of HMG-CoA reductase.

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FIG. 3. Growth of wild-type T. kodakaraensis KOD1 and ${Delta}$ apu_Tk and ${Delta}$ mal_Tk mutant strains in the presence of various concentrations of simvastatin. Open circle, wild-type strain; filled square, ${Delta}$ apu_Tk strain; filled triangle, ${Delta}$ mal_Tk strain; OD, optical density.

Phenotype analyses of the gene disruption strains. We examined the growth characteristics of the ${Delta}$ apu_Tk and ${Delta}$ mal_Tkstrains in various media and compared them with those of thewild-type strain (Fig. 4). No change in phenotype was observedwhen the three strains were grown on amino acids (ASW-YT-S⁰)or amino acids and pyruvate (ASW-YT-pyruvate) as carbon sources.However, disruption of apu_Tk and mal_Tk brought about dramaticchanges in phenotype when the strains were grown on varioussugars. Disruption of the Mal_Tk transporter abolished growthon all sugars examined. Although the ${Delta}$ apu_Tk strain displayedgrowth on a number of maltooligosaccharides, the strain couldnot grow on pullulan (Fig. 4I). Interestingly, while several ${alpha}$ -amylase homologs (including TK1884) are present on the genome,we found that disruption of apu_Tk led to a significant decreasein growth rates when strains were grown on amylose, a maltopolysaccharideconsisting of only ${alpha}$ -1,4-linkages (Fig. 4H). Another intriguingfinding was that the disruption of apu_Tk had a greater detrimentaleffect on growth with shorter maltooligosaccharides, which wasrather surprising as amylopullulanases are presumed to functionin the breakdown of poly- or oligosaccharides. In contrast tothe wild-type strain, no growth was observed for ${Delta}$ apu_Tk in themedium supplemented with maltotriose (Fig. 4C).

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FIG. 4. Growth of wild-type T. kodakaraensis KOD1 and ${Delta}$ apu_Tk and ${Delta}$ mal_Tk mutant strains on various carbon sources. The carbon sources examined are indicated above each panel. Glucose and maltose were not examined, as the wild-type strain cannot utilize these sugars. Open circle, wild-type strain; filled square, ${Delta}$ apu_Tk strain; filled triangle, ${Delta}$ mal_Tk strain; OD, optical density.

Recombination at the hmg_Tk locus. As the plasmid used in this study harbors the endogenous hmg_Tkand P_gdh, there will always be a possibility of these regionsrecombining with the corresponding native loci present on thegenome. We have actually applied this property in developinga single-crossover insertion/pop-out recombination system usingthe pyrF gene as a selective marker (52). We therefore examinedwhether recombination events had occurred at the hmg_Tk and/orP_gdh glutamate dehydrogenase locus. We observed that in somestrains the native hmg_Tk locus had been disturbed (data notshown). As the locus of the target gene is stable in a disruptedform via double-crossover recombination, the recombination atthe hmg_Tk locus does not directly pose a problem. Nevertheless,we examined possibilities to prevent or decrease the frequenciesof unintended recombination. Linearizing the plasmids priorto introducing them into the cells would prevent single-crossoverrecombination. Utilizing an hmg gene from a heterologous hostwould also decrease the possibilities of single-crossover recombinationas well as further recombination due to the presence of twoidentical regions on the genome. We found that both methodscould be used for gene disruption. In examining the latter possibility,we used the hmg gene from the closely related P. furiosus andconstructed plasmids to disrupt the apu_Tk gene. In five randomlychosen transformants with resistance against simvastatin, weclearly observed that double-crossover recombination had occurredat the apu_Tk locus (Fig. 5A and B) along with the appearanceof a single copy of the hmg_Pf gene (Fig. 5C) and an additionalcopy of the gdh promoter (Fig. 5E), whereas neither the nativehmg_Tk locus nor the gdh promoter regions were disturbed (Fig.5D and E). We further confirmed that the strains obtained usinghmg_Pf as a marker gene (i) were able to grow in the presenceof 20 µM simvastatin, (ii) exhibited significant HMG-CoAreductase activities in the cell extracts (720 nmol min^–1mg^–1), and (iii) displayed the same growth characteristicstoward various carbon sources as those shown in Fig. 4 (datanot shown).

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FIG. 5. Southern blot analyses on ${Delta}$ apu_Tk strains obtained with the hmg_Pf gene as a selectable marker. Genomic DNA from five selected ${Delta}$ apu_Tk strains and from wild-type T. kodakaraensis KOD1 (W) was subjected to Southern blot analyses using probes within the regions indicated below each membrane.

DISCUSSION

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Discussion

In this study, we have developed in T. kodakaraensis a genedisruption system based on resistance against antibiotics usingsimvastatin and an overexpression cassette of hmg_Tk. The systemhas many advantages for initiating gene disruption studies inhyperthermophilic archaea. First, one does not need to constructa host strain with a particular defect or auxotrophy towardan amino acid. There is also no need for selection to be carriedout in minimal medium. Positive selection of mutant strainsis possible in nutrient-rich medium. If the genome sequenceis available, the possibilities to disrupt genes with this systemcan be examined immediately. It should be noted that we havenot examined the stability of simvastatin under acidic conditions,which should be examined prior to application of the methodon strains such as Sulfolobus. When the genome sequence is notavailable, it may be possible to use the genes of a closelyrelated strain whose genome has been sequenced. As demonstratedin this study, the heterologous hmg_Pf gene was applicable forgene disruption in T. kodakaraensis. In order to avoid initialsingle-crossover recombination and possible recombination eventsafterwards due to the presence of two identical regions on thesame chromosome, a heterologous marker may be more advantageousthan an endogenous marker. We hope this methodology will promotegene disruption studies in a broader range of hyperthermophilicarchaea.

