DOI: 10.15468/4kazx2
title: Halococcoides cellulosivorans gen. nov., sp. nov., an extremely halophilic cellulose-utilizing haloarchaeon from hypersaline lakes

description: This dataset contains the digitized treatments in Plazi based on the original journal article Sorokin, Dimitry Y., Khijniak, Tatiana V., Elcheninov, Alexander G., Toshchakov, Stepan V., Kostrikina, Nadezhda A., Bale, Nicole J., Sinninghe Damsté, Jaap S., Kublanov, Ilya V. (2019): Halococcoides cellulosivorans gen. nov., sp. nov., an extremely halophilic cellulose-utilizing haloarchaeon from hypersaline lakes. International Journal of Systematic and Evolutionary Microbiology 69 (5): 1327-1335, DOI: 10.1099/ijsem.0.003312, URL: http://dx.doi.org/10.1099/ijsem.0.003312AbstractAn extremely halophilic euryarchaeon, strain HArcel1 T, was enriched and isolated in pure culture from the surface brines and sediments of hypersaline athalassic lakes in the Kulunda Steppe (Altai region, Russia) using amorphous cellulose as the growth substrate. The colonies of HArcel1 T are pale-orange, and form large zones of cellulose hydrolysis around them. The cells are non-motile cocci of variable size with a thin monolayer cell wall. The isolate is an obligate aerobic heterotroph capable of growth with only three substrates: various forms of insoluble cellulose, xylan and cellobiose. Strain HArcel1 T is an extremely halophilic neutrophile, growing within the salinity range from 2.5 to 5 M NaCl (optimum at 3.5–4 M). The core archaeal lipids are dominated by C20–C20 and C25–C20 dialkyl glycerol ethers, in approximately 6:1 proportion. The 16S rRNA and rpoB ′ gene analysis indicated that HArcel1 T forms a separate lineage within the family Haloarculaceae, order Halobacteriales, with the genera Halorhabdus and Halopricus as closest relatives. On the basis of the unique phenotypic properties and distinct phylogeny of the 16S rRNA and rpoB ′ genes, it is suggested that strain HArcel1 T is classified into a new genus and species Halococcoides cellulosivorans gen. nov., sp. nov. (JCM 31941 T = UNIQEM U975 T).Extremely halophilic euryarchaea of the class Halobacteria form dense blooms in inland salt lakes and sea solar salterns with salt concentrations close to saturation. Most of the cultured species are aerobic heterotrophs, utilizing simple soluble organic monomers, such as sugars and organic acids, or complex rich amino acid-based substrates, such as various peptons and yeast extract [1 – 6].The polymer mineralizing function at hypersaline conditions is usually attributed to halophilic bacteria [3, 4]. There are only few published examples of the utilization of polymeric substances, such as starch, proteins or olive oil, as growth substrates among the haloarchaeal species [7 – 11]. In particular, nearly nothing is known about the ability of haloarchaea to hydrolyse and utilize insoluble recalcitrant polysaccharides, such as cellulose or chitin, for growth. The glycosidase genes encoding putative cellulases (GH family 3, 5 and 9) are present in many haloarchaeal genomes (Haloarcula, Halobacterium, Halalkalicoccus, Haloferax, Halorhabdus, Halovivax, Halostagnicola, Haloterrigena– Natrinema group, Natronococcus), while the presence of functional beta-1,4 endoglucanases has been, to date, demonstrated only in two genera of neutrophilic haloarchaea, i.e. Haloarcula and Halorhabdus [12 – 14]. However, it remains to be investigated whether these haloarchaea are actually capable of using native forms of cellulose as a carbon and energy source.So far, only two studies have focused on the functional aspect of cellulose degradation by haloarchaea [15, 16]. In those works we were able, for the first time, to enrich and isolate in pure culture a number of haloarchaeal strains utilizing various forms of native insoluble cellulose as a carbon and energy source both in neutral and alkaline saturated salt brines. The cellulotrophic natronoarchaea from hypersaline alkaline lakes included two subgroups: two strains with relative weak cellulase activity, belonging to a known species Natronolimnobius baerhaense (for which the capacity for cellulose hydrolysis had not previously been demonstrated) [15] and six strains with high cellulose-degrading capacity described recently as Natronobiforma cellulositropha gen. nov., sp. nov. [16]. The group of neutrophilic cellulotrophic haloarchaeal isolated from various hypersaline chloride-sulfate lakes, included Halomicrobium sp. strain HArel3, Halosimplex sp. strain HArcel2 and a novel lineage, strain HArcel1 T [15]. In this paper we describe the phenotypic and phylogenetic properties of strain HArcel1 T and suggest its assignment into a novel genus and species Halococcoides cellulosivorans.Surface sediments and near-bottom brines from three hypersaline lakes in the Kulunda Steppe (Altai region, Russia) with salt concentration of 280–350 g l ‒1 and pH from 7.5 to 8.1 were used to enrich for cellulotrophic haloarchaea [15]. The brine-sediment slurries from three lakes were mixed, homogenized by vortexing, and the resulting mix was briefly centrifuged at low speed to remove the course sediment fraction, while the remaining colloidal fraction was used as an inoculum.The basic mineral medium used for the enrichment and cultivation of haloarchaea contained (g l ‒1): 240 NaCl, 5 KCl, 0.25 NH 4 Cl and 3 K 2 HPO 4 /KH 2 PO 4, pH 6.8. After sterilization, the base was supplemented with vitamin and trace metal mix [17], 1 mM MgSO 4, 20 mg l ‒1 yeast extract and 10 mM filter-sterilized NaHCO 3. Various forms of insoluble cellulose obtained from Sigma or synthesized as described previously (amorphous cellulose, [15]) were used as the only carbon and energy source at a final concentration of 1 g l ‒1. For the enrichment, 1 ml colloidal sediment was used to inoculate 20 ml medium containing 1 g l ‒1 amorphous cellulose in 100 ml closed serum bottles placed on a rotary shaker at 37 Ǫ C and at 120 r.p.m. The development of cells was monitored by the visual extent of cellulose degradation, the appearance of pink-orange colour and by microscopy. After visible cellulose degradation and cell growth (30–40 days), the culture was serially diluted in the same medium containing amorphous cellulose as substrate and the maximal positive dilutions were plated onto a solid medium prepared by mixing the liquid medium (with additional solid NaCl to compensate for dilution with agar) and 5 % extensively washed agar 3: 2 at 55 Ǫ C. The plates were incubated at 37 Ǫ C in closed plastic bags for 40–60 days. The appearance of coloured colonies with large cellulose clearance zones around them was used as an indicator of growth of cellulolytic haloarchaea. It needs to be stressed here that such colonies were never dominating on the plates, even obtained from final positive serial dilutions, indicating a presence of high proportion of satellites probably feeding on the cellulose hydrolysis products. The cellulolytic colonies (Fig. 1a) were transferred to the liquid medium with amorphous cellulose and the positive cultures were further purified by several rounds of plating-liquid culture cultivation with amorphous cellulose. This yielded three pure cultures of cellulotrophic haloarchaea with identical 16S rRNA gene sequences, of which strain HArcel1 T was chosen for further characterization.The phase contrast microscopy was done using the Zeiss Axioplan Imaging 2 microscope. For the electron microscopy of thin sections, cells of strain HArcel1 T grown with amorphous cellulose were fixed in 1 % (w/v) OsO 4 containing 3.0 M NaCl for 1 week at 4 Ǫ C, washed and resuspended in 3 M NaCl, stained overnight with 1 % (w/v) uranyl acetate, dehydrated in ethanol series, and embedded in Epon resin. After thin sectioning, the preparations were post-stained with 1 % (w/v) lead citrate and examined using the JEOL-100 TEM.Cells of HArcel1 T were non-motile cocci of variable size from 0.8 to 3 µm (Fig. 1b). During the first stage of growth on insoluble celluloses most of the cells aggregated with cellulose particles/fibres (Fig. 1c), while free cells appeared only after massive cellulose hydrolysis. Electron microscopy revealed the presence of a large