Rhodothalassium (Rts.) salexigens is a halophilic purple nonsulfur bacterium and the sole species in the genus Rhodothalassium, which is itself the sole genus in the family Rhodothalassiaceae and sole family in the order Rhodothalassiales (class Alphaproteobacteria). The genome of this phylogenetically unique phototroph comprises 3.35 Mb and is highly chimeric, with nearly half of its genes originating from families other than the Rhodothalassiaceae, many of which lack phototrophic species. Photosynthesis genes in Rts. salexigens are not arranged in a typical photosynthesis gene cluster but are scattered across the genome, suggesting an origin from horizontal transfers. Despite an encoded RuBisCO, autotrophy has not been observed in Rts. salexigens, and enzymes that oxidize common inorganic electron donors are not encoded. Phospholipid biosynthesis in Rts. salexigens is restricted, and phosphoglycerolipids are the only phospholipids present in its intracytoplasmic membranes. Rts. salexigens fixes nitrogen using a Mo-containing nitrogenase and uses ammonia despite previous results that indicated it was a glutamate auxotroph. Glycine betaine is the sole osmolyte in Rts. salexigens, and enzymes are encoded that facilitate both its uptake and its biosynthesis from glycine. The genomic data also support chemotactic swimming motility, growth over a range of salinities, and the production of membrane-strengthening hopanoids.

Among the many ice-binding proteins (IBPs) found in microorganisms (bacteria, archaea, fungi and algae), the canonical DUF3494 beta-barrel type is the most common. Until now, little variation has been found in this structure: an initial coil leads into an alpha helix that directs the following coils into a reverse stack, with the final coil ending up next to the initial coil. Here, I show that there exist many bacterial proteins whose AlphaFold-predicted structures deviate from the DUF3494 structure so that they are not recognized as belonging to an existing DUF or Pfam family. In these non-canonical DUF3494 (ncDUF3494) proteins, the number of coils in the alpha helix is highly variable, often being as high as 14. The putative ice-binding sides of each of 13 proteins modeled have a well-aligned row of hydrophilic residues, with spacings that are close to the repeat distance on the ice a-axis. A recombinant protein made for one of the proteins showed that it had ice-binding activity, even in the µg/ml range. The ncDUF3494 proteins appear to be found only in bacteria, the great majority of which live in icy habitats. C-terminal PEP-Cterm motifs, which are rare in DUF3494s, are present in most of the ncDUF3494s, possibly indicating a secretory function. The relatively narrow distribution of ncDUF3494 proteins suggests that they are a later development in DUF3494 evolution.

The novel esterase gene lipP1, which encodes HjEstP1, was discovered in the genome of the extremely halophilic archaeon Haloarcula japonica. A homology search and sequence alignment revealed that HjEstP1 is a member of family IV esterases with conserved GXSXG and HGGG motifs. lipP1 was expressed in its parental strain, and recombinant HjEstP1 was purified and characterized. Optimal pH and temperature of HjEstP1 were 6.0 and > 60 °C, respectively. HjEstP1 showed higher activity with increasing NaCl concentration, and optimal NaCl concentration was > 4.5 M. Furthermore, HjEstP1 preferentially hydrolyzed pNP and glycerol esters with short chain fatty acids. To our knowledge, this is the first report of an esterase from an extremely halophilic archaeon obtained via homologous expression.

The halophilic archaeon Haloarcula hispanica utilizes the sugar alcohols mannitol and sorbitol as carbon and energy sources. Genes, enzymes, and transcriptional regulators involved in uptake and degradation of these sugar alcohols were identified by growth experiments with deletion mutants and enzyme characterization. It is shown that both mannitol and sorbitol are taken up via a single ABC transporter of the CUT1 transporter family. Then, mannitol and sorbitol are oxidized to fructose by two distinct dehydrogenases. Fructose is further phosphorylated to fructose-1-phosphate by a haloarchaeal ketohexokinase, providing the first evidence for a physiological function of ketohexokinase in prokaryotes. Finally, fructose-1-phosphate is phosphorylated via fructose-1-phosphate kinase to fructose-1,6-bisphosphate, which is cleaved to triosephosphates by a Class I fructose-1,6-bisphosphate aldolase. Two distinct transcriptional regulators, acting as activators, have been identified: an IclR-like regulator involved in activating genes for sugar alcohol uptake and oxidation to fructose, and a GfcR-like regulator that likely activates genes involved in the degradation of fructose to pyruvate. This is the first comprehensive analysis of a sugar alcohol degradation pathway in Archaea.

