Different Glycolytic Pathways for Glucose and Fructose in the Halophilic Archaeon Halococcus Saccharolyticus

Arch Microbiol (2001) 175:52Ð61 DOI 10.1007/s002030000237

ORIGINAL PAPER

Ulrike Johnsen á Martina Selig á Karina B. Xavier á Helena Santos · Peter Schönheit Different glycolytic pathways for glucose and fructose in the halophilic archaeon Halococcus saccharolyticus

Received: 14 August 2000 / Revised: 24 October 2000 / Accepted: 26 October 2000 / Published online: 9 December 2000 © Springer-Verlag 2000

Abstract The glucose and fructose degradation path- oses. The data indicate that, in the archaeon H. saccha- ways were analyzed in the halophilic archaeon Halococ- rolyticus, the isomeric hexoses glucose and fructose are cus saccharolyticus by 13C-NMR labeling studies in degraded via inducible, functionally separated glycolytic growing cultures, comparative enzyme measurements and pathways: glucose via a modified ED pathway, and fruc- cell suspension experiments. H. saccharolyticus grown on tose via a modified EM pathway. complex media containing glucose or fructose specifically 13C-labeled at C1 and C3, formed acetate and small Keywords Halococcus saccharolyticus á Archaea á amounts of lactate. The 13C-labeling patterns, analyzed by Modified Embden-Meyerhof pathway á Modified 1H- and 13C-NMR, indicated that glucose was degraded Entner-Doudoroff pathway á 13C-NMR á Ketohexokinase á via an Entner-Doudoroff (ED) type pathway (100%), Fructose-1-phosphate kinase á Glucose dehydrogenase á whereas fructose was degraded almost completely via an Gluconate dehydratase á 2-Keto-3-deoxy-gluconate Embden-Meyerhof (EM) type pathway (96%) and only to kinase a small extent (4%) via an ED pathway. Glucose-grown and fructose-grown cells contained all the enzyme activi- Abbreviations KDG 2-Keto-3-deoxygluconate á ties of the modified versions of the ED and EM pathways KDPG 2-Keto-3-deoxy-6-phosphogluconate á recently proposed for halophilic archaea. Glucose-grown FBP Fructose-1,6-bisphosphate á TIM Triosephosphate cells showed increased activities of the ED enzymes glu- isomerase á GAP Glyceraldehyde-3-phosphate á conate dehydratase and 2-keto-3-deoxy-gluconate kinase, PEP Phosphoenolpyruvate á PTS Phosphotransferase á whereas fructose-grown cells contained higher activities 1-PFK Fructose 1-phosphate kinase of the key enzymes of a modified EM pathway, ketohexokinase and fructose-1-phosphate kinase. During growth of H. saccharolyticus on media containing both glucose Introduction and fructose, diauxic growth kinetics were observed. Af- ter complete consumption of glucose, fructose was de- Various halophilic archaea, including species of the graded after a lag phase, in which fructose-1-phosphate genera Halobacterium, Haloarcula, Haloferax and Halo- kinase activity increased. Suspensions of glucose-grown coccus (for taxonomy, see Kamekura 1998), have been cells consumed initially only glucose rather than fructose, shown to grow on sugars, including glucose, fructose and those of fructose-grown cells degraded fructose rather sucrose, as carbon and energy sources (Hochstein 1988; than glucose. Upon longer incubation times, glucose- and Rawal et al. 1988; Danson 1993). The glucose degrada- fructose-grown cells also metabolized the alternate hex- tion pathway was first analyzed for Halobacterium saccharaovorum by Tomlinson et al. (1974). Based on the identification of enzyme activities in crude extracts, a modified Entner-Doudoroff (ED) pathway was postulated U. Johnsen · M. Selig · P. Schönheit (✉) for glucose conversion to pyruvate. Accordingly, glucose Institut für Allgemeine Mikrobiologie, is oxidized to gluconate via NADP+-dependent glucose Christian-Albrechts-Universität Kiel, dehydrogenase, followed by gluconate dehydratase yield- Am Botanischen Garten 1Ð9, 24118 Kiel, Germany e-mail: [email protected], ing 2-keto-3-deoxygluconate (KDG). KDG is then phos- Tel.: +49-431-8804328/4330, Fax: +49-431-880-2194 phorylated by KDG kinase to 2-keto-3-deoxy-6-phospho- K. B. Xavier á H. Santos gluconate (KDPG), which is cleaved by KDPG aldolase Instituto de Tecnologia Quimica e Biológica, to pyruvate and glyceraldehyde 3-phosphate. The latter Apartato 127, 2780 Oeiras, Portugal compound is oxidized to pyruvate, a process that involves 53 e.g. glucose dehydrogenase from Haloferax mediterranei (Bonete et al. 1996), and ketohexokinase and 1-phosphofructokinase from Haloarcula vallismortis (Rangaswamy and Altekar 1994a, b), have been purified. However, con- clusive evidence for the operation of these glycolytic pathways in vivo has not been demonstrated. A recent comparative enzyme analysis of H. vallismortis and H. mediterranei grown on either glucose or fructose re- vealed the presence of enzymes of the modified ED pathway and of the modified EM pathway (Altekar and Ran- gaswamy 1992). In vivo evidence for the attribution of both glycolytic pathways to the degradation of the particular hexose has not been given. One method to analyze glycolytic pathways in vivo is to use 13C-NMR to identify the products derived after fermentation of specifically labeled 13C-glucose in growing cultures or cell suspensions. This approach was recently successfully applied to elucidate glycolytic pathways in various hyperthermophilic archaea (see Selig et al. 1997; Schönheit and Schäfer 1995; Kengen et al. 1996). Fig.1 Proposed pathways of glucose degradation via modified In the present communication, glucose and fructose Entner-Doudoroff pathway and of fructose degradation via modi- degradation pathways in the halophilic archaeon Halo- fied Embden-Meyerhof pathway in halophilic archaea. For litera- coccus saccharolyticus (Montero et al. 1989) were ana- ture see text. 1 Glucose dehydrogenase, 2 Gluconate dehydratase, lyzed by 13C-labeling studies in growing cultures, com- 3 2-Keto-3-deoxygluconate kinase, 4 2-Keto-3-deoxy-6-phospho- parative enzyme measurements, and cell suspensions ex- gluconate aldolase, 5 Ketohexokinase, 6 1-Phosphofructokinase, 7 Fructose-1,6-bisphosphate aldolase, 8 Triosephosphate isomerase, periments. H. saccharolyticus was chosen for this com- 9 Glycerinaldehyde-3-phosphate dehydrogenase, 10 3-Phospho- parative study because initial growth experiments indi- glycerate kinase, 11 Phosphoglycerate mutase, 12 Enolase, cated that this organism grew equally well on glucose and 13 Pyruvate kinase fructose and formed about the same amount of acetate from both hexoses, a prerequisite for the planned 13C-labeling experiments. The data obtained indicate that the two isomeric hexoses are catabolized in vivo via function- conventional steps, including glyceraldehyde-3-phosphate ally separated, inducible glycolytic pathways, glucose via dehydrogenase, phosphoglycerate kinase, phosphoglycer- an ED pathway and fructose via an EM pathway. The ate mutase, enolase, and pyruvate kinase. Evidence for the pathways showed modifications recently proposed for operation of this pathway has also been demonstrated for other halophilic archaea (Fig.1). other species of Halobacterium, Haloferax, and Halo- coccus (Hochstein 1988; Rawal et al. 1988; Severina