Deep Conservation of Histone Variants in Thermococcales Archaea.

Histones are ubiquitous in eukaryotes where they assemble into nucleosomes, binding and wrapping DNA to form chromatin. One process to modify chromatin and regulate DNA accessibility is the replacement of histones in the nucleosome with paralogous variants. Histones are also present in archaea but whether and how histone variants contribute to the generation of different physiologically relevant chromatin states in these organisms remains largely unknown. Conservation of paralogs with distinct properties can provide prima facie evidence for defined functional roles. We recently revealed deep conservation of histone paralogs with different properties in the Methanobacteriales, but little is known experimentally about these histones. In contrast, the two histones of the model archaeon Thermococcus kodakarensis, HTkA and HTkB, have been examined in some depth, both in vitro and in vivo. HTkA and HTkB exhibit distinct DNA-binding behaviors and elicit unique transcriptional responses when deleted. Here, we consider the evolution of HTkA/B and their orthologs across the order Thermococcales. We find histones with signature HTkA- and HTkB-like properties to be present in almost all Thermococcales genomes. Phylogenetic analysis indicates the presence of one HTkA- and one HTkB-like histone in the ancestor of Thermococcales and long-term maintenance of these two paralogs throughout Thermococcales diversification. Our results support the notion that archaea and eukaryotes have convergently evolved histone variants that carry out distinct adaptive functions. Intriguingly, we also detect more highly diverged histone-fold proteins, related to those found in some bacteria, in several Thermococcales genomes. The functions of these bacteria-type histones remain unknown, but structural modeling suggests that they can form heterodimers with HTkA/B-like histones.

Phylogenetic Diversity of Lhr Proteins and Biochemical Activities of the Thermococcales aLhr2 DNA/RNA Helicase.

Helicase proteins are known to use the energy of ATP to unwind nucleic acids and to remodel protein-nucleic acid complexes. They are involved in almost every aspect of DNA and RNA metabolisms and participate in numerous repair mechanisms that maintain cellular integrity. The archaeal Lhr-type proteins are SF2 helicases that are mostly uncharacterized. They have been proposed to be DNA helicases that act in DNA recombination and repair processes in Sulfolobales and Methanothermobacter. In Thermococcales, a protein annotated as an Lhr2 protein was found in the network of proteins involved in RNA metabolism. To investigate this, we performed in-depth phylogenomic analyses to report the classification and taxonomic distribution of Lhr-type proteins in Archaea, and to better understand their relationship with bacterial Lhr. Furthermore, with the goal of envisioning the role(s) of aLhr2 in Thermococcales cells, we deciphered the enzymatic activities of aLhr2 from Thermococcus barophilus (Tbar). We showed that Tbar-aLhr2 is a DNA/RNA helicase with a significant annealing activity that is involved in processes dependent on DNA and RNA transactions.

H2-dependent formate production by hyperthermophilic Thermococcales: an alternative to sulfur reduction for reducing-equivalents disposal.

Removal of reducing equivalents is an essential catabolic process for all microorganisms to maintain their internal redox balance. The electron disposal by chemoorganotrophic Thermococcales generates H2 by proton reduction or H2S in presence of S0. Although in the absence of S0 growth of these (hyper)thermopiles was previously described to be H2-limited, it remains unclear how Thermococcales could be present in H2-rich S0-depleted habitats. Here, we report that 12 of the 47 strains tested, distributed among all three orders of Thermococcales, could grow without S0 at 0.8 mM dissolved H2 and that tolerance to H2 was always associated with formate production. Two conserved gene clusters coding for a formate hydrogenlyase (FHL) and a putative formate dehydrogenase-NAD(P)H-oxidoreductase were only present in H2-dependent formate producers, and were both systematically associated with a formate dehydrogenase and a formate transporter. As the reaction involved in this alternative pathway for disposal of reducing equivalents was close to thermodynamic equilibrium, it was strongly controlled by the substrates-products concentration ratio even in the presence of S0. Moreover, experimental data and thermodynamic modelling also demonstrated that H2-dependent CO2 reduction to formate could occur within a large temperature range in contrasted hydrothermal systems, suggesting it could also provide an adaptive advantage.

The Effect of Crowding on Protein Stability, Rigidity, and High Pressure Sensitivity in Whole Cells.

