Structure and function of archaeal histones.

The genomes of all organisms throughout the tree of life are compacted and organized in chromatin by association of chromatin proteins. Eukaryotic genomes encode histones, which are assembled on the genome into octamers, yielding nucleosomes. Post-translational modifications of the histones, which occur mostly on their N-terminal tails, define the functional state of chromatin. Like eukaryotes, most archaeal genomes encode histones, which are believed to be involved in the compaction and organization of their genomes. Instead of discrete multimers, in vivo data suggest assembly of "nucleosomes" of variable size, consisting of multiples of dimers, which are able to induce repression of transcription. Based on these data and a model derived from X-ray crystallography, it was recently proposed that archaeal histones assemble on DNA into "endless" hypernucleosomes. In this review, we discuss the amino acid determinants of hypernucleosome formation and highlight differences with the canonical eukaryotic octamer. We identify archaeal histones differing from the consensus, which are expected to be unable to assemble into hypernucleosomes. Finally, we identify atypical archaeal histones with short N- or C-terminal extensions and C-terminal tails similar to the tails of eukaryotic histones, which are subject to post-translational modification. Based on the expected characteristics of these archaeal histones, we discuss possibilities of involvement of histones in archaeal transcription regulation.

Complementary Tendencies in the Use of Regulatory Elements (Transcription Factors, Sigma Factors, and Riboswitches) in Bacteria and Archaea.

In prokaryotes, the key players in transcription initiation are sigma factors and transcription factors that bind to DNA to modulate the process, while premature transcription termination at the 5' end of the genes is regulated by attenuation and, in particular, by attenuation associated with riboswitches. In this study, we describe the distribution of these regulators across phylogenetic groups of bacteria and archaea and find that their abundance not only depends on the genome size, as previously described, but also varies according to the phylogeny of the organism. Furthermore, we observed a tendency for organisms to compensate for the low frequencies of a particular type of regulatory element (i.e., transcription factors) with a high frequency of other types of regulatory elements (i.e., sigma factors). This study provides a comprehensive description of the more abundant COG, KEGG, and Rfam families of transcriptional regulators present in prokaryotic genomes.IMPORTANCE In this study, we analyzed the relationship between the relative frequencies of the primary regulatory elements in bacteria and archaea, namely, transcription factors, sigma factors, and riboswitches. In bacteria, we reveal a compensatory behavior for transcription factors and sigma factors, meaning that in phylogenetic groups in which the relative number of transcription factors was low, we found a tendency for the number of sigma factors to be high and vice versa. For most of the phylogenetic groups analyzed here, except for Firmicutes and Tenericutes, a clear relationship with other mechanisms was not detected for transcriptional riboswitches, suggesting that their low frequency in most genomes does not constitute a significant impact on the global variety of transcriptional regulatory elements in prokaryotic organisms.

High level of intergenera gene exchange shapes the evolution of haloarchaea in an isolated Antarctic lake.

Deep Lake in Antarctica is a globally isolated, hypersaline system that remains liquid at temperatures down to -20 °C. By analyzing metagenome data and genomes of four isolates we assessed genome variation and patterns of gene exchange to learn how the lake community evolved. The lake is completely and uniformly dominated by haloarchaea, comprising a hierarchically structured, low-complexity community that differs greatly to temperate and tropical hypersaline environments. The four Deep Lake isolates represent distinct genera (∼85% 16S rRNA gene similarity and ∼73% genome average nucleotide identity) with genomic characteristics indicative of niche adaptation, and collectively account for ∼72% of the cellular community. Network analysis revealed a remarkable level of intergenera gene exchange, including the sharing of long contiguous regions (up to 35 kb) of high identity (∼100%). Although the genomes of closely related Halobacterium, Haloquadratum, and Haloarcula (>90% average nucleotide identity) shared regions of high identity between species or strains, the four Deep Lake isolates were the only distantly related haloarchaea to share long high-identity regions. Moreover, the Deep Lake high-identity regions did not match to any other hypersaline environment metagenome data. The most abundant species, tADL, appears to play a central role in the exchange of insertion sequences, but not the exchange of high-identity regions. The genomic characteristics of the four haloarchaea are consistent with a lake ecosystem that sustains a high level of intergenera gene exchange while selecting for ecotypes that maintain sympatric speciation. The peculiarities of this polar system restrict which species can grow and provide a tempo and mode for accentuating gene exchange.

Role of 4-hydroxybutyrate-CoA synthetase in the CO2 fixation cycle in thermoacidophilic archaea.

