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 H 2 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


Introduction
Our previous research into anaerobic haloarchaea growing by sulfur respiration resulted in the discovery of two functional groups in this novel ecotype of extremely halophilic archaea, including obligately anaerobic acetate-and formate-utilizing genera Halanaeroarchaeum and Halodesulfurarchaeum [1][2][3][4][5].Such haloarchaea seem to specialize on the final anaerobic mineralization of fermentation products -a metabolism not previously known among multiple pure cultures of predominantly aerobic haloarchaea, suggesting that extremely halophilic euryarchaea may play an active role in anoxic sulfidic sediments of hypersaline systems, such as athalassic salt lakes and thalassic evaporative salt crystallizers.However, to be self-sufficient, this new functional system of sulfur-respiring haloarchaea lacked one metabolic group, namely primary anaerobes, which would decompose carbohydrates and provide electron donors for the aforementioned subgroups.Such organisms have recently been discovered and their metabolism characterized [6].They are facultatively anaerobic, in contrast to the two previously characterized groups of obligate anaerobes, fermenting sugars and glycerol in the absence of electron acceptors with formation of H 2 .In the presence of sulfur or sulfoxides, they switch to anaerobic respiration and can also grow by aerobic respiration at microoxic conditions [6].Here we provide a formal taxonomic description of these organisms as Halapricum desulfuricans sp.nov.

Enrichment and cultivation conditions
The sources of the inocula were subsurface (3-30 cm deep) anaerobic sulfidic sediments from Russian hypersaline salt lakes in Kulunda Steppe (Altai region) and Volgograd region (Table 1).https://doi.org/10.1016/j.syapm.2021.1262490723-2020/Ó 2021 The Authors.Published by Elsevier GmbH.This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).The samples were taken by a stratometer corer tube with 25 mm internal diameter into sterile 100 ml Schott bottles and filled to the top with the near-bottom brines.After transportation, part of the brine was removed and the head space was flushed with argon.For the inoculation, the 1:1 mixture of brine-sediment was homogenized by vortexing and subjected to a brief low speed centrifugation to remove a coarse sandy fraction which left a fine colloidal fraction containing most of the cells.The enrichment and isolation procedures, the medium composition, cultivation conditions and analyses of growth and sulfide/polysulfide formation have been described previously [5].In short, 4 M NaCl base medium was supplemented with (g l À1 ): g l À1 of HEPES 5, K 2 HPO 4 2.5; NH 4 Cl 0.5, KCl 5 and adjusted to pH 7 with 1 M NaOH.After sterilization, the medium was supplemented with trace metal and vitamin solutions, 1 mM MgSO 4 and 40 mM of NaHCO 3 (from 1 M filtersterilized stock solution) to balance acidification by fermentation products.Sulfur was added at approximately 5 g l À1 directly into incubation vials, thiosulfate was used at 10 mM and sulfoxidesat 5-10 mM.The medium was reduced with 0.5 mM sulfide and the cultivation was done in serum bottles varying in volumes from 12 to 115 ml after creating anoxic conditions by evacuation-argon flushing.Microoxic cultivation was performed in 115 ml serum bottles with 20 ml medium at 2% O 2 in the gas phase.The isolation of pure cultures was achieved by several rounds of serial dilution to extinction in anaerobic conditions and the purity was confirmed by microscopy and by 16S rRNA gene and genome sequencing

Pure culture characterization
Cell morphology was examined by using phase contrast microscopy (Zeiss Axioplane Imaging 2, Germany) and two types of electron microscopy.For the flagella detection, the paraformaldehydefixed cells in 4 M NaCl were positively stained with 1% uranyl acetate and the salts were removed with a brief emersion into demineralized water.For thin section electron microscopy, the cells, resuspended in 4 M NaCl, were mixed 1:1 with 2% OsO 4 , incubated at 4 °C for 1 week, embedded into agar, dehydrated in alcohol series and finally in acetone, incorporated into Epon resin and thin sectioned on ultramicrotome.The sections were contrasted in 1% (w/w) uranyl acetate and lead citrate solutions.
For the cell suspension activity tests strain HSR12-1 was grown in 200 ml volume anaerobically with glucose and three different sulfoxides -dimethylsulfoxide (DMSO), methionine sulfoxide (MSO) and tetramethylene sulfoxide (TMSO).The cells were collected by centrifugation, washed and resuspended in mineral medium containing 4 M NaCl and 50 mM K-phosphate buffer at pH 7 at cell protein concentration of 0.12-0.15mg ml À1 . 2 ml of the cell suspensions were incubated at 37 °C for 48 h anoxically in 9 ml serum bottles with 5 mM glucose or under H 2 atmosphere with 5 mM of each sulfoxides.

