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1.  The archaeal origins of the eukaryotic translational system 
Archaea  2005;2(1):1-9.
Among the 78 eukaryotic ribosomal proteins, eleven are specific to Eukarya, 33 are common only to Archaea and Eukarya and 34 are homologous (at least in part) to those of both Bacteria and Archaea. Several other translational proteins are common only to Eukarya and Archaea (e.g., IF2a, SRP19, etc.), whereas others are shared by the three phyla (e.g., EFTu/EF1A and SRP54).
Although this and other analyses strongly support an archaeal origin for a substantial fraction of the eukaryotic translational machinery, especially the ribosomal proteins, there have been numerous unique and ubiquitous additions to the eukaryotic translational system besides the 11 unique eukaryotic ribosomal proteins. These include peptide additions to most of the 67 archaeal homolog proteins, rRNA insertions, the 5.8S RNA and the Alu extension to the SRP RNA. Our comparative analysis of these and other eukaryotic features among the three different cellular phylodomains supports the idea that an archaeal translational system was most likely incorporated by means of endosymbiosis into a host cell that was neither bacterial nor archaeal in any modern sense. Phylogenetic analyses provide support for the timing of this acquisition coinciding with an ancient bottleneck in prokaryotic diversity.
PMCID: PMC2685589  PMID: 16877317
Crenarchaea; eukaryotic origin; evolution; ribosome
2.  Oligomerization of Sulfolobus solfataricus signature amidase is promoted by acidic pH and high temperature 
Archaea  2005;1(6):411-423.
The recombinant amidase from the hyperthermophylic archaeon Sulfolobus solfataricus (SSAM) a signature amidase, was cloned, purified and characterized. The enzyme is active on a large number of aliphatic and aromatic amides over the temperature range 60–95 °C and at pH values between 4.0 and 9.5, with an optimum at pH 5.0. The recombinant enzyme is in the form of a dimer of about 110 kD that reversibly associates into an octamer in a pH-dependent reaction. The pH dependence of the state of association was studied using gel permeation chromatography, analytical ultracentrifugation and dynamic light scattering techniques. At pH 7.0 all three techniques show the presence of two species, in about equal amounts, which is compatible with the existence of a dimeric and an octameric form. In decreasing pH, the dimers formed the octameric species and in increasing pH, the octameric species was converted to dimers. Above pH 8.0, only dimers were present, below pH 3.0 only octamers were present. The association of dimers into octamers decreased in non-polar solvents and increased with temperature. A mutant (Y41C) was obtained that did not show this behavior.
PMCID: PMC2685583  PMID: 16243781
amidase signature; amide metabolism; hyperthermophile
3.  A study of archaeal enzymes involved in polar lipid synthesis linking amino acid sequence information, genomic contexts and lipid composition 
Archaea  2005;1(6):399-410.
Cellular membrane lipids, of which phospholipids are the major constituents, form one of the characteristic features that distinguish Archaea from other organisms. In this study, we focused on the steps in archaeal phospholipid synthetic pathways that generate polar lipids such as archaetidylserine, archaetidylglycerol, and archaetidylinositol. Only archaetidylserine synthase (ASS), from Methanothermobacter thermautotrophicus,has been experimentally identified. Other enzymes have not been fully examined. Through database searching, we detected many archaeal hypothetical proteins that show sequence similarity to members of the CDP alcohol phosphatidyltransferase family, such as phosphatidylserine synthase (PSS), phosphatidylglycerol synthase (PGS) and phosphatidylinositol synthase (PIS) derived from Bacteria and Eukarya. The archaeal hypothetical proteins were classified into two groups, based on the sequence similarity. Members of the first group, including ASS from M. thermautotrophicus, were closely related to PSS. The rough agreement between PSS homologue distribution within Archaea and the experimentally identified distribution of archaetidylserine suggested that the hypothetical proteins are ASSs. We found that an open reading frame (ORF) tends to be adjacent to that of ASS in the genome, and that the order of the two ORFs is conserved. The sequence similarity of phosphatidylserine decarboxylase to the product of the ORF next to the ASS gene, together with the genomic context conservation, suggests that the ORF encodes archaetidylserine decarboxylase, which may transform archaetidylserine to archaetidylethanolamine. The second group of archaeal hypothetical proteins was related to PGS and PIS. The members of this group were subjected to molecular phylogenetic analysis, together with PGSs and PISs and it was found that they formed two distinct clusters in the molecular phylogenetic tree. The distribution of members of each cluster within Archaea roughly corresponded to the experimentally identified distribution of archaetidylglycerol or archaetidylinositol. The molecular phylogenetic tree patterns and the correspondence to the membrane compositions suggest that the two clusters in this group correspond to archaetidylglycerol synthases and archaetidylinositol synthases. No archaeal hypothetical protein with sequence similarity to known phosphatidylcholine synthases was detected in this study.
