Phyla Bdellovibrionota, Fusobacteriota, and Myxococcota were present in the green microbial mat but in negligible quantities in the brown mat. The unique phyla detected in the brown mat, but not in the green microbial mat, included Caldatribacteriota, Thermodesulfobacteriota, Dictyoglomota, Elusimicrobiota, Thermotogota, Candidatus Calescamantes, Fervidibacteria, Hydrothermae, GAL15 and TA06. The Candidatus Caldatribacterium (phyla Caldatribacteriota), earlier named OP9 was also detected in this work. Using single-cell and metagenome sequencing, data elucidated that Ca. Caldatribacterium conducts anaerobic sugar fermentation and exhibited diverse glycosyl hydrolases, including endoglucanase37.
Cyanobacteria and Chloroflexota were the main identified phyla in the green microbial mat. Because the hot spring is almost stagnant, undisturbed, and the water surface temperature (< 64 °C) is below the maximum threshold of the bacteria photosynthesis process38, together these factors favor the growth of the microorganisms. Chloroflexota Thermoflexus hugenholtzii39 (opt. growth temp. [OGT] 72–75 °C) constituted 31% of B1 microbiota (Fig. 2). The complete genome of T. hugenholtzii JAD2T and several associated metagenome-assembled genomes are available40, and they harbored multiple cellulosic degrading enzymes. When we extracted the DNA materials from sample B3, approximately half of the working materials was reddish-brown jelly-type microbial mat while the remaining were heterogeneous materials. 63% of the total ASVs in the B3 mat were dominated a taxon related to Roseiflexus, another Chloroflexota member. At the time of writing, Roseiflexus castenholzii HLO8T (DSM 13941) is the only described type strain. Bacterium HLO8T, a photosynthetic strain, formed a reddish-brown microbial mat in a Japanese hot spring41. We anticipate that the Chloroflexota associated taxon that formed reddish-brown jelly-type microbial mat in the spring head of SKY hot spring (71–74 °C) is different from strain HLO8T (OGT 55 °C) as the latter could not thrive at a higher temperature41.
Fervidobacterium, under the Thermotogota phylum, was a major genus in sample B2. Fervidobacterium species, for instance, F. islandicum and F. changbaicum, exhibit a broad range of CAZymes. The percentage of Fervidobacterium in hot spring microbiota would be increased if the water was enriched with switchgrass inoculum32. Some Firmicutes ASVs were detected in the brown mats. Firmicutes’ members, i.e., Geobacillus and other Firmicutes bacilli thermophiles, may dominate cellulose-degrading consortium in a heated lab setup42. Caldicellulosiruptor thermophilum, another member of Firmicutes, has been targeted as one potential thermophile for consolidated bioprocessing of lignocellulose2. We detected Caldicellulosiruptor and other Firmicutes in relatively small quantities in SKY hot spring mats.
Crenarchaeota was the dominated Archaea phylum in brown mats, with Nitrososphaeria being the main class and consisted of Ca. Caldiarchaeum and Ca. Nitrosocaldus (Fig. 2). The knowledge on these candidates is very limited43,44,45. The remaining classes in brown samples were Bathyarchaeia and Thermoprotei. ASVs stated above were also present in green microbial mats with the exception of Nitrosocaldales which was the main order in green biofilm datasets but existed in relatively smaller quantity in brown mats.
The study of eukaryotes is scarce for hot springs around the globe and is often neglected compared to prokaryotes. Oliverio et al. examined the presence of protists (microbial eukaryotes) in 160 New Zealand geothermal springs and suggested that the main protists possibly thrived in elevated temperatures are Amoebozoa, Archaeplastida, Alveolata, Excavata, Rhizaria, and Stramenopiles20. In SKY hot spring green microbial mat, we noticed ASVs of Amoebozoa, Ciliophora (protozoa algae that feed bacteria), Protalveolata, and Ochrophyta. The Amoebozoa Echinamoeba thermarum was a common thermophilic protist in New Zealand geothermal springs20. E. thermarum was positively identified in the green mat but absent in the brown mat. We spotted other Amoebozoa, for instance, taxa from order Euamoebida and Leptomyxida. Additionally, thermophilic protist Protalveolata (mainly from class Syndiniales) was approximately 1% of total eukaryotes ASVs in the B1b sample. Thermophilic protist Ochrophyta (particularly class Chrysophyceae) was detected in very small quantity in brown samples.
