Symbiosis between nanohaloarchaeon and haloarchaeon is based on utilization of different polysaccharides.

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.

Cultivation of a vampire: 'Candidatus Absconditicoccus praedator'.

Halorhodospira halophila, one of the most-xerophilic halophiles, inhabits biophysically stressful and energetically expensive, salt-saturated alkaline brines. Here, we report an additional stress factor that is biotic: a diminutive Candidate-Phyla-Radiation bacterium, that we named 'Ca. Absconditicoccus praedator' M39-6, which predates H. halophila M39-5, an obligately photosynthetic, anaerobic purple-sulfur bacterium. We cultivated this association (isolated from the hypersaline alkaline Lake Hotontyn Nur, Mongolia) and characterized their biology. 'Ca. Absconditicoccus praedator' is the first stably cultivated species from the candidate class-level lineage Gracilibacteria (order-level lineage Absconditabacterales). Its closed-and-curated genome lacks genes for the glycolytic, pentose phosphate- and Entner-Doudoroff pathways which would generate energy/reducing equivalents and produce central carbon currencies. Therefore, 'Ca. Absconditicoccus praedator' is dependent on host-derived building blocks for nucleic acid-, protein-, and peptidoglycan synthesis. It shares traits with (the uncultured) 'Ca. Vampirococcus lugosii', which is also of the Gracilibacteria lineage. These are obligate parasitic lifestyle, feeding on photosynthetic anoxygenic Gammaproteobacteria, and absorption of host cytoplasm. Commonalities in their genomic composition and structure suggest that the entire Absconditabacterales lineage consists of predatory species which act to cull the populations of their respective host bacteria. Cultivation of vampire : host associations can shed light on unresolved aspects of their metabolism and ecosystem dynamics at life-limiting extremes.

Carbohydrate-dependent sulfur respiration in halo(alkali)philic archaea.

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.

Reclassification of the genus Natronolimnobius: proposal of two new genera, Natronolimnohabitans gen. nov. to accommodate Natronolimnobius innermongolicus and Natrarchaeobaculum gen. nov. to accommodate Natronolimnobius aegyptiacus and Natronolimnobius sulfurireducens.

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.

Natronolimnobius sulfurireducens sp. nov. and Halalkaliarchaeum desulfuricum gen. nov., sp. nov., the first sulfur-respiring alkaliphilic haloarchaea from hypersaline alkaline lakes.

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).

Microbial community of the deep-sea brine Lake Kryos seawater-brine interface is active below the chaotropicity limit of life as revealed by recovery of mRNA.

Within the complex of deep, hypersaline anoxic lakes (DHALs) of the Mediterranean Ridge, we identified a new, unexplored DHAL and named it 'Lake Kryos' after a nearby depression. This lake is filled with magnesium chloride (MgCl2 )-rich, athalassohaline brine (salinity > 470 practical salinity units), presumably formed by the dissolution of Messinian bischofite. Compared with the DHAL Discovery, it contains elevated concentrations of kosmotropic sodium and sulfate ions, which are capable of reducing the net chaotropicily of MgCl2 -rich solutions. The brine of Lake Kryos may therefore be biologically permissive at MgCl2 concentrations previously considered incompatible with life. We characterized the microbiology of the seawater-Kryos brine interface and managed to recover mRNA from the 2.27-3.03 M MgCl2 layer (equivalent to 0.747-0.631 water activity), thereby expanding the established chaotropicity window-for-life. The primary bacterial taxa present there were Kebrit Deep Bacteria 1 candidate division and DHAL-specific group of organisms, distantly related to Desulfohalobium. Two euryarchaeal candidate divisions, Mediterranean Sea Brine Lakes group 1 and halophilic cluster 1, accounted for > 85% of the rRNA-containing archaeal clones derived from the 2.27-3.03 M MgCl2 layer, but were minority community-members in the overlying interface-layers. These findings shed light on the plausibility of life in highly chaotropic environments, geochemical windows for microbial extremophiles, and have implications for habitability elsewhere in the Solar System.

The gut microbiota of larvae of Rhynchophorus ferrugineus Oliver (Coleoptera: Curculionidae).