Analyses
Sulfide formation was measured with methylene blue method after fixation with 10% Zn acetate [7] and products of sulfoxide reduction were analyzed as described previously either by HPLC in liquid (methionine) or by the GC in the gas phase (dimethyl sulfide and tetrahydrothiophene) [8].Catalase and oxidase activity were tested with 3% (v/v) H 2 O 2 and 0.1% N,N,N,N tetramethyl-pphenylenediamine hydrochloride, respectively, using cell-free extract from cells of HSR12-1 and HSR12-2 T grown microaerophilically.The protease, esterase and lipase activities were tested in microaerobic cultures of the type strains HSR12-2 T grown on plates with casein/gelatin (after flooding with 10% TCA) and emulsified tributyrin/olive oil (turbidity clearance), respectively.Antibiotic sensitivity of HSR12-2 T was tested aerobically in liquid medium at pH 7 with glucose as substrate.
The intact polar lipids (IPLs) and respiratory quinones were ultrasonically extracted twice for 10 min from freeze-dried biomass in methanol, dichloromethane (DCM) and phosphate buffer (2:1:0.8,v:v:v).The extracts were phase-separated by adding additional DCM and buffer to a final solvent ratio of 1:1:0.9(v:v:v) The organic phase containing the IPLs was collected and the aqueous phase re-extracted twice with DCM.All steps of the extraction were then repeated, but with a solvent mixture of methanol, DCM and trichloroacetic acid pH 2-3 (2:1:0.8,v:v:v).Finally, the combined extract was dried under a stream of N 2 gas [9].Before analysis, the extracts were redissolved in MeOH:DCM (9:1, v:v) and filtered through 0.45 lm cellulose syringe filters (4 mm diameter; Grace Alltech, Deerfield, IL, United States).Analysis of extracts was carried out using an Ultra High-Pressure Liquid Chromatography-High Resolution Mass Spectrometry (UHPLC-HRMS) according to the reversed phase method with modifications of [9,10].Identification was carried out by comparison of accurate masses and mass spectral fragmentation with published data for IPLs [11] and for quinones [12].

Genomic and phylogenomic analyses
The genomes of four representative isolates, HSR12-1, HSR12-2 T , HSR-Bgl and HSR-Est, were sequenced, and analyzed as described previously [6].The genomes of all four strains were assembled as a single circular chromosome.Isolates HSR-Bgl and HSR-Est also contained a plasmid.The genome size was ca.3.0 Mbp.The details of genome statistics are given in the Supplementary Table S1.The G + C % was calculated from the four whole genome sequences.
For phylogenetic reconstructions, 122 archaeal single copy conservative marker genes were used according to the Genome Taxonomy Data Base [13] as well as 16S rRNA gene.The trees were built using the IQ-TREE 2 program [14] with fast model selection via ModelFinder [15] and ultrafast bootstrap approximation [16] as well as approximate likelihood-ratio test for branches [17].Whole genome comparison was conducted by using three different methods: Average Nucleotide Identity (ANIb and ANIm), using JSpe-ciesWS web server; Average Amino acid Identity (AAI) by the AAI calculator online of Kostas lab (http://enve-omics.ce.gatech.edu/aai/index) and DDH by the Genome-to-Genome Distance Calculator 2.1 online tool (http://ggdc.dsmz.de/ggdc.php)[18,19].