18S rRNA metataxonomic sample datasets elucidated that thermophilic fungi were present only as the minority (< 1% ASVs). That included Chaetomium (Tmax 61 °C), Paecilomyces (55 °C), Chrysosporium (60 °C), Trichothecium (57 °C), Paecilomyces (55 °C), Torula (58 °C), Talaromyces (50 °C), Paecilomyces (50 °C), Geosmithia (55 °C), and Thermomyces (60 °C)46. We were doubtful if all detected fungi ASVs could grow pleasantly in SKY hot spring. Also, the water level was low during the first field trip, and the green mat was not submerged completely, water temperature was 58 °C, as measured ~ 5 cm below the G1 green mat. The actual temperature was expected to be lower in the floating green microbial mat; therefore, certain thermophilic fungi or some heat-tolerant mesophilic fungi may survive. However, none of the currently known thermophilic fungi can develop beyond 70 °C; therefore, detected taxa are not likely to survive without sporulation at the SKY brown mat at the spring head. Moreover, it is suspected that most of the detected mesophilic fungi ASVs were originated from fallen plant litter, and we expect them to be in their dormant form. For the first time, microscopic water bear Tardigrada (Macrobiotus hufelandi) was detected in a Malaysian hot spring. This small eight-legged animal feed on microorganisms, decomposed leaf, and survive in extreme temperatures. We also detected high background of plant phyla Phragmoplastophyta that likely originated from plant debris. Other background ASVs included flies, grasshoppers, and fringed winged insects, mites, and ticks. We confirmed that eukaryotes were the minority group in the SKY hot spring microbial mats by putting together the shotgun and amplicon data. We think that 18S rRNA primers excessively amplified the background of plant residuals or chromosomal fragments from the dead organisms, spores, inactivated eggs or larva in particular mesophilic fungi or insects. Collectively, we concluded that relatively high background noise was observed using 18S rRNA primer set.
We performed shotgun metagenomic sequencing using two green- and two brown microbial mats. Negligible amounts (~ 0.1%) of virus reads were detected in all the mats, and it is quite common to see a trace quantities of viruses in hot springs47. Judged using contigs generated from shotgun sequencing data, a greater percentage of archaea present in the brown mat was probably related to a higher temperature at the spring head (Fig. 3). We also think that temperature is the main abiotic factor that differentiate the microbial profile in green- and brown mats. In addition, data elucidated that bacteria are the main microbiota in green and brown mats, and they are the primary plant-biomass degraders and consumers in SKY hot spring. This observation was also noticed earlier in a separate report16. Despite lower abundance and diversity, archaea and some candidate taxa may exhibit some functional role on lignocellulosic decomposition in SKY hot spring. Several sequences of CAZymes from GH families (i.e., GH1, 3, 5, 10, etc.) were identified from Candidatus Bathyarchaeota, Candidatus Brockarchaeota, Thaumarchaeota, Nitrososphaeria archaeon, and Thermoprotei archaeon.
On average, more than 10,000 CAZymes ORFs were found in each type of microbial mat (Table S2). The annotated glycoside hydrolases sequences included cellulases, hemicellulases, CEs, GTs, AAs, and enzymes acting on carbohydrates such as starch. More cellulase and hemicellulase sequences were identified in SKY hot spring than the counterpart numbers detected in an Indian hot spring metagenomic study using water–sediment samples lacking in-situ plant litters3. Besides, the metataxonomic described in this current study differed from Deulajhari hot springs and Obsidian Pool that contained Pandanus leaf litters and heat-tolerant plant Juncus tweedyi, respectively16,28. The microbial and enzyme diversity in SKY hot springs are far more complex than other heated in-situ or ex-situ studies supplemented with insoluble cellulosic biomass31,32,33,42.
