Fabrice Confalonieri

The complete genome of the crenarchaeon Sulfolobus solfataricus P2

The genome of the crenarchaeon Sulfolobus solfataricus P2 contains 2,992,245 bp on a single chromosome and encodes 2,977 proteins and many RNAs. One-third of the encoded proteins have no detectable homologs in other sequenced genomes. Moreover, 40% appear to be archaeal-specific, and only 12% and 2.3% are shared exclusively with bacteria and eukarya, respectively. The genome shows a high level of plasticity with 200 diverse insertion sequence elements, many putative nonautonomous mobile elements, and evidence of integrase-mediated insertion events. There are also long clusters of regularly spaced tandem repeats. Different transfer systems are used for the uptake of inorganic and organic solutes, and a wealth of intracellular and extracellular proteases, sugar, and sulfur metabolizing enzymes are encoded, as well as enzymes of the central metabolic pathways and motility proteins. The major metabolic electron carrier is not NADH as in bacteria and eukarya but probably ferredoxin. The essential components required for DNA replication, DNA repair and recombination, the cell cycle, transcriptional initiation and translation, but not DNA folding, show a strong eukaryal character with many archaeal-specific features. The results illustrate major differences between crenarchaea and euryarchaea, especially for their DNA replication mechanism and cell cycle processes and their translational apparatus.

Silk fibroin: Structural implications of a remarkable amino acid sequence

The amino acid sequence of the heavy chain of Bombyx mori silk fibroin was derived from the gene sequence. The 5,263‐residue (391‐kDa) polypeptide chain comprises 12 low‐complexity “crystalline” domains made up of Gly–X repeats and covering 94% of the sequence; X is Ala in 65%, Ser in 23%, and Tyr in 9% of the repeats. The remainder includes a nonrepetitive 151‐residue header sequence, 11 nearly identical copies of a 43‐residue spacer sequence, and a 58‐residue C‐terminal sequence. The header sequence is homologous to the N‐terminal sequence of other fibroins with a completely different crystalline region. In Bombyx mori, each crystalline domain is made up of subdomains of ∼70 residues, which in most cases begin with repeats of the GAGAGS hexapeptide and terminate with the GAAS tetrapeptide. Within the subdomains, the Gly–X alternance is strict, which strongly supports the classic Pauling–Corey model, in which β‐sheets pack on each other in alternating layers of Gly/Gly and X/X contacts. When fitting the actual sequence to that model, we propose that each subdomain forms a β‐strand and each crystalline domain a two‐layered β‐sandwich, and we suggest that the β‐sheets may be parallel, rather than antiparallel, as has been assumed up to now. Proteins 2001;44:119–122.

Nanoarchaea: representatives of a novel archaeal phylum or a fast-evolving euryarchaeal lineage related to Thermococcales?

BackgroundCultivable archaeal species are assigned to two phyla - the Crenarchaeota and the Euryarchaeota - by a number of important genetic differences, and this ancient split is strongly supported by phylogenetic analysis. The recently described hyperthermophile Nanoarchaeum equitans, harboring the smallest cellular genome ever sequenced (480 kb), has been suggested as the representative of a new phylum - the Nanoarchaeota - that would have diverged before the Crenarchaeota/Euryarchaeota split. Confirming the phylogenetic position of N. equitans is thus crucial for deciphering the history of the archaeal domain.ResultsWe tested the placement of N. equitans in the archaeal phylogeny using a large dataset of concatenated ribosomal proteins from 25 archaeal genomes. We indicate that the placement of N. equitans in archaeal phylogenies on the basis of ribosomal protein concatenation may be strongly biased by the coupled effect of its above-average evolutionary rate and lateral gene transfers. Indeed, we show that different subsets of ribosomal proteins harbor a conflicting phylogenetic signal for the placement of N. equitans. A BLASTP-based survey of the phylogenetic pattern of all open reading frames (ORFs) in the genome of N. equitans revealed a surprisingly high fraction of close hits with Euryarchaeota, notably Thermococcales. Strikingly, a specific affinity of N. equitans and Thermococcales was strongly supported by phylogenies based on a subset of ribosomal proteins, and on a number of unrelated molecular markers.ConclusionWe suggest that N. equitans may more probably be the representative of a fast-evolving euryarchaeal lineage (possibly related to Thermococcales) than the representative of a novel and early diverging archaeal phylum.

