MAG binning and general features
From the four hot springs, we assembled four associated metagenomes and then binned a total of 42 MAGs. We recovered 12 MAGs from RB10 hot spring, 13 from RB13, 14 from RB32 and 3 from RB108. Out of these 42 MAGs, 7 were of high-quality, 25 of nearly-high quality, 9 of medium quality and 1 of low quality (Table 1) based on metagenomic standards26. The GC% was quite variable, ranging from 25.76 to 70.35% among all MAGs and between 32.15 and 69.21% only among the high- and near high-quality MAGs. With the exception of RB108 from which we only recovered bacterial MAGs, we retrieved both bacterial and archaeal MAGs in the other hot springs. Two thirds of the MAGs (26/42) were assigned to the domain Bacteria and the rest to the domain Archaea (16/42) (Table 2).
Taxonomic and phylogenomic analyses of MAGs
The taxonomic affiliation of the MAGs was investigated in detail through the workflow classify of GTDB-Tk (v 2.1.0; GTDB reference tree 07-RS207) (Table 2) and through de novo phylogenomic analyses (Fig. S1a–i). We also tried to classify MAGs on the basis of overall genome relatedness indices (OGRI), which is detailed in supplementary material (Text S1, Table S2, Fig. S2).
De novo phylogenomic analyses globally confirmed the positioning of MAGs provided by GTDB-Tk, with high branching support. For Bacteria, GTDB-Tk analyses allowed us to place the MAGs in the following clades: six in the phylum Aquificota from the four different springs, comprising four MAGs belonging to the genus Hydrogenivirga (family Aquificaceae) (RB10-MAG07, RB13-MAG10, RB32-MAG07, RB108-MAG02), and two belonging to the family ‘Hydrogenobaculaceae’ (RB10-MAG12, RB32-MAG11) (Table 2, Fig. S1a). Their closest cultured relatives originated either from hot springs or from deep-sea hydrothermal vents27. Three MAGs from three geothermal springs belonged to the phylum Armatimonadota (RB10-MAG03, RB13-MAG04, RB32-MAG03) and had no close cultured relatives. Seven MAGs have been classified into the phylum Chloroflexota: three MAGs belonging to the genus Thermoflexus from three different springs (RB10-MAG04, RB13-MAG05, RB32-MAG02), one affiliating with the genus Thermomicrobium (RB32-MAG08), one falling into the family Ktedonobacteraceae (RB108-MAG03), one belonging to the class Dehalococcoidia (RB32-MAG04) and another one whose phylogenetic position is more difficult to assert because it is a MAG of medium quality (RB32-MAG14). Six MAGs from four various hot springs belonged to the phylum Deinococcota, and to the genera Thermus (RB10-MAG08, RB10-MAG11, RB13-MAG09, RB32-MAG10, RB108-MAG01) and Meiothermus (RB13-MAG13). One MAG belonged to the family ‘Sulfurifustaceae’ (RB13-MAG01), in the phylum Proteobacteria (Gamma-class). The MAG referenced as RB32-MAG13 was classified into the phylum ‘Patescibacteria’, in the class ‘Paceibacteria’, and was distantly related to MAGs originating from groundwater and from hot springs. Finally, two MAGs from two different springs belonged to the phylum WOR-3, in the Candidatus genus ‘Caldipriscus’ (RB32-MAG12, RB10-MAG09).
For Archaea, almost all the MAGs reconstructed in this study, e.g. 15 of the 16 archaeal MAGs, belonged to the phylum Thermoproteota. Among them, four belonged to the genus Ignisphaera (RB10-MAG05, RB13-MAG08, RB13-MAG11, RB32-MAG05), three to the genus Infirmifilum (RB10-MAG06, RB13-MAG03, RB32-MAG09), two to the genus Zestosphaera (RB10-MAG02, RB13-MAG06), three to the family Acidilobaceae (RB10-MAG01, RB13-MAG02, RB32-MAG01) and two to the order Geoarchaeales (RB10-MAG10, RB32-MAG06). Additionally, one belonged to the family Thermocladiaceae (RB13-MAG07). Lastly, the MAG belonging to another phylum (RB13-MAG12) was affiliated with the ‘Aenigmatarchaeota’, class ‘Aenigmatarchaeia’, and was distantly related to MAGs from hot springs and from deep-sea hydrothermal vent sediments28,29.