PMCID: PMC2685579  PMID: 16243780
archaetidylcholine; archaetidylglycerol; archaetidylinositol; archaetidylserine; genomic context; phylogenetic tree
4.  Succession of methanogenic archaea in rice straw incorporated into a Japanese rice field: estimation by PCR-DGGE and sequence analyses 
Archaea  2005;1(6):391-397.
The succession and phylogenetic profiles of methanogenic archaeal communities associated with rice straw decomposition in rice-field soil were studied by polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) analysis followed by 16S rDNA sequencing. Nylon bags containing either leaf sheaths or blades were buried in the plowed layer of a Japanese rice field under drained conditions during the off-crop season and under flooded conditions after transplanting. In addition, rice straw samples that had been buried in the rice field under drained conditions during the off-crop season were temporarily removed during spring plowing and then re-buried in the same rice field under flooded conditions at transplanting. Populations of methanogenic archaea were examined by amplification of the 16S rRNA genes in the DNA extracted from the rice straw samples. No PCR product was produced for samples of leaf sheath or blade prior to burial or after burial under drained conditions, indicating that the methanogen population was very small during decomposition of rice straw under oxic conditions. Many common bands were observed in rice straw samples of leaf sheath and blade during decomposition of rice straw under flooded conditions. Cluster analysis based on DGGE patterns divided methanogenic archaeal communities into two groups before and after the mid-season drainage. Sequence analysis of DGGE bands that were commonly present were closely related to Methanomicrobiales and Rice cluster I. Methanomicrobiales, Rice cluster I and Methanosarcinales were major members before the mid-season drainage, whereas the DGGE bands that characterized methanogenic archaeal communities after the mid-season drainage were closely related to Methanomicrobiales. These results indicate that mid-season drainage affected the methanogenic archaeal communities irrespective of their location on rice straw (sheath and blade) and the previous history of decomposition during the off-crop season.
PMCID: PMC2685580  PMID: 16243779
decomposition; methanogen; paddy field soil; phylogenetic profile
5.  Characterization of the Family I inorganic pyrophosphatase from Pyrococcus horikoshii OT3 
Archaea  2005;1(6):385-389.
A gene encoding for a putative Family inorganic pyrophosphatase (PPase, EC 3.6.1.1) from the hyperthermophilic archaeon Pyrococcus horikoshii OT3 was cloned and the biochemical characteristics of the resulting recombinant protein were examined. The gene (Accession No. 1907) from P. horikoshii showed some identity with other Family I inorganic pyrophosphatases from archaea. The recombinant PPase from P. horikoshii (PhPPase) has a molecular mass of 24.5 kDa, determined by SDS-PAGE. This enzyme specifically catalyzed the hydrolysis of pyrophosphate and was sensitive to NaF. The optimum temperature and pH for PPase activity were 70 °C and 7.5, respectively. The half-life of heat inactivation was about 50 min at 105 °C. The heat stability of PhPPase was enhanced in the presence of Mg2+. A divalent cation was absolutely required for enzyme activity, Mg2+ being most effective; Zn2+, Co2+ and Mn2+ efficiently supported hydrolytic activity in a narrow range of concentrations (0.05– 0.5 mM). The Km for pyrophosphate and Mg2+ were 113 and 303 µM, respectively; and maximum velocity, Vmax, was estimated at 930 U mg–1.
PMCID: PMC2685581  PMID: 16243777
archaea; hyperthermophile
6.  Role of the precorrin 6-X reductase gene in cobamide biosynthesis in Methanococcus maripaludis  
Archaea  2005;1(6):375-384.
In Methanococcus maripaludis strain JJ, deletion of the homolog to cbiJ, which encodes the corrin biosynthetic enzyme precorrin 6-X reductase, yielded an auxotroph that required either cobamide or acetate for good growth. This phenotype closely resembled that of JJ117, a mutant in which tandem repeats were introduced into the region immediately downstream of the homolog of cbiJ. Mutant JJ117 also produced low quantities of cobamides, about 15 nmol g–1 protein or 1–2% of the amount found in wild-type cells. These results confirm the role of the cbiJ homolog in cobamide biosynthesis in the Archaea and suggest the presence of low amounts of a bypass activity in these organisms.
PMCID: PMC2685584  PMID: 16243778
archaea; cbiJ; vitamin B12 biosynthesis
7.  New experimental approaches for investigating interactions between Pyrococcus furiosus carbamate kinase and carbamoyltransferases, enzymes involved in the channeling of thermolabile carbamoyl phosphate 
Archaea  2005;1(6):365-373.