Metallosphaera sedula is an extremely thermoacidophilic archaeon that grows heterotrophically on peptides and chemolithoautotrophically on hydrogen, sulfur, or reduced metals as energy sources. During autotrophic growth, carbon dioxide is incorporated into cellular carbon via the 3-hydroxypropionate/4-hydroxybutyrate cycle (3HP/4HB). To date, all of the steps in the pathway have been connected to enzymes encoded in specific genes, except for the one responsible for ligation of coenzyme A (CoA) to 4HB. Although several candidates for this step have been identified through bioinformatic analysis of the M. sedula genome, none have been shown to catalyze this biotransformation. In this report, transcriptomic analysis of cells grown under strict H(2)-CO(2) autotrophy was consistent with the involvement of Msed_0406 and Msed_0394. Recombinant versions of these enzymes catalyzed the ligation of CoA to 4HB, with similar affinities for 4HB (K(m) values of 1.9 and 1.5 mm for Msed_0406 and Msed_0394, respectively) but with different rates (1.69 and 0.22 µmol × min(-1) × mg(-1) for Msed_0406 and Msed_0394, respectively). Neither Msed_0406 nor Msed_0394 have close homologs in other Sulfolobales, although low sequence similarity is not unusual for acyl-adenylate-forming enzymes. The capacity of these two enzymes to use 4HB as a substrate may have arisen from simple modifications to acyl-adenylate-forming enzymes. For example, a single amino acid substitution (W424G) in the active site of the acetate/propionate synthetase (Msed_1353), an enzyme that is highly conserved among the Sulfolobales, changed its substrate specificity to include 4HB. The identification of the 4-HB CoA synthetase now completes the set of enzymes comprising the 3HP/4HB cycle.

Archaeal DNA polymerase D but not DNA polymerase B is required for genome replication in Thermococcus kodakarensis.

Three evolutionarily distinct families of replicative DNA polymerases, designated polymerase B (Pol B), Pol C, and Pol D, have been identified. Members of the Pol B family are present in all three domains of life, whereas Pol C exists only in Bacteria and Pol D exists only in Archaea. Pol B enzymes replicate eukaryotic chromosomal DNA, and as members of the Pol B family are present in all Archaea, it has been assumed that Pol B enzymes also replicate archaeal genomes. Here we report the construction of Thermococcus kodakarensis strains with mutations that delete or inactivate key functions of Pol B. T. kodakarensis strains lacking Pol B had no detectable loss in viability and no growth defects or changes in spontaneous mutation frequency but had increased sensitivity to UV irradiation. In contrast, we were unable to introduce mutations that inactivated either of the genes encoding the two subunits of Pol D. The results reported establish that Pol D is sufficient for viability and genome replication in T. kodakarensis and argue that Pol D rather than Pol B is likely the replicative DNA polymerase in this archaeon. The majority of Archaea contain Pol D, and, as discussed, if Pol D is the predominant replicative polymerase in Archaea, this profoundly impacts hypotheses for the origin(s), evolution, and distribution of the different DNA replication enzymes and systems now employed in the three domains of life.

MCM structure and mechanics: what we have learned from archaeal MCM.

Minichromosome maintenance (MCM) complexes have been identified as the primary replicative helicases responsible for unwinding DNA for genome replication. The focus of this chapter is to discuss the current structural and functional understanding of MCMs and their role at origins of replication, which are based mostly on the studies of MCM proteins and MCM complexes from archaeal genomes.

Genomic analysis of Acidianus hospitalis W1 a host for studying crenarchaeal virus and plasmid life cycles.

The Acidianus hospitalis W1 genome consists of a minimally sized chromosome of about 2.13 Mb and a conjugative plasmid pAH1 and it is a host for the model filamentous lipothrixvirus AFV1. The chromosome carries three putative replication origins in conserved genomic regions and two large regions where non-essential genes are clustered. Within these variable regions, a few orphan orfB and other elements of the IS200/607/605 family are concentrated with a novel class of MITE-like repeat elements. There are also 26 highly diverse vapBC antitoxin-toxin gene pairs proposed to facilitate maintenance of local chromosomal regions and to minimise the impact of environmental stress. Complex and partially defective CRISPR/Cas/Cmr immune systems are present and interspersed with five vapBC gene pairs. Remnants of integrated viral genomes and plasmids are located at five intron-less tRNA genes and several non-coding RNA genes are predicted that are conserved in other Sulfolobus genomes. The putative metabolic pathways for sulphur metabolism show some significant differences from those proposed for other Acidianus and Sulfolobus species. The small and relatively stable genome of A. hospitalis W1 renders it a promising candidate for developing the first Acidianus genetic systems.

Biosynthetic capacity, metabolic variety and unusual biology in the CPR and DPANN radiations.