and Pimenov 1988; Danson 1993). This modified ED path- Materials and methods way, with the exception of glucose dehydrogenase, has also been shown to be involved in the degradation of glu- Growth of the organism conate in the anaerobic eubacterium Clostridium aceticum (Andreesen and Gottschalk 1969; Bender et al. 1971). Halococcus saccharolyticus (Montero et al. 1989) was grown in a complex medium containing yeast extract, casamino acids, and the Glucose degradation in this organism, however, follows sugars glucose or fructose or a mixture of both. The medium con- the conventional Embden-Meyerhof (EM) pathway. tained (per liter): 25 mM fructose or 25 mM glucose, 2.5 g yeast The pathway of fructose degradation has been ana- extract, 5 g casamino acids, 250 g NaCl, 19.5 g MES, 2 g KCl, 1 g lyzed for Haloarcula vallismortis. From enzyme measure- Na-glutamate, 3 g Na-citrate, 20 g MgSO4á7H20, 200 ml trace elements, it has been concluded that fructose is degraded to ment solution and 10 ml vitamin solution. The pH was adjusted to 7.35 with 5 N NaOH. The trace element solution contained (per pyruvate via a modified EM pathway (Altekar and Ran- liter): 1.5 g EDTA, 0.01 g Na2MoO4á2H2O, 0.5 g MnSO4áH2O, gaswamy 1990, 1992). Accordingly, fructose is phospho- 0.1 g FeSO4á7H2O, 0.1 g CoCl, 0.1 g ZnSO4á7H20, 0.01 g CuSO4á rylated by ketohexokinase to fructose 1-phosphate, which 5H2O. The vitamin solution contained (per liter): 2 mg biotin, in turn is phosphorylated to fructose 1,6-bisphosphate 2 mg folic acid, 11 mg pyridoxine-HCl, 5 mg riboflavin, 5 mg thi- by the activity of fructose-1-phosphate kinase. Fructose amine-HCl, 5 mg nicotinamide, 5 mg calcium panthothenate, 0.1 mg B12, 5 mg p-aminobenzoic acid. In some growth experi- 1,6-bisphosphate is then converted to 2 mol of pyruvate ments, the concentration of casamino acids, yeast extract, glu- via the conventional enzymes of the EM pathway. cose,or fructose was changed as indicated in the text and figure The proposed pathways for glucose or fructose degra- legends. Routinely, cells were grown aerobically at 37¡C in Erlen- meyer flasks (0.5Ð2 l) containing 10% medium under continuous dation (Fig.1) were mainly concluded from enzyme shaking at 200 rpm. Growth was followed by measuring optical analyses, in particular from the identification of key en- ∆ ∆ densities at 578 nm against the medium blank ( OD). An OD578 zymes of these modified pathways. Some key enzymes, of 1 corresponded to a protein content of about 0.6 mg protein/ml. 54 Preparation of cell suspensions and cell extracts Gluconate dehydratase was determined by measuring the formation of KDG as described by De Rosa et al. (1984). The amount Cell suspensions of H. saccharolyticus were prepared after growth of KDG formed was determined as described by Weissbach and on complex medium containing either glucose or fructose or in the Hurwitz (1959). The assay mixture contained 100 mM Tris/HCl, ∆ absence of added sugars. Late-exponential-phase cells ( OD578 of pH 8.5, 1 M KCl, 10 mM gluconate, and extract. ∆ 5Ð6 after growth on glucose or fructose, and OD578 of 3 after KDG kinase was determined at 37¡C by measuring the ATP- growth in the absence of sugars) were harvested by centrifugation and gluconate-dependent formation of pyruvate, which was cou- (9,000×g, 20 min), washed once with buffer (suspension buffer: pled to the oxidation of NADH via lactate dehydrogenase. The as- 100 mM Tris/HCl, pH 7.35, 4.3 M NaCl, 0.03 M KCl) and were say mixture contained 100 mM Tris/HCl, pH 8.5, 1 M KCl, suspended in the same buffer at a concentration of 25Ð30 mg pro- 10 mM MgCl2, 2 mM ATP, 0.3 mM NADH, 10 mM gluconate, tein/ml. Cell suspensions experiments were performed at 37¡C in 7 U lactate dehydrogenase, and extract. 20-ml tubes filled with 7 ml cell suspension (25 mg protein/ml) in KDPG aldolase was tested by measuring KDPG formation suspension buffer. The tubes were shaken at 200 rpm. For the from glyceraldehyde 3-phosphate and pyruvate. The assay mixture preparation of cell extracts, cells were washed and suspended in contained 100 mM Tris/HCl, pH 8.5, 1 M KCl, 100 mM pyruvate, 100 mM Tris/HCl, pH 7.5, and 4.3 M NaCl. The suspensions 1 mM glyceraldehyde 3-phosphate and extract (5Ð17 mg protein). (25Ð30 mg protein/ml) were disrupted by sonication followed by The formation of KDPG was followed at 37¡C over a period of centrifugation (10,000×g, 10 min). The protein content of suspen- 60 min and quantitated in the thiobarbituric acid assay at 546 nm sions and cell extract was determined by the Biuret method (Bode (Weissbach and Hurwitz 1959). et al. 1968) with bovine serum albumin as standard. Hexokinase was determined by measuring the ATP-dependent formation of glucose 6-phosphate from glucose in a discontinuous assay. The reaction was performed in 250-µl assay mixture con- 13C-labeling experiments taining 100 mM Tris/HCl, pH 8.3, 1.5 M KCl, 2 mM ATP, 10 mM MgCl2, 10 mM glucose, and extract. The amount of glucose-6- 13 13 13 phosphate formed after 30 min incubation was quantitated by mea- C-labeling experiments with C-glucose or C-fructose labeled µ on C1 or C3 were carried out in growing cultures of H. saccha- suring the oxidation of NADH upon addition of 750 l of detection rolyticus to identify the glycolytic pathway for the degradation of solution containing 100 mM Tris/HCl, pH 7.0, 2 mM ATP, 10 mM glucose and fructose, respectively. The cells were grown at 37¡C MgCl2, 0.5 mM NADH, 0.5 U 6-phosphofructokinase, 0.18 U in 15-ml test tubes filled with 6 ml medium containing 25 mM of fructose-1,6-bisphosphate aldolase, 10 U triosephosphate iso- each [1-13C]- or [3-13C]-labeled glucose or fructose. Cultures were merase, 0.34 U glycerol-3-phosphate dehydrogenase. Glucose-6-phosphate dehydrogenase was determined by mea- shaken at 220 rpm (Certomat R, Braun Melsungen, Germany), and + at the end of growth (after about 50 h) the medium was centrifuged suring the glucose-6-phosphate-dependent reduction of NADP . × 13 The assay mixture contained 100 mM Tris/HCl, pH 8.3, 1 M KCl, (9,000 g, 20 min). The C-labeling pattern was determined from + 13C- and 1H-NMR spectra of the products acetate and lactate, 10 mM glucose 6-phosphate, 1 mM NADP , and extract. 