Composition of the lipids of <Emphasis Type="Italic">Nanoarchaeum equitans</Emphasis> and their origin from its host <Emphasis Type="Italic">Ignicoccus</Emphasis> sp. strain KIN4/I

The contents and nature of the membrane lipids of Nanoarchaeum equitans and Ignicoccus sp. strain KIN4/I, grown at 90°C, and Ignicoccus sp. strain KIN4/I, cultivated at its lowest and highest growth temperatures (75°C and 95°C) were analyzed. Both organisms contained very simple and qualitatively identical assemblages of glycerol ether lipids, showing only differences in the amounts of certain components. LC–MS analyses of the total lipid extracts revealed that archaeol and caldarchaeol were the main core lipids. The predominant polar headgroups consisted of one or more sugar residues attached either directly to the core lipid or via a phosphate group. GC–MS analyses of hydrolyzed total lipid extracts revealed that the co-culture of N. equitans and Ignicoccus sp. strain KIN4/I, as well as Ignicoccus sp. strain KIN4/I grown at 90°C, contained phytane and biphytane in a ratio of approximately 4:1. Purified N. equitans cells and Ignicoccus sp. strain KIN4/I cultivated at 75°C and 95°C had a phytane to biphytane ratio of 10:1. Sugar residues were mainly mannose and small amounts of glucose. Consistent ¹³C fractionation patterns of isoprenoid chains of N. equitans and its host indicated that the N. equitans lipids were synthesized in the host cells.     

Gene transfers from nanoarchaeota to an ancestor of diplomonads and parabasalids

Rare evolutionary events, such as lateral gene transfers and gene fusions, may be useful to pinpoint, and correlate the timing of, key branches across the tree of life. For example, the shared possession of a transferred gene indicates a phylogenetic relationship among organismal lineages by virtue of their shared common ancestral recipient. Here, we present phylogenetic analyses of prolyl-tRNA and alanyl-tRNA synthetase genes that indicate lateral gene transfer events to an ancestor of the diplomonads and parabasalids from lineages more closely related to the newly discovered archaeal hyperthermophile Nanoarchaeum equitans (Nanoarchaeota) than to Crenarchaeota or Euryarchaeota. The support for this scenario is strong from all applied phylogenetic methods for the alanyl-tRNA sequences, whereas the phylogenetic analyses of the prolyl-tRNA sequences show some disagreements between methods, indicating that the donor lineage cannot be identified with a high degree of certainty. However, in both trees, the diplomonads and parabasalids branch together within the Archaea, strongly suggesting that these two groups of unicellular eukaryotes, often regarded as the two earliest independent offshoots of the eukaryotic lineage, share a common ancestor to the exclusion of the eukaryotic root. Unfortunately, the phylogenetic analyses of these two aminoacyl-tRNA synthetase genes are inconclusive regarding the position of the diplomonad/parabasalid group within the eukaryotes. Our results also show that the lineage leading to Nanoarchaeota branched off from Euryarchaeota and Crenarchaeota before the divergence of diplomonads and parabasalids, that this unexplored archaeal diversity, currently only represented by the hyperthermophilic organism Nanoarchaeum equitans, may include members living in close proximity to mesophilic eukaryotes, and that the presence of split genes in the Nanoarchaeum genome is a derived feature.     

Structural Basis for a Unique ATP Synthase Core Complex from Nanoarcheaum equitans

ATP synthesis is a critical and universal life process carried out by ATP synthases. Whereas eukaryotic and prokaryotic ATP synthases are well characterized, archaeal ATP synthases are relatively poorly understood. The hyperthermophilic archaeal parasite, Nanoarcheaum equitans, lacks several subunits of the ATP synthase and is suspected to be energetically dependent on its host, Ignicoccus hospitalis. This suggests that this ATP synthase might be a rudimentary machine. Here, we report the crystal structures and biophysical studies of the regulatory subunit, NeqB, the apo-NeqAB, and NeqAB in complex with nucleotides, ADP, and adenylyl-imidodiphosphate (non-hydrolysable analog of ATP). NeqB is ∼20 amino acids shorter at its C terminus than its homologs, but this does not impede its binding with NeqA to form the complex. The heterodimeric NeqAB complex assumes a closed, rigid conformation irrespective of nucleotide binding; this differs from its homologs, which require conformational changes for catalytic activity. Thus, although N. equitans possesses an ATP synthase core A₃B₃ hexameric complex, it might not function as a bona fide ATP synthase.     

