Different Glycolytic Pathways for Glucose and Fructose in the Halophilic Archaeon Halococcus Saccharolyticus
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Arch Microbiol (2001) 175:52–61 DOI 10.1007/s002030000237 ORIGINAL PAPER Ulrike Johnsen · Martina Selig · Karina B. Xavier · 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 · Archaea · amounts of lactate. The 13C-labeling patterns, analyzed by Modified Embden-Meyerhof pathway · Modified 1H- and 13C-NMR, indicated that glucose was degraded Entner-Doudoroff pathway · 13C-NMR · Ketohexokinase · via an Entner-Doudoroff (ED) type pathway (100%), Fructose-1-phosphate kinase · Glucose dehydrogenase · whereas fructose was degraded almost completely via an Gluconate dehydratase · 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 · ties of the modified versions of the ED and EM pathways KDPG 2-Keto-3-deoxy-6-phosphogluconate · recently proposed for halophilic archaea. Glucose-grown FBP Fructose-1,6-bisphosphate · TIM Triosephosphate cells showed increased activities of the ED enzymes glu- isomerase · GAP Glyceraldehyde-3-phosphate · conate dehydratase and 2-keto-3-deoxy-gluconate kinase, PEP Phosphoenolpyruvate · PTS Phosphotransferase · whereas fructose-grown cells contained higher activities 1-PFK Fructose 1-phosphate kinase of the key enzymes of a modified EM pathway, ketohex- okinase 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 1–9, 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 · 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-phospho- fructokinase 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 path- way 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 partic- ular hexose has not been given. One method to analyze glycolytic pathways in vivo is to use 13C-NMR to identify the products derived after fer- mentation 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-la- beling 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 MgSO4·7H20, 200 ml trace ele- ments, 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 Na2MoO4·2H2O, 0.5 g MnSO4·H2O, gaswamy 1990, 1992). Accordingly, fructose is phospho- 0.1 g FeSO4·7H2O, 0.1 g CoCl, 0.1 g ZnSO4·7H20, 0.01 g CuSO4· 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 (0.5–2 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