Out of these 42 MAGs, at least 19 MAGs corresponded to different taxa at the taxonomic rank of species or higher according to GTDB (Table 2). Eighteen of them belonged to lineages with several cultivated representatives including the species Thermus thermophilus. 13 new genomic species within the GTDB genera Hydrogenivirga, HRBIN17, Thermoflexus, SpSt-223, CADDYT01, Zestosphaera, Ignisphaera, Infirmifilum, Thermus, Thermus_A, Meiothermus_B, JAHLMO01 and Caldipriscus, and 6 putative new genomic genera belonging to the GTDB families Hydrogenobaculaceae, Acidilobaceae, WAQG01, Thermocladiaceae, Sulfurifustaceae and HR35 could be identified (Table 2). In addition, 9 MAGs belonged to lineages that are predominantly or exclusively known through environmental DNA sequences. Thus, these 42 MAGs comprised a broad phylogenetic range of Bacteria and Archaea at different levels of taxonomic organization, of which a large majority were not reported before.
The approaches implemented here were not intended to describe the microbial diversity present in these sources in an exhaustive way or to compare them in a fine way, and cannot allow it because of a 2-year storage at 4 °C. This long storage has probably led to changes in the microbial communities and to the selective loss or enrichment of some taxa. As a result, no analysis of abundance or absence of taxa can be conducted from these metagenomes and the results are discussed taking this bias into account. However, they do provide an overview of the microbial diversity effectively present. If we compare the phylogenetic diversity of the MAGs found in the four hot springs, we can observe that 3 shared phyla (Deinococcota, Aquificota and Chloroflexota: phyla names according to GTDB), 2 shared families (Thermaceae and Aquificaceae), and one shared genus (Hydrogenivirga) were found among the four sources (Fig. 2). In addition, hot springs RB10, RB13 and RB32, that are geographically close (< 60 m), also share 2 other phyla (Thermoproteota and Armatimonadota) and 5 other families in common (Acidilobaceae, Ignisphaeraceae, Thermofilaceae, Thermoflexaceae, and HRBIN17) (Fig. 2). These phyla and families that are shared between sources are widespread lineages in terrestrial geothermal habitats (e.g.4,5,6,12). Phyla and families detected in the hot environments of Antarctica are also found here, such as Patescibacteria15. In summary, this metagenomic analysis highlighted the presence of bacterial and archaeal lineages commonly found in hot springs, and lineages found in hot habitats from polar areas (e.g.4,5,6,15,30). The microbial communities in these Kerguelen Islands hot springs samples were diverse, particularly in RB10, RB13, and RB32 hot springs. Within these lineages previously reported to occur in geothermal environments, a majority of the genomic taxa detected here were novel. Those results were obtained considering their taxonomic affiliation by GTDB-Tk, and their phylogenomic position with respect to closest relatives and the OGRI thresholds (16S rRNA gene sequence similarity, average nucleotide identity, and average amino acid identity) classically used to delineate different taxonomic ranks in cultured strains, used here as indicators of taxonomic differentiation (Table 2, Table S2).
Venn diagrams showing the shared phyla, families and genera according to GTDB classification in the reconstructed MAGs from the hot springs RB10, RB13, RB32 and RB108.
