Untersuchungen zur transkriptionellen Regulation des zentralen Kohlenhydrat-Metabolismus sowie zum Trehalose-Metabolismus in dem hyperthermophilen Crenarchaeum Thermoproteus tenax

Evolution of life led to three major domains of living organisms: the Eucarya and two distinct prokaryotic domains, the Bacteria and Archaea. Originally, the domain of the Archaea was identified by Carl Woese and Geoge E. Fox (Woese and Fox, 1977; Woese et al., 1990) as being the third major line of life based on 16S rRNA sequence analyses. The metabolic pathways of the central carbohydrate metabolism (CCM) of the hyperthermophilic crenarchaeote Thermoproteus tenax reflect the complexity and variety of central metabolic pathways that is found as a general feature in several Archaea. Although many unusual pathways have been unravelled in different Archaea, the knowledge about their regulation is rather limited. The sulphur-dependent T. tenax is a facultatively heterotrophic organism and therefore represents an ideal organism to study the carbon flux in response to autotrophic and heterotrophic growth conditions (glycolytic/gluconeogenic carbon switch). Within the present work, the DNA microarray technology (focussed approach; 105 different CCM genes) has been established for T. tenax in order to analyse the mode and significance of transcriptional regulation of the CCM. Changes in transcript levels of the CCM-related genes of T. tenax in response to autotrophic growth on CO2/H2 in comparison to transcript levels under heterotrophic growth on glucose were followed. The results of the microarray analysis reflect a highly coordinated transcription of the genes involved in the reversible Embden-Meyerhof-Parnas (EMP) pathway and the reversible citric acid cycle (CAC) for controlling the catabolic and anabolic carbon flux. In contrast, the genes of the catabolic branched Entner-Doudoroff (ED) pathway exhibit no strong regulation at the gene level under the chosen growth conditions (glucose and CO2/H2). The catabolic flux (heterotrophic growth) is enforced by the enhanced expression of the three EMP genes pfp, fba and gor encoding pyrophosphate-dependent phosphofructokinase, fructose-bisphosphate aldolase and ferredoxin-dependent glyceraldehyde-3-phosphate (GAP) oxidoreductase (GAPOR) as well as the CAC genes acn, idhA, gltA-2, sdhA-B-C-D coding for aconitase, isocitrate dehydrogenase and for the key enzymes citrate synthase 2 and succinate dehydrogenase. The anabolic flux (autotrophic growth) is driven by induction of the EMP genes gap, pgk and pps encoding classical, phosphorylating GAP dehydrogenase (GAPDH), phosphoglycerate kinase and phosphoenolpyruvate (PEP) synthetase (PEPS) as well as the CAC genes oorA-B-C-D and frdA-B coding for the reversible 2-oxoglutarate-ferredoxin oxidoreductase and fumarate reductase. This study in combination with available biochemical data (Brunner et al., 1998, 2001; Schramm et al., 2000; Tjaden et al., 2006) spot key regulation points of the T. tenax EMP variant at the level of GAP and PEP/pyruvate conversion. At both regulation sites three different genes/enzymes are responsible for the control of the carbon flux: GAPDH (gap), non-phosphorylating GAPDH (GAPN; gapN), GAPOR (gor) and pyruvate kinase (PK; pyk), PEPS (pps), pyruvate phosphate dikinase (PPDK; ppdk), respectively. From comparable studies of two other hyperthermophilic, heterotrophic Archaea, Pyrococcus furiosus and Sufolobus solfataricus as well as of the halophile Haloferax volcanii it can be concluded that GAP conversion seems to represents a conserved key regulation point in Archaea, whereas regulation at PEP/pyruvate conversion seems to be less conserved. Interestingly, another conserved regulation site might be situated at the upper part of the EMP pathway (fructose 6-phosphate/fructose 1,6-bisphosphate conversion), which is exclusively executed on gene level. To get more insights into the molecular background of CCM regulation in T. tenax the functional genome organisation of CCM genes was analysed in order to identify transcriptional regulators. The gene coding for a Lrp homolog (leucine-responsive regulatory protein, bacterial-type global transcription regulator) was identified downstream of the gad gene coding for gluconate dehydratase belonging to the ED gene cluster of T. tenax and the properties of its gene product have been analysed. DNA binding studies with the recombinant protein demonstrated that Lrp binds to its own promoter region and also binds to the promoter region of the ED gene cluster, thus suggesting an involvement in transcriptional regulation of the ED genes. In addition to the regulation of the CCM in dependence of the carbon source, it is also a matter of interest how T. tenax adapts to environmental stress, e.g. high temperature and osmolarity or oxidative stress. Therefore, the metabolism of the compatible solute trehalose was further investigated. Initial studies revealed that trehalose is synthesised via the OtsA/OtsB pathway in T. tenax. The genes coding for trehalose-6-phosphate synthase/phosphatase (tpsp) and glycosyl transferase (gt) are part of the trehalose operon of T. tenax. The clustering of the tpsp and gt gene with an additional ORF coding for a putative mechanosensitive channel (msc; MscTTX) in the trehalose operon of T. tenax (msc-gt-tpsp), suggests a functional relation of all three gene products. Functional analysis of the recombinant proteins shows that the pathway is characterised by the first reported bifunctional trehalose-6-phosphate synthase/phosphatase (TPSP), which is activated by the putative glycosyl transferase (GT; TPSP activating protein). However, the mode of activation is still unclear. The results of the present study lead to a proposed model of stress response in T. tenax that comprehends regulation of cell turgor, e.g. under osmotic stress. The current work supports the role of trehalose as compatible solute rather than as carbon and energy source in T. tenax