Candidate phyla radiation (CPR) bacteria and DPANN (an acronym of the names of the first included phyla) archaea are massive radiations of organisms that are widely distributed across Earth's environments, yet we know little about them. Initial indications are that they are consistently distinct from essentially all other bacteria and archaea owing to their small cell and genome sizes, limited metabolic capacities and often episymbiotic associations with other bacteria and archaea. In this Analysis, we investigate their biology and variations in metabolic capacities by analysis of approximately 1,000 genomes reconstructed from several metagenomics-based studies. We find that they are not monolithic in terms of metabolism but rather harbour a diversity of capacities consistent with a range of lifestyles and degrees of dependence on other organisms. Notably, however, certain CPR and DPANN groups seem to have exceedingly minimal biosynthetic capacities, whereas others could potentially be free living. Understanding of these microorganisms is important from the perspective of evolutionary studies and because their interactions with other organisms are likely to shape natural microbiome function.

Reverse gyrase: an unusual DNA manipulator of hyperthermophilic organisms.

Reverse gyrase is the only DNA topoisomerase capable of introducing positive supercoiling into DNA molecules. This unique activity reflects a distinctive arrangement of the protein, which is composed of a topoisomerase IA module fused to a domain containing sequence motives typical of helicases; however, reverse gyrase works neither like a canonical topoisomerase IA nor like a helicase. Extensive genomic analysis has shown that reverse gyrase is present in all organisms living above 70 degrees C and in some of those living at 60- 70 degrees C, but is invariably absent in organisms living at mesophilic temperatures. For its peculiar distribution and biochemical activity, the enzyme has been suggested to play a role in maintenance of genome stability at high temperature. We review here recent phylogenetic, biochemical and structural data on reverse gyrase and discuss the possible role of this enzyme in the biology of hyperthermophilic organisms.

The interplay between nucleoid organization and transcription in archaeal genomes.

The archaeal genome is organized by either eukaryotic-like histone proteins or bacterial-like nucleoid-associated proteins. Recent studies have revealed novel insights into chromatin dynamics and their effect on gene expression in archaeal model organisms. In this Progress article, we discuss the interplay between chromatin proteins, such as histones and Alba, and components of the basal transcription machinery, as well as between chromatin structure and gene-specific transcription factors in archaea. Such an interplay suggests that chromatin might have a role in regulating gene expression on both a global and a gene-specific level. Moreover, several archaeal transcription factors combine a global gene regulatory role with an architectural role, thus contributing to chromatin organization and compaction, as well as gene expression. We describe the emerging principles underlying how these factors cooperate in nucleoid structuring and gene regulation.

Sequencing of seven haloarchaeal genomes reveals patterns of genomic flux.

We report the sequencing of seven genomes from two haloarchaeal genera, Haloferax and Haloarcula. Ease of cultivation and the existence of well-developed genetic and biochemical tools for several diverse haloarchaeal species make haloarchaea a model group for the study of archaeal biology. The unique physiological properties of these organisms also make them good candidates for novel enzyme discovery for biotechnological applications. Seven genomes were sequenced to ∼20×coverage and assembled to an average of 50 contigs (range 5 scaffolds-168 contigs). Comparisons of protein-coding gene compliments revealed large-scale differences in COG functional group enrichment between these genera. Analysis of genes encoding machinery for DNA metabolism reveals genera-specific expansions of the general transcription factor TATA binding protein as well as a history of extensive duplication and horizontal transfer of the proliferating cell nuclear antigen. Insights gained from this study emphasize the importance of haloarchaea for investigation of archaeal biology.

The genome characteristics and predicted function of methyl-group oxidation pathway in the obligate aceticlastic methanogens, Methanosaeta spp.

