Natrarchaeobius versutus sp. nov. and Natrarchaeobius oligotrophus sp. nov., chitinotrophic natronoarchaea from hypersaline soda lakes, and functional genome analysis of the Natrarchaeobius species

Abstract

Polysaccharide-degrading natronoarchaea have been poorly studied to date. However, over the past decade, significant progress has been made in understanding their diversity and metabolic potential. In this study, two natronoarchaeal strains, enriched from oxic sediment samples of the soda lakes of Wadi an Natrun in Egypt (AArcel7) and Kulunda steppe in Russia (A-rgal3), were characterized. Strain AArcel7 was enriched with amorphous cellulose, while strain A-rgal3 dominated an enrichment culture using rhamnogalacturonan. Cells of both strains are polymorphic, from motile flat rods to nonmotile cocci. They are aerobic heterotrophs that are able to grow on chitin and several other carbohydrates. Both strains thrive within a salinity range of 2.5 to 4.5 M total Na⁺, with optimal growth at 3.5–4 M, and are moderately alkaliphilic with an optimum pH at 8.5–9.0 (AArcel7) and 9.2–9.5 (A-rgal3). Genome-based phylogenetic analysis demonstrated that these isolates form a new species lineage in the chitin-specialized genus Natrarchaeobius. An in-depth study of Natrarchaeobius genomes allowed us to identify several genes that potentially enable them to hydrolyze chitin and to metabolize N-acetylglucosamine (GlcNAc), which has not been investigated previously in the chitin-utilizing natronoarchaea. Based on physiological, phylogenetic, and genomic analyses, strains AArcel7 and A-rgal3 are suggested to form a novel species, Natrarchaeobius versutus sp. nov., with AArcel7^T (DSM 119357 = UNIQEM U973) as the type strain. Furthermore, strain AArcht7^T, formerly classified as the type species of the genus Natrarchaeobius, is proposed to be reclassified as Natrarchaeobius oligotrophus (DSM 119677 = UNIQEM U967).

Keywords: Natrarchaeobius; chitin; chitinase; glycoside hydrolases; natronoarchaea; soda lakes.

Figure 1

Cell morphology of strains AArcel7^T (a,c,d) and A-rgal3 (b) grown with cellobiose at 4 M total Na⁺ and pH 9.5: (a,b) phase contrast microphotographs; (c) transmission electron microscopy of the whole cell showing flagellation and (d) thin-section electron microscopy showing S-layer thin cell wall (S) and extended nucleoid (N). CPM, cytoplasmic membrane.

Figure 2

Maximum-likelihood phylogenetic tree based on the “ar122” set of archaeal proteins revealed the position of strains AArcel7 and A-rgal3, and AArcht7 (in bold font) within Natrialbaceae family. The numbers at nodes indicate the percentage values of rapid bootstrap (from 1,000 replicates). The blue box indicates the Natrarchaeobius genus. Methanothermobacter thermautotrophicus DeltaH (GCA 000008645.1), Archaeoglobus fulgidus DSM 4304 (GCA 000008665.1), and Methanocella paludicola SANAE (GCA 000011005.1) were used as the outgroup (not shown).

Figure 3

Genes of carbohydrate-active enzyme families found in the genomes of Natrarchaeobius species.

Figure 4

Domain organization of chitinases, found in the genus Natrarchaeobius. CBM5 (Carbohydrate-Binding Module family 5), GH18 (catalytic domain of chitinase from Glycoside Hydrolase family 18), PKD (Polycystic Kidney Disease-like domain).

Figure 5

Gene clusters encoding chitinases in the genomes of Natrarchaeobius species: DGA (putative D-glucosaminate-6-phosphate ammonia lyase), GAD (galactonate dehydratase), GDH (glucose dehydrogenase), IclR (HTH-type transcriptional regulator from the IclR family), RutC (transcriptional repressor from the RutC family), S8 (serine peptidase from the S8 family). Hypothetical genes marked with red stars indicate the presence of the ambiguous chitinase.

Figure 6

Predicted pathway of N-acetylglucosamine metabolism in representatives of the genus Natrarchaeobius: CE4/CE14, carbohydrate esterase from CE4/CE14 family; GDH, glucose dehydrogenase; DGA, putative D-glucosaminate-6-phosphate ammonia lyase; GclNAc, N-acetylglucosamine; GlcN, D-glucosamine; KDPG, 2-keto-3-deoxy-6-P-gluconate. The asterisks indicate that target enzymes are not characterized; however, there may be homologs having broad substrate specificity that perform these reactions.