Biochemical studies have suggested that, in hyperthermophilic archaea, the metabolic conversion of glucose via the ED (Entner-Doudoroff) pathway generally proceeds via a non-phosphorylative variant. A key enzyme of the non-phosphorylating ED pathway of Sulfolobus solfataricus, KDG (2-keto-3-deoxygluconate) aldolase, has been cloned and characterized previously. In the present study, a comparative genomics analysis is described that reveals conserved ED gene clusters in both Thermoproteus tenax and S. solfataricus. The corresponding ED proteins from both archaea have been expressed in Escherichia coli and their specificity has been identified, revealing: (i) a novel type of gluconate dehydratase (gad gene), (ii) a bifunctional 2-keto-3-deoxy-(6-phospho)-gluconate aldolase (kdgA gene), (iii) a 2-keto-3-deoxygluconate kinase (kdgK gene) and, in S. solfataricus, (iv) a GAPN (non-phosphorylating glyceraldehyde-3-phosphate dehydrogenase; gapN gene). Extensive in vivo and in vitro enzymatic analyses indicate the operation of both the semi-phosphorylative and the non-phosphorylative ED pathway in T. tenax and S. solfataricus. The existence of this branched ED pathway is yet another example of the versatility and flexibility of the central carbohydrate metabolic pathways in the archaeal domain.

In response to K+ availability or medium osmolality, the sensor kinase KdpD and the response regulator KdpE control the expression of the kdpFABC operon, coding for the high affinity K+-translocating Kdp ATPase of Escherichia coli. The stimulus for KdpD to undergo autophosphorylation is believed to be a change in turgor or some effect thereof, reflecting the role of K+ as an important cytoplasmic osmotic solute. The membrane-bound sensor kinase KdpD was overproduced as a fusion protein containing six contiguous histidine residues two amino acids before the C terminus. This KdpD-His6 protein was functional in vitro and in vivo. KdpD-His6 was purified from everted membrane vesicles by solubilization with the zwitterionic detergent lauryldimethylamine oxide followed by nickel chelate chromatography and ion exchange chromatography to >99% homogeneity. The solubilized protein was not active with respect to autophosphorylation, but retained the ability to bind 2-azido-ATP. KdpD-His6 was reconstituted into proteoliposomes in a unidirectional inside-out orientation as revealed by ATP accessibility and protease susceptibility. Purified and reconstituted KdpD-His6 exhibited autokinase activity, and the phosphoryl group could be transferred to KdpE. Furthermore, KdpD-His6 was found to be the only protein that mediates dephosphorylation of KdpE approximately P.

b CRISPR (clustered regularly interspaced short palindromic repeats) elements and cas (CRISPR-associated) genes are widespread in Bacteria and Archaea. The CRISPR/Cas system operates as a defense mechanism against mobile genetic elements (i.e., viruses or plasmids). Here, we investigate seven CRISPR loci in the genome of the crenarchaeon Thermoproteus tenax that include spacers with significant similarity not only to archaeal viruses but also to T. tenax genes. The analysis of CRISPR RNA (crRNA) transcription reveals transcripts of a length between 50 and 130 nucleotides, demonstrating the processing of larger crRNA precursors. The organization of identified cas genes resembles CRISPR/Cas subtype I-A, and the core cas genes are shown to be arranged on two polycistronic transcripts: cascis (cas4, cas1/2, and csa1) and cascade (csa5, cas7, cas5a, cas3, cas3=, and cas8a2). Changes in the environmental parameters such as UV-light exposure or high ionic strength modulate cas gene transcription. Two reconstitution protocols were established for the production of two discrete multipartite Cas protein complexes that correspond to their operonic gene arrangement. These data provide insights into the specialized mechanisms of an archaeal CRISPR/ Cas system and allow selective functional analyses of Cas protein complexes in the future.

