The genus Natronolimnobius, currently including four species, is a member of the order Natrialbales, class Halobacteria, and consists of obligately alkaliphilic and extremely halophilic members found exclusively in highly alkaline hypersaline soda lakes. The species were classified into this genus mostly based on phylogenetic analysis of the 16S rRNA gene. However, a more advanced phylogenomic reconstruction based on 122 conserved single-copy archaeal protein markers clearly indicates a polyphyletic origin of the species included into this genus, thus warranting its reclassification into three separate genera. We therefore propose to transfer Nlb. innermongolicus (type strain N-1311) to a new genus Natronolimnohabitans as Nlh. innermongolicus comb. nov. and to transfer Nlb. aegyptiacus (type strain JW/NM-HA 15) and Nlb. sulfurireducens (type strain AArc1) to a new genus Natrarchaeobaculum as Nbl. aegyptiacum comb. nov. and Nbl. sulfurireducens comb. nov. The phylogenomic differentiation of these four species is also supported by the ANI/AAI distances and unique phenotypes. The most important physiological differences includes a previously unreported ability for cellulose and xylan utilization in Nlb. baerhuensis, thermophily in Nbl. aegyptiacus and anaerobic sulfur respiration in Nbl. sulfurireducens. We further present an emended description of Natronolimnobius baerhuensis.@en

Nine pure cultures of neutrophilic haloaloarchaea capable of anaerobic growth by carbohydrate dependent sulfur respiration were isolated from hypersaline lakes in southwestern Siberia and southern Russia. According to phylogenomic analysis the isolates were closely related to each other and formed a new species within the genus Halapricum (family Haloarculaceae). They have three types of catabolism: fermentative, resulting in H2 formation; anaerobic respiration using sulfur compounds as e-acceptors and aerobic respiration. Apart from elemental sulfur, all isolates can also use three different sulfoxides as acceptors and the type strain also grows with thiosulfate, reducing it partially to sulfide and sulfite. All strains utilized sugars and glycerol as the e-donors and C source for anaerobic growth and some can also grow with alpha-glucans, such as starch and dextrins. The major respiratory menaquinones are MK-8:8 and MK-8:7, but 5–19% consists of ‘‘thermoplasmata” quinones (MMK-8:8 and MMK-8:7), whose occurrence in haloarchaea is unprecedented. On the basis of their unique physiological properties and results of phylogenomic analysis, the isolates are suggested to be classified into a novel species Halapricum desulfuricans sp. nov. (type strain HSR12-2T = JCM 34032T = UNIQEM U1001T).@en

Strain M27-SA2 was isolated from the deep-sea salt-saturated anoxic lake Medee, which represents one of the most hostile extreme environments on our planet. On the basis of physiological studies and phylogenetic positioning this extremely halophilic euryarchaeon belongs to a novel genus 'Halanaeroarchaeum' within the family Halobacteriaceae. All members of this genus cultivated so far are strict anaerobes using acetate as the sole carbon and energy source and elemental sulfur as electron acceptor. Here we report the complete genome sequence of the strain M27-SA2 which is composed of a 2,129,244-bp chromosome and a 124,256-bp plasmid. This is the second complete genome sequence within the genus Halanaeroarchaeum. We demonstrate that genome of 'Halanaeroarchaeum sulfurireducens' M27-SA2 harbors complete metabolic pathways for acetate and sulfur catabolism and for de novo biosynthesis of 19 amino acids. The genomic analysis also reveals that 'Halanaeroarchaeum sulfurireducens' M27-SA2 harbors two prophage loci and one CRISPR locus, highly similar to that of Kulunda Steppe (Altai, Russia) isolate 'H. sulfurireducens' HSR2T. The discovery of sulfur-respiring acetate-utilizing haloarchaeon in deep-sea hypersaline anoxic lakes has certain significance for understanding the biogeochemical functioning of these harsh ecosystems, which are incompatible with life for common organisms. Moreover, isolations of Halanaeroarchaeum members from geographically distant salt-saturated sites of different origin suggest a high degree of evolutionary success in their adaptation to this type of extreme biotopes around the world.@en