nucleoid and a thin, singlelayer cell wall, typical for many haloarchaeal species (Fig. 1d). The cells lysed after resuspension in solutions containing less than 10 % NaCl.Genomic DNA was isolated by Isolate II Genomic DNA Kit (Bioline Reagents) according to the manufacturer’ s instructions. Fragment genomic libraries were prepared from 1 µg genomic DNA with the NEBNext Ultra DNA library preparation kit (New England Biolabs) according to the manufacturer’ s instructions to obtain mean library size of 600– 700 bp. The library was sequenced with the MiSeq Illumina apparatus using paired-end 250 bp reads. After sequencing all reads were subjected to stringent quality filtering and trimming with CLC Genomics Workbench 10.0 (Qiagen). Sequencing adapters were trimmed with the SeqPrep tool (https://github.com/jstjohn/SeqPrep). Finally, 925 497 read pairs were used for de novo assembly. Reads were assembled with SPAdes 3.10.0 [18]. Initial assembly consisted of 166 scaffolds of total length 2 793 855 nt and an N50 of 2 525 738 nt. In parallel, reads were assembled with the MIRA 4.0.2 genome assembler [19], resulting in an assembly of total length 2 726 789 nt and N50 43 612 nt. After manual curation and comparison of two assemblies using CLC Genomics Workbench 10.0 software (Qiagen) circular ungapped chromosome of strain HArcel1 T was obtained. The total length of the strain HArcel1 T chromosome was 2 723 120 bp, the DNA G+C content was 65.74 mol%. Validation of an assembly was performed by analysis of mapping of all obtained reads back to chromosome sequence performed with the CLC Genomics Workbench. 99.76 % of the reads were mapped resulting in final genome coverage of 88.3±22.6×. Additionally, the integrity of the assembly was checked by the analysis of unaligned read ends with InDel analysis tool of CLC Genomics Workbench. No regions that were significantly enriched by partially aligned reads were found. Due to these results, our genomic assembly can be considered as the finalized complete genome sequence. Annotation with the IMG/ER server pipeline [20] resulted in the prediction of 2641 protein-coding genes, 60 tRNA genes and one complete rRNA operon. Genomic assembly and related metadata have been deposited in the NCBI database under accession numbers XCP028858, PRJNA449302 and SAMN08826612 for the genomic assembly, Bioproject and Biosample, respectively.Fig. 1. Morphology of strain HArcel1 T growing at 4 M total NaCl and 37 Ǫ C. (a) Colonies on amorphous cellulose plates forming large hydrolysis zones; (b) phase contrast microphotograph of cells grown with amorphous cellulose in liquid culture; (c) phase contrast microphotograph of cells forming biofilm on a cellulose fibre; (d) electron microscopy of thin sections of cells grown with amorphous cellulose. CW, cell wall; CM, cytoplasmic membrane; N, nucleoid.16S rRNA and rpoB ′ gene sequences were obtained from the draft genome assemblies of strain HArcel1 T. The phylogenetic analysis was performed in the MEGA 7 package [21]. The 16S rRNA gene sequences of all species of the order Halobacteriales with validly described names obtained from the GenBank were aligned together with the complete sequence of strain HArcel1 T using G-INS-i method in MAFFT server version 7 [22]. The phylogenetic analysis was performed using the maximum-likelihood algorithm and the general time reversible (GTR) model (G+I, four categories) [23]. The rpoB ′-based phylogenetic analysis was performed in the same way as used for 16S rRNA gene. For ribosomal protein phylogenetic analysis, 17 single-copy conserved ribosomal protein sequences (S2, S3, S11, S12, S17, S19, L3, L4, L5, L10, L11, L13, L14, L15, L23, L24, L29) were obtained from the 39 available in the IMG/M-ER [20] genomes of Halobacteriales representatives with Natronomonas as an outgroup. The protein sequences were aligned in MAFFT version 7 [22] using the L- INS-i algorithm and then concatenated using FaBox joiner alignment [24]. The phylogenetic tree based on concatenated alignment of the proteins was reconstructed using the maximum-likelihood method and