The phenotypes of the ${Delta}$ apu_Tk and ${Delta}$ mal_Tk strains in various medianot only provide valuable information on the physiological rolesof the disrupted genes themselves but also allow us to estimatethe contribution of other genes on the genome. The growth characteristicsof the ${Delta}$ mal_Tk strain indicated that it is the only transporterin T. kodakaraensis involved in the uptake of the poly- andoligosaccharides examined in this study. As the ${Delta}$ apu_Tk straincould not grow at all on pullulan, there is a possibility thatApu_Tk is the only relevant enzyme responsible for the initialhydrolysis of pullulan, meaning that TK0977, annotated as atype II pullulan hydrolase, is not directly involved in thedegradation of extracellular pullulan. Another possibility isthat Apu_Tk and the TK0977 product display activity specifiedtoward pullulan substrates of different lengths and are unableto complement each other. A third possibility would be thatthe lack of growth is due to the fact that the ${Delta}$ apu_Tk strainis defective in the uptake of maltotriose (Fig. 4C), a majorproduct of pullulan hydrolysis. From the results shown in Fig.4H, we also found that Apu_Tk plays a much greater role thanexpected in the cleavage of ${alpha}$ -1,4-glycosidic linkages, suggestingthat the experimentally verified ${alpha}$ -amylase TK1884 protein (59)may not be the major amylose-degrading enzyme in T. kodakaraensis.This agrees well with the results of a transcriptome analysisof P. furiosus grown on starch, which revealed that PF1935*,the homolog of TK1774*, is the protein most up-regulated inthe presence of starch (39).

An intriguing change in phenotype was observed in the ${Delta}$ apu_Tkstrain grown on maltooligosaccharides. As the amylopullulanasewas presumed to function in the degradation of poly- or oligosaccharides,we expected that gene disruption would have little effect ongrowth with maltooligosaccharides. Even if phenotypic changeswere to be observed, the effects brought about by disruptingapu_Tk were expected to be greater with longer substrates. However,the results obtained with ${Delta}$ apu_Tk were just the opposite. Thismay be the result of polar effects brought about by insertionof the hmg_Tk overexpression cassette. With our disruption strategy,the downstream genes, in particular, malK_Tk (TK1775), wouldbe under the control of the gdh promoter and would thereby disturbthe stoichiometric expression of the transporter subunits. Anotherpossibility is that the up-regulation of transporter expressionwas disturbed by the absence of apu_Tk. Further, Apu_Tk itselfmay also be a component of the sugar transporter complex, resultingin a decrease in stability or efficiency of the complex whenApu_Tk is absent. This is consistent with the fact that the apu_Tkgene is clustered within the subunit genes of the transporteritself. The possibility that amylopullulanase resides on thecell surface has been proposed in closely related hyperthermophilicarchaea (1, 23, 32). The enzyme from Thermococcus hydrothermalishas been reported to harbor a large C-terminal extension withthree domains in addition to the central catalytic domain (23).One of the domains consists of two sequence repeats, each containingmotifs with similarity to the S-layer homology signature (42),which is considered responsible for the anchoring of proteinsto the cell surface in several bacteria (40, 42, 46, 51). Thesecond region is extremely rich in threonine residues and isfollowed by the third, putative transmembrane domain. This architectureresembles the C-terminal regions of S-layer proteins of thehaloarchaea, in which the Thr-rich regions are targets for O-linkedglycosylation and the transmembrane domain serves as eithera transmembrane anchor or hydrophobic cell wall anchor (38,58). Moreover, as pointed out by Albers et al. (1), the tworegions are also found in a number of proteins of the Thermococcalesannotated as periplasmic components of ABC-type dipeptide transportsystems, further supporting the involvement of these domainsin cell surface attachment (Fig. 6). As amylopullulanases fromP. furiosus, P. abyssi, and T. kodakaraensis also harbor thesedomains, it can be presumed that the amylopullulanases fromthe Thermococcales are attached to the cell surface. Furtherbiochemical examination will be necessary to clarify whetherthese amylopullulanases have any additional function besidestheir roles in poly- and oligosaccharide hydrolysis.

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FIG. 6. Carboxy-terminal regions of various amylopullulanase proteins from the Thermococcales, along with the corresponding regions of periplasmic components of two putative ABC-type dipeptide transport systems of T. kodakaraensis. All of these proteins harbor a threonine (or serine)-rich region, followed by a putative transmembrane domain and a stretch of basic residues (indicated by circles) at the extreme C terminus. The subscripts of the amylopullulanase proteins identify the source organism as follows (accession number): Apu_Tl, Thermococcus litoralis (BAC10983); Apu_Th, Thermococcus hydrothermalis (AAD28552); Apu_Pf, P. furiosus (ABA33719); Apu_Pa, P. abyssi (CAB49104). Accession numbers for TK1760 and TK1804 are BAD85949 and BAD85993, respectively.

ACKNOWLEDGMENTS

This study was supported by a Grant-in-Aid for Scientific Researchto T.I. (no. 14103011), a Grant-in-Aid for Scientific Researchon Priority Areas, "Applied Genomics," to H.A. (no. 18018026),and a Grant-in-Aid for Scientific Research to R.M. (no. 18608002),from the Ministry of Education, Culture, Sports, Science andTechnology of Japan.

FOOTNOTES

* Corresponding author. Mailing address: Department of Synthetic Chemistry and Biological Chemistry, Graduate School of Engineering, Kyoto University, Katsura, Nishikyo-ku, Kyoto 615-8510, Japan. Phone: 81 75 383 2777. Fax: 81 75 383 2778. E-mail: imanaka{at}sbchem.kyoto-u.ac.jp.

Published ahead of print on 26 January 2007.

REFERENCES

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