BACKGROUND: The red palm weevil (RPW) Rhynchophorus ferrugineus Olivier (Coleoptera: Curculionidae) is one of the major pests of palms. The larvae bore into the palm trunk and feed on the palm tender tissues and sap, leading the host tree to death. The gut microbiota of insects plays a remarkable role in the host life and understanding the relationship dynamics between insects and their microbiota may improve the biological control of insect pests. The purpose of this study was to analyse the diversity of the gut microbiota of field-caught RPW larvae sampled in Sicily (Italy). RESULTS: The 16S rRNA gene-based Temporal Thermal Gradient Gel Electrophoresis (TTGE) of the gut microbiota of RPW field-trapped larvae revealed low bacterial diversity and stability of the community over seasons and among pools of larvae from different host trees. Pyrosequencing of the 16S rRNA gene V3 region confirmed low complexity and assigned 98% of the 75,564 reads to only three phyla: Proteobacteria (64.7%) Bacteroidetes (23.6%) and Firmicutes (9.6%) and three main families [Enterobacteriaceae (61.5%), Porphyromonadaceae (22.1%) and Streptococcaceae (8.9%)]. More than half of the reads could be classified at the genus level and eight bacterial genera were detected in the larval RPW gut at an abundance ≥1%: Dysgonomonas (21.8%), Lactococcus (8.9%), Salmonella (6.8%), Enterobacter (3.8%), Budvicia (2.8%), Entomoplasma (1.4%), Bacteroides (1.3%) and Comamonas (1%). High abundance of Enterobacteriaceae was also detected by culturing under aerobic conditions. Unexpectedly, acetic acid bacteria (AAB), that are known to establish symbiotic associations with insects relying on sugar-based diets, were not detected. CONCLUSIONS: The RPW gut microbiota is composed mainly of facultative and obligate anaerobic bacteria with a fermentative metabolism. These bacteria are supposedly responsible for palm tissue fermentation in the tunnels where RPW larvae thrive and might have a key role in the insect nutrition, and other functions that need to be investigated.

Metagenomic analysis of hadopelagic microbial assemblages thriving at the deepest part of Mediterranean Sea, Matapan-Vavilov Deep.

The marine pelagic zone situated > 200 m below the sea level (bls) is the largest marine subsystem, comprising more than two-thirds of the oceanic volume. At the same time, it is one of the least explored ecosystems on Earth. Few large-scale environmental genomics studies have been undertaken to examine the phylogenetic diversity and functional gene repertoire of planktonic microbes present in mesopelagic and bathypelagic environments. Here, we present the description of the deep-sea microbial community thriving at > 4900 m depth in Matapan-Vavilov Deep (MVD). This canyon is the deepest site of Mediterranean Sea, with a deepest point located at approximately 5270 m, 56 km SW of city Pylos (Greece) in the Ionian Sea (36°34.00N, 21°07.44E). Comparative analysis of whole-metagenomic data revealed that unlike other deep-sea metagenomes, the prokaryotic diversity in MVD was extremely poor. The decline in the dark primary production rates, measured at 4908 m depth, was coincident with overwhelming dominance of copiotrophic Alteromonas macleodii'deep-ecotype' AltDE at the expense of other prokaryotes including those potentially involved in both autotrophic and anaplerotic CO(2) fixation. We also demonstrate the occurrence in deep-sea metagenomes of several clustered regularly interspaced short palindromic repeats systems.

Functional diversity of nanohaloarchaea within xylan-degrading consortia.

