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