Methanogenic archaea are chemolithotrophic prokaryotes that can reduce carbon dioxide with hydrogen gas to form methane. These microorganisms make a significant contribution to the global carbon cycle, with methanogenic archaea from anoxic environments estimated to contribute > 500 million tons of global methane annually. Archaeal methanogenesis is dependent on the methanofurans; aminomethylfuran containing coenzymes that act as the primary C1 acceptor molecule during carbon dioxide fixation. Although the biosynthetic pathway to the methanofurans has been elucidated, structural adaptations which confer thermotolerance to Mfn enzymes from extremophilic archaea are yet to be investigated. Here we focus on the methanofuran biosynthetic enzyme MfnB, which catalyses the condensation of two molecules of glyceralde-3-phosphate to form 4‑(hydroxymethyl)-2-furancarboxaldehyde-phosphate. In this study, MfnB enzymes from the hyperthermophile Methanocaldococcus jannaschii and the mesophile Methanococcus maripaludis have been recombinantly overexpressed and purified to homogeneity. Thermal unfolding studies, together with steady-state kinetic assays, demonstrate thermoadaptation in the M. jannaschii enzyme. Molecular dynamics simulations have been used to provide a structural explanation for the observed properties. These reveal a greater number of side chain interactions in the M. jannaschii enzyme, which may confer protection from heating effects by enforcing spatial residue constraints.

Knufia petricola is a black fungus that colonizes sun-exposed surfaces as extreme and oligotrophic environments. As ecologically important heterotrophs and biofilm-formers on human-made surfaces, black fungi form one of the most resistant groups of biodeteriorating organisms. Due to its moderate growth rate in axenic culture and available protocols for its transformation and CRISPR/Cas9-mediated genome editing, K.petricola is used for studying the morpho-physiological adaptations shared by extremophilic and extremotolerant black fungi. In this study, the bacteria-derived tetracycline (TET)-dependent promoter (Tet-on) system was implemented to enable controllable gene expression in K. petricola. The functionality i.e., the dose-dependent inducibility of TET-regulated constructs was investigated by using GFP fluorescence, pigment synthesis (melanin and carotenoids) and restored uracil prototrophy as reporters. The newly generated cloning vectors containing the Tet-on construct, and the validated sites in the K. petricola genome for color-selectable or neutral insertion of expression constructs complete the reverse genetics toolbox. One or multiple genes can be expressed on demand from different genomic loci or from a single construct by using 2A self-cleaving peptides, e.g., for localizing proteins and protein complexes in the K.petricola cell or for using K. petricola as host for the expression of heterologous genes.

The heterotrophic cultivation of extremophilic archaea still heavily relies on complex media. However, complex media are associated with unknown composition, high batch-to-batch variability, potential inhibiting and interfering components, as well as regulatory challenges, hampering advancements of extremophilic archaea in genetic engineering and bioprocessing. For Metallosphaera sedula, a widely studied organism for biomining and bioremediation and a potential production host for archaeal ether lipids, efforts to find defined cultivation conditions have still been unsuccessful. This study describes the development of a novel chemically defined growth medium for M. sedula. Initial experiments with commonly used complex casein-derived media sources deciphered Casamino Acids as the most suitable foundation for further development. The imitation of the amino acid composition of Casamino Acids in basal Brock medium delivered the first chemically defined medium. We could further simplify the medium to 5 amino acids based on the respective specific substrate uptake rates. This first defined cultivation medium for M. sedula allows advanced genetic engineering and more controlled bioprocess development approaches for this highly interesting archaeon.

The extremophile bacterium Deinococcus radiodurans is characterized by its ability to survive and sustain its activity at high levels of radiation and is considered an organism that might survive in extraterrestrial environments. In the present work, we studied the combined effects of temperature and chlorine-containing salts, with focus on perchlorate salts which have been detected at high concentrations in Martian regolith, on D. radiodurans activity (CO2 production rates) and viability after incubation in liquid cultures for up to 30 days. Reduced CO2 production capacity and viability was observed at high perchlorate concentrations (up to 10% w/v) during incubation at 0 or 25 °C. Both the metabolic activity and viability were reduced as the perchlorate and chloride salt concentration increased and temperature decreased, and an interactive effect of temperature and salt concentration on the metabolic activity was found. These results indicate the ability of D. radiodurans to remain metabolically active and survive in low temperature environments rich in perchlorate.

Extremophiles and their products have been a major focus of research interest for over 40 years. Through this period, studies of these organisms have contributed hugely to many aspects of the fundamental and applied sciences, and to wider and more philosophical issues such as the origins of life and astrobiology. Our understanding of the cellular adaptations to extreme conditions (such as acid, temperature, pressure and more), of the mechanisms underpinning the stability of macromolecules, and of the subtleties, complexities and limits of fundamental biochemical processes has been informed by research on extremophiles. Extremophiles have also contributed numerous products and processes to the many fields of biotechnology, from diagnostics to bioremediation. Yet, after 40 years of dedicated research, there remains much to be discovered in this field. Fortunately, extremophiles remain an active and vibrant area of research. In the third decade of the twenty-first century, with decreasing global resources and a steadily increasing human population, the world’s attention has turned with increasing urgency to issues of sustainability. These global concerns were encapsulated and formalized by the United Nations with the adoption of the 2030 Agenda for Sustainable Development and the presentation of the seventeen Sustainable Development Goals (SDGs) in 2015. In the run-up to 2030, we consider the contributions that extremophiles have made, and will in the future make, to the SDGs.