Fructose-1,6-bisphosphate (FBP) aldolase activity has been detected previously in several Archaea. However, no obvious orthologs of the bacterial and eucaryal Class I and II FBP aldolases have yet been identified in sequenced archaeal genomes. Based on a recently described novel type of bacterial aldolase, we report on the identification and molecular characterization of the first archaeal FBP aldolases. We have analyzed the FBP aldolases of two hyperthermophilic Archaea, the facultatively heterotrophic Crenarchaeon Thermoproteus tenax and the obligately heterotrophic Euryarchaeon Pyrococcus furiosus. For enzymatic studies the fba genes of T. tenax and P. furiosus were expressed in Escherichia coli. The recombinant FBP aldolases show preferred substrate specificity for FBP in the catabolic direction and exhibit metal-independent Class I FBP aldolase activity via a Schiff-base mechanism. Transcript analyses reveal that the expression of both archaeal genes is induced during sugar fermentation. Remarkably, the fbp gene of T. tenax is co-transcribed with the pfp gene that codes for the reversible PP i -dependent phosphofructokinase. As revealed by phylogenetic analyses, orthologs of the T. tenax and P. furiosus enzyme appear to be present in almost all sequenced archaeal genomes, as well as in some bacterial genomes, strongly suggesting that this new enzyme family represents the typical archaeal FBP aldolase. Because this new family shows no significant sequence similarity to classical Class I and II enzymes, a new name is proposed, archaeal type Class I FBP aldolases (FBP aldolase Class IA).Fructose-1,6-bisphosphate (FBP) 1 aldolase (EC 4.1.2.13) catalyzes the reversible aldol condensation of glyceraldehyde 3-phosphate (GAP) and dihydroxyacetone phosphate (DHAP) yielding FBP. The enzyme fulfills an amphibolic function being involved in catabolic (glycolysis) as well as anabolic pathways (gluconeogenesis and Calvin cycle). In spite of this central function in carbohydrate metabolism, up to now no archaeal genes coding for the respective enzyme activities have been analyzed.Two distinct classes of FBP aldolases occur in nature, which differ in their enzymatic mechanisms (1-4). Class I FBP aldolases form a Schiff-base intermediate between the carbonyl substrate (FBP and DHAP) and the ⑀-amino group of the active site lysine residue and are inactivated by borohydride (NaBH 4 ), whereas Class II FBP aldolases depend on divalent metal ions to stabilize the carbanion intermediate and are, therefore, inhibited by EDTA. Class II enzymes of bacterial and eucaryal origin generally form dimers with a subunit molecular mass of ϳ40 kDa, whereas the Class I pendants are heterogeneous. Eucaryal aldolases are homomeric tetramers with a subunit molecular mass of ϳ40 kDa, and for bacterial enzymes oligomeric arrangements from monomer to decamer and subunit molecular masses of 27-40 kDa have been described (5, 6).Sequence comparisons of Class I and II FBP aldolases revealed no detectable sequence homology, suggesting convergent evolut...

Pyruvate kinase (PK; EC 2.7.1.40) of Thermoproteus tenax was purified to homogeneity, and its coding gene was cloned and expressed in Escherichia coli. It represents a homomeric tetramer with a molecular mass of 49 kDa per subunit. PK exhibits positive binding cooperativity with respect to phosphoenolpyruvate and metal ions such as Mg 2؉ and Mn 2؉ . Heterotropic effects, as commonly found for PKs from bacterial and eucaryal sources, could not be detected. The enzyme does not depend on K ؉ ions. Heterotrophically grown cells exhibit specific activity of PK four times higher than autotrophically grown cells. Since the mRNA level of the PK coding gene is also accordingly higher in heterotrophic cells, we conclude that the PK activity is adjusted to growth conditions mainly on the transcript level. The enzymic properties of the PK and the regulation of its expression are discussed with respect to the physiological framework given by the T. tenax-specific variant of the Embden-Meyerhof-Parnas pathway. T. tenax PK shows moderate overall sequence similarity (25 to 40% identity) to its bacterial and eucaryal pendants. Phylogenetic analyses of the known PK sequences result in a dichotomic tree topology that divides the enzymes into two major PK clusters, probably diverged by an early gene duplication event. The phylogenetic divergence is paralleled by a striking phenotypic differentiation of PKs: PKs of cluster I, which occur in eucaryal cytoplasm, some gamma proteobacteria, and low-GC grampositive bacteria, are only active in the presence of fructose-1,6-bisphosphate or other phosphorylated sugars, whereas PKs of cluster II, found in various bacterial phyla, plastids, and in Archaea, show activity without effectors but are commonly regulated by the energy charge of the cell.As shown by several investigations, Archaea use the Embden-Meyerhof-Parnas (EMP) pathway for carbon metabolism, and in some archaeal species (the genera Pyrococcus, Thermococcus, Desulfurococcus, and Thermoproteus) the catabolic EMP pathway represents the main route for glucose degradation (37, 40). So far, in thermophilic Archaea only variants of the classical EMP pathway have been found. Common to these variants is the irreversible oxidation step transforming glyceraldehyde-3-phosphate directly to 3-phosphoglycerate by a ferredoxin-dependent glyceraldehyde-3-phosphate oxidoreductase (GAP:FdOR) found in Pyrococcus, Thermococcus, and Desulfurococcus strains (37, 43) or by a nonphosphorylating NAD ϩ -dependent glyceraldehyde-3-phosphate dehydrogenase (GAPN) found in Thermoproteus tenax (4,15,38). These enzymes substitute for the combined action of conventional phosphorylating-but reversible-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and 3-phosphoglycerate kinase, which, however, allows ATP formation.We chose T. tenax, a hyperthermophilic, facultatively heterotrophic member of the kingdom Crenarchaeota, as an object for regulatory studies on archaeal carbohydrate metabolism. T. tenax is able to grow chemolithoautotrophically on CO 2 , H 2 and S ...