Genome analysis and genome-wide proteomics of Thermococcus gammatolerans, the most radioresistant organism known amongst the Archaea

BackgroundThermococcus gammatolerans was isolated from samples collected from hydrothermal chimneys. It is one of the most radioresistant organisms known amongst the Archaea. We report the determination and annotation of its complete genome sequence, its comparison with other Thermococcales genomes, and a proteomic analysis.ResultsT. gammatolerans has a circular chromosome of 2.045 Mbp without any extra-chromosomal elements, coding for 2,157 proteins. A thorough comparative genomics analysis revealed important but unsuspected genome plasticity differences between sequenced Thermococcus and Pyrococcus species that could not be attributed to the presence of specific mobile elements. Two virus-related regions, tgv1 and tgv2, are the only mobile elements identified in this genome. A proteogenome analysis was performed by a shotgun liquid chromatography-tandem mass spectrometry approach, allowing the identification of 10,931 unique peptides corresponding to 951 proteins. This information concurrently validates the accuracy of the genome annotation. Semi-quantification of proteins by spectral count was done on exponential- and stationary-phase cells. Insights into general catabolism, hydrogenase complexes, detoxification systems, and the DNA repair toolbox of this archaeon are revealed through this genome and proteome analysis.ConclusionsThis work is the first archaeal proteome investigation done at the stage of primary genome annotation. This archaeon is shown to use a large variety of metabolic pathways even under a rich medium growth condition. This proteogenomic study also indicates that the high radiotolerance of T. gammatolerans is probably due to proteins that remain to be characterized rather than a larger arsenal of known DNA repair enzymes.

The nature of the last universal ancestor and the root of the tree of life, still open questions

The nature of the last universal ancestor to all extent cellular organisms and the rooting of the universal tree of life are fundamental questions which can now be addressed by molecular evolutionists. Several scenarios have been proposed during the last years, based on the phylogenies of ribosomal RNA and of duplicated proteins, which suggest that the last universal ancestor was either an RNA progenote or an hyperthermophilic prokaryote. We discuss these hypotheses in the light of new data on the evolution of DNA metabolizing enzymes and of contradictions between different protein phylogenies. We conclude that the last universal ancestor was a member of the DNA world already containing several DNA polymerases and DNA topoisomerases. Furthermore, we criticize current data which suggest that the rooting of the universal tree of life is located in the eubacterial branch and we conclude that both rooting the universal tree and the nature of the last universal ancestor are still open questions.

Identification of the gene encoding archeal‐specific DNA‐binding proteins of the Sac10b family