6-Phosphogluconate deydrogenase activity was determined by which were formed during growth. The compounds were isolated + from the supernatants of the growth medium by ether perforation measuring the 6-phosphogluconate-dependent reduction of NADP . The assay mixture contained 100 mM Tris/HCl, pH 9.0, 1.5 M as previously described (Schäfer et al. 1994; Selig et al. 1997). + 13C-NMR spectra were acquired in a Bruker DRX500 spectrome- KCl, 10 mM 6-phosphogluconate, 1 mM NADP , and extract. ter operating at 125.77 MHz, and 1H-NMR spectra were acquired Fructose-1,6-bisphosphatase activity was determined by mea- in a AMX300 spectrometer operating at 300.13 MHz. suring fructose-1,6-bisphosphate-dependent formation of fructose 6-phosphate coupled to the reduction of NADP+ via phosphoglucose isomerase and glucose-6-phosphate dehydrogenase. The assay mixture contained 100 mM Tris/HCl, pH 8.3, 30 mM MgCl2, Enzyme studies 1 mM NADP+, 10 mM fructose 1,6-bisphosphate, 2.5 U phosphoglucose isomerase, 1.5 U glucose-6-phosphate dehydrogenase, and All enzyme assays were carried out aerobically at 37¡C in glass extract. cuvettes filled with 1 ml assay mixtures. Enzyme assays monitor- Fructose-1-phosphate mutase activity was determined in the ing NADH oxidation were carried out in stoppered cuvettes (N2 same assay as described for fructose-1,6-bisphosphatase, except gas phase) under anoxic conditions. It was ensured that, in coupled that fructose 1,6-bisphosphate was replaced by 10 mM fructose enzymatic assays, which were performed either at 1 M or 1.5 M 1-phosphate. KCl, or at 30 mM MgCl2, as indicated, the auxiliary enzymes were 6-Phosphoglucose isomerase activity was determined by mea- not rate-limiting. One unit (U) of enzyme activity is defined as µ suring the fructose-6-phosphate-dependent formation of glucose 6- 1 mol substrate consumed or product formed per min. phosphate coupled to NADP+ reduction via glucose-6-phosphate Ketohexokinase was determined by measuring the ATP-depen- dehydrogenase. The assay mixture contained 100 mM Tris/HCl, dent decrease of fructose. The amount of fructose formed upon in- pH 8.3, 1 M KCl, 20 mM fructose-6-phosphate, 1 mM NADP+, cubation up to 20 min was quantitated by measuring the reduction 1.4 U glucose-6-phosphate dehydrogenase, and extract. + of NADP via hexokinase, glucose-6-phosphate isomerase and Fructose-1,6-bisphosphate aldolase (EC 4.1.2.13) (30 mM glucose-6-phosphate dehydrogenase. The assay mixture contained MgCl2), triosephosphate isomerase (EC 5.3.1.1) (1 M KCl), glyc- 100 mM Tris/HCl, pH 9.0, 1.5 M KCl, 10 mM MgCl2, 20 mM eraldehyde-3-phosphate dehydrogenase (EC 1.2.1.13) (1 M KCl), + ATP, 20 mM fructose, 2 mM NADP , 7.5 U hexokinase, 3.5 U phosphoglycerate kinase (EC 2.7.2.3) (1 M KCl), phosphoglycer- glucose-6-phosphate isomerase, 0.7 U glucose-6-phosphate dehy- ate mutase (EC 2.7.5.3) (1 M KCl), enolase (EC 4.2.1.11) (30 mM drogenase, and extract. MgCl2) and pyruvate kinase (EC 2.7.1.40) (30 mM MgCl2) were 1-Phosphofructokinase was determined at 37¡C by measuring measured as described previously (Schäfer and Schönheit 1992, the ATP-dependent formation of fructose 1,6-bisphosphate (FBP) 1993; Schröder et al. 1994). The assay mixtures contained 100 mM from fructose 1-phosphate, which was coupled to the oxidation of Tris/HCl, pH 8.5, and KCl or MgCl as indicated. NADH via FBP aldolase, triosephosphate isomerase (TIM), and 2 glycerol-3-phosphate dehydrogenase. The assay mixture contained 100 mM Tris/HCl, pH 8.5, 30 mM MgCl2, 10 mM fructose 1-phosphate, 2 mM ATP, 0.3 mM NADH, 0.54 U FBP-aldolase, Source of material 20 U TIM, 0.34 U glycerol-3-phosphate dehydrogenase, and extract. All commercially available chemicals were reagent grade and were Glucose dehydrogenase was tested at 37¡C by the reduction of obtained from Merck (Darmstadt, Germany), Roth (Karlsruhe, NADP+. The assay mixture contained 100 mM Tris/HCl, pH 9.0, Germany), or Sigma-Aldrich Fluka Chemie (Deisenhofen, Ger- 1.5 M KCl, 1 mM NADP+, 10 mM glucose, and extract. many). Yeast extract was from GIBCO BRL (Eggenstein, Ger- 55 many) and casamino acids from Difco (Stuttgart, Germany). En- Glucose and fructose degradation pathways zymes and coenzymes were from Boehringer (Mannheim, Ger- in growing cultures studied by 13C-NMR many). N2 was from Messer Griesheim (Hamburg, Germany). [1-13]Glucose, [3-13]glucose, and [1-13]fructose, [3-13]fructose, each with 99% isotopic enrichment, were purchased from Omicron Halococcus saccharolyticus was grown in 15-ml tubes (South Bend, Ind. USA). Halococcus saccharolyticus (DSM 5350) with 6 ml complex medium (0.25% yeast extract; 0.5% was obtained from the Deutsche Sammlung von Mikroorganismen casamino acids) supplemented with 25 mM 13C-hexoses und Zellkulturen (Braunschweig, Germany). labeled at either C1 or C3 (see Methods section). At the end of the exponential growth phase (after about 50 h), Results acetate (7Ð10 mM) and small amounts of lactate (0.5Ð 2 mM) were detected. Growth of H. saccharolyticus on glucose or fructose Glucose degradation pathway Halococcus saccharolyticus was grown in 500-ml flasks with 50 ml medium containing complex constituents The 13C-labeling patterns in acetate and lactate were ana- (0.25% yeast; 0.5% casamino acids) and 25 mM of either lyzed after fermentation of [1-13C]glucose and [3-13C]glu- glucose or fructose (Fig.2A, B). On both sugars, the cells cose. When [3-13C]glucose was metabolized, the methyl grew exponentially, with a doubling time of about 15 h, groups of acetate and lactate were labeled, whereas the la- ∆ up to an OD578 of about 6 (3.6 mg protein/ml). During bel was found only on the carboxylic group of lactate growth, both hexoses were completely consumed, after when [1-13C]glucose was supplied (Fig.3A,B). In each which growth ceased. Acetate (5Ð7 mM) was formed at cluster of proton resonances, the central resonance (sin- the end of the exponential growth phase. In the absence of glet at 1.9 ppm for acetate, and doublet at 1.3 ppm for lac- sugars, the cells grew on the complex constituents of the tate) is due to unlabeled methyl groups; the two lateral ∆ medium up to an OD578 of about 3; acetate was not signals are due to isotopomers of lactate or acetate in formed in significant amounts under these conditions (not which the methyl group is labeled. When [3-13C]glucose shown). On media containing reduced complex con- was metabolized, the ratio between the areas of lateral and stituents, e.g. 0.05% yeast extract, the cells grew on either central signals was approximately 0.8 (spectrum A of Fig. glucose or fructose (each 25 mM) at higher doubling 3). In contrast, when [1-13C]glucose was metabolized, that times (about 20 h) to less maximal cell densities. Under ratio was approximately 0.01 (spectrum B of Fig.3) indi- these conditions, acetate was not formed in significant cating the absence of isotopic enrichment on the methyl amounts. In the complete absence of complex con- groups of both products. However, the central multiplet in stituents, no growth of H.saccharolyticus on sugars was the lactate pattern was split by a small coupling constant observed. (4 Hz) due to coupling with C1, ruling out the presence of The findings indicate that growth of H. saccharolyticus lactate labeled on the carboxylic group only. The 13C-NMR on glucose and fructose is dependent on complex con- spectra of the same samples corroborated the labeling stituents and that only in the presence of higher concen- pattern derived from 1H-NMR and also showed that the trations of yeast extract/casamino acids is acetate formed CH-group of lactate was not labeled; therefore, the pen- from sugars in significant amounts. Thus, the latter me- tose phosphate pathway did not operate under the experi- dium composition was used for 13C-labeling experiments mental conditions used. We conclude that in this organism in growing cultures, since acetate can be analyzed by 13C glucose is fermented to acetate and lactate via an ED-type NMR. glycolytic pathway.