“Hot standards” for the thermoacidophilic archaeon Sulfolobus solfataricus

Within the archaea, the thermoacidophilic crenarchaeote Sulfolobus solfataricus has become an important model organism for physiology and biochemistry, comparative and functional genomics, as well as, more recently also for systems biology approaches. Within the Sulfolobus Systems Biology (“SulfoSYS”)-project the effect of changing growth temperatures on a metabolic network is investigated at the systems level by integrating genomic, transcriptomic, proteomic, metabolomic and enzymatic information for production of a silicon cell-model. The network under investigation is the central carbohydrate metabolism. The generation of high-quality quantitative data, which is critical for the investigation of biological systems and the successful integration of the different datasets, derived for example from high-throughput approaches (e.g., transcriptome or proteome analyses), requires the application and compliance of uniform standard protocols, e.g., for growth and handling of the organism as well as the “–omics” approaches. Here, we report on the establishment and implementation of standard operating procedures for the different wet-lab and in silico techniques that are applied within the SulfoSYS-project and that we believe can be useful for future projects on Sulfolobus or (hyper)thermophiles in general. Beside established techniques, it includes new methodologies like strain surveillance, the improved identification of membrane proteins and the application of crenarchaeal metabolomics

The Complete Genome Sequence of Thermoproteus tenax: A Physiologically Versatile Member of the Crenarchaeota

Here, we report on the complete genome sequence of the hyperthermophilic Crenarchaeum Thermoproteus tenax (strain Kra 1, DSM 2078(T)) a type strain of the crenarchaeotal order Thermoproteales. Its circular 1.84-megabase genome harbors no extrachromosomal elements and 2,051 open reading frames are identified, covering 90.6% of the complete sequence, which represents a high coding density. Derived from the gene content, T. tenax is a representative member of the Crenarchaeota. The organism is strictly anaerobic and sulfur-dependent with optimal growth at 86 degrees C and pH 5.6. One particular feature is the great metabolic versatility, which is not accompanied by a distinct increase of genome size or information density as compared to other Crenarchaeota. T. tenax is able to grow chemolithoautotrophically (CO2/H-2) as well as chemoorganoheterotrophically in presence of various organic substrates. All pathways for synthesizing the 20 proteinogenic amino acids are present. In addition, two presumably complete gene sets for NADH:quinone oxidoreductase (complex I) were identified in the genome and there is evidence that either NADH or reduced ferredoxin might serve as electron donor. Beside the typical archaeal A(0)A(1)-ATP synthase, a membrane-bound pyrophosphatase is found, which might contribute to energy conservation. Surprisingly, all genes required for dissimilatory sulfate reduction are present, which is confirmed by growth experiments. Mentionable is furthermore, the presence of two proteins (ParA family ATPase, actin-like protein) that might be involved in cell division in Thermoproteales, where the ESCRT system is absent, and of genes involved in genetic competence (DprA, ComF) that is so far unique within Archaea