Enrichment and isolation of pure cultures
The primary anaerobic sulfur-reducing enrichments were performed with three e-donors (2 mM) including glucose, soluble starch and glycerol.Microbial growth, dominated by archaea (judged from the lack of inhibition by a mixture of kanamycin/strepromycin and vancomicin, 100 mg l À1 each), and sulfide formation (up to 6 mM) was observed with all three e-donors, and these efforts resulted eventually in nine pure cultures after several rounds of dilution to extinction (Table 1).Colony formation was not observed with sulfur as the electron acceptor, probably because of its practical insolubility at neutral pH.The isolates had mostly spheric nonmotile cells of variable size often containing refractive storage granules, most probably polyhydroxyalkanoates (as confirmed by Nile-Blue staining and inferred from genome analysis) (Fig. 1a-b); [6].Thin section electron microscopy showed that the cells are not actually round but, rather, angular coccoids with a thin S-layer type of cell wall covered with EPS and often containing several large intracellular PHA-like granules (Fig. 1c-d).It is noteworthy that, in contrast to the previously described genera of obligate anaerobic sulfur-reducing haloarchaea, whose biomass was black, the cells of sulfur/thiosulfate and, especially, sulfoxide-reducing carbohydrate-utilizing isolates were red, suggesting production of carotenoids under strictly anoxic conditions.

Chemotaxonomy
In the two analyzed neutrophilic isolates HSR12-1 and HSR12-2 T and in the reference strain H. salinum [21] the core lipids were represented by archaeol (AR; C 20 -C 20 ) and extended archaeol (Ext-AR; C 20 -C 25 ) in roughly equal proportion (Supplementary Table S2), as is usual for haloarchaea.In all three strains the majority of IPLs had a PGP-Me or PG head groups.Furthermore, two glycolipids were also detected, with one or two hexose moieties.Strain HSR12-1 also contained trace levels of AR and Ext-AR with no polar head group, or with just a phosphate moiety.These are possibly precursors or break down products of the IPLs.In general, H. salinum profile was more similar to HSR12-2 T than to HSR12-1.

Phylogenetic and genomic analyses
The previous 16S rRNA gene-based phylogenetic analysis showed that the nine carbohydrate-utilizing isolates from salt lakes clustered together and all had two highly dissimilar 16S rRNA genes (91.4-92.7%sequence identity to each other) [6], with one of them related to the genus Halapricum [21][22] and the other to the genus Halosimplex [23] (Fig. S1).Both these genera are classified in the family Haloarculaceae.To clarify the phylogeny of the novel isolates, an extended phylogenomic analysis based on 122 archaeal conserved single-copy protein markers was performed using the four sequenced genomes of the HSR isolates.The result showed that the novel haloarchaea belonged to the genus Halapricum, despite the significant difference in their rrn structure, forming a distinct novel species lineage (Fig. 2).The genus, apparently, includes one more yet uncharacterized species represented by Halapricum sp.CBA1109.This conclusion was also apparent from calculations of the whole genome comparison indexes, ANI, AAI and DDH (Supplementary Table S4).These calculations as well as Fig. 2. Phylogenomic placement of carbohydrate-utilizing sulfur-reducing haloarchaea Halapricum desulfuricans within the Haloarculaceae family based on concatenated partial amino acid sequences of 122 archaeal single copy conserved marker proteins with taxonomic designations according to the Genome Taxonomy Data Base.Bootstrap consensus tree is shown with values above 85% placed at the nodes.Bar, 0.1 changes per position.

Table 2
Comparative properties of carbohydrate-utilizing sulfur-reducing haloarchaea with the type species of the nearest phylogenetically related genus Halapricum salinum [21,22].

Property
Halapricum desulfuricans (9 isolates) phylogenomic analysis also suggested that among the four genome-analyzed HSR strains, HSR-Est might represent a separate species.However, we could not sufficiently differentiate this strain phenotypically from the other neutrophilic isolates and, therefore, suggest to classify all HSR isolates in a single species.