Protein trans-splicing and characterization of a split family B-type DNA polymerase from the hyperthermophilic archaeal parasite Nanoarchaeum equitans

Nanoarchaeum equitans family B-type DNA polymerase (Neq DNA polymerase) is encoded by two separate genes, the large gene coding for the N-terminal part (Neq L) of Neq DNA polymerase and the small gene coding for the C-terminal part (Neq S), including a split mini-intein sequence. The two Neq DNA polymerase genes were cloned and expressed in Escherichia coli individually, together (for the Neq C), and as a genetically protein splicing-processed form (Neq P). The protein trans-spliced Neq C was obtained using the heating step at 80 degrees C after the co-expression of the two genes. The protein trans-splicing of the N-terminal and C-terminal parts of Neq DNA polymerase was examined in vitro using the purified Neq L and Neq S. The trans-splicing was influenced mainly by temperature, and occurred only at temperatures above 50 degrees C. The trans-splicing reaction was inhibited in the presence of zinc. Neq S has no catalytic activity and Neq L has lower 3'-->5' exonuclease activity; whereas Neq C and Neq P have polymerase and 3'-->5' exonuclease activities, indicating that both Neq L and Neq S are needed to form the active DNA polymerase that possesses higher proofreading activity. The genetically protein splicing-processed Neq P showed the same properties as the protein trans-spliced Neq C. Our results are the first evidence to show experimentally that natural protein trans-splicing occurs in an archaeal protein, a thermostable protein, and a family B-type DNA polymerase.     

The non-monophyletic origin of the tRNA molecule and the origin of genes only after the evolutionary stage of the last universal common ancestor (LUCA)

A model has been proposed suggesting that the tRNA molecule must have originated by direct duplication of an RNA hairpin structure [Di Giulio, M., 1992. On the origin of the transfer RNA molecule. J. Theor. Biol. 159, 199-214]. A non-monophyletic origin of this molecule has also been theorized [Di Giulio, M., 1999. The non-monophyletic origin of tRNA molecule. J. Theor. Biol. 197, 403-414]. In other words, the tRNA genes evolved only after the evolutionary stage of the last universal common ancestor (LUCA) through the assembly of two minigenes codifying for different RNA hairpin structures, which is what the exon theory of genes suggests when it is applied to the model of tRNA origin. Recent observations strongly corroborate this theorization because it has been found that some tRNA genes are completely separate in two minigenes codifying for the 5' and 3' halves of this molecule [Randau, L., et al., 2005a. Nanoarchaeum equitans creates functional tRNAs from separate genes for their 5'- and 3'-halves. Nature 433, 537-541]. In this paper it is shown that these tRNA genes codifying for the 5' and 3' halves of this molecule are the ancestral form from which the tRNA genes continuously codifying for the complete tRNA molecule are thought to have evolved. This, together with the very existence of completely separate tRNA genes codifying for their 5' and 3' halves, proves a non-monophyletic origin for tRNA genes, as a monophyletic origin would exclude the existence of these genes which have, on the contrary, been observed. Here the polyphyletic origin of genes codifying for proteins is also suggested and discussed. Moreover, a hypothesis is advanced to suggest that the LUCA might have had a fragmented genome made up of RNA and the possibility that 'Paleokaryotes' may exist is outlined. Finally, the characteristic of the indivisibility of homology that these polyphyletic origins seem to remove at the sequence level is discussed.     

Permuted tRNA genes of Cyanidioschyzon merolae, the origin of the tRNA molecule and the root of the Eukarya domain