Extremely halophilic representatives of the phylum Candidatus Nanohaloarchaeota (members of the DPANN superphyla) are obligately associated with extremely halophilic archaea of the phylum Halobacteriota (according to the GTDB taxonomy). Using culture-independent molecular techniques, their presence in various hypersaline ecosystems around the world has been confirmed over the past decade. However, the vast majority of nanohaloarchaea remain uncultivated, and thus their metabolic capabilities and ecophysiology are currently poorly understood. Using the (meta)genomic, transcriptomic, and DNA methylome platforms, the metabolism and functional prediction of the ecophysiology of two novel extremely halophilic symbiotic nanohaloarchaea (Ca. Nanohalococcus occultus and Ca. Nanohalovita haloferacivicina) stably cultivated in the laboratory as members of a xylose-degrading binary culture with a haloarchaeal host, Haloferax lucentense, was determined. Like all known DPANN superphylum nanoorganisms, these new sugar-fermenting nanohaloarchaea lack many fundamental biosynthetic repertoires, making them exclusively dependent on their respective host for survival. In addition, given the cultivability of the new nanohaloarchaea, we managed to discover many unique features in these new organisms that have never been observed in nano-sized archaea both within the phylum Ca. Nanohaloarchaeota and the entire superphylum DPANN. This includes the analysis of the expression of organism-specific non-coding regulatory (nc)RNAs (with an elucidation of their 2D-secondary structures) as well as profiling of DNA methylation. While some ncRNA molecules have been predicted with high confidence as RNAs of an archaeal signal recognition particle involved in delaying protein translation, others resemble the structure of ribosome-associated ncRNAs, although none belong to any known family. Moreover, the new nanohaloarchaea have very complex cellular defense mechanisms. In addition to the defense mechanism provided by the type II restriction-modification system, consisting of Dcm-like DNA methyltransferase and Mrr restriction endonuclease, Ca. Nanohalococcus encodes an active type I-D CRISPR/Cas system, containing 77 spacers divided into two loci. Despite their diminutive genomes and as part of their host interaction mechanism, the genomes of new nanohaloarchaea do encode giant surface proteins, and one of them (9,409 amino acids long) is the largest protein of any sequenced nanohaloarchaea and the largest protein ever discovered in cultivated archaea.

Nanohaloarchaea as beneficiaries of xylan degradation by haloarchaea.

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.

Anaerobic carboxydotrophy in sulfur-respiring haloarchaea from hypersaline lakes.

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.

Natranaeroarchaeum sulfidigenes gen. nov., sp. nov., carbohydrate-utilizing sulfur-respiring haloarchaeon from hypersaline soda lakes, a member of a new family Natronoarchaeaceae fam. nov. in the order Halobacteriales.

A pure culture of alkaliphilic haloarchaeon strain AArc-ST capable of anaerobic growth by carbohydrate-dependent sulfur respiration was obtained from hypersaline lakes in southwestern Siberia. According to phylogenetic analysis, AArc-ST formed a new genus level branch most related to the genus Natronoarchaeum in the order Halobacteriales. The strain is facultatively anaerobic with strictly respiratory metabolism growing either by anaerobic respiration with elemental sulfur and thiosulfate as the electron acceptors or by aerobic respiration at microoxic conditions. Thiosulfate is reduced partially to sulfide and sulfite. It is a first sulfur-reducing alkaliphilic haloarchaeon utilizing sugars, starch and glycerol as substrates for anaerobic growth. It is extremely halophilic (optimum at 3.5 M total Na+) and obligately alkaliphilic (optimum at pH 9.5). The dominant polar lipids include PG and PGP-Me with the archaeol (C20-C20) or extended archaeol (C20-C25) cores. The dominant respiratory lipoquinone is MK-8:8. On the basis of unique physiological properties and results of phylogenetic analysis, the soda lake isolate is suggested to be classified into a novel genus and species Natranaeroarchaeum sulfidigenes gen. nov., sp. nov. (=JCM 34033T = UNIQEM U1000T). Furthermore, on the bases of phylogenomic reconstruction, a new family Natronoarchaeaceae fam. nov. is proposed within the order Halobacteriales incorporating Natranaeroarchaeum and three related genera: Natronoarchaeum, Salinarchaeum and Halostella.

Unveiling microbial life in new deep-sea hypersaline Lake Thetis. Part I: Prokaryotes and environmental settings.