In this work, we report the complete genome sequence of an obligate aceticlastic methanogen, Methanosaeta harundinacea 6Ac. Genome comparison indicated that the three cultured Methanosaeta spp., M. thermophila, M. concilii and M. harundinacea 6Ac, each carry an entire suite of genes encoding the proteins involved in the methyl-group oxidation pathway, a pathway whose function is not well documented in the obligately aceticlastic methanogens. Phylogenetic analysis showed that the methyl-group oxidation-involving proteins, Fwd, Mtd, Mch, and Mer from Methanosaeta strains cluster with the methylotrophic methanogens, and were not closely related to those from the hydrogenotrophic methanogens. Quantitative PCR detected the expression of all genes for this pathway, albeit ten times lower than the genes for aceticlastic methanogenesis in strain 6Ac. Western blots also revealed the expression of fwd and mch, genes involved in methyl-group oxidation. Moreover, (13)C-labeling experiments suggested that the Methanosaeta strains might use the pathway as a methyl oxidation shunt during the aceticlastic metabolism. Because the mch mutants of Methanosarcina barkeri or M. acetivorans failed to grow on acetate, we suggest that Methanosaeta may use methyl-group oxidation pathway to generate reducing equivalents, possibly for biomass synthesis. An fpo operon, which encodes an electron transport complex for the reduction of CoM-CoB heterodisulfide, was found in the three genomes of the Methanosaeta strains. However, an incomplete protein complex lacking the FpoF subunit was predicted, as the gene for this protein was absent. Thus, F(420)H(2) was predicted not to serve as the electron donor. In addition, two gene clusters encoding the two types of heterodisulfide reductase (Hdr), hdrABC, and hdrED, respectively, were found in the three Methanosaeta genomes. Quantitative PCR determined that the expression of hdrED was about ten times higher than hdrABC, suggesting that hdrED plays a major role in aceticlastic methanogenesis.

The impact of extremophiles on structural genomics (and vice versa).

The advent of the complete genome sequences of various organisms in the mid-1990s raised the issue of how one could determine the function of hypothetical proteins. While insight might be obtained from a 3D structure, the chances of being able to predict such a structure is limited for the deduced amino acid sequence of any uncharacterized gene. A template for modeling is required, but there was only a low probability of finding a protein closely-related in sequence with an available structure. Thus, in the late 1990s, an international effort known as structural genomics (SG) was initiated, its primary goal to "fill sequence-structure space" by determining the 3D structures of representatives of all known protein families. This was to be achieved mainly by X-ray crystallography and it was estimated that at least 5,000 new structures would be required. While the proteins (genes) for SG have subsequently been derived from hundreds of different organisms, extremophiles and particularly thermophiles have been specifically targeted due to the increased stability and ease of handling of their proteins, relative to those from mesophiles. This review summarizes the significant impact that extremophiles and proteins derived from them have had on SG projects worldwide. To what extent SG has influenced the field of extremophile research is also discussed.

Biochemical characterization of DNA-binding proteins from Pyrobaculum aerophilum and Aeropyrum pernix.

Several representatives of the Crenarchaeal branch of the Archaea contain highly abundant, small, positively charged proteins exemplified by the Sso7d protein from Sulfolobus solfataricus. These proteins bind to DNA in a non-sequence-specific manner. Using publicly available genomic sequence information, we identified a second class of small Crenarchaeal DNA-binding proteins represented by the Pyrobaculum aerophilum open reading frame 3192-encoded (Pae3192) protein and its paralogs. We investigated the biochemical properties of the Pae3192 protein and an orthologous protein (Ape1322b) from Aeropyrum pernix in side-by-side experiments with the Sso7d protein. We demonstrate that the recombinant Ape1322b, Pae3192 and Sso7d proteins bind to DNA and that the DNA-protein complexes formed are slightly different for each protein. We show that like Sso7d, Pae3192 constrains negative supercoils in DNA. In addition, we show that all three proteins raise the melting temperature of duplex DNA upon binding. Finally, we present the equilibrium affinity constants and kinetic association constants of each protein for single-stranded and double-stranded DNA.

Metabolism of halophilic archaea.

In spite of their common hypersaline environment, halophilic archaea are surprisingly different in their nutritional demands and metabolic pathways. The metabolic diversity of halophilic archaea was investigated at the genomic level through systematic metabolic reconstruction and comparative analysis of four completely sequenced species: Halobacterium salinarum, Haloarcula marismortui, Haloquadratum walsbyi, and the haloalkaliphile Natronomonas pharaonis. The comparative study reveals different sets of enzyme genes amongst halophilic archaea, e.g. in glycerol degradation, pentose metabolism, and folate synthesis. The carefully assessed metabolic data represent a reliable resource for future system biology approaches as it also links to current experimental data on (halo)archaea from the literature.

Genome dynamics in a natural archaeal population.

Evolutionary processes that give rise to, and limit, diversification within strain populations can be deduced from the form and distribution of genomic heterogeneity. The extent of genomic change that distinguishes the acidophilic archaeon Ferroplasma acidarmanus fer1 from an environmental population of the same species from the same site, fer1(env), was determined by comparing the 1.94-megabase (Mb) genome sequence of the isolate with that reconstructed from 8 Mb of environmental sequence data. The fer1(env) composite sequence sampled approximately 92% of the isolate genome. Environmental sequence data were also analyzed to reveal genomic heterogeneity within the coexisting, coevolving fer1(env) population. Analyses revealed that transposase movement and the insertion and loss of blocks of novel genes of probable phage origin occur rapidly enough to give rise to heterogeneity in gene content within the local population. Because the environmental DNA was derived from many closely related individuals, it was possible to quantify gene sequence variability within the population. All but a few gene variants show evidence of strong purifying selection. Based on the small number of distinct sequence types and their distribution, we infer that the population is undergoing frequent genetic recombination, resulting in a mosaic genome pool that is shaped by selection. The larger genetic potential of the population relative to individuals within it and the combinatorial process that results in many closely related genome types may provide the basis for adaptation to environmental fluctuations.