Genome-Scale Reconstruction and Analysis of the Metabolic Network in the Hyperthermophilic Archaeon Sulfolobus Solfataricus

<div><p>We describe the reconstruction of a genome-scale metabolic model of the crenarchaeon <em>Sulfolobus solfataricus</em>, a hyperthermoacidophilic microorganism. It grows in terrestrial volcanic hot springs with growth occurring at pH 2–4 (optimum 3.5) and a temperature of 75–80°C (optimum 80°C). The genome of <em>Sulfolobus solfataricus P2</em> contains 2,992,245 bp on a single circular chromosome and encodes 2,977 proteins and a number of RNAs. The network comprises 718 metabolic and 58 transport/exchange reactions and 705 unique metabolites, based on the annotated genome and available biochemical data. Using the model in conjunction with constraint-based methods, we simulated the metabolic fluxes induced by different environmental and genetic conditions. The predictions were compared to experimental measurements and phenotypes of <em>S. solfataricus</em>. Furthermore, the performance of the network for 35 different carbon sources known for <em>S. solfataricus</em> from the literature was simulated. Comparing the growth on different carbon sources revealed that glycerol is the carbon source with the highest biomass flux per imported carbon atom (75% higher than glucose). Experimental data was also used to fit the model to phenotypic observations. In addition to the commonly known heterotrophic growth of <em>S. solfataricus</em>, the crenarchaeon is also able to grow autotrophically using the hydroxypropionate-hydroxybutyrate cycle for bicarbonate fixation. We integrated this pathway into our model and compared bicarbonate fixation with growth on glucose as sole carbon source. Finally, we tested the robustness of the metabolism with respect to gene deletions using the method of Minimization of Metabolic Adjustment (MOMA), which predicted that 18% of all possible single gene deletions would be lethal for the organism.</p> </div

Central carbon metabolism in <i>S. solfataricus</i> with flux distribution for growth on glucose.

<p>Solid arrows show the used pathways and their directions. Dashed arrows show available but not used pathways. Arrow size represents predicted flux though the pathways. Enzymes in this pathway: (1) glucose 1-dehydrogenase, (2) gluconolactonase, (3) gluconate dehydratase (4) 2-dehydro-3-deoxygluconokinase, (5) 2-dehydro-3-deoxy-phosphogluconate aldolase, (6) glyceraldehyde-3-phosphate dehydrogenase, (7) phosphoglycerate mutase, (8) phosphopyruvate hydratase, (9) pyruvate kinase, (10) pyruvate synthase, (11) citrate (Si)-synthase, (12) aconitate hydratase, (13) isocitrate dehydrogenase, (14) 2-oxoglutarate synthase, (15) succinate-CoA ligase, (16) succinate dehydrogenase, (17) fumarate hydratase, (18) malate dehydrogenase, (19) glucose 1-dehydrogenase, (20) galactonate dehydratase, (21) 2-keto-3-deoxygluconate aldolase, (22) aldehyde dehydrogenase, (23) glycerate kinase, (24) malate dehydrogenase, (25) phosphoenolpyruvate carboxykinase (GTP), (26) phosphoenolpyruvate carboxylase, (27) pyruvate carboxylase, (28) triose-phosphate isomerase, (29) fructose-bisphosphate aldolase, (30) fructose-bisphosphatase, (31) glucose-6-phosphate isomerase, (32) beta-phosphoglucomutase, (33) glucose-1-phosphate adenylyltransferase, (34) starch synthase, (35) glucan 1,4-alpha-glucosidase, (36) 6-phospho-3-hexuloisomerase, (37) 3-hexulose-6-phosphate synthase, (38) ribose-5-phosphate isomerase, (39) ribose-phosphate diphosphokinase, (40) transketolase, (41) transketolase, (42) sulfite reductase, (43) phosphoadenylyl-sulfate reductase, (44) sulfate adenylyltransferase, (45) adenylyl-sulfate kinase, (46) 3′(2′),5′-bisphosphate nucleotidase, (47) phosphoglycerate kinase, (48) glyceraldehyde-3-phosphate dehydrogenase.</p

Hydroxypropionate-hydroxybutyrate cycle and tricarboxylic acid cycle in <i>S. solfataricus</i> with flux distribution for growth on HCO₃⁻.