Functional potential of MAGs: putative metabolisms and adaptations
A genomic characterization of the 42 MAGs has been performed to explore the possible metabolic pathways and adaptations of the microbial populations from which these MAGs originate. KEGG Decoder visualization highlighted various pathways associated with carbohydrate degradation, oxidative phosphorylation and sulfur, nitrogen, and amino-acid metabolisms, among others (Fig. 3). To confirm these initial metabolic predictions, a further annotation was performed by combining data generated by Prokka with the MetaCyc database. Efforts have been directed at studying catabolic pathways, particularly those involving inorganic electron donors and acceptors. These results are not representative of the metabolic diversity of all the hot spring ecosystems studied, but they do reflect some of the microbial catabolism likely to be used in situ to produce energy and, by assumption, the most abundant ones. Metabolic predictions are presented hereafter, at different taxonomic ranks and have been compared to the known phenotypes of the closest cultivated taxa, and in some cases to genomic content of the closest relatives.
Metabolic pathway diagram of the 42 MAGs based on KEGG Decoder annotations, showing MAG classification according to GTDB-Tk and estimated genome completion.
MAGs belonging to the genus Thermoflexus (RB10-MAG04, RB13-MAG05, RB32-MAG02) encode pathways for carbon monoxide oxidation (via aerobic carbon monoxide dehydrogenase), hydrogen oxidation and nitrate respiration; the only cultivated known representative of this genus is a heterotrophic bacterium31. The same pathways, except for the nitrate reduction pathway, are encoded in the complete genome of Thermoflexus hugenholtzii (NCBI: ASM1877156v1). In contrast, the genome of T. hugenholtzii, a strain isolated from a terrestrial hot spring in Nevada31, encodes the tetrathionate reduction and thiosulfate disproportionation pathways, which are not encoded in the three Kerguelen Island MAGs. The Dehalococcoidia’s MAG (RB32-MAG04) encodes only a carbon monoxide oxidation pathway; the cultivated members of this genus are strict anaerobic hydrogenotrophic, organohalide-respiring bacteria32. In the MAG associated with the genus Thermomicrobium (RB32-MAG08), we predicted pathways for dimethylsulfide degradation, thiosulfate disproportionation and carbon monoxide oxidation; only carboxydotrophic growth has been reported in this genus and demonstrated by culture33. The same pathways are encoded in the complete genome of Thermomicrobium roseum, a strain isolated from a hot spring in Yellowstone National Park (NCBI: ASM2168v1)34. In the Chloroflexota’s MAG (RB32-MAG14) (belonging to the order Chloroflexales, Table S2), carbon monoxide oxidation and thiosulfate disproportionation pathways are present but no coding DNA sequence associated with phototrophy could be find, which may suggest a chemoorganotrophy mode of energy production35. The Ktedonobacteraceae’s MAG (RB108-MAG03) encodes enzymes for hydrogen oxidation (aerobic) pathways, carbon monoxide oxidation, dimethylsulfide degradation, selenate reduction, thiosulfate oxidation and disproportionation and finally tetrathionate oxidation; yet, the few taxa of this family isolated so far are mesophilic heterotrophic bacteria36. Within Hydrogenobaculaceae MAGs (RB10-MAG12, RB32-MAG11), we predicted a thiosulfate disproportionation pathway; most of the species within this family are capable of chemolithotrophic microaerophilic or anaerobic growth37. MAGs belonging to the genus Hydrogenivirga (RB10-MAG07, RB13-MAG10, RB32-MAG07, RB108-MAG02) possess genes encoding enzymes of aerobic respiration, thiosulfate oxidation, thiosulfate disproportionation, tetrathionate reduction, and hydrogen oxidation (aerobic and anaerobic); which is consistent with what is known about the genus (nitrate and oxygen respiration combined to hydrogen, sulfur, or thiosulfate oxidation)37. The same pathways, with the exception of the hydrogen oxidation pathway, are encoded in the genome of Hydrogenivirga caldilitoris (NCBI: ASM366400v1), a close relative isolated from a coastal hot spring in Japan27. In MAGs associated with the genus Thermus (RB10-MAG08, RB10-MAG11, RB13-MAG09, RB32-MAG10, RB108-MAG01), we