In the 1980s, several abundant, small and basic DNA-binding proteins were isolated from extreme thermophilic archaea of the genus Sulfolobus (Grote et al., 1986, Biochim Biophys Acta, 873: 405–413). They were grouped into three classes according to their molecular sizes (7, 8 and 10 kDa respectively) (Djick and Reinhardt, 1986, Bacterial Chromatin. Gualerzi and Pon (eds). New York: Springer-Verlag, pp. 185–218). Each of these classes comprises several members that can be discriminated according to their electrophoretic properties: 7a-e, 8a,b and 10a,b. Biochemical and structural characterization of several of these various putative archaeal histone-like proteins, especially those of the 7 kDa and the 10 kDa families (Lurz et al., 1986, EMBO J 5: 3715–3721), was first performed in the mid-1980s by Reinhardt and co-workers. Further work concentrated exclusively on two proteins of the 7 kDa group, the protein Sso7d from Sulfolobus solfataricus and Sac7d from Sulfolobus acidocaldarius. Their genes were cloned and sequenced, and their three-dimensional structures were determined by both NMR and X-ray crystallography (Agabeck et al.,1998, Nature Struct Biol 5: 579–584; Gao et al., 1998, Nature Struct Biol 5: 782– 786; Robinson et al., 1998, Nature 392: 202–205). These proteins have no sequence similarity with archaeal histones or histone-like proteins discovered in other archaea (Grayling et al., 1994, Syst Appl Microbiol 16: 582–590). Both Sac7d and Sso7d contain extensive beta-sheet structures and bind double-stranded DNA with micromolar affinity in a non-co-operative manner (Baumann et al., 1994, Nature Struct Biol 1: 808–819; Lundbäck et al., 1998, J Mol Biol 276: 775–786). Binding of proteins of this family leads to significant bending and unwinding of DNA and induces negative supercoils, suggesting a role in DNA compaction in Sulfolobus (Lopez-Garcia et al., 1998; Nucleic Acids Res 26: 2322–2328). In contrast, the 8 kDa and the 10 kDa proteins have been largely ignored since their discovery, and the genes encoding these proteins are unknown. However, we recently noticed that short internal peptide sequences of 17 and 28 amino acids were reported for the proteins 8b and 10b of S. acidocaldarius (Djick and Reinhardt, 1986, Bacterial Chromatin. Gualerzi and Pon (eds). New York: Springer-Verlag, pp. 185–218). Using these sequences as queries, we searched for the putative genes encoding these two proteins concurrently and found in Sulfolobus shibatae a gene encoding a protein with a region of very high similarity to the peptide isolated from Sac10b. This gene (ssh10b) is predicted to encode a protein of 10 578 Da with a deduced isoelectric point of 11.24. Interestingly, ssh10b is located at the 58 end of the S. shibatae reverse gyrase topR gene (Jaxel et al., 1996, Nucleic Acids Res 24: 4668–4675; Fig. 1). Whereas proteins Sac7d and Sso7d have no homologues in other archaea whose genomes have been completely sequenced, we found an homologue of ssh10b in all archaeal genomes (see alignment in Fig. 2), but not in either bacteria or eukarya. In addition, S. Shibatae was found to have a paralogue of ssh10b that, surprisingly, overlaps the last 245 nucleotides of the topR gene in the reverse orientation (Fig. 1). This gene (ssh10b2) was predicted to encode a protein of 10 258 Da with an isoelectric point of 10.23 (Fig. 2). This protein probably does not correspond to Ssh10a because, according to Djick and Reinhardt (1986), Sac10a and Sac10b are unrelated in terms of amino-acid sequence. A dendrogram of all archaeal protein homologues to Ssh10b shows a clear-cut separation between the protein Ssh10b2 from S. Shibatae and all other archaeal 10b proteins (not shown). Archaeal proteins of the Sac10b family are the first known, small, basic DNA-binding proteins that are both ubiquitous in archae and specific to this domain. This suggests an important physiological role for these proteins. Preliminary biochemical and structural analyses have shown that Sac10b is a dimer in solution and has a greater affinity for DNA than its two counterparts of 7 and 8 kDa (Djick and Reinhardt, 1986, Bacterial Chromatin. Gualerzi and Pon (eds). New York: Springer-Verlag, pp. 185–218). It was shown by electron microscopy that the fixation of Sac10b to DNA is co-operative and that it can envelop two strands of duplex into a helical protein structure in a RecA-like manner (Lurz et al., 1986, Molecular Microbiology (1999) 32(3), 669–670

SAV, an archaebacterial gene with extensive homology to a family of highly conserved eukaryotic ATPases

Nucleotide sequencing of a region of the hyperthermophilic archaebacterium Sulfolobus acidocaldarius allowed us to identify an open reading frame of 780 amino acids strikingly similar to a family of eukaryotic ATPases, involved in a variety of biological functions. Sequence analysis of the predicted polypeptide revealed 63 to 66% similarity with S. cerevisiae CDC 48p and its related genes in amphibians (p97ATPase) and mammals (Valosin Containing Protein, VCP), all possibly involved in the regulation of the cell cycle. The finding of an archaebacterial equivalent of these proteins with a high degree of similarity suggests that it represents the same gene in these various species. The new archaebacterial ORF, called SAV (S. acidocaldarius VCP-like) exhibited the usual signature of all members of the family, a highly conserved domain of about 200 amino acids, which is duplicated. Thus, apart from the VCP-like proteins, SAV also appeared similar, although less clearly, to other ATPases, members of the family, involved in vesicle-mediated transport (NSF, Sec18p), peroxysome assembly (PAS1p), and gene expression in yeast (SUG1p) and in human immunodeficiency virus (TBP-1). Finally, the discovery of the archaebacterial gene could enlighten not only the evolutionary relationships between the members of this complex ATPase family, but also the cellular function of these proteins, that is presently obscure.