Fig.2 Growth of Halococcus saccharolyticus on either glucose (A) or fructose (B) in the presence of yeast extract (0.25%) and casamino acids (0.5%). Cultures were incubated in 500-ml Erlenmeyer flasks filled with 50 ml medium and shaken at 200 rpm. Growth (), glucose or fructose consumption () and acetate formation () were followed over time 56 Fructose degradation pathway

The 13C-labeling patterns in acetate and lactate were analyzed after fermentation of [1-13C]fructose and [3-13C]fructose. When [3-13C]fructose was supplied, isotopic enrichment was detected not only on the carboxyl group of lactate, but a small enrichment was also observed on the methyl carbon of acetate and lactate (Fig.4, spectrum A). From [1-13C]fructose, the majority of the methyl groups of acetate and lactate were labeled, as made evident by the two satellite resonances centered at 1.9 and 1.3 ppm, respectively (Fig.4, spectrum B). These data show that this organism ferments fructose mainly via an EM-type pathway, with a small contribution by an ED-type pathway. Contribution of the pentose phosphate pathway would lead to labeling of the CH group on lactate from [3-13C]glucose, an observation that is not found (data not shown). Therefore, contribution of the pentose phosphate pathway can be ruled out. The relative contributions of the EM- and ED-type pathways can be determined from the analysis of the pro- 13 Ð Fig.3 1H-NMR spectra of acetate and lactate derived from the ton spectra. The molar ratio between CH3COO and 13 13 Ð 13 metabolism of [3- C]glucose (A) or [1- C]glucose (B) in grow- CH3COO was 0.488 when [1- C]fructose was metabo- ing cultures of H. saccharolyticus. A schematic presentation of lized (Fig.4, trace B) and 0.033 when [3-13C]fructose was isotopomer populations of acetate and lactate is included fermented (Fig.4, trace A). The relative contributions of EM- and ED-type glycolytic pathways were 94% and 6%, respectively. These values were determined taking into account that the two experiments with labeled fructose were run in parallel and discounting the natural abun- dance of 13C (1.1%). An independent experiment led to the values of 96% (EM) and 4% (ED).

