Halococcoides cellulosivorans gen. nov., sp. nov., an extremely halophilic cellulose-utilizing haloarchaeon from hypersaline lakes

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.0.003312 Abstract An 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