Using the threshold of ≥ 90% subject coverage and ≥ 90% protein sequence identity, the three main phyla essential for biomass degradation were Chloroflexota, Armatimonadota, and Deinococcota. The primary contributors (phylum Chloroflexota) were taxa related to Roseiflexus castenholzii, candidatus taxa, and a few unclassified Chloroflexota bacteria. Other important Chloroflexota taxa for high-temperature lignocellulosic degraders included Thermomicrobium roseum, Anaerolineae, Ardenticatenia, Caldilinea aerophile, Ca. Thermofonsia Clade 1, Chloroflexus islandicus, Thermoflexales, and Thermoflexia bacterium. According to the online CAZy genome databases, members of the Chloroflexota phylum exhibited a broad range of GH enzymes. For instance, the anaerobe photoheterotrophic thermophilic R. castenholzii DSM 13941 (NCBI genome accession CP000804.1) has 22 GH families and 3 CE families that accounted for 60 different protein sequences41.
Armatimonadota is another phylum spotted with multiple sequences from GH1, 5, 10, 43, and 51, CE7, and AA12 (threshold: ≥ 90% subject coverage and ≥ 90% protein sequence identity). Using the metataxonomy dataset, at least four Armatimonadia ASVs were detected in SKY hot spring mats, and an ASV was closely related to class Chthonomonadetes while the rest were unresolved at the lower taxonomy level. This phylum, earlier designated as candidate division OP10, was initially found in Yellowstone National Park Obsidian Pool48. Currently, Chthonomonas calidirosea T49T is the only thermophilic type strain48. The complete genome of strain T49T harbored 64 glycosyl hydrolases and eight carbohydrate esterases. To the best of our knowledge, the characteristics of these enzymes are still undescribed.
Deinococcota is the third-largest phylum with 28 sequences with ≥ 90% subject coverage and identity to the CAZy database. These putative sequences have high identity to counterpart proteins from Calidithermus, Thermus, and Meiothermus species. The draft genome of Calidithermus timidus DSM17022 indicated that the bacterium harbor seven AA, 38 GH, 16 CE sequences49. So far, only a GH57-glycogen branching enzyme50 and GH13 amylosucrase51 from this bacterium have been analysed in detail. Meiothermus spp. may help break down plant litter in SKY because a representative, M. taiwanensis WR-220 (PRJNA205607), had enzymes such as xylanase, β-xylosidase, endoglucanase, and polysaccharide deacetylase. More than a dozen Thermus spp. have completely curated genomes in the CAZyme genome database. For an example, the genome of Thermus thermophilus (http://www.cazy.org/b12268.html) encoded sequence of 15 types of enzymes particularly from families GH1, 13, 23, 36, 57, 63 and 77; however, the essential enzyme for lignocellulose hydrolysis is missing. Therefore, Thermus spp. are sugar consumers in the SKY community.
We are interested in mining novel CAZymes from the shotgun contigs (Fig. S1). A protein sequence may be considered novel if the primary sequence has ≥ 90% subject coverage and 50–70% identity to the deposited protein sequences. We spotted a 1036-residues β-xylanase B1_109149 with approximately 60% identity with an endo-1,4-β-xylanase (WP_012584062.1) from thermophilic Dictyoglomus turgidum. Both protein sequences formed a cluster with β-xylanase from Thermotoga maritima (Q60037) (Fig. 5). All three sequences contained a signal peptide, two N-terminal β-sandwich fold CBM4_9, followed by a TIM barrel GH10-catalytic domain consisting of four conserved motifs and with two β-sandwich CBM9_1 at the C-terminal. The putative protein structure of β-xylanase G1_109149 was predicted using AlpaFold v.252 and is shown in Fig. 6a. Additionally, another twelve GH10 family putative novel β-xylanase sequences were present in the dataset. These proteins sequences were probably related to phyla Armatimonadota, Bacteroidota/Chlorobiota, Ca. Bipolaricaulota, Ca. Solibacter, Ignavibacteriota, Planctomycetota, or Verrucomicrobiota (Fig. 5). The identified β-xylanase sequences have a single domain of the GH10_2 family where the four conserved motifs were located. The predicted structures of the selected xylanases are shown in Fig. 6b–d.