predicted pathways for aerobic respiration, assimilatory sulfate reduction, hydrogen oxidation, selenate reduction, thiosulfate oxidation and thiosulfate disproportionation; cultivated species of this genus grow mainly chemoorganoheterotrophically by aerobic respiration, but some have genes coding for chemolithotrophic and anaerobic respiration enzymes38. The MAG belonging to the genus Meiothermus (RB13-MAG13) encodes pathways for carbon monoxide oxidation, hydrogen oxidation, thiosulfate oxidation and thiosulfate disproportionation; Meiothermus strains are known to grow chemoorganotrophically by oxygen or nitrate respiration39. For the RB13-MAG01 belonging to the Sulfurifustaceae, we predicted the genetic potential for aerobic respiration, ammonia oxidation, dissimilatory sulfate reduction, sulfite oxidation, sulfide oxidation (to sulfur globules), tetrathionate reduction, thiosulfate oxidation and thiosulfate disproportionation; Sulfurifustaceae (referenced as Acidiferrobacteraceae in the LPSN taxonomy) are known to be able to oxidize sulfur and iron, and the microorganism corresponding to this MAG may possess a larger panel of chemolithotrophic abilities40. For members of the Armatimonadota (RB10-MAG03, RB13-MAG04, RB32-MAG03), we predicted pathways for assimilatory sulfate reduction, carbon monoxide oxidation, selenate reduction and thiosulfate disproportionation; the members of the phylum have a phenotype of aerobic heterotrophs41. In Zestosphaera’s (RB10-MAG02, RB13-MAG06) and Ignisphaera’s (RB10-MAG05, RB13-MAG08, RB13-MAG11, RB32-MAG05) MAGs, we predicted sulfur and polysulfide reduction pathways; those MAGs could be classified as Desulfurococcaceae (LPSN taxonomy, Table S2) which are known as heterotrophs respiring sulfur species42,43. MAGs belonging to the class Thermoproteia (RB10-MAG10, RB13-MAG07, RB32-MAG06) encode dissimilatory sulfate reduction pathway; various catabolic pathways are described in this class44. In MAGs related to the genus Caldipriscus (RB10-MAG09, RB32-MAG12), phylum Patescibacteria (RB32-MAG13), family Acidilobaceae (RB10-MAG01, RB13-MAG02, RB32-MAG01), family Thermofilaceae (RB10-MAG06, RB13-MAG03, RB32-MAG09) and class Aenigmatarchaeia (RB13-MAG12), we did not predict any catabolic pathway of inorganic nutrients among those reported in the MetaCyc database. This could be explained by the low completion of the MAGs and/or the fact that only well-known pathways are documented in this database. However, all these MAGs have pathways associated with carbohydrate and protein degradation. This may indicate that these taxa are chemoheterotrophs, which has already been reported in geothermal environments and already described for relatives of some of these taxa45,46.
Sulfide oxidation may be a possible energy production pathway for 28 MAGs based on KEGG Decoder (Fig. 3), since they code for a sulfide:quinone oxidoreductase (KEGG:K17218) and a flavoprotein chain of sulfide dehydrogenase (KEGG:K17229), but this hypothesis was not confirmed by MetaCyc except for RB13-MAG01. Due to high representations of sulfur metabolisms, genes encoded in MAGs were evaluated with DiSCo, which gave similar results to those obtained when analyzed with Pathway tools. DiSCo confirmed complete dissimilatory sulfate reduction pathways for two MAGs, predicted to be associated to sulfate reduction processes (RB13-MAG07) or sulfide oxidation processes by reverse sulfate reduction pathway (RB13-MAG01). The assimilatory sulfate reduction pathway is more represented in the overall dataset formed by all MAGs than the dissimilatory pathway, which is consistent with the low sulfate concentration measured in the four hot springs (Table S1). The thiosulfate disproportionation pathway predicted by MetaCyc in many MAGs simply refers to the detection of an enzyme, the rhodanese-type thiosulfate sulfurtransferase. However, in the current state of knowledge on the disproportionation pathways of inorganic sulfur compounds47,48, this enzyme alone does not allow the implementation of this catabolic pathway. If we consider all the genes present in these MAGs, nothing indicates that the microorganisms from which these MAGs originate can achieve the disproportionation of inorganic sulfur compounds.
