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.
Table 1.
Key features of the population genome bins (MAGs) constructed from the assembled metagenome of Lotus Pond’s vent-water.
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).
Table 2.
Bacterial genera that were detected in the present exploration of the Lotus Pond vent-water as well as in one or more previous studies of the same habitat using 16S rRNA gene sequence analysis.