the LG model (G+I, four categories) [25]. A BLAST search of the 16S rRNA gene of strain HArcel1 T against nucleotide sequences from cultured haloarchaeal species revealed Halorhabdus species and Halapricum salinum as being the closest relatives with 94.0–92.9 and 92.5 % sequence identity, respectively. This level of relation indicates a separate genus status. Further phylogenetic analysis based of the 16S rRNA gene comparison demonstrated that strain HArcel1 T forms a separate lineage within the family Haloarcelaceae [26] with the genera Halorhabdus and Halapricus as the closest relatives (Fig. 2a). Since the divergence point of ‘strain HArcel1– Halorhabdus ’ and Halapricum clusters was not supported by the bootstrap test, the additional markers (rpoB ′ gene and ribosomal proteins) were used to infer the phylogenetic position of strain HArcel1 T (Fig. 2b, c). The results support a separation of strain HArcel1 T, Halorhabdus and Halapricum in a distinct cluster, whereby strain HArcel1 T forms the longest branch, suggesting its novel genus level.Fig. 2. Phylogeny of strain HArcel1 T. (a) Maximum-likelihood 16S rRNA gene sequence-based phylogenetic tree showing the position of strain HArcel1 T (in bold) within the order Halobacteriales. Branch lengths (see scale) correspond to the number of substitutions per site with corrections, associated with the model (GTR, G+I, four categories). All positions with less than 95 % site coverage were eliminated. In total, 1435 positions were used in the alignment of 119 sequences. Numbers at nodes indicate bootstrap values of 1000 repetitions, bootstrap values below 50 % are not shown. The genus Halomarina was used as an outgroup. (b) Maximum-likelihood rpoB ′ gene sequence-based tree showing the position of strain HArcel1 T (in bold) within the order Halobacteriales. All parameters were the same as in 16S rRNA gene-based phylogeny. In total, 1827 positions were used in the alignment of 81 sequences. The genus Halomarina was used as an outgroup. (c) Maximum-likelihood tree based on alignment of 17 ribosomal proteins showing the position of strain HArcel1 T (in bold) within the order Halobacteriales. Branch lengths (see scale) correspond to the number of substitutions per site with corrections, associated with the model (LG, G+I, four categories). All positions with less than 95 % site coverage were eliminated. In total, 2938 positions were used in the alignment of 40 amino acid sequences. The genus Natronomonas was used as an outgroup.Fig. 2. (cont.)Pairwise average nucleotide identity (ANI) comparison was performed using the IMG built-in tool [27]. The calculated ANI values were 74.1 % between strain HArcel1 T and Halapricum salinum; 74.8 % between strain HArcel1 T and Halorhabdus utahensis; and 75.1 % between strain HArcel1 T and Halorhabdus tiamatea (Table 1). For digital DNA– DNA hybridization (DDH) we used the Genome-to- Genome Distance Calculator 2.1 [28]. BLAST+ was selected as local alignment tool and three formulae were used: 1, length of all HSPs divided by total genome length; 2, sum of all identities found in HSPs divided by overall HSP length (recommended); and 3, sum of all identities found in HSPs divided by total genome length. The average in silico DDH values calculated from the three formulas between strain HArcel1 T and Halapricum salinum, Halorhabdus utahensis and Halorhabdus tiamatea were 15.7, 16.4 and 16.6 %, respectively (Table 1). Thus the calculated values of both ANI and DDH were significantly below the recognized species separation (96 and 70 %, respectively), [29].Table 1. Average pairwise genomic nucleotide identity (ANI-P) and digital DNA–DNA hybridization analyses (% similarity) of strain HArcel1 T with the nearest phylogenetic relatives from the family HaloarculaceaeCompared with: ANI-P Digital DDH (average from Strain HArcel1 T Halorhabdus tiamatea Halothabdus utahensis Halapricum salinum three formulas) Strain HArcel1 T Halorhabdus tiamatea SARL4B T 75.1 85.6 75.7 16.6 Halorhabdus utahensis AX-2T 74.8 85.6 75.3 16.4 Halapricum salinum CBA1105 T 74.1 75.7 75.2 