Enzyme studies

The 13C-labeling experiments indicated that glucose was degraded to acetate and lactate via an ED glycolytic pathway, whereas fructose was almost completely degraded (96%) via an EM pathway and only to a small extent by an ED pathway. A comparative analysis of enzyme activities of the ED and EM pathways was performed in cells of H. saccharolyticus after growth on either glucose or fructose. It was found that glucose- and fructose-grown cells contained enzymes of both the ED and the EM pathway with the same modifications recently proposed for other halophilic archaea (Hochstein 1988; Altekar and Rangaswamy 1990). The specific enzyme activities and some of the kinetic properties are listed in Table 1.

Enzymes of the modified ED pathway

Fig.4 1H-NMR spectra of acetate and lactate derived from the The following enzyme activities catalyzing glucose metabolism of [3-13C]fructose (A) or [1-13C]fructose (B) in grow- degradation to glyceraldehyde 3-phosphate (GAP) and ing cultures of H. saccharolyticus. A schematic presentation of pyruvate were detected in cell extracts of both glucose- isotopomer populations of acetate and lactate is included grown and fructose-grown cells: glucose-dehydrogenase, gluconate dehydratase, KDG kinase, 2-keto-3-deoxy-6- phosphogluconate aldolase. KDG aldolase could not be detected. The activity of gluconate dehydratase was three- 57

Table 1 Specific activities and apparent Km values of enzymes of 1-phosphate, F-1,6-BP fructose 1,6-bisphosphate, Pi inorganic a modified Embden-Meyerhof pathway and Entner-Doudoroff phosphate, 2-PG 2-phosphoglycerate, Ð not determined. Apparent pathway in cell extracts of Halococcus saccharolyticus at 37¡C. Km values were obtained with glucose-grown cells (1) or fructose- KDG 2-Keto-3-deoxygluconate, KDPG 2-keto-3-deoxy-6-phos- grown cells (2) phogluconate, GAP glyceraldehyde 3-phosphate, F-1-P fructose