An evolutionary analysis is conducted on the permuted tRNA genes of Cyanidioschyzon merolae, in which the 5′ half of the tRNA molecule is codified at the 3′ end of the gene and its 3′ half is codified at the 5′ end. This analysis has shown that permuted genes cannot be considered as derived traits but seem to possess characteristics that suggest they are ancestral traits, i.e. they originated when tRNA molecule genes originated for the first time. In particular, if the hypothesis that permuted genes are a derived trait were true, then we should not have been able to observe that the most frequent class of permuted genes is that of the anticodon loop type, for the simple reason that this class would derive by random permutation from a class of non-permuted tRNA genes, which instead is the rarest. This would not explain the high frequency with which permuted tRNA genes with perfectly separate 5′ and 3′ halves were observed. Clearly the mechanism that produced this class of permuted genes would envisage the existence, in an advanced stage of evolution, of minigenes codifying for the 5′ and 3′ halves of tRNAs which were assembled in a permuted way at the origin of the tRNA molecule, thus producing a high frequency of permuted genes of the class here referred. Therefore, this evidence supports the hypothesis that the genes of the tRNA molecule were assembled by minigenes codifying for hairpin-like RNA molecules, as suggested by one model for the origin of tRNA [Di Giulio, M., 1992. On the origin of the transfer RNA molecule. J. Theor. Biol. 159, 199–214; Di Giulio, M., 1999. The non-monophyletic origin of tRNA molecule. J. Theor. Biol. 197, 403–414]. Moreover, the late assembly of the permuted genes of C. merolae, as well as their ancestrality, strengthens the hypothesis of the polyphyletic origins of these genes. Finally, on the basis of the uniqueness and the ancestrality of these permuted genes, I suggest that the root of the Eukarya domain is in the super-ensemble of the Plantae and that the Rhodophyta to which C. merolae belongs are the first line of divergence.     

Superoxide reduction by Nanoarchaeum equitans neelaredoxin, an enzyme lacking the highly conserved glutamate iron ligand

Superoxide reductases (SORs) are antioxidant enzymes present in many prokaryotes, either anaerobes or microaerophiles, which detoxify superoxide by reducing it to hydrogen peroxide. The reaction mechanism involves the diffusion-limited encounter of superoxide with the reduced iron site and concomitant formation of an Fe³⁺–(hydro)peroxo adduct that, upon protonation, leads to the formation of hydrogen peroxide. By the end of this process, a glutamate residue coordinates the ferric ion, acting as a sixth ligand. Although this residue is able to shuttle protons to the intermediate at low pH, it seems to have a minor relevance to the overall reduction mechanism. Nevertheless, this ligand is conserved in most SORs known thus far, with the notable exception of neelaredoxin from Nanoarchaeum equitans. The protein of this organism was cloned and overexpressed, and its spectroscopic characterization revealed distinct pH-equilibrium properties in comparison with those of glutamate-containing SORs. A three-dimensional model of this protein was generated in an effort to identify structural properties that could explain these distinct features. Pulse radiolysis measurements showed that the efficiency of this enzyme in reducing superoxide is comparable to that of glutamate-containing SORs, thus definitely ruling out the requirement for such a ligand in the reduction mechanism. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

<Emphasis Type="Italic">Ignicoccus hospitalis</Emphasis> and <Emphasis Type="Italic">Nanoarchaeum equitans</Emphasis>: ultrastructure,cell–cell interaction,and 3D reconstruction from serial sections of freeze-substituted cells and by electron cryotomography

Ultrastructure and intercellular interaction of Ignicoccus hospitalis and Nanoarchaeum equitans were investigated using two different electron microscopy approaches, by three-dimensional reconstructions from serial sections, and by electron cryotomography. Serial sections were assembled into 3D reconstructions, for visualizing the unusual complexity of I. hospitalis, its huge periplasmic space, the vesiculating cytoplasmic membrane, and the outer membrane. The cytoplasm contains fibres which are reminiscent to a cytoskeleton. Cell division in I. hospitalis is complex, and different to that in Euryarchaeota or Bacteria. An irregular invagination of the cytoplasmic membrane is followed by separation of the two cytoplasms. Simultaneous constriction of cytoplasmic plus outer membrane is not observed. Cells of N. equitans show a classical mode of cell division, by constriction in the mid-plane. Their cytoplasm exhibits two types of fibres, elongated and ring-shaped. Electron micrographs of contact sites between I. hospitalis and N. equitans exhibit two modes of interaction. One is indirect and mediated by thin fibres; in other cells the two cell surfaces are in direct contact. The two membranes of I. hospitalis cells are frequently seen in direct contact, possibly a prerequisite for transporting metabolites or substrates from the cytoplasm of one cell to the other. Rarely, a transport based on cargo vesicles is observed between I. hospitalis and N. equitans.