<p>Solid arrows show the used pathways and their directions. Dashed arrows show available but not used pathways. Arrow size represents predicted flux though the pathways. Enzymes in the hydroxypropionate-hydroxybutyrate cycle are: (1) acetyl-CoA C-acetyltransferase, (2) acetyl-CoA carboxylase, (3) malonyl-CoA reductase (NADPH), (4) malonic semialdehyde reductase (NADPH), (5) 3-hydroxypropionate-CoA ligase, (6) 3-hydroxypropionyl-CoA dehydratase, (7) acryloyl-CoA reductase (NADPH), (8) propionyl-CoA carboxylase, (9) methylmalonyl-CoA epimerase, (10) methylmalonyl-CoA mutase, (11) succinyl-CoA reductase, (12) succinic semialdehyde reductase (NADPH), (13) 4-hydroxybutyrate-CoA ligase, (14) 4-hydroxybutyryl-CoA dehydratase, (15) enoyl-CoA hydratase, (16) (S)-3-hydroxybutyryl-CoA dehydrogenase (NAD⁺).</p

Scatter plot of the flux distributions during growth on glucose and the phenol-utilizing scenario.

<p>Red dots show significant differences and blue dots show not significant differences between both scenarios. Blue labels describe not significant differences. The numbers indicate the number of involved reactions.</p

Comparison of the properties of the metabolic reconstructions of <i>S. solfataricus</i>, <i>M. barkeri</i>, <i>C. salexigens</i>, <i>M. tuberculosis</i>, and <i>E. coli</i>.

<p>Comparison of the properties of the metabolic reconstructions of <i>S. solfataricus</i>, <i>M. barkeri</i>, <i>C. salexigens</i>, <i>M. tuberculosis</i>, and <i>E. coli</i>.</p

Central carbon metabolism in <i>S. solfataricus</i> with flux distribution for growth on HCO₃⁻.

<p>Solid arrows show the used pathways and their directions. Dashed arrows show available but not used pathways. Arrow size represents predicted flux though the pathways. Enzymes in this pathway: (1) glucose 1-dehydrogenase, (2) gluconolactonase, (3) gluconate dehydratase (4) 2-dehydro-3-deoxygluconokinase, (5) 2-dehydro-3-deoxy-phosphogluconate aldolase, (6) glyceraldehyde-3-phosphate dehydrogenase, (7) phosphoglycerate mutase, (8) phosphopyruvate hydratase, (9) pyruvate kinase, (10) pyruvate synthase, (11) citrate (Si)-synthase, (12) aconitate hydratase, (13) isocitrate dehydrogenase, (14) 2-oxoglutarate synthase, (15) succinate-CoA ligase, (16) succinate dehydrogenase, (17) fumarate hydratase, (18) malate dehydrogenase, (19) glucose 1-dehydrogenase, (20) galactonate dehydratase, (21) 2-keto-3-deoxygluconate aldolase, (22) aldehyde dehydrogenase, (23) glycerate kinase, (24) malate dehydrogenase, (25) phosphoenolpyruvate carboxykinase (GTP), (26) phosphoenolpyruvate carboxylase, ((27) pyruvate carboxylase, (28) triose-phosphate isomerase, (29) fructose-bisphosphate aldolase, (30) fructose-bisphosphatase, (31) glucose-6-phosphate isomerase, (32) beta-phosphoglucomutase, (33) glucose-1-phosphate adenylyltransferase, (34) starch synthase, (35) glucan 1,4-alpha-glucosidase, (36) 6-phospho-3-hexuloisomerase, (37) 3-hexulose-6-phosphate synthase, (38) ribose-5-phosphate isomerase, (39) ribose-phosphate diphosphokinase, (40) transketolase, (41) transketolase, (42) sulfite reductase, (43) phosphoadenylyl-sulfate reductase, (44) sulfate adenylyltransferase, (45) adenylyl-sulfate kinase, (46) 3′(2′),5′-bisphosphate nucleotidase, (47) phosphoglycerate kinase, (48) glyceraldehyde-3-phosphate dehydrogenase.</p