Enzyme activity Glucose-grown Fructose-grown Apparent Km cells cells (mM) Specific activity Specific activity (mU/mg) (mU/mg) Glucose dehydrogenase 25 20 2.0 (Glucose)1, 0.28 (NADP+)1 [Glucose+NADP+→Gluconate+NADPH+H+] → 1 Gluconate dehydratase [Gluconate KDG+H2O] 60 20 3.0 (Gluconate) KDG kinase [KDG+ATP→KDPG+ADP] 5 2 Ð KDPG aldolase [KDPG→pyruvate+GAP] 1 1 Ð Ketohexokinase [Fructose+ATP→F-1-P+ADP] 4 12 2.7 (Fructose)2, 3.0 (ATP)2 1-Phosphofructokinase [F-1-P+ATP→F-1,6-BP+ADP] 4 22 0.2 (F-1-P)2, 0.13 (ATP)2 Fructose-1,6-bisphosphate aldolase 0.7 3 1.4 (F-1,6-BP)2 Triosephosphate isomerase 68 77 Ð + 2 Glyceraldehyde-3-phosphate dehydrogenase 32 (Pi), 95 (Pi), 0.2 (NAD ) 47 (Arsenate) 155 (Arsenate) Phosphoglycerate kinase 14 19 Ð Phosphoglycerate mutase 158 167 Ð Enolase 65 55 0.6 (2-PG)1 Pyruvate kinase 4 4 Ð fold higher in glucose-grown cells, suggesting a glucose Enzymes involved in pentose-phosphate formation specific induction. Also, KDG kinase and glucose dehy- from hexoses drogenase showed higher activities in glucose-grown cells. Glucose dehydrogenase exhibited a 14-fold lower appar- In glucose- and fructose-grown cells, the following en- + + ent Km for NADP (0.28 mM) than for NAD (4 mM), in- zyme activities (Table 2) were detected, catalyzing glu- dicating that the physiological electron acceptor is NADP+, cose or fructose conversion to pentose phosphates (ribu- as has been found in other halophilic archaea (see Bonete lose 5-phosphate) and derived compounds in the an- et al. 1996). abolism. Extracts of glucose-grown cells contained low activities of hexokinase, glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase. A catabolic Enzymes of a modified EM pathway role for hexokinase and glucose-6-phosphate dehydrogenase in glucose degradation via a classical ED pathway Both glucose- and fructose-grown cells contained the fol- appears unlikely, since 6-phosphogluconate dehydratase lowing enzyme activities of fructose conversion to GAP: could not be detected. Extract of fructose-grown cells ketohexokinase, fructose-1-phosphate kinase, fructose- contained the following enzyme activities catalyzing fruc- 1,6-bisphosphate aldolase, TIM. 6-Phosphofructose ki- tose 1,6-bisphosphate conversion to ribulose 5-phosphate: nase was not detected. The specific activities of ketohex- Fructose-1,6-bisphosphatase, glucose-6-phosphate isomer- okinase and fructose-1-phosphate kinase were up to six- ase, glucose-6-phosphate dehydrogenase and 6-phospho- fold higher in fructose-grown cells than in glucose-grown gluconate dehydrogenase. Activities of fructose-1-phos- cells, indicating fructose-specific induction of the key en- phate mutase and 6-phosphogluconate dehydratase could zymes of the modified EM pathway. not be detected (Table 2).

Enzymes of GAP conversion to pyruvate Hexose-specific induction of glycolytic pathways in H. saccharolyticus Glucose- and fructose-grown cells contained all enzymes of GAP conversion to pyruvate, which are operative in The comparative enzyme analyses showed that cells both the modified ED and EM pathways: GAP-DH + grown either on glucose or on fructose contained enzymes (NAD reducing), phosphoglycerate kinase, phosphoglyc- of both a modified ED and a modified EM pathway. The erate mutase, enolase, pyruvate kinase (Table 1). The ac- functional separation of ED and EM pathways during tivity of GAP-DH was stimulated by arsenate and was fructose and glucose utilization in vivo, as demonstrated four-fold higher in fructose-grown cells. by the 13C-NMR studies, might be due to a glucose- or fructose-specific induction of key enzymes of the particu- 58

Table 2 Enzyme activities (at 37¡C) in cell extracts of Enzyme activity Glucose-grown cells Fructose-grown cells H. saccharolyticus involved in Specific activity (mU/mg) Specific activity (mU/mg) pentose phosphate (ribulose 5-phosphate) formation from Hexokinase 3 Ð hexoses. n.d. Not detectable, Glucose-6-phosphate dehydrogenase 7 5 Ð not determined 6-Phosphogluconate dehydrogenase 2 1 6-Phosphogluconate dehydratase n.d. n.d. Glucose-6-phosphate isomerase 12 11 Fructose-1,6-bisphosphatase Ð 3 Fructose-1-phosphate mutase Ð n.d.