15.7Taken together, the phylogenetic analysis and genomebased comparison demonstrated a separate genus-level status of strain HArcel1 T within the family Haloarculaceae.The core membrane lipids were obtained by acid hydrolysis (5 % HCl in methanol by reflux for 3 h) of the freeze-dried cells and subsequent analysis by HPLC–MS for Glycerol Diphytanyl Glycerol Tetraether (GDGTs) and archaeol derivatives according to [30]. Intact polar lipids were obtained by Bligh–Dyer extraction of freeze-dried cells and subsequent HPLC–MS analysis as described in [31].Table 2. Comparative properties of cellulotrophic haloarchaeon strain HArcel1 T with the nearest phylogenetic relatives in the Haloarculaceae: Halorhabdus tiamatea [14, 37] and Halopricum salinum [40]Phospholipids: PGP-Me, phosphatidylglycerophosphate methylester; PG, phosphatidylglycerol; GL-PG, phosphatidylglycose; DGD, diglycosyl glycerol diether; PGS, phosphatidylglycerol sulfate; PGP, phosphatidylglycerophosphate. Glycolipids: S 1 -DGD, monosulfated diglycosyl diether; TGD, triglycosyl glycerol diether. ND, Not determined.Feature Strain HArcel1 T Halorhabdus tiamatea Halapricum salinum JCM 14471 T CBA1105 T Cell morphology Non-motile coccoids Pleomorphic, non-motile Pleomorphic cocci, nonmotile Pigmentation Pale orange – Red Growth substrates, polymers Insoluble celluloses, xylan Pullulan†, starch, xyloglycan†#, xylane†, arabinoxylane", – sugars others cellobiose glycomannan†, beta-mannan (weak)† Glucose, mannose maltose, Galactose, maltose, mannose†, xylose† sucrose glutamate Number of cellulase genes (GH GH5 (24); GH9 (3); GH12 (2) GH5 (6); GH9 (1); GH12 (1) None families) in the genome Anaerobic growth – + (Fermentative, denitrification) – Esterase/lipase ‒ (Tributyrin/olive oil) + (C8)/ND Tweens/ND Protease activity ‒ (Casein, gelatin) + (Gelatin) – Oxidase/catalase weak/+ ‒/+ +/‒ NaCl tolerance range for growth 2.5–5 (3.5–4.0) 1.6–5 (4.5) 2.5–6.0 (3.2) (optimum) (M) pH range for growth (optimum) 6.5–8.0 (7.0–7.2) 6.0–8.5 (7.0–7.5) 7.0–8.0 (7.0) Temperature range for growth max. 50 (opt. 43) max. 55 (opt. 45) max. 45 (37) (optimum)(Ǫ C) Core lipids C20-C20, C25-C20 DGE (undefined) ND Intact membrane polar lipids PGP-Me, PG, DGD, PGP, PGS; PG, PGP-Me, TGD, S1-DGD PG, PGP-Me, unknown sulfolipid three unidentified glycolipids DNA G+C (mol%) 65.7 (genome) 61.7 (Tm) 66.0 (Tm) Habitat Hypersaline salt lakes in south- Deep-sea hypersaline brine Solar saltern west Siberia (Red Sea)*Based on the genomic data and activity measurements but not yet validated by growth experiments.†Determined in this work; negative results for H. tiamatea included amylopectin, dextrans, inulin, galactan, galactomannan, beta-1,3 glycans, arabinan, arabinogalactan and various forms of native insoluble cellulose.The core membrane lipids were dominated by archaeol [C 20 -C 20 dialkyl glycerol ether (DGE), 81 % of the total] with lesser amounts of extended archaeol (C 25 -C 20 DGE, 13 % of the total). Traces of the monoglycerol ether (MGE) lipids (1 C 20 MGE, 2 C 20 MGE, and 2 C 25 MGE) were also detected. The intact polar lipid profile (identified using multistage mass spectrometry) was quite complex, including (in order of abundance) phosphatidylglycerophosphate methylester (PGP-Me), phosphatidylglycerol (PG), a sulfophospholipid with an unknown sulfur-containing headgroup, a diglycosyl (2 GL), phosphatidylglycerophosphate (PGP) and phosphatidylglycerosulfate (PGS) (Fig. S1, available in the online version of this article). When compared with the two closest phylogenetic neighbours (Table 2), only first two most abundant lipids were present in all three species: PGP- Me and PG. These phospholipids are most common in the members of Halobacteria and, in particular, the domination of the PGP-Me is considered to be related to extreme salt tolerance [32]. The less abundant lipids in strain HArcel1 T included a glycolipid phosphatidyldiglycoside and two sulfolipids. Lipids belonging to the glycolipid and sulfolipid classes are also present in the two closest relatives of HArcel1 T. For example, the closest relative, Halorhabdus