The primary sequence of β-xylanase G1_35826 is unique because it has a domain related to the secretion system C-terminal sorting domain and is absent in other counterparts displayed in Fig. 5. The other name for that domain is por-secretion system or the T9SS type IX secretion system. The putative protein structure of β-xylanase G1_35826 is displayed in Fig. 6c, and the C-terminal domain resembled a β-sandwich fold structure. There is little research exploring how annotated xylanase is incorporated with a T9SS. We observed such domain in xylanase XynRA2 from halo-thermophilic Roseithermus sp., and xylanase Xyn10A from Rhodothermus marinus, and xylanase Xyl2091 from Melioribacter roseus53. All these thermo-halophilic bacteria are from Bacteroidota/Chlorobiota group. Based on a recent review, certain microorganisms, especially those from Bacteroidota utilise the T9SS system for secreting proteins54.
Subsequently, we data-mined novel cellulase sequences in the SKY hot spring dataset. Fourteen unique sequences are putative cellulases, and each of the sequences contained a GH5 domain. Putative cellulase G3_96404 was 55% homologous to cellulase Cel5A of T. maritima (PDB 3AMC)55. 3AMC structure has a classic TIM barrel fold that resembles endoglucanase TM1752 (1VJZ) from T. maritima, endocellulase EGPh (3W6M) from Pyrococcus horikoshii and endoglucanase (6GJF) from a synthetic construct56. Cellulase EGPh has the optimum activity at 100 °C57. Putative cellulase B3_136450 (Figs. 6e and 7) has identical domain setups with the endo-β-1,4-glucanase BlCel5B sequence (4YZP) from Bacillus licheniformis58. BlCel5B was catalytically actived on CMC, β-glucan, lichenan, and xyloglucan. Protein BlCel5B has tri-modular structure with an N-terminal catalytic GH5 domain (18–320 amino acid stretch), an immunoglobulin-like module (345–428), and a C-terminal CBM46 (432–533)58. The immunoglobulin-like module with two β-sheets resembles an earlier known CBM_X2 that may be involved with cellulosome59.
Additionally, we annotated three cellulases that belong to non-GH5 groups (Fig. 7). B3_230401 (238 aa) was 50% homologous with the primary sequence of endoglucanase Cel12A (PDB 2BW8, 227 aa) from Rhodothermus marinus60. Both sequences have a single GH12 catalytic domain. Often, that domain is similar to the concanavalin-like glucanase domain superfamily that looks like a sandwich structure with 12–14 β-strands (Fig. 6f). Another putative cellulase G1_45801 sequence was 69% homologous to the sequence of a crystal structure CelM2 (3FW6), and Interproscan indicates that both sequences have identical domain arrangements. The gene of CelM2 was cloned from a metagenomic library60. GH44 domain (β-sandwich structure) was found in the N-terminal while a galactose-binding domain (TIM-like barrel structure) was present at the C-terminal, where the acid/base Glu 221 and nucleophilic Glu393 are located61. Enzyme CelM2 actively hydrolysed multiple substrates, including birchwood xylan, barley glucan and cellulosic CMC, respectively having β-1,3/4-glucan and β-1,4-glucan linkages61. The binding ability of multi-substrates is possibly related to the broad and deep pocket. Due to relatively close sequence identity, G1_45801 may exhibit a similar bifunctional glucanase-xylanase activity as CelM2. Lastly, putative cellulase B3_106662 (615 aa) was detected in a brown microbial dataset. The stretch 42–238 resembles a GH114-family domain having a typical aldolase-type TIM-barrel structure (Fig. 6g). The latter half of the sequence (residue 273–587) is the GH5 family domain, resembling the second TIM-barrel. A short loop (residue 247–254) joint both TIM-barrels. As shown in Fig. 6g, a long loop protruding from the GH5-TIM barrel that points towards the GH114-TIM barrel. We expect that the protruding loop has some structural role. So far, there are no closely related crystal structures to the sequence of B3_106662.
Source: Ecology - nature.com