lar pathways (see Table 1). Further evidence for hexose- Glucose and fructose conversion in cell suspensions specific induction was obtained in growth and cell suspension experiments. Functionally separated, inducible glycolytic pathways could also be demonstrated in cell suspension experiments. Suspensions of H. saccharolyticus cells previously Diauxic growth on glucose and fructose grown on either glucose or fructose or yeast extract/ casamino acids in the absence of sugars, were incubated Halococcus saccharolyticus previously grown on yeast with glucose or fructose and consumption of the hexoses extract in the absence of sugars was incubated on a me- was followed over time. Initially, up to an incubation pe- dium containing low amounts of yeast extract (0.05%) riod of about 4 h, glucose-grown cells consumed only glu- and both glucose and fructose (each 12.5 mM). Growth cose (3 nmoláminÐ1ámgÐ1) rather than fructose (Fig.6A); showed a biphasic profile (Fig.5). After complete con- conversely, fructose-grown cells consumed only fructose sumption of glucose, growth leveled off but resumed upon (3 nmoláminÐ1ámgÐ1) rather than glucose. Upon longer in- degradation of fructose after a lag phase, in which fruc- cubation (10Ð40 h) of the suspensions in the presence of tose-1-phosphate kinase was induced about four-fold. We sugars, e.g., glucose-grown cells started to consume also conclude that H. saccharolyticus preferentially metabo- fructose at significant rates. During this period an increase lized glucose over fructose and that fructose degradation in ketohexokinase activity was observed (Fig.6B), sug- started after the induction of the modified EM pathway. gesting fructose-specific induction of the modified EM pathway. Similarly, after an incubation time of about 5 h, fructose-adapted cells consumed glucose; after about 10 h the rates of glucose consumption were almost equal to those of fructose consumption. Suspensions of H. saccharolyticus cells, adapted to yeast extract/casamino acids, did not consume either glucose or fructose at significant rates up to incubation periods of 4 h (not shown). After 10 h these suspensions also started to consume either glucose or fructose (data not shown). From the data it is concluded that the pathways for glucose and fructose degradation can be induced by the particular hexose in cell suspensions and, after induction, are in vivo functionally separated.

Discussion

In the present communication the glycolytic pathways of the isomeric hexoses glucose and fructose were analyzed in the halophilic archaeon H. saccharolyticus by 13C-NMR analyses in growing cultures, comparative enzyme measurements and cell suspension experiments. The data indicate that glucose and fructose were degraded via different, functionally completely separated, inducible glycolytic Fig.5 Diauxic growth of H. saccharolyticus on media containing pathways, glucose via a modified ED pathway and fruc- both glucose and fructose, 12 mM each, and yeast extract (0.05%). Growth (), glucose () or fructose () consumption and the spe- tose via a modified EM pathway. Participation of the pen- cific activity of 1-phosphofructose kinase (1-PFK) () were fol- tose phosphate pathway in hexose degradation could be lowed over time excluded. 59 Fig.6A,B Consumption of glucose and fructose in cell suspensions (25 mg protein/ml) of H. saccarolyticus adapted to glucose. The suspensions were incubated with either glucose () or fructose () and consumption of the hexoses and the specific activity of ketohexokinase (KHK) () were followed up to 240 min (A) and up to 50 h (B)