In live cells, high concentrations up to 300-400 mg/mL, as in Eschericia coli (Ellis, R. J. Curr. Opin. Struct. Biol. 2001, 11, 114) are achieved which have effects on their proper functioning. However, in many experiments only individual parts of the cells as proteins or membranes are studied in order to get insight into these specific components and to avoid the high complexity of whole cells, neglecting by the way the influence of crowding. In the present study, we investigated cells of the order of Thermococcales, which are known to live under extreme conditions, in their intact form and after cell lysis to extract the effect of crowding on the molecular dynamics of the proteome and of water molecules. We found that some parameters characterizing the dynamics within the cells seem to be intrinsic to the cell type, as flexibility typical for the proteome, others are more specific to the cellular environment, as bulk water's residence time and some fractions of particles participating to the different motions, which make the lysed cells' dynamics similar to the one of another Thermococcale adapted to live under high hydrostatic pressure. In contrast to studies on the impact of crowding on pure proteins we show here that the release of crowding constraints on proteins leads to an increase in the rigidity and a decrease in the high pressure sensitivity. In a way similar to high pressure adaptation in piezophiles, the hydration water layer is decreased for the lysed cells, demonstrating a first link between protein adaptation and the impact of crowding or osmolytes on proteins.

Greigite nanocrystals produced by hyperthermophilic archaea of Thermococcales order.

Interactions between hyperthermophilic archaea and minerals occur in hydrothermal deep-sea vents, one of the most extreme environments for life on Earth. These interactions occur in the internal pores and at surfaces of active hydrothermal chimneys. In this study, we show that, at 85°C, Thermococcales, the predominant hyperthermophilic microorganisms inhabiting hot parts of hydrothermal deep-sea vents, produce greigite nanocrystals (Fe3S4) on extracellular polymeric substances, and that an amorphous iron phosphate acts as a precursor phase. Greigite, although a minor component of chimneys, is a recognized catalyst for CO2 reduction thus implying that Thermococcales may influence the balance of CO2 in hydrothermal ecosystems. We propose that observation of greigite nanocrystals on extracellular polymeric substances could provide a signature of hyperthermophilic life in hydrothermal deep-sea vents.

Physiological, metabolic and biotechnological features of extremely thermophilic microorganisms.

The current upper thermal limit for life as we know it is approximately 120°C. Microorganisms that grow optimally at temperatures of 75°C and above are usually referred to as 'extreme thermophiles' and include both bacteria and archaea. For over a century, there has been great scientific curiosity in the basic tenets that support life in thermal biotopes on earth and potentially on other solar bodies. Extreme thermophiles can be aerobes, anaerobes, autotrophs, heterotrophs, or chemolithotrophs, and are found in diverse environments including shallow marine fissures, deep sea hydrothermal vents, terrestrial hot springs-basically, anywhere there is hot water. Initial efforts to study extreme thermophiles faced challenges with their isolation from difficult to access locales, problems with their cultivation in laboratories, and lack of molecular tools. Fortunately, because of their relatively small genomes, many extreme thermophiles were among the first organisms to be sequenced, thereby opening up the application of systems biology-based methods to probe their unique physiological, metabolic and biotechnological features. The bacterial genera Caldicellulosiruptor, Thermotoga and Thermus, and the archaea belonging to the orders Thermococcales and Sulfolobales, are among the most studied extreme thermophiles to date. The recent emergence of genetic tools for many of these organisms provides the opportunity to move beyond basic discovery and manipulation to biotechnologically relevant applications of metabolic engineering. WIREs Syst Biol Med 2017, 9:e1377. doi: 10.1002/wsbm.1377 For further resources related to this article, please visit the WIREs website.

Starting up microbial enhanced oil recovery.

This chapter gives the reader a practical introduction into microbial enhanced oil recovery (MEOR) including the microbial production of natural gas from oil. Decision makers who consider the use of one of these technologies are provided with the required scientific background as well as with practical advice for upgrading an existing laboratory in order to conduct microbiological experiments. We believe that the conversion of residual oil into natural gas (methane) and the in situ production of biosurfactants are the most promising approaches for MEOR and therefore focus on these topics. Moreover, we give an introduction to the microbiology of oilfields and demonstrate that in situ microorganisms as well as injected cultures can help displace unrecoverable oil in place (OIP). After an initial research phase, the enhanced oil recovery (EOR) manager must decide whether MEOR would be economical. MEOR generally improves oil production but the increment may not justify the investment. Therefore, we provide a brief economical assessment at the end of this chapter. We describe the necessary state-of-the-art scientific equipment to guide EOR managers towards an appropriate MEOR strategy. Because it is inevitable to characterize the microbial community of an oilfield that should be treated using MEOR techniques, we describe three complementary start-up approaches. These are: (i) culturing methods, (ii) the characterization of microbial communities and possible bio-geochemical pathways by using molecular biology methods, and (iii) interfacial tension measurements. In conclusion, we hope that this chapter will facilitate a decision on whether to launch MEOR activities. We also provide an update on relevant literature for experienced MEOR researchers and oilfield operators. Microbiologists will learn about basic principles of interface physics needed to study the impact of microorganisms living on oil droplets. Last but not least, students and technicians trying to understand processes in oilfields and the techniques to examine them will, we hope, find a valuable source of information in this review.