A somewhat neglected but essential aspect of the molecular physiology of hyperthermophiles is the protection of thermolabile metabolites and coenzymes. An example is carbamoyl phosphate (CP), a precursor of pyrimidines and arginine, which is an extremely labile and potentially toxic intermediate. The first evidence for a biologically significant interaction between carbamate kinase (CK) and ornithine carbamoyltransferase (OTC) from Pyrococcus furiosus was provided by affinity electrophoresis and co-immunoprecipitation in combination with cross-linking (Massant et al. 2002). Using the yeast two-hybrid system, Hummel-Dreyer chromatography and isothermal titration calorimetry, we obtained additional concrete evidence for an interaction between CK and OTC, the first evidence for an interaction between CK and aspartate carbamoyltransferase (ATC) and an estimate of the binding constant between CK and ATC.The physical interaction between CK and OTC or ATC may prevent thermodenaturation of CP in the aqueous cytoplasmic environment. Here we emphasize the importance of developing experimental approaches to investigate the mechanism of thermal protection of metabolic intermediates by metabolic channeling and the molecular basis of transient protein–protein interactions in the physiology of hyperthermophiles.
PMCID: PMC2685582  PMID: 16243776
carbamoyl phosphate metabolism; hyperthermophiles; metabolic channeling; protein–protein interactions
8.  Higher-level classification of the Archaea: evolution of methanogenesis and methanogens 
Archaea  2005;1(5):353-363.
We used a phylogenetic approach to analyze the evolution of methanogenesis and methanogens. We show that 23 vertically transmitted ribosomal proteins do not support the monophyly of methanogens, and propose instead that there are two distantly related groups of extant archaea that produce methane, which we have named Class I and Class II. Based on this finding, we subsequently investigated the uniqueness of the origin of methanogenesis by studying both the enzymes of methanogenesis and the proteins that synthesize its specific coenzymes. We conclude that hydrogenotrophic methanogenesis appeared only once during evolution. Genes involved in the seven central steps of the methanogenic reduction of carbon dioxide (CO2) are ubiquitous in methanogens and share a common history. This suggests that, although extant methanogens produce methane from various substrates (CO2, formate, acetate, methylated C-1 compounds), these archaea have a core of conserved enzymes that have undergone little evolutionary change. Furthermore, this core of methanogenesis enzymes seems to originate (as a whole) from the last ancestor of all methanogens and does not appear to have been horizontally transmitted to other organisms or between members of Class I and Class II. The observation of a unique and ancestral form of methanogenesis suggests that it was preserved in two independent lineages, with some instances of specialization or added metabolic flexibility. It was likely lost in the Halobacteriales, Thermoplasmatales and Archaeoglobales. Given that fossil evidence for methanogenesis dates back 2.8 billion years, a unique origin of this process makes the methanogenic archaea a very ancient taxon.
PMCID: PMC2685549  PMID: 15876569
phylogeny; taxonomy
9.  Characterization of an archaeal malic enzyme from the hyperthermophilic archaeon Thermococcus kodakaraensis KOD1 
Archaea  2005;1(5):293-301.
Although the interconversion between C4 and C3 compounds has an important role in overall metabolism, limited information is available on the properties and regulation of enzymes acting on these metabolites in hyperthermophilic archaea. Malic enzyme is one of the enzymes involved in this interconversion, catalyzing the oxidative decarboxylation of malate to pyruvate as well as the reductive carboxylation coupled with NAD(P)H. This study focused on the enzymatic properties and expression profile of an uncharacterized homolog of malic enzyme identified in the genome of a heterotrophic, hyperthermophilic archaeon Thermococcus kodakaraensis KOD1 (Tk-Mae). The amino acid sequence of Tk-Mae was 52–58% identical to those of malic enzymes from bacteria, whereas the similarities to the eukaryotic homologs were lower. Several catalytically important regions and residues were conserved in the primary structure of Tk-Mae. The recombinant protein, which formed a homodimer, exhibited thermostable malic enzyme activity with strict divalent cation dependency. The enzyme preferred NADP+ rather than NAD+, but did not catalyze the decarboxylation of oxaloacetate, unlike the usual NADP-dependent malic enzymes. The apparent Michaelis constant (Km) of Tk-Mae for malate (16.9 mM) was much larger than those of known enzymes, leading to no strong preference for the reaction direction. Transcription of the gene encoding Tk-Mae and intracellular malic enzyme activity in T. kodakaraensis were constitutively weak, regardless of the growth substrates. Possible roles of Tk-Mae are discussed based on these results and the metabolic pathways of T. kodakaraensis deduced from the genome sequence.
PMCID: PMC2685551  PMID: 15876562
carbon metabolism; hyperthermophile; malate; pyruvate; tricarboxylic acid cycle

Results 1-9 (9)