13C-NMR analyses with position-labeled glucose and growth on media containing both glucose and fructose, fructose showed that glucose was degraded via an ED glucose was metabolized first, and Ð after a lag phase Ð pathway (100%), whereas fructose was degraded almost fructose was degraded. Degradation was paralleled by an completely (96%) via an EM-type glycolytic pathway. It increase of fructose-1-phosphate kinase, suggesting in- should be mentioned that these data were obtained for duction of the modified EM pathway. Diauxic growth ki- glucose or fructose conversion to the fermentation prod- netics, as shown here for H. saccharolyticus, were also re- ucts acetate and lactate, which accounts for 10Ð20% of ported for H. vallismortis during growth on sucrose (Al- the carbon flux of hexose degradation. It is, however, rea- tekar and Rangaswamy 1992). However, preferred utiliza- sonable to assume that the same glycolytic pathways are tion of either hexose, glucose or fructose has not been de- operative during complete oxidation of the hexoses to termined. (2) Suspensions of cells adapted to yeast ex- CO2. This is the first in vivo demonstration of the opera- tract/casamino acids did not degrade either glucose or tion of the ED and EM pathways in glucose and fructose fructose, suggesting that both glycolytic pathways were degradation in halophilic archaea. Surprisingly, compara- repressed by these complex constituents. (3) Glucose and tive enzyme analysis with glucose- or fructose-adapted fructose degradation was only observed in glucose- and cells indicate the presence of all enzymes of both a modi- fructose-grown cells, respectively, indicating that a func- fied ED pathway and a modified EM pathway (Table 1). tional glycolytic pathway requires specific induction by The modifications of the EM and ED pathways corre- its corresponding hexoses. Furthermore, induction of the sponded to those previously proposed for other halophilic alternate glycolytic pathway in cell suspensions was archaea (Hochstein 1988; Altekar and Rangaswamy 1990; demonstrated upon longer incubation in the presence of Danson 1993). Accordingly, glucose is converted to GAP the alternate hexose. The preferential utilization of glu- and pyruvate via glucose dehydrogenase, gluconate dehy- cose over fructose shown for H. saccharolyticus indicates dratase, KDG kinase and KDPG aldolase. Fructose con- a sort of “catabolite repression”, which has been inten- version involves the enzymes ketohexokinase, fructose-1- sively studied in the domain of Bacteria. To date, the mol- phosphate kinase and fructose-1,6-bisphosphate aldolase ecular basis of catabolite-repression-like phenomena in (see Fig.1). Archaea has not been reported. Despite the fact that both enzyme sets were present The most important result of this study is the demon- in glucose- and fructose-grown cells, the 13C analyses stration of two different and functionally completely sep- clearly demonstrated that in vivo the pathways are com- arated pathways for the degradation of two isomeric hex- pletely separate. This functional separation is probably oses, glucose and fructose, in one organism. To date, sim- due to sugar-specific induction of key enzymes of the par- ilar data have been reported only for the phototrophic bac- ticular pathways, with up to four-fold higher specific ac- terium Rhodobacter capsulatus (Conrad and Schlegel tivities, e.g. of gluconate dehydratase and KDG kinase in 1977). In this organism, functionally different pathways glucose-grown cells, and up to six-fold higher activities of for glucose and fructose degradation were demonstrated ketohexokinase and fructose-1-phosphate kinase in fruc- in cell suspensions by radiorespirometric experiments tose-adapted cells. The molecular basis of this regulation and enzyme measurements. In Rba. capsulatus, glucose of enzyme activity, either at the transcriptional or post- was degraded mainly by the conventional ED pathway, translational level, remains to be determined. typical for bacteria, in which the formation of KDPG in- Further evidence for sugar-specific induction of the ED volves hexokinase, glucose-6-phosphate dehydrogenase and EM pathways in H. saccarolyticus was concluded and 6-phosphogluconate dehydratase (Conway 1992). De- from growth and cell suspension experiments. (1) During gradation of fructose in this organism proceeded mainly 60 by the EM pathway. Accordingly, fructose catabolism was erate, a process involving non-phosphorylated intermedi- initiated by the fructose-inducible phosphoenolpyruvate ates (De Rosa et al. 1984; Danson 1993; Selig et al. 1997). (PEP):fructose phosphotransferase system (PTS) catalyz- Furthermore, glucose and maltose degradation in the ing vectorial phosphorylation of fructose to fructose anaerobic hyperthermophilic archaea Thermococcus celer 1-phosphate, which in turn is phosphorylated to FBP by and Pyrococcus furiosus proceeds via a modified EM inducible fructose-1-phosphate kinase (1-PFK). pathway, again different from that in halophiles. It con- Functional separation of fructose degradation by the tains unusual enzymes such as ADP-dependent hexoki- EM pathway and glucose degradation by the ED pathway nase, ADP-dependent 6-phosphofructokinase and GAP in Rba. capsulatus were explained by enzyme analyses as ferredoxin oxidoreductase (see in Schönheit and Schäfer follows: (1) Fructose specifically induced PEP:fructose 1995; Kengen et al. 1996; Selig et al. 1997). In halophilic PTS and 1-PFK to yield FBP, the characteristic intermedi- archaea, the transport of glucose (Severina et al. 1991; ate of the EM pathway. (2) Glucose is degraded by the ED Tawara and Kamo 1991) and fructose (Takano et al. 1995) pathway due to the presence of hexokinase and enzymes has been reported. Transport activity was dependent on involved in KDPG formation. Degradation of glucose by sodium ions. Both transport of glucose and fructose in the EM pathway is apparently prevented, since FBP for- Haloferax volcanii was found to be induced by the re- mation from fructose 6-phosphate (formed from glucose spective hexose (Tawara and Kamo 1991; Takona et al. 6-phosphate by glucose-6-phosphate isomerase) is limited 1995). due to extremely low 6-phosphofructokinase activity in Here, we also report on enzyme activities (Table 2) the cells (Conrad and Schlegel 1977). involved in hexose conversion to pentose phosphates In Pseudomonas species, glucose is degraded com- required for anabolic purposes. The respective enzymes pletely via the ED pathway; however, in contrast to Rba. are compatible with the following pathways of ribulose capsulatus, fructose catabolism in Pseudomonas species 5-phosphate formation from either hexose: (1) glu- proceeds mainly by the ED pathway and only to a minor cose→glucose 6-phosphate→6-phosphogluconate→ribu- extent by the EM pathway, despite the fact that fructose lose 5-phosphate. A participation of hexokinase and glu- degradation also started with inducible PEP:fructose PTS cose-6-phosphate dehydrogenase in catabolic glucose and 1-PFK (Sawyer et al 1977; Lessie and Phibbs 1984; degradation appears unlikely due to the complete absence Conway 1992). of 6-phosphogluconate dehydratase. (2) Fructose→fruc- The modified EM pathway found in various halophilic tose 1-phosphate→fructose 1,6-bisphosphate→fructose 6- archaea, including H. saccharolyticus, differs from the phosphate→glucose 6-phosphate→6-phosphogluconate→ fructose degradation pathways in bacteria only in the ribulose 5-phosphate. According to this route, the first en- mechanisms of fructose 1-phosphate formation. In halo- zymes, ketohexokinase and fructose-1-phosphate kinase, philic archaea, fructose 1-phosphate is formed by keto- are also involved in fructose catabolism. Fructose-1-phos- hexokinase, whereas in most bacteria fructose is phospho- phate mutase could not be detected, suggesting that fruc- rylated to fructose 1-phosphate during transport by tose 6-phosphate formation proceeds only via fructose PEP:fructose PTS. (Romano and Saier 1992). Thus, keto- 1,6-bisphosphate. Low activities of hexokinase, glucose- hexokinase is the key enzyme in fructose catabolism in 6-phosphate isomerase, glucose-6-phosphate dehydroge- halophilic archaea, and possibly also in other archaeal nase and fructose-1,6-bisphosphatase have also been re- species. The enzyme has long been known in eukaryotes, ported for other halophilic archaea, but their exact phy- but has so far not been found in bacteria. Besides its pres- siological role has not been defined (Rawal et al. 1988). ence in halophilic archaea, ketohexokinase has also been demonstrated in the hyperthermophilic archaeon Pyro- Acknowledgements The work was supported by grants from the coccus furiosus (Schäfer et al. 1994). European Union (“Extremophiles as cell factories”) and the Fonds der Chemischen Industrie. The authors thank A. Brandenburger for To date, there are only a few reports on sugar transport expert technical assistance. The help of Dr. T. Schäfer (Novo in archaea, including halophiles. 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