In September 2008, an expedition of the RV Urania was devoted to exploration of the genomic richness of deep hypersaline anoxic lakes (DHALs) located in the Western part of the Mediterranean Ridge. Approximately 40 nautical miles SE from Urania Lake, the presence of anoxic hypersaline lake, which we named Thetis, was confirmed by swath bathymetry profiling and through immediate sampling casts. The brine surface of the Thetis Lake is located at a depth of 3258 m with a thickness of ≈ 157 m. Brine composition was found to be thalassohaline, saturated by NaCl with a total salinity of 348‰, which is one of highest value reported for DHALs. Similarly to other Mediterranean DHALs, seawater-brine interface of Thetis represents a steep pycno- and chemocline with gradients of salinity, electron donors and acceptors and posseses a remarkable stratification of prokaryotic communities, observed to be more metabolically active in the upper interface where redox gradient was sharper. [(14) C]-bicarbonate fixation analysis revealed that microbial communities are sustained by sulfur-oxidizing chemolithoautotrophic primary producers that thrive within upper interface. Besides microaerophilic autotrophy, heterotrophic sulfate reduction, methanogenesis and anaerobic methane oxidation are likely the predominant processes driving the ecosystem of Thetis Lake.

Halapricum desulfuricans sp. nov., carbohydrate-utilizing, sulfur-respiring haloarchaea from hypersaline lakes.

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).

Author Correction: The discovery of Lake Hephaestus, the youngest athalassohaline deep-sea formation on Earth.

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has been fixed in the paper.

The discovery of Lake Hephaestus, the youngest athalassohaline deep-sea formation on Earth.

Hydrated, magnesium-rich minerals and subglacial brines exist on the martian surface, so the habitability of high-Mg2+ environments on Earth has extraterrestrial (as well as terrestrial) implications. Here, we report the discovery of a MgCl2-dominated (4.72 M) brine lake on the floor of the Mediterranean Ridge that underlies a 3500-m water column, and name it Lake Hephaestus. Stable isotope analyses indicated that the Hephaestus brine is derived from interactions between ancient bishofite-enriched evaporites and subsurface fluids. Analyses of sediment pore waters indicated that the Hephaestus depression had contained the MgCl2 brine for a remarkably short period; only 700 years. Lake Hephaestus is, therefore, the youngest among currently known submarine athalassohaline brine lakes on Earth. Due to its biologically hostile properties (low water-activity and extreme chaotropicity), the Hephaestus brine is devoid of life. By contrast, the seawater-Hephaestus brine interface has been shown to act as refuge for extremely halophilic and magnesium-adapted stratified communities of microbes, even at MgCl2 concentrations that approach the water-activity limit for life (0.653).

Sulfur Respiration in a Group of Facultatively Anaerobic Natronoarchaea Ubiquitous in Hypersaline Soda Lakes.

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.

Discovery of anaerobic lithoheterotrophic haloarchaea, ubiquitous in hypersaline habitats.

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.

Analysis of defence systems and a conjugative IncP-1 plasmid in the marine polyaromatic hydrocarbons-degrading bacterium Cycloclasticus sp. 78-ME.

Marine prokaryotes have evolved a broad repertoire of defence systems to protect their genomes from lateral gene transfer including innate or acquired immune systems and infection-induced programmed cell suicide and dormancy. Here we report on the analysis of multiple defence systems present in the genome of the strain Cycloclasticus sp. 78-ME isolated from petroleum deposits of the tanker 'Amoco Milford Haven'. Cycloclasticus are ubiquitous bacteria globally important in polyaromatic hydrocarbons degradation in marine environments. Two 'defence islands' were identified in 78-ME genome: the first harbouring CRISPR-Cas with toxin-antitoxin system, while the second was composed by an array of genes for toxin-antitoxin and restriction-modification proteins. Among all identified spacers of CRISPR-Cas system only seven spacers match sequences of phages and plasmids. Furthermore, a conjugative plasmid p7ME01, which belongs to a new IncP-1θ ancestral archetype without any accessory mobile elements was found in 78-ME. Our results provide the context to the co-occurrence of diverse defence mechanisms in the genome of Cycloclasticus sp. 78-ME, which protect the genome of this highly specialized PAH-degrader. This study contributes to the further understanding of complex networks established in petroleum-based microbial communities.

Complete genome sequence of 'Halanaeroarchaeum sulfurireducens' M27-SA2, a sulfur-reducing and acetate-oxidizing haloarchaeon from the deep-sea hypersaline anoxic lake Medee.

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' HSR2(T). 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.