Culture-Independent Exploration of the Hypersaline Ecosystem Indicates the Environment-Specific Microbiome Evolution

Abstract

Sambhar Salt Lake, situated in the state of Rajasthan, India is a unique temperate hypersaline ecosystem. Exploration of the salt lake microbiome will enable us to understand microbiome functioning in nutrient-deprived extreme conditions, as well as enrich our understanding of the environment-specific microbiome evolution. The current study has been designed to explore the Sambhar Salt Lake microbiome with a culture-independent multi-omics approach to define its metagenomic features and prevalent metabolic functionaries. The rRNA feature and protein feature-based phylogenetic reconstruction synchronously (R = 0.908) indicated the dominance of the archaea (Euryarchaeota) and bacteria (Firmicutes, Proteobacteria, Bacteroidetes, and Actinobacteria). Metabolic reconstruction identified selective enrichment of the protein features associated with energy harvesting and stress tolerance (osmotic, oxidative, metal/metalloid, heat/cold, antibiotic, and desiccation). Metabolites identified with metabolome analysis confirmed physiological adaptation of the lake microbiome within a hypersaline and nutrient-deprived environment. Comparative metagenomics of the 212 metagenomes representing freshwater, alkaline, and saline ecosystem microbiome indicated the selective enrichment of the microbial groups and genetic features. The current study elucidates microbiome functioning within the nutrient-deprived harsh ecosystems. In summary, the current study harnessing the strength of multi-omics and comparative metagenomics indicates the environment-specific microbiome evolution.

Keywords: extremophiles; hypersaline lake; metagenome; microbiome; microbiome physiology; multi-omics analysis; stress response and adaptation.

FIGURE 1

Overview of osmotic stress tolerance mechanisms prevalent within Sambhar hypersaline lake microbiome. Sambhar salt lake microbiome has genes clusters encoding proteins for the biosynthesis of osmolytes like Glycine Betaine from glycine (1) & choline (2), Ectoine and Hydroxyectoine (3); transport of various osmolytes (proline, glycine betaine, choline, etc.) (4), water (5), ions (6); and metabolism of glycine betaine to generate energy-rich metabolic substrate (7). GMT, glycine N-methyltransferase; SMT, sarcosine N-methyltransferase; DGMT, dimethylglycine N-methyltransferase; DGDH, dimethylglycine dehydrogenase; SDH, sarcosine dehydrogenase; CDH, choline dehydrogenase; BADH, betaine aldehyde dehydrogenase; EctABCD, ectoine biosynthetic enzymes.

FIGURE 2

Principal component analysis (PCA) of the diverse metagenomes with rRNA-based taxonomic features. Principal component analysis showing the clustering of the freshwater, alkaline water, and saline water metagenomes with taxonomic features identified at the phylum level of taxonomic hierarchy (A) and class level of taxonomic hierarchy (B).

FIGURE 3

Principal component analysis (PCA) of the diverse metagenomes with subsystem annotated protein features. Principal component analysis showing the clustering of the freshwater, alkaline water, and saline water metagenomes with subsystem database annotated metabolic at the hierarchy level L1 (A) and hierarchy level L2 (B).

FIGURE 4

Ternary plots showcasing the differential microbiome composition using rRNA features phylogenetically affiliated at phylum (A) and class level (B) of the taxonomic hierarchy.

FIGURE 5

Ternary plot indicating the differentially abundant metabolic features of saline, freshwater, and alkaline ecosystem identified with subsystem annotated metabolic features at hierarchy level 1.

FIGURE 6

Differential abundance of the protein features associated Dormancy and Sporulation, Motility and Chemotaxis, Metabolism of aromatic compounds, and Photosynthesis (p < 0.05) among saline, freshwater, and alkaline metagenomes (A). Comparative analysis shows metabolic features associated with the protection from osmotic stress, oxidative stress, antibiotic, and metal/metalloid toxicity among saline, freshwater, and alkaline ecosystems indicating the significantly diverse abundance (p < 0.05) of these metabolic features in these ecosystems (B).