Reconstruction of the Central Carbohydrate Metabolism of Thermoproteus tenax by Use of Genomic and Biochemical Data

Abstract

The hyperthermophilic, facultatively heterotrophic crenarchaeum Thermoproteus tenax was analyzed using a low-coverage shotgun-sequencing approach. A total of 1.81 Mbp (representing 98.5% of the total genome), with an average gap size of 100 bp and 5.3-fold coverage, are reported, giving insights into the genome of T. tenax. Genome analysis and biochemical studies enabled us to reconstruct its central carbohydrate metabolism. T. tenax uses a variant of the reversible Embden-Meyerhof-Parnas (EMP) pathway and two different variants of the Entner-Doudoroff (ED) pathway (a nonphosphorylative variant and a semiphosphorylative variant) for carbohydrate catabolism. For the EMP pathway some new, unexpected enzymes were identified. The semiphosphorylative ED pathway, hitherto supposed to be active only in halophiles, is found in T. tenax. No evidence for a functional pentose phosphate pathway, which is essential for the generation of pentoses and NADPH for anabolic purposes in bacteria and eucarya, is found in T. tenax. Most genes involved in the reversible citric acid cycle were identified, suggesting the presence of a functional oxidative cycle under heterotrophic growth conditions and a reductive cycle for CO 2 fixation under autotrophic growth conditions. Almost all genes necessary for glycogen and trehalose metabolism were identified in the T. tenax genome.Archaea were recognized as a distinct phylogenetic group more than 25 years ago (75). Their importance as the third major evolutionary line is well established, but our knowledge of their physiological capabilities remains limited. Central metabolic pathways within these organisms are far from fully understood (43,47,72).Thermoproteus tenax was the first hyperthermophilic archaeum described (76). It is able to grow chemolithoautotrophically on H 2 , CO 2 , and S o as well as chemoorganoheterotrophically in the presence of S o and various organic substrates such as glucose, starch, amylose, glycerate, glycerol, ethanol, and malate (12, 76). Physiological and biochemical studies revealed T. tenax as a physiologically versatile organism with numerous archaeon-specific metabolic capabilities, regulation, and thermoadaptive traits.Comparative studies of carbohydrate metabolism in hyperthermophilic archaea indicate that sugars are generally metabolized by variants of the Entner-Doudoroff (ED) and Embden-Meyerhof-Parnas (EMP) pathways. The so-called nonphosphorylative ED pathway (phosphorylation takes place only at the stage of glycerate) is the only pathway that has been identified for sugar degradation in the aerobes Sulfolobus solfataricus (9) and Thermoplasma acidophilum (7), whereas the anaerobes Pyrococcus furiosus (27,28,31,66,69), Thermococcus spp. (31,44,50), Desulfurococcus amylolyticus (20), and Archaeoglobus fulgidus (33) use modified versions of the EMP pathway. In contrast to findings for other hyperthermophilic archaea, T. tenax uses both variants-the ED and EMP pathways-for glucose metabolism, as shown by in vitro studies identifying specific intermediates...

The non-phosphorylating glyceraldehyde-3-phosphate dehydrogenase (GAPN) of Sulfolobus solfataricus: a key-enzyme of the semi-phosphorylative branch of the Entner–Doudoroff pathway

“…Separate transcription start sites for the internal kdgA and kdgK genes could not be detected (data not shown). The assignment of crenarchaeal consensus promoter sequences in front of the first gene of an operon (gad) and in front of downstream located single genes (gapN), the absence of Shine-Dalgarno sequences upstream of the first gene and subsequent translation via leaderless transcripts is in good agreement with previous studies in S. solfataricus (Condo et al 1999;Tolstrup et al 2000) and other Crenarchaea [T. tenax (Schramm et al 2000;Siebers et al 2001Siebers et al , 2004, Pyrobaculum aerophilum (Slupska et al 2001)]. The transcription start points were confirmed by analysis of RNA samples derived from cultures grown on different carbon sources (S. solfataricus cells grown on D-glucose, D-arabinose and tryptone; data not shown).…”

“…Comparative studies of the carbohydrate metabolism in Archaea indicate that sugars are generally metabolized by modifications of the classical Entner-Doudoroff (ED) and Embden-Meyerhof-Parnas (EMP) pathways that are operative in Bacteria and Eukarya (Ronimus and Morgan 2002;Siebers et al 2004;Siebers and Schönheit 2005;Verhees et al 2003). The archaeal pathways are characterized by the presence of numerous novel enzymes and enzyme families.…”

“…However, recent combinations of genomics-based and biochemical analyses demonstrated the presence of the branched ED pathway in all Archaea that utilize the ED pathway (Siebers et al 2004;Ahmed et al 2005;Jung and Lee 2005;Kehrer et al 2007). The only exception known so far is Halobacterium sp.…”

Carbohydrate Metabolism in Archaea: Current Insights into Unusual Enzymes and Pathways and Their Regulation

“…acidophilum (see below). The coding sequences show low but detectable sequence similarities to the classical PGIs, especially in those residues involved in catalysis (82)(83)(84). However, in contrast to the G6P/F6P-specific conventional PGIs, the corresponding enzymes show promiscuous PGI and phosphomannose isomerase (PMI) activities, converting both G6P and mannose 6-phosphate (M6P) to F6P.…”

“…Thus, only in the anaerobe Tpt. tenax do GAPN and GAPOR operate simultaneously in sugar degradation via both the modified EMP and the modified branched ED pathways, respectively (84). All three enzymes are described above in the section discussing the lower common shunt of the EMP pathway.…”