Sorokin, Dimitry Y.

Significance We report on cultivation and characterization of an association between Candidatus Nanohalobium constans and its host, the chitinotrophic haloarchaeon Halomicrobium LC1Hm, obtained from a crystallizer pond of marine solar salterns. High-quality nanohaloarchael genome sequence in conjunction with electron- and fluorescence microscopy, growth analysis, and proteomic and metabolomic data revealed mutually beneficial interactions between two archaea, and allowed dissection of the mechanisms for these interactions. Owing to their ubiquity in hypersaline environments, Nanohaloarchaeota may play a role in carbon turnover and ecosystem functioning, yet insights into the nature of this have been lacking. Here, we provide evidence that nanohaloarchaea can expand the range of available substrates for the haloarchaeon, suggesting that the ectosymbiont increases the metabolic capacity of the host.

Abstract This protocol describes a rapid protein extraction method for the archaeon Candidatus Vulcanisaeta moutnovskia, which can be also implemented for other archaea. The utilization of two different methods for protein extraction constitute the main step of the protocol. Method I involves the extraction with a multi-chaotropic lysis buffer containing a non-denaturing zwitterionic detergent, most efficient for extracting cytosolic proteins. Method II involves a denaturing anionic detergent allowing total disruption of the membranes and capable of extracting both membrane (hydrophobic) and non-membrane (water-soluble, hydrophilic) proteins. The big advantage of the methods is to use general laboratory chemicals to make powerful extraction buffers, resulting in high quality and quantity of proteins. The methods probably are usable for any other archaea or microbial cells, and takes about 14-22 h. Following extraction and further protein digestion, 1D-nano Liquid Chromatography Electrospray Ionization Tandem Mass Spectrometric (LC ESI-MSMS) analysis with Triple TOF 5600 and Orbitrap technologies were used for protein identification and further quantification.

Abstract Me.tha.no.hal'ar.chae.um N.L. neut. n. methanum methane; N.L. pref. methano ‐ pertaining to methane; Gr. masc. n. hals, halos salt; N.L. neut. n. archaeum (pertaining to archaios, ‐e, ‐on) ancient; N.L. neut. n. Methanohalarchaeum a salt‐loving archaeon forming methane. The candidate genus Ca . Methanohalarchaeum includes obligately anaerobic, extremely halophilic, neutrophilic, and moderately thermophilic methanogens with small coccoid cells producing methane by reduction of C 1 ‐methylated compounds using either H 2 or formate as electron donors. Amino acids or acetate is required as C‐source for assimilation. Core membrane lipids are represented by diphytanyl glycerol tetra‐ and diethers. Cells accumulate potassium as a compatible solute. They inhabit hypersaline lakes or salterns with neutral pH. The genus contains a single candidate species Ca . M. thermophilum, which is the type species. It is a member of the class Methanonatronarchaeia within a newly proposed phylum Halobacteriota (formerly part of the phylum Euryarchaeota ). DNA G + C content (mol%) : 35.4 (genome of the type strain). Type species : Ca . Methanohalarchaeum thermophilum Sorokin, Merkel, Abbas, Makarova, Rijpstra et al. 2018, 2206. Taxonomic and Nomenclature Notes According to the List of Prokaryotic names with Standing in Nomenclature (LPSN), the taxonomic status of the genus Candidatus Methanohalarchaeum is: preferred name (not correct name) (last update, February 2025) * . LPSN classification: Archaea / Methanobacteriati / Methanobacteriota / Methanonatronarchaeia / Methanonatronarchaeales / Methanonatronarchaeaceae / Candidatus Methanohalarchaeum The genus Candidatus Methanohalarchaeum can also be recovered in the Genome Taxonomy Database (GTDB) as g__Methanohalarchaeum (version v220) ** . GTDB classification: d__Archaea / p__Halobacteriota / c__Methanonatronarchaeia / o__Methanonatronarchaeales / f__Methanonatronarchaeaceae / g__Methanohalarchaeum * Meier‐Kolthoff et al. ( 2022 ). Nucleic Acids Res , 50 , D801 – D807 ; DOI: 10.1093/nar/gkab902 ** Parks et al. ( 2022 ). Nucleic Acids Res , 50 , D785 – D794 ; DOI: 10.1093/nar/gkab776

Summary Until now anaerobic oxidation of VFA at high salt‐pH has been demonstrated only at sulfate‐reducing conditions. Here, we present results of a microbiological investigation of anaerobic conversion of organic acids and alcohols at methanogenic conditions by syntrophic associations enriched from hypersaline soda lakes in Central Asia. Sediment incubation experiments showed active, albeit very slow, methane formation from acetate, propionate, butyrate and C 2 C 4 alcohols at pH 10 and various levels of salinity. Enrichments of syntrophic associations using hydrogenotrophic members of the genus Methanocalculus from soda lakes as partners resulted in several highly enriched cultures converting acetate, propionate, butyrate, benzoate and EtOH to methane. Most syntrophs belonged to Firmicutes , while the propionate‐oxidizer formed a novel lineage within the family Syntrophobacteraceae in the Deltaproteobacteria . The acetate‐oxidizing syntroph was identified as ‘ Ca . Syntrophonatronum acetioxidans’ previously found to oxidize acetate at sulfate‐reducing conditions up to salt‐saturating concentrations. Butyrate and a benzoate‐degrading syntrophs represent novel genus‐level lineages in Syntrophomonadales which are proposed as Candidatus taxons ‘Syntrophobaca’, ‘Syntrophocurvum’ and ‘Syntropholuna’. Overall, despite very slow growth, the results indicated the presence of a functionally competent syntrophic community in hypersaline soda lakes, capable of efficient oxidation of fermentation products to methane at extremely haloalkaline conditions.