Biochemical evidence of a physical interaction between Sulfolobus solfataricus B-family and Y-family DNA polymerases.

The hyper-thermophilic archaeon Sulfolobus solfataricus possesses two functional DNA polymerases belonging to the B-family (Sso DNA pol B1) and to the Y-family (Sso DNA pol Y1). Sso DNA pol B1 recognizes the presence of uracil and hypoxanthine in the template strand and stalls synthesis 3-4 bases upstream of this lesion ("read-ahead" function). On the other hand, Sso DNA pol Y1 is able to synthesize across these and other lesions on the template strand. Herein we report evidence that Sso DNA pol B1 physically interacts with DNA pol Y1 by surface plasmon resonance measurements and immuno-precipitation experiments. The region of DNA pol B1 responsible for this interaction has been mapped in the central portion of the polypeptide chain (from the amino acid residue 482 to 617), which includes an extended protease hyper-sensitive linker between the N- and C-terminal modules (amino acid residues Asn482-Ala497) and the alpha-helices forming the "fingers" sub-domain (alpha-helices R, R' and S). These results have important implications for understanding the polymerase-switching mechanism on the damaged template strand during genome replication in S. solfataricus.

Characterization of an ATP-dependent DNA ligase from the acidophilic archaeon "Ferroplasma acidarmanus" Fer1.

Analysis of the genome of "Ferroplasma acidarmanus" Fer1, an archaeon that is an extreme acidophile, identified an open reading frame encoding a putative ATP-dependent DNA ligase, which we termed FaLig. The deduced amino acid sequence of FaLig contains 595 amino acids, with a predicted molecular mass of 67.8 kDa. "F. acidarmanus" Fer1 is classified as a Euryarchaeote, but phylogenetic analysis using amino acid sequences showed that FaLig is more similar to DNA ligases from Crenarchaeota, suggesting that lateral transfer of these genes has occurred among archaea. The gene sequence encoding FaLig was cloned into a bacterial expression vector harbouring an upstream His-tag to aid purification. Conditions for expression and purification from Escherichia coli were identified and recombinant FaLig was confirmed to be an ATP-dependent DNA ligase. Optimal conditions for nick-joining by the protein were pH 6-7, 0.5 mM ATP, in the presence of either Mg(2+) or Mn(2+). Using a range of nicked, double-stranded nucleic acids, ligation was detected with the same substrates as previously determined for other DNA ligases. Although FaLig is the DNA ligase from one of the most extreme acidophilic organism yet studied, this characterization suggests that its biochemical mechanism is analogous to that of enzymes from other cellular systems.

The Tmc complex from Desulfovibrio vulgaris hildenborough is involved in transmembrane electron transfer from periplasmic hydrogen oxidation.

Three membrane-bound redox complexes have been reported in Desulfovibrio spp., whose genes are not found in the genomes of other sulfate reducers such as Desulfotalea psycrophila and Archaeoglobus fulgidus. These complexes contain a periplasmic cytochrome c subunit of the cytochrome c(3) family, and their presence in these organisms probably correlates with the presence of a pool of periplasmic cytochromes c(3), also absent in the two other sulfate reducers. In this work we report the isolation and characterization of the first of such complexes, Tmc from D. vulgaris Hildenborough, which is associated with the tetraheme type II cytochrome c(3). The isolated Tmc complex contains four subunits, including the TpIIc(3) (TmcA), an integral membrane cytochrome b (TmcC), and two cytoplasmically predicted proteins, an iron-sulfur protein (TmcB) and a tryptophan-rich protein (TmcD). Spectroscopic studies indicate the presence of eight hemes c and two hemes b in the complex pointing to an alpha(2)betagammadelta composition (TmcA(2)BCD). EPR analysis reveals the presence of a [4Fe4S](3+) center and up to three other iron-sulfur centers in the cytoplasmic subunit. Nearly full reduction of the redox centers in the Tmc complex could be obtained upon incubation with hydrogenase/TpIc(3), supporting the role of this complex in transmembrane transfer of electrons resulting from periplasmic oxidation of hydrogen.