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.@en

Nano-sized archaeota, with their small genomes and limited metabolic capabilities, are known to associate with other microbes, thereby compensating for their own auxotrophies. These diminutive and yet ubiquitous organisms thrive in hypersaline habitats that they share with haloarchaea. Here, we reveal the genetic and physiological nature of a nanohaloarchaeon-haloarchaeon association, with both microbes obtained from a solar saltern and reproducibly cultivated together in vitro. The nanohaloarchaeon Candidatus Nanohalobium constans LC1Nh is an aerotolerant, sugar-fermenting anaerobe, lacking key anabolic machinery and respiratory complexes. The nanohaloarchaeon cells are found physically connected to the chitinolytic haloarchaeon Halomicrobium sp. LC1Hm. Our experiments revealed that this haloarchaeon can hydrolyze chitin outside the cell (to produce the monosaccharide N-acetylglucosamine), using this beta-glucan to obtain carbon and energy for growth. However, LC1Hm could not metabolize either glycogen or starch (both alpha-glucans) or other polysaccharides tested. Remarkably, the nanohaloarchaeon's ability to hydrolyze glycogen and starch to glucose enabled growth of Halomicrobium sp. LC1Hm in the absence of a chitin. These findings indicated that the nanohaloarchaeon-haloarchaeon association is both mutualistic and symbiotic; in this case, each microbe relies on its partner's ability to degrade different polysaccharides. This suggests, in turn, that other nano-sized archaeota may also be beneficial for their hosts. Given that availability of carbon substrates can vary both spatially and temporarily, the susceptibility of Halomicrobium to colonization by Ca Nanohalobium can be interpreted as a strategy to maximize the long-term fitness of the host.@en

Eight pure cultures of alkaliphilic haloaloarchaea capable of growth by dissimilatory sulfur reduction (previously only shown for neutrophilic haloarchaea) were isolated from hypersaline alkaline lakes in different geographic locations. These anaerobic enrichments, inoculated with sediments and brines, used formate, butyrate and peptone as electron donors and elemental sulfur as an electron acceptor 4 M total Na+ and at pH 9-10. According to 16S rRNA gene sequencing, the isolates fell into two distinct groups. A major group, comprising seven obligate alkaliphilic isolates from highly alkaline soda lakes, represents a new species-level branch within the genus Natronolimnobius (order Natrialbales), while a single moderately alkaliphilic isolate from the less alkaline Searles Lake forms a novel genus-level lineage within the order Haloferacales. The cells of the isolates are either flat rods or coccoid. They are facultative anaerobes using formate or H2 (in the presence of acetate or yeast extract as carbon source), C4-C9 fatty acids or peptone (the major group) as electron donors and either sulfur or DMSO (the major group) as electron acceptors. Aerobic growth is only possible with organic acids and peptone-yeast extract. All isolates are extreme halophiles, growing optimally at 4 M total Na+. On the basis of their unique physiological properties and distinct phylogeny, we propose that the seven isolates from the soda lakes are placed into a novel species, Natronolimnobiussulfurireducens sp. nov. (type strain AArc1T=JCM 30663T=UNIQEM U932T), and the Searles Lake isolate, AArc-SlT, into a new genus and species Halalkaliarchaeum desulfuricum (=JCM 30664T=UNIQEM U999T).@en

Ha.lo.de.sul.fur.ar.chae’um. Gr. n. hals, halos salt of the sea; L. pref. de-, from; L. n. sulfur , sulfur; N.L. neut. n. archaeum archaeon from Gr. adj. archaios-ê-on ancient; N.L. neut. n. Halodesulfurarchaeum sulfur-reducing haloarchaeon. The genus Halodesulfurarchaeum, classified within the family Halobacteriaceae, order Halobacteriales, and in the class Halobacteria, consists of obligately anaerobic extremely halophilic euryarchaea that grow by oxidation of H2 or formate with elemental sulfur, DMSO, or thiosulfate (some strains) as the electron acceptor, representing a first example of lithoheterotrophy in the class Halobacteria. Strains have been isolated from sediments of various terrestrial hypersaline chloride-sulfate lakes, marine solar salterns, and deep-sea hypersaline brines in the Mediterranean. The genus consists of one species Halodesulfurarchaeum formicicum, which is the type species of the genus. DNA G+C content (mol%): 63.6–63.8 mol% (genome). Type species: Halodesulfurarchaeum formicicum.@en