An expansion of age constraints for microbial clades that lack a conventional fossil record using phylogenomic dating.

Most microbial taxa lack a conventional microfossil or biomarker record, and so we currently have little information regarding how old most microbial clades and their associated traits are. Building on the previously published oxygen age constraint, two new age constraints are proposed based on the ability of microbial clades to metabolize chitin and aromatic compounds derived from lignin. Using the archaeal domain of life as a test case, phylogenetic analyses, along with published metabolic and genetic data, showed that members of the Halobacteriales and Thermococcales are able to metabolize chitin. Ancestral state reconstruction combined with phylogenetic analysis of the genes underlying chitin degradation predicted that the ancestors of these two groups were also likely able to metabolize chitin or chitin-related compounds. These two clades were therefore assigned a maximum age of 1.0 Ga (when chitin likely first appeared). Similar analyses also predicted that the ancestor to the Sulfolobus solfataricus-Sulfolobus islandicus clade was able to metabolize phenol using catechol dioxygenase, so this clade was assigned a maximum age of 475 Ma. Inferred ages of archaeal clades using relaxed molecular clocks with the new age constraints were consistent with those inferred with the oxygen age constraints. This work expands our current toolkit to include Paleoproterozoic, Neoproterozoic, and Paleozoic age constraints, and should aid in our ability to phylogenetically reconstruct the antiquity of a wide array of microbial clades and their associated morphological and biogeochemical traits, spanning deep geologic time. Such hypotheses-although built upon evolutionary inferences-are fundamentally testable.

Evolution of mal ABC transporter operons in the Thermococcales and Thermotogales.

BACKGROUND: The mal genes that encode maltose transporters have undergone extensive lateral transfer among ancestors of the archaea Thermococcus litoralis and Pyrococcus furiosus. Bacterial hyperthermophiles of the order Thermotogales live among these archaea and so may have shared in these transfers. The genome sequence of Thermotoga maritima bears evidence of extensive acquisition of archaeal genes, so its ancestors clearly had the capacity to do so. We examined deep phylogenetic relationships among the mal genes of these hyperthermophiles and their close relatives to look for evidence of shared ancestry. RESULTS: We demonstrate that the two maltose ATP binding cassette (ABC) transporter operons now found in Tc. litoralis and P. furiosus (termed mal and mdx genes, respectively) are not closely related to one another. The Tc. litoralis and P. furiosus mal genes are most closely related to bacterial mal genes while their respective mdx genes are archaeal. The genes of the two mal operons in Tt. maritima are not related to genes in either of these archaeal operons. They are highly similar to one another and belong to a phylogenetic lineage that includes mal genes from the enteric bacteria. A unique domain of the enteric MalF membrane spanning proteins found also in these Thermotogales MalF homologs supports their relatively close relationship with these enteric proteins. Analyses of genome sequence data from other Thermotogales species, Fervidobacterium nodosum, Thermosipho melanesiensis, Thermotoga petrophila, Thermotoga lettingae, and Thermotoga neapolitana, revealed a third apparent mal operon, absent from the published genome sequence of Tt. maritima strain MSB8. This third operon, mal3, is more closely related to the Thermococcales' bacteria-derived mal genes than are mal1 and mal2. F. nodosum, Ts. melanesiensis, and Tt. lettingae have only one of the mal1-mal2 paralogs. The mal2 operon from an unknown species of Thermotoga appears to have been horizontally acquired by a Thermotoga species that had only mal1. CONCLUSION: These data demonstrate that the Tc. litoralis and P. furiosus mdx maltodextrin transporter operons arose in the Archaea while their mal maltose transporter operons arose in a bacterial lineage, but not the same lineage as the two maltose transporter operons found in the published Tt. maritima genome sequence. These Tt. maritima maltose transporters are phylogenetically and structurally similar to those found in enteric bacteria and the mal2 operon was horizontally transferred within the Thermotoga lineage. Other Thermotogales species have a third mal operon that is more closely related to the bacterial Thermococcales mal operons, but the data do not support a recent horizontal sharing of that operon between these groups.