Metabolic properties
The key physiological property of all isolates is their ability to utilize sugars and glycerol as the energy and carbon source during sulfidogenic growth in presence of elemental sulfur and sulfoxides as the electron acceptor [6].To the best of our knowledge, anaerobic growth with glycerol as an electron donor has not been previously demonstrated in any pure haloarchaeal culture, and anaerobic growth by sugar fermentation has been reported for only a few known species of extremely halophilic members of Halobacteria.
The HSR isolates were able to grow anaerobically by fermentation (i.e.without external electron acceptor) producing H 2 as one of the major products, while in the presence of sulfur, the formation of H 2 drastically decreased in favor of H 2 S accompanied by a significant increase in the biomass yield [6].Such behavior is commonly observed in fermentative sulfur-reducing archaea using sulfur reductase-hydrogenase for cytoplasmic dump of excessive electrons to prevent H 2 accumulation [24], but the HSR isolates used a different mechanism, based on the respiratory membranebound PsrABC system [6].Moreover, external H 2 also served as a direct electron donor for sulfur reduction in HSR strains pregrown on glucose or glycerol due to the presence of a membrane-bound Ni,Fe hydrogenase type Ia [6].In contrast, formate was not utilized for sulfur respiration in the novel sulfurreducing isolates.The potential ability for elemental sulfur respiration has already been proposed previously for Halapricum salinum due to the presence of a putative PsrABC-encoding operon in its genome [6].Therefore, the type species, H. salinum, was tested for the in vivo capability to grow anaerobically with glucose and sulfur or DMSO as acceptors.Although the results can be considered as positive, only 3 mM sulfide and trace amount of DMS were produced in a month incubation, which is much lower than in the HSR strains.Apart from elemental sulfur, the type strain HSR12-2 T was able to use thiosulfate as an e-acceptor partially reducing it to sulfide and sulfite.This property has previously been detected only in members of the sulfur-respiring genus Halodesulfurarchaeum [4,5].Finally, the novel isolates, in addition to DMSO, which until recently was the only sulfoxide known to serve as the e-acceptor for haloarchaea, also used methionine sulfoxide (MSO) and tetramethylene sulfoxide (TMSO) for anaerobic growth, converting them into methionine and tetrahydrothiophene, respectively (Supplementary Fig. S2).Such potential has recently also been demonstrated in other sulfur-reducing genera of halo(natrono)archaea, including Halanaeroarchaeum, Natrarchaeobaculum and Halobiforma [8].
Concerning the utilized spectrum of carbohydrates as the edonor/C source, apart from a limited number of mono-and disugars and glycerol, three strains enriched with starch were able to grow anaerobically with soluble starch and some other homologous alpha-glucans (Table 1).This is a first example of the polysaccharide-dependent sulfur respiration in haloarchaea.This potential is confirmed by the presence of multiple gene copies encoding the extracellular alpha-amylase of the GH13 family in the starch-utilizing strains HSR-Est (Supplementary Table S1).
The four sequenced genomes of HSR strains do not encode catalase/peroxidase proteins but, instead, contain several genes for peroxiredoxins (cysteine-containing H 2 O 2 -reactive proteins involved in oxidative stress response) and a superoxide dismutase.Testing confirmed the absence of a catalase reaction and also showed a weak-positive result for oxidase.This probably reflects the fact that it was difficult to adapt HSR strains to grow aerobically from sulfidogenic cultures.The cells are nonmotile angular coccoids highly variable in size from 0.8 to 3 lm, depending on the growth conditions often accumulating PHA-like storage granules.The cell wall consists of a thin monolayer covered with an extracellar matrix.The cells lyze in hypotonic solutions below 1 M NaCl.Red carotenoids are produced both during aerobic and anaerobic growth.The core membrane diether lipids are composed of C 20 -C 20 DGE (archaeol) and C 20 -C 25 DGE (extended archaeol) with 0-4 double bonds.The polar lipid head groups include phosphatidylglycerolphosphate methyl ester (PGP-Me), phosphatidylglycerol (PG) and mono-and diglycosyl ether glycolipids.The dominant respiratory quinone is MK-8:7 with the MK-8:8 second in abundance.Furthermore methylmenaquinones MMK-8:7 and MMK-8:8 (''thermoplasma quinones") also present in lesser