Additionally, no enzymes clearly associated with photosystems I and II were found. Nevertheless, it cannot be ruled out that these energy production pathways are absent in microorganisms indigenous to these sources, due to sample storage bias and low completion of some MAGs. On the other hand, our results show that these sources host chemolithoautotrophic taxa involved in the carbon and sulfur cycle, and to a lesser extent in the hydrogen and nitrogen cycles. Several taxa are likely to be involved in the primary production of these sources through chemolithoautotrophy, but in addition, heterotrophs appear to be present and diverse in the collected samples. Additional studies will be required to better understand the metabolic diversity and trophic webs of these hot springs, in order to better understand the ecology of the microbial communities of the Kerguelen hot springs.
Regarding thermophily, we found that all MAGs encode heat shock proteins, mainly associated with the HSP20 family, with the exception of RB10-MAG12 and RB13-MAG12. The absence of Hsp encoding genes in these two MAGs is possibly due to the low genome completeness of these two MAGs. Under conditions of heat stress, it has been shown that the small heat shock proteins Hsp20, protect cellular proteins from aggregation and membrane lipids from destabilization, in some thermophilic archaea49. In taxa of these geothermal sources, these proteins could help the cells to counteract the deleterious effects of environmental stress and in particular of thermal stress. In addition, reverse gyrase coding sequences were found in 29 out of the 42 MAGs; these enzymes are known to be exclusive to hyperthermophiles and involved in DNA protection and repair at high temperatures50. Only MAGs RB10-MAG04, RB10-MAG09, RB10-MAG11, RB13-MAG01, RB13-MAG05, RB13-MAG09, RB13-MAG13, RB32-MAG02, RB32-MAG04, RB32-MAG08, RB32-MAG10, RB32-MAG12 and RB108-MAG03, belonging to the phylum Chloroflexota, the family Sulfurifustaceae or the genus Caldipriscus (GTDB taxonomy), do not encode any reverse gyrase gene. These results suggest the presence of numerous thermophilic and hyperthermophilic prokaryotes in these high temperature hot springs. Further cultural and physiological investigations from samples of these Kerguelen hot springs will be necessary to confirm these statements.
In conclusion, this first metagenomic overview of the microbial diversity of Kerguelen hot springs allowed the assembly of 42 MAGs, from four hot springs. Several MAGs correspond to putative new taxa, namely 13 new putative genomic species and 6 new putative genera affiliated to Bacteria and Archaea according to GTDB. Based on their genetic potential, these taxa appear to be chemolithoautotrophs and chemoheterotrophs and thus probably involved in the carbon, sulfur, hydrogen and nitrogen cycle. Many of these MAGs are likely to be derived from populations of thermophilic/hyperthermophilic bacteria and archaea. As geographically isolated sites, the Kerguelen Islands are reservoirs of diversity and taxa of novel microorganisms that should be interesting to study the evolution of microbial life and speciation processes. It has been difficult to fully assess the microbial metabolic diversity in these geothermal pools due to the inherent limitations of MAG reconstruction and the state of knowledge of microbial pathways that remains limited. However, these geothermal ecosystems could be reservoirs of biological and genomic novelty. The physiological properties and adaptive mechanisms of microorganisms inhabiting these unique environments will deserve to be examined in detail in the future by implementing large-scale metagenomics, metatranscriptomics and cultural analyses.
Source: Ecology - nature.com