tiamatea, contains a three glycosyl (3 GL) glycolipid and a monosulfated diglycosyl diether (S1-DGD) sulfolipid. It is probable that the structurally homologues different glyco- and sulfolipids play a similar function in maintaining membrane homeostasis at extreme salinity [33, 34] [33, 34]. Sulfolipids are also commonly found in neutrophilic haloarchaea, and in particular in the members of the family Haloarculaceae [26].Strain HArcel1 T is an obligately aerobic saccharolytic haloarchaeon. Anaerobic growth with cellobiose as substrate was tested in 10 ml liquid cultures placed into 23 ml serum bottles, closed with butyl rubber stoppers and made anoxic by sterile evacuation-flushing with argon. The results were negative either for fermentation, or with elemental sulfur, thiosulfate, DMSO, TMA and nitrate as e-acceptors. During aerobic growth, strain HArcel1 T utilized only three substrates as their carbon and energy source: insoluble celluloses with different degree of crystallinity, including an amorphous form, Sigma celluloses, filter paper; xylan (from birch wood); and cellobiose. Weak and irregular growth was noticed with lichenan (beta-1,4/–1,3 glycan). No growth was detected with the following polysaccharides: CMC, beta 1,3/1,6 and alpha glucans, beta-mannan, beta-galactan, chitin, chitosan, pectin; heteropolysaccharides, such as beta gluco- and galacto-mannans, alginate. The soluble sugar compounds tested negative included glucose, fructose, galactose, mannose, arabinose, rhamnose, N -acetylglucosamine, glucosamine, glucuronic and galacturonic acids, maltose, lactose, trehalose, melibioze, melizitose, xylose, ribose, sorbitol, mannitol, and glycerol. Likewise, no growth was observed with organic acids (C 2 -C 10 fatty acids, lactate, pyruvate, malate, succinate, fumarate) and complex organic amino acid substrates, such as various peptones and yeast extract. The extremely narrow specialization on cellulose polymers of the neutraphilic haloarchaeon HArcel1 T is only the second example among known species of haloaerchae, resembling its recently described alkaliphilic counterpart, Natronobiforma cellulositropha, found in various hyperslaine soda lakes [16].Recommended enzymatic activity tests [35] included plate assays for amylase (soluble starch), protease (casein, gelatin), esterase (tributyrin) and lipase (emulsified olive oil) using a low background of cellobiose (1 mM). Amylase activity was detected by flooding the plate with Lugol solution, for protease activity the plate was flooded with 10 % TCA to denature undegraded protein, while esterase and lipase activities were evident from the visual clearance of turbid background around the colonies. All of these activities were negative. Strain HArcel1 T was strongly catalase positive (colony test with 3 % H 2 O 2), but only weak-positive for oxidase activity (colony test with 1 % tetramethyl- p -phenylenediamine hydrochloride on filter paper). Sulfide formation from thiosulfate or sulfur during aerobic growth with cellobiose (lead acetate paper test) and indole formation from tryptophan (Kovac’ s reagent test, [36]) were all negative. While growing with cellobiose, strain HArcel1 T used only ammonium salts as the N source (urea, nitrate, nitrite were negative).The salt profile for growth in strain HArcel1 T culture was investigated using cellobiose as the substrate in medium buffered at pH 7 with potassium phosphate buff in liquid culture incubated at 37 Ǫ C. Growth was observed within NaCl range from 2.5 to 5 M with an optimum at 3.5– 4 M. The pH for growth with cellobiose at 4 M NaCl was investigated within the range from 5 to 9 using a combination of HEPES (4 g l ‒1) and potassium phosphates (5 g l ‒ 1 in total) as buffers for the pH range from 5 to 8 and a combination of potassium phosphates and 0.5 M Na 2 CO 3 for the pH 8.5–9. The pH during growth was also maintained either by adding CO 2 into the gas phase (to decrease the actual pH) or 1 M filter-sterilized NaHCO 3 (to increase the actual pH). Strain HArcel1 T was able to grow within the pH range of pH 6.5–8.0 