Aquificae overcomes competition by archaeal thermophiles, and crowding by bacterial mesophiles, to dominate the boiling vent-water of a Trans-Himalayan sulfur-borax spring

Abstract

Trans-Himalayan hot spring waters rich in boron, chlorine, sodium and sulfur (but poor in calcium and silicon) are known based on PCR-amplified 16S rRNA gene sequence data to harbor high diversities of infiltrating bacterial mesophiles. Yet, little is known about the community structure and functions, primary productivity, mutual interactions, and thermal adaptations of the microorganisms present in the steaming waters discharged by these geochemically peculiar spring systems. We revealed these aspects of a bacteria-dominated microbiome (microbial cell density ~8.5 × 104 mL-1; live:dead cell ratio 1.7) thriving in the boiling (85°C) fluid vented by a sulfur-borax spring called Lotus Pond, situated at 4436 m above the mean sea-level, in the Puga valley of eastern Ladakh, on the Changthang plateau. Assembly, annotation, and population-binning of >15-GB metagenomic sequence illuminated the numeral predominance of Aquificae. While members of this phylum accounted for 80% of all 16S rRNA-encoding reads within the metagenomic dataset, 14% of such reads were attributed to Proteobacteria. Post assembly, only 25% of all protein-coding genes identified were attributable to Aquificae, whereas 41% was ascribed to Proteobacteria. Annotation of metagenomic reads encoding 16S rRNAs, and/or PCR-amplified 16S rRNA genes, identified 163 bacterial genera, out of which 66 had been detected in past investigations of Lotus Pond's vent-water via 16S amplicon sequencing. Among these 66, Fervidobacterium, Halomonas, Hydrogenobacter, Paracoccus, Sulfurihydrogenibium, Tepidimonas, Thermus and Thiofaba (or their close phylogenomic relatives) were presently detected as metagenome-assembled genomes (MAGs). Remarkably, the Hydrogenobacter related MAG alone accounted for ~56% of the entire metagenome, even though only 15 out of the 66 genera consistently present in Lotus Pond's vent-water have strains growing in the laboratory at >45°C, reflecting the continued existence of the mesophiles in the ecosystem. Furthermore, the metagenome was replete with genes crucial for thermal adaptation in the context of Lotus Pond's geochemistry and topography. In terms of sequence similarity, a majority of those genes were attributable to phylogenetic relatives of mesophilic bacteria, while functionally they rendered functions such as encoding heat shock proteins, molecular chaperones, and chaperonin complexes; proteins controlling/modulating/inhibiting DNA gyrase; universal stress proteins; methionine sulfoxide reductases; fatty acid desaturases; different toxin-antitoxin systems; enzymes protecting against oxidative damage; proteins conferring flagellar structure/function, chemotaxis, cell adhesion/aggregation, biofilm formation, and quorum sensing. The Lotus Pond Aquificae not only dominated the microbiome numerically but also acted potentially as the main primary producers of the ecosystem, with chemolithotrophic sulfur oxidation (Sox) being the fundamental bioenergetic mechanism, and reductive tricarboxylic acid (rTCA) cycle the predominant carbon fixation pathway. The Lotus Pond metagenome contained several genes directly or indirectly related to virulence functions, biosynthesis of secondary metabolites including antibiotics, antibiotic resistance, and multi-drug efflux pumping. A large proportion of these genes being attributable to Aquificae, and Proteobacteria (very few were ascribed to Archaea), it could be worth exploring in the future whether antibiosis helped the Aquificae overcome niche overlap with other thermophiles (especially those belonging to Archaea), besides exacerbating the bioenergetic costs of thermal endurance for the mesophilic intruders of the ecosystem.

Fig 1

Topography of the study site: (A) the Lotus Pond hot spring in the context of the river Rulang and the valley of Puga, (B) the boiling vent-water explored for microbiome structure and function, (C) boratic sinters, and fresh condensates of sulfur and boron minerals covering the broken wall of the old crater within which Lotus Pond is embedded.

Fig 2

Representative picture of the fluorescence microscopic fields based on which microbial cell density was calculated in the vent-water sample of Lotus Pond: (A) sample stained with DAPI, (B) sample stained with FDA, (C) sample stained with PI. Scale bars in all the three micrographs indicate 50 μm length.

