Anaerobic carboxydotrophy is a widespread catabolic trait in bacteria, with two dominant pathways: hydrogenogenic and acetogenic. The marginal mode by direct oxidation to CO₂ using an external e-acceptor has only a few examples. Use of sulfidic sediments from two types of hypersaline lakes in anaerobic enrichments with CO as an e-donor and elemental sulfur as an e-acceptor led to isolation of two pure cultures of anaerobic carboxydotrophs belonging to two genera of sulfur-reducing haloarchaea: Halanaeroarchaeum sp. HSR-CO from salt lakes and Halalkaliarchaeum sp. AArc-CO from soda lakes. Anaerobic growth of extremely halophilic archaea with CO was obligatory depended on the presence of elemental sulfur as the electron acceptor and yeast extract as the carbon source. CO served as a direct electron donor and H₂ was not generated from CO when cells were incubated with or without sulfur. The genomes of the isolates encode a catalytic Ni,Fe-CODH subunit CooS (distantly related to bacterial homologs) and its Ni-incorporating chaperone CooC (related to methanogenic homologs) within a single genomic locus. Similar loci were also present in a genome of the type species of Halalkaliarchaeum closely related to AArc-CO, and the ability for anaerobic sulfur-dependent carboxydotrophy was confirmed for three different strains of this genus. Moreover, similar proteins are encoded in three of the four genomes of recently described carbohydrate-utilizing sulfur-reducing haloarchaea belonging to the genus Halapricum and in two yet undescribed haloarchaeal species. Overall, this work demonstrated for the first time the potential for anaerobic sulfur-dependent carboxydotrophy in extremely halophilic archaea.

The ubiquity of strictly anaerobic sulfur-respiring haloarchaea in hypersaline systems with circumneutral pH has shaken a traditional concept of this group as predominantly aerobic heterotrophs. Here, we demonstrated that this functional group of haloarchaea also has its representatives in hypersaline alkaline lakes. Sediments from various hypersaline soda lakes showed high activity of sulfur reduction only partially inhibited by antibiotics. Eight pure cultures of sulfur-reducing natronoarchaea were isolated from such sediments using formate and butyrate as electron donors and sulfur as an electron acceptor. Unlike strict anaerobic haloarchaea, these novel sulfur-reducing natronoarchaea are facultative anaerobes, whose metabolic capabilities were inferred from cultivation experiments and genomic/proteomic reconstruction. While sharing many physiological traits with strict anaerobic haloarchaea, following metabolic distinctions make these new organisms be successful in both anoxic and aerobic habitats: the recruiting of heme-copper quinol oxidases as terminal electron sink in aerobic respiratory chain and the utilization of formate, hydrogen or short-chain fatty acids as electron donors during anaerobic growth with elemental sulfur. Obtained results significantly advance the emerging concept of halo(natrono)archaea as important players in the anaerobic sulfur and carbon cycling in various salt-saturated habitats.

Methanogenic archaea are major players in the global carbon cycle and in the biotechnology of anaerobic digestion. The phylum Euryarchaeota includes diverse groups of methanogens that are interspersed with non-methanogenic lineages. So far, methanogens inhabiting hypersaline environments have been identified only within the order Methanosarcinales. We report the discovery of a deep phylogenetic lineage of extremophilic methanogens in hypersaline lakes and present analysis of two nearly complete genomes from this group. Within the phylum Euryarchaeota, these isolates form a separate, class-level lineage 'Methanonatronarchaeia' that is most closely related to the class Halobacteria. Similar to the Halobacteria, 'Methanonatronarchaeia' are extremely halophilic and do not accumulate organic osmoprotectants. The high intracellular concentration of potassium implies that 'Methanonatronarchaeia' employ the 'salt-in' osmoprotection strategy. These methanogens are heterotrophic methyl-reducers that use C 1 -methylated compounds as electron acceptors and formate or hydrogen as electron donors. The genomes contain an incomplete and apparently inactivated set of genes encoding the upper branch of methyl group oxidation to CO₂ as well as membrane-bound heterodisulfide reductase and cytochromes. These features differentiate 'Methanonatronarchaeia' from all known methyl-reducing methanogens. The discovery of extremely halophilic, methyl-reducing methanogens related to haloarchaea provides insights into the origin of methanogenesis and shows that the strategies employed by methanogens to thrive in salt-saturating conditions are not limited to the classical methylotrophic pathway.

Hypersaline anoxic habitats harbour numerous novel uncultured archaea whose metabolic and ecological roles remain to be elucidated. Until recently, it was believed that energy generation via dissimilatory reduction of sulfur compounds is not functional at salt saturation conditions. Recent discovery of the strictly anaerobic acetotrophic Halanaeroarchaeum compels to change both this assumption and the traditional view on haloarchaea as aerobic heterotrophs. Here we report on isolation and characterization of a novel group of strictly anaerobic lithoheterotrophic haloarchaea, which we propose to classify as a new genus Halodesulfurarchaeum. Members of this previously unknown physiological group are capable of utilising formate or hydrogen as electron donors and elemental sulfur, thiosulfate or dimethylsulfoxide as electron acceptors. Using genome-wide proteomic analysis we have detected the full set of enzymes required for anaerobic respiration and analysed their substrate-specific expression. Such advanced metabolic plasticity and type of respiration, never seen before in haloarchaea, empower the wide distribution of Halodesulfurarchaeum in hypersaline inland lakes, solar salterns, lagoons and deep submarine anoxic brines. The discovery of this novel functional group of sulfur-respiring haloarchaea strengthens the evidence of their possible role in biogeochemical sulfur cycling linked to the terminal anaerobic carbon mineralisation in so far overlooked hypersaline anoxic habitats.