with an optimum at pH 7.0–7.2. Based on the data, the isolate can be classified as an extremely halophilic neutrophile. At pH 7 and 4 M NaCl, the strain grew equally well at Mg concentrations from 1 to 20 mM, thus belonging to a low Mg-requiring type. The temperature profiling during growth on cellobiose at pH 7 and 4 M NaCl was done starting from 20 and up to 60 Ǫ C at increments of 5 Ǫ C. Growth was possible from 25 to 50 Ǫ C with an optimum between 40 and 45 Ǫ C.Antibiotic resistance of strain HArcel1 T was tested at optimal growth conditions in liquid culture using cellobiose as substrate. The following antibiotics (100 mg l ‒1) did not inhibit growth: penicillin G, ampicillin, kanamycin, streptomycin, erythromycin, gentamicine and vancomicin. No growth was observed in the presence of chloramphenicol and rifampicin at concentrations above 50 and 30 mg l ‒1, respectively.A phenotypic comparison of strain HArcel1 T with the closest haloarchaeal relatives from Haloarcelaceae is shown in Table 2. Interestingly, the closest relatives of HArcel1 T, the Halorhabdus species, are apparently polysaccharide degraders, according to the presence of multiple GH genes in the genome and activity tests in H. tiamatea [14, 37] and the proven ability of H. utahensis to grow with xylan [38]). Our tests with the type strain of H. tiamatea JCM 14471 T and also with our own isolates closely related to this species demonstrated that these haloarchaea are, indeed, potent polysaccharide degraders capable of growth with a range of glycans as sole source of carbon and energy (Table 2). Especially interesting is the ability (albeit weak with never a complete utilization) of H. tiamatea to grow with beta-1,4 mannan. So far, only two such cases have been found among the extremely halophilc euryarchaea – in Natronoarchaeum mannanilyticum and recently described cellulose-utilizing Natronobiforma cellulositropha [16, 39]. However, the major difference between the Halorhabdus species and strain HArcel1 T is the ability of the latter to use cellulose as growth substrate: none of the tested forms of insoluble celluloses with different degree of crystallinity, including amorphous, four types of Sigma celluloses, filter paper and Avicell, supported growth of H. tiamatea. On the other hand, tests on CMC plates showed a presence of beta-1,4 endoglucanase activity in colonies of H. tiamatea. This is another demonstration that what is often claimed on the basis of test with soluble artificial analogue of cellulose (CMC) as the ability to grow with cellulose should not be considered as valid. Since the genome of another closest relative of strain HArcel1 T, Halapricum salinum [40], completely lacks genes encoding the GH-family glycosidases, it might be concluded that it differs significantly in its key physiological specialization, most probably being an ordinary saccharolytic utilizing products of polymer hydrolysis. Taking into account that three other members of the family Haloarculaceae, the genera Haloarcula, Halomicrobium and Halosimplex do have species with confirmed ability to degrade glycans, including cellulose [12, 13, 15] and chitin (Halomicrobium) [15], it might be speculated that such potential has already been acquired in the common ancestor of this radiation of Halobacteria but lost later on in some members, such as Halapricum, and proliferated in the others, of which strain HArcel1 T seems to be the most narrowly specialized. Further phylogenomic reconstructions might be able to substantiate this interesting question.In conclusion, strain HArcel1 T is the first example of an extremely halophilic euryarchaeon directly enriched and isolated from hypersaline lakes using insoluble celluloses as the growth substrate. Taking into account its unique phenotypic properties and distant phylogenetic position, as inferred from the robust phylogenetic reconstruction based on 19 conservative markers, and ANI and in silico DDH values far below the recognized intragenus levels, we propose to classify strain HArcel1 T in a novel genus and species Halococcoides cellulovorans.