Fig 3

Taxonomic distribution of (A) the CDSs identified in the assembled metagenome of Lotus Pond, and (B) the metagenomic reads which corresponded to 16S rRNA genes (actual CDS count or metagenomic read count recorded for each taxon is given in parenthesis). In (A) “minor phyla” included the following: Acidobacteria, Chlamydiae, Chlorobi, Deferribacteres, Fusobacteria, Gemmatimonadetes, Nitrospirae, Planctomycetes, Spirochaetes, Synergistetes, Tenericutes, Thermomicrobia and Verrucomicrobia. In (B) “other phyla”, also marginal in representation, included the following: Cyanobacteria, Thermotogae, Bacteroidetes, Chloroflexi, Actinobacteria, Armatimonadetes and Synergistetes.

Fig 4. Phylogenetic tree (number of bootstrap tests carried out = 10000) based on 92 universal bacterial core genes that shows the evolutionary relationships among the 14 bacterial MAGs reconstructed from the Lotus Pond vent-water metagenome and their closest relatives identified based on dDDH and/or rRNA gene sequence relationships.

Scale bar denotes a distance equivalent to 10% nucleotide substitution. In the phylogeny reconstructed, nucleotide substitutions were interpreted using the generalized time-reversible model, which considers the proportion of invariable sites and/or the rate of variation across the sites.

Fig 5. Simplified schematic view of the carbon assimilation pathways for which genes were identified in the assembled metagenome of Lotus Pond.

The relevant genes were identified based on the annotation of CDSs with reference to the published literature, and biochemical modules curated under the KEGG Pathway Maps database located at https://www.genome.jp/kegg/pathway.html: (A) reductive citrate cycle, (B) reductive pentose phosphate cycle, (C) dicarboxylate/4-hydroxybutyrate cycle, (D) 3-hydroxypropionate/4-hydroxybutyrate cycle, (E) 3-hydroxypropionate bi-cycle, and (F) reductive acetyl Co-A pathway. Names of the pathways for which one or more genes were undetectable in the metagenome have been written in pink fonts. The active inorganic carbon substrates assimilated by the different pathways are highlighted in yellow. The biochemical conversions (steps) of the different pathways for which CDSs could be identified in the assembled metagenome are indicated by the roman numerals I through L. While the roman numerals shaded green indicate the key enzymatic steps of the pathway in question, the numbers given in parentheses denote the numbers of CDSs identified for the individual enzymatic steps. The enzyme-coding genes actually detected in the metagenome for the biochemical steps labeled as I through L are enumerated below by their KEGG orthology identifiers. I—K00024; II—K01676, K01677, K01678, K01679; III—K18556, K18557, K18558, K18559, K18560; IV—K01902, K01903; V—K00174, K00175, K00176, K00177; VI—K00031; VII—K01681, K01682; VIIIA—K15232, K15233; VIIB—K15234; IX—K15230, K15231; X—K00169, K00170, K00171, K00172, K03737; XI—K01006, K01007; XII—K01595; XIII—K01958, K01959, K01960; XIV—K00855; XV—K01601, K01602; XVI—K00927; XVII—K00134, K00150; XVIII—K01623, K01624; XIX—K01086, K02446, K03841, K11532; XX—K00615; XXI—K01086, K11532; XXII—K01807, K01808; XXIII—K15038; XXIV—K14534; XXV—K15016; XXVI—K00626; XXVII—K00169, K00170, K00171, K00172; XXVIII—K01007; XXIX—K01677, K01678; XXX—K00239, K00240, K00241, K18860; XXXI—K01902, K01903; XXXII—K15037; XXXIII—K15020; XXXIV—K05606, K01848, K01849; XXXV—K02160, K01961, K01962, K01963; XXXVI—K14468; XXXVII—K14469; XXXVIII—K08691; XXXIX—K14449; XL—K14470; XLI—K09709; XLII—K15052; XLIII—K05606, K01847, K01848, K01849; XLIV—K14471, K14472; XLV—K00239, K00240, K00241, K01679; XLVI—K05299, K22015; XLVII—K00194, K00195, K00196, K00197, K00198, K14138; XLVIII—K01938; XLIX—K01491; L—K00297. In the panel A showing the different steps of the rTCA cycle, dotted lines indicate the reactions of the modified rTCA cycle which are mediated by the enzymes citryl-CoA synthetase (VIIIA) and citryl-CoA lyase (VIIIB).