Recovery of ionizing-radiation damage after high doses of gamma ray in the hyperthermophilic archaeon Thermococcus gammatolerans

The recently discovered hyperthermophilic and radioresistant archaeon Thermococcus gammatolerans is of great interest to compare and contrast the impact of its physiology on radioresistance and its ability to repair damaged chromosomes after exposure to gamma irradiation with radioresistant bacteria. We showed that, in contrast to other organisms, cell survival was not modified by the cellular growth phase under optimal growth conditions but nutrient-limited conditions did affect the T. gammatolerans radioresistance. We determined the first kinetics of damaged DNA recovery in an archaeon after exposure to massive doses of gamma irradiation and compared the efficiency of chromosomal DNA repair according to the cellular growth phase, nutrient availability and culture conditions. Chromosomal DNA repair kinetics showed that stationary phase cells reconstitute disrupted chromosomes more rapidly than exponential phase cells. Our data also revealed that this radioresistant archaeon was proficient to reconstitute shattered chromosomes either slowly or rapidly without any loss of viability. These results suggest that rapid DNA repair is not required for the extreme radioresistance of T. gammatolerans.

Evolution of DNA Topoisomerases and DNA Polymerases: a Perspective from Archaea

Summary We review our present knowledge on DNA topoisomerase and DNA polymerase evolution, with emphasis on information obtained by studying these enzymes in Archaea. Two archaeal DNA topoisomerase genes have been sequenced: the reverse gyrase from Sulfolobus acidocaldarius turns out to be a completely novel type of enzyme, likely originating from the fusion of a helicase and a type I DNA topoisomerase, whereas a novobiocin sensitive type II DNA topoisomerase from Haloferax is closely related to bacterial DNA gyrases. Beside reverse gyrase, we recently isolated from Sulfolobus shibatae a type II DNA topoisomerase which has no gyrase activity and exhibits the pattern of drug sensitivity specific for its eukaryotic counterpart. Several archaeal DNA polymerase genes have now been sequenced: they all belong to the DNA polymerase B family, together with the three eucaryal DNA replicases and Escherichia coli DNA pol II, a repair enzyme. All DNA polymerases from family B described so far are sensitive to aphidicolin. We present recent data suggesting that archaeal DNA polymerases resistant to aphidicolin also belong to family B. Phylogenetic trees of DNA topoisomerases and DNA polymerases turn out to be all noncongruent with the rRNA tree and exhibit different and unusual topologies. We discuss several hypotheses which can explain this observation and their implications concerning the nature of the last common ancestor to the three domains. We conclude that this universal ancestor was probably an advanced member of the DNA world that already contained several DNA polymerases and DNA topoisomerases.

Genome-Wide Transcriptional Response of the Archaeon Thermococcus gammatolerans to Cadmium

Thermococcus gammatolerans, the most radioresistant archaeon known to date, is an anaerobic and hyperthermophilic sulfur-reducing organism living in deep-sea hydrothermal vents. Knowledge of mechanisms underlying archaeal metal tolerance in such metal-rich ecosystem is still poorly documented. We showed that T. gammatolerans exhibits high resistance to cadmium (Cd), cobalt (Co) and zinc (Zn), a weaker tolerance to nickel (Ni), copper (Cu) and arsenate (AsO4) and that cells exposed to 1 mM Cd exhibit a cellular Cd concentration of 67 µM. A time-dependent transcriptomic analysis using microarrays was performed at a non-toxic (100 µM) and a toxic (1 mM) Cd dose. The reliability of microarray data was strengthened by real time RT-PCR validations. Altogether, 114 Cd responsive genes were revealed and a substantial subset of genes is related to metal homeostasis, drug detoxification, re-oxidization of cofactors and ATP production. This first genome-wide expression profiling study of archaeal cells challenged with Cd showed that T. gammatolerans withstands induced stress through pathways observed in both prokaryotes and eukaryotes but also through new and original strategies. T. gammatolerans cells challenged with 1 mM Cd basically promote: 1) the induction of several transporter/permease encoding genes, probably to detoxify the cell; 2) the upregulation of Fe transporters encoding genes to likely compensate Cd damages in iron-containing proteins; 3) the induction of membrane-bound hydrogenase (Mbh) and membrane-bound hydrogenlyase (Mhy2) subunits encoding genes involved in recycling reduced cofactors and/or in proton translocation for energy production. By contrast to other organisms, redox homeostasis genes appear constitutively expressed and only a few genes encoding DNA repair proteins are regulated. We compared the expression of 27 Cd responsive genes in other stress conditions (Zn, Ni, heat shock, γ-rays), and showed that the Cd transcriptional pattern is comparable to other metal stress transcriptional responses (Cd, Zn, Ni) but not to a general stress response.