proportions.Facultatively anaerobic.Anaerobic growth is possible by fermentation of carbohydrates with the formation of acetate, lactate and H 2 .Also grow by anaerobic respiration with sulfur compounds as acceptors, including elemental sulfur and sulfoxides (DMSO, MSO and TMSO) and some strains can also perform 2-electron reduction of thiosulfate to sulfide and sulfite.In the presence of electron acceptors, the H 2 formation decreases with a concomitant biomass yield increase.Aerobic growth occurs at microoxic conditions.The utilized substrates include hexoses (glucose, fructose, mannose, raffinose, trehalose, maltose, sucrose), starch, pullulane, dextrin and cyclodextrin (three out of the nine isolates) and glycerol.Ammonium and yeast extract are utilized as the N-source.Oxidase is weakly positive, catalase is negative.Maximum growth temperature is 50 °C.Extremely halophilic with a range of total Na + for growth from 3 to 5 M (optimum at 4 M) and neutraphilic, with a pH range for growth from 6.5 to 7.8 (optimum at 7.0-7.2).The G + C content of the DNA is 63.7-64.6 % (four genomes).Habitat -anaerobic sediments of hypersaline salt lakes and salterns.The type strain (HSR12-2 T = JCM 34032 T = UNIQEM U1001 T ) was isolated sediments of hypersaline salt lakes in Kulunda Steppe (Altai, Russia).The species also includes other eight closely related strains isolated from various hypersaline salt lakes in Russia.The four genomes of strains belonging to this species (with two of them containing a plasmid) are deposited in the GenBank under accession numbers CP064787-CP064792.The protease, esterase and lipase activities in microaerobic cultures of HSR12-2 T grown on plates were negative.Ammonium and yeast extract (but not nitrate or urea) can serve as the N-source in cultures grown anaerobically with glucose.Indole formation from tryptophan (Kovac's reagent test) showed negative results for HSR12-2 T .Antibiotic sensitivity was tested aerobically in liquid medium for HSR12-2 T grown with glucose.It was sensitive to rifampicin and chloramphenicol above 25 mg l À1 , but resistant to streptomycin, ampicillin, kanamycin, vancomycin and gentamicin up to 100 mg l À1 .
All isolates belonged to low Mg-requiring extreme halophiles, with optimal growth occurring at 4 M total Na + and the range from 3 to 5 M (tested aerobically from 1 to 5 M).
The pH profiling in aerobic cultures at 4 M total Na + showed that HSR12-2 T is a typical neutrophile with the pH range for growth from 6.5 to 7.8 and an optimum at 7-7.2 (buffer system based on 50 mM HEPES/potassium phosphate/NaHCO 3 ; tested range from pH 6 to 8.5).The temperature range for aerobic growth of HSR12-2 T was from 20 to 50 °C with an optimum at 37-40 °C.
Comparative properties of the HSR isolates strains with the only known (type) species of the genus Halapricum in Table 2.The main difference of the new isolates from the related Halapricum salinum is their better adaptation for anaerobic growth, the ability to grow anaerobically with alpha-glucans and the presence of a second highly dissimilar rrn operon in the genome.Overall, on the basis of distinct phenotypic and genomic features, the nine carbohydrate-utilizing sulfur-reducing haloarchaeal isolates from hypersaline salt lakes are suggested to be classified as a novel species in the genus Halapricum, i.e.Halapricum desulfuricans sp.nov.The species protologue is presented in Table 3.

Emended description of the genus Halapricum Song et al. 2014
In addition to the genus description given in the original publication by the Song et al. (2014) [21], both species of the genus are capable of anaerobic respiration using sugars as the e-donor and elemental sulfur or DMSO as the e-acceptor.Furthermore, one species can also ferment sugars, starch or glycerol.The major respiratory lipoquinones are MK-8:

Fig. 1 .
Fig. 1. Cell morphology of carbohydrate-utilizing sulfur-respiring haloarchaea growing anaerobically with sulfur as electron acceptor at 4 M NaCl, pH 7 (a-b), phase contrast microscopy of HSR12-1 and HSR12-2 T cells, respectively.(c-d), electron microphotographs of thin sections of HSR12-2 T cells showing the cell angularity, a thin monolayer cell wall covered with extracellular matrix and intracellular PHA-like storage granules.

Table S2 .
Intact polar lipids identified and their abundance (in percent of lipid peak area) in carbohydrate-utilizing haloarchaea grown anaerobically with sulfur and glucose at 37 o C and 4 M NaCl until late exponential growth phase : 1strain HSR12-1; 2 -strain HSR12-2 T ; 3 -Halapricum salinum JCM 19729 T