Ha.lo.de.sul.fur.ar.chae’um. Gr. n. hals, halos salt of the sea; L. pref. de-, from; L. n. sulfur , sulfur; N.L. neut. n. archaeum archaeon from Gr. adj. archaios-ê-on ancient; N.L. neut. n. Halodesulfurarchaeum sulfur-reducing haloarchaeon. The genus Halodesulfurarchaeum, classified within the family Halobacteriaceae, order Halobacteriales, and in the class Halobacteria, consists of obligately anaerobic extremely halophilic euryarchaea that grow by oxidation of H2 or formate with elemental sulfur, DMSO, or thiosulfate (some strains) as the electron acceptor, representing a first example of lithoheterotrophy in the class Halobacteria. Strains have been isolated from sediments of various terrestrial hypersaline chloride-sulfate lakes, marine solar salterns, and deep-sea hypersaline brines in the Mediterranean. The genus consists of one species Halodesulfurarchaeum formicicum, which is the type species of the genus. DNA G+C content (mol%): 63.6–63.8 mol% (genome). Type species: Halodesulfurarchaeum formicicum.@en

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.@en

Climate change, desertification, salinisation of soils and the changing hydrology of the Earth are creating or modifying microbial habitats at all scales including the oceans, saline groundwaters and brine lakes. In environments that are saline or hypersaline, the biodegradation of recalcitrant plant and animal polysaccharides can be inhibited by salt-induced microbial stress and/or by limitation of the metabolic capabilities of halophilic microbes. We recently demonstrated that the chitinolytic haloarchaeon Halomicrobium can serve as the host for an ectosymbiont, nanohaloarchaeon ‘Candidatus Nanohalobium constans’. Here, we consider whether nanohaloarchaea can benefit from the haloarchaea-mediated degradation of xylan, a major hemicellulose component of wood. Using samples of natural evaporitic brines and anthropogenic solar salterns, we describe genome-inferred trophic relations in two extremely halophilic xylan-degrading three-member consortia. We succeeded in genome assembly and closure for all members of both xylan-degrading cultures and elucidated the respective food chains within these consortia. We provide evidence that ectosymbiontic nanohaloarchaea is an active ecophysiological component of extremely halophilic xylan-degrading communities (although by proxy) in hypersaline environments. In each consortium, nanohaloarchaea occur as ectosymbionts of Haloferax, which in turn act as scavenger of oligosaccharides produced by xylan-hydrolysing Halorhabdus. We further obtained and characterised the nanohaloarchaea–host associations using microscopy, multi-omics and cultivation approaches. The current study also doubled culturable nanohaloarchaeal symbionts and demonstrated that these enigmatic nano-sized archaea can be readily isolated in binary co-cultures using an appropriate enrichment strategy. We discuss the implications of xylan degradation by halophiles in biotechnology and for the United Nation's Sustainable Development Goals.@en

Archaea are environmentally ubiquitous on Earth, and their extremophilic and metabolically versatile phenotypes make them useful as model systems for astrobiology. Here, we reveal a new functional group of halo(natrono)archaea able to utilize alpha-d-glucans (amylopectin, amylose and glycogen), sugars, and glycerol as electron donors and carbon sources for sulfur respiration. They are facultative anaerobes enriched from hypersaline sediments with either amylopectin, glucose or glycerol as electron/carbon sources and elemental sulfur as the terminal electron acceptor. They include 10 strains of neutrophilic haloarchaea from circum pH-neutral lakes and one natronoarchaeon from soda-lake sediments. The neutrophilic isolates can grow by fermentation, although addition of S0 or dimethyl sulfoxide increased growth rate and biomass yield (with a concomitant decrease in H2). Natronoarchaeal isolate AArc-S grew only by respiration, either anaerobically with S0 or thiosulfate as the terminal electron acceptor, or aerobically. Through genome analysis of five representative strains, we detected the full set of enzymes required for the observed catabolic and respiratory phenotypes. These findings provide evidence that sulfur-respiring haloarchaea partake in biogeochemical sulfur cycling, linked to terminal anaerobic carbon mineralization in hypersaline anoxic habitats. We discuss the implications for life detection in analogue environments such as the polar subglacial brine-lakes of Mars.@en

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.@en