Microbial communities colonising plastics during transition from the wastewater treatment plant to marine waters.

BACKGROUND: Plastics pollution and antimicrobial resistance (AMR) are two major environmental threats, but potential connections between plastic associated biofilms, the 'plastisphere', and dissemination of AMR genes are not well explored. RESULTS: We conducted mesocosm experiments tracking microbial community changes on plastic surfaces transitioning from wastewater effluent to marine environments over 16 weeks. Commonly used plastics, polypropylene (PP), high density polyethylene (HDPE), low density polyethylene (LDPE) and polyethylene terephthalate (PET) incubated in wastewater effluent, river water, estuarine water, and in the seawater for 16 weeks, were analysed via 16S rRNA gene amplicon and shotgun metagenome sequencing. Within one week, plastic-colonizing communities shifted from wastewater effluent-associated microorganisms to marine taxa, some members of which (e.g. Oleibacter-Thalassolituus and Sphingomonas spp., on PET, Alcanivoracaceae on PET and PP, or Oleiphilaceae, on all polymers), were selectively enriched from levels undetectable in the starting communities. Remarkably, microbial biofilms were also susceptible to parasitism, with Saprospiraceae feeding on biofilms at late colonisation stages (from week 6 onwards), while Bdellovibrionaceae were prominently present on HDPE from week 2 and LDPE from day 1. Relative AMR gene abundance declined over time, and plastics did not become enriched for key AMR genes after wastewater exposure. CONCLUSION: Although some resistance genes occurred during the mesocosm transition on plastic substrata, those originated from the seawater organisms. Overall, plastic surfaces incubated in wastewater did not act as hotspots for AMR proliferation in simulated marine environments.

Microbiome-derived cobalamin and succinyl-CoA as biomarkers for improved screening of anal cancer.

Human papillomavirus can cause preinvasive, high-grade squamous intraepithelial lesions (HSILs) as precursors to cancer in the anogenital area, and the microbiome is suggested to be a contributing factor. Men who have sex with men (MSM) living with human immunodeficiency virus (HIV) have a high risk of anal cancer, but current screening strategies for HSIL detection lack specificity. Here, we investigated the anal microbiome to improve HSIL screening. We enrolled participants living with HIV, divided into a discovery (n = 167) and validation cohort (n = 46), and who were predominantly (93.9%) cisgender MSM undergoing HSIL screening with high-resolution anoscopy and anal biopsies. We identified no microbiome composition signatures associated with HSILs, but elevated levels of microbiome-encoded proteins producing succinyl coenzyme A and cobalamin were significantly associated with HSILs in both cohorts. Measurement of these candidate biomarkers alone in anal cytobrushes outperformed anal cytology as a diagnostic indicator for HSILs, increasing the sensitivity from 91.2% to 96.6%, the specificity from 34.1% to 81.8%, and reclassifying 82% of false-positive results as true negatives. We propose that these two microbiome-derived biomarkers may improve the current strategy of anal cancer screening.

Evolutionary patterns of archaea predominant in acidic environment.

BACKGROUND: Archaea of the order Thermoplasmatales are widely distributed in natural acidic areas and are amongst the most acidophilic prokaryotic organisms known so far. These organisms are difficult to culture, with currently only six genera validly published since the discovery of Thermoplasma acidophilum in 1970. Moreover, known great diversity of uncultured Thermoplasmatales represents microbial dark matter and underlines the necessity of efforts in cultivation and study of these archaea. Organisms from the order Thermoplasmatales affiliated with the so-called "alphabet-plasmas", and collectively dubbed "E-plasma", were the focus of this study. These archaea were found predominantly in the hyperacidic site PM4 of Parys Mountain, Wales, UK, making up to 58% of total metagenomic reads. However, these archaea escaped all cultivation attempts. RESULTS: Their genome-based metabolism revealed its peptidolytic potential, in line with the physiology of the previously studied Thermoplasmatales isolates. Analyses of the genome and evolutionary history reconstruction have shown both the gain and loss of genes, that may have contributed to the success of the "E-plasma" in hyperacidic environment compared to their community neighbours. Notable genes among them are involved in the following molecular processes: signal transduction, stress response and glyoxylate shunt, as well as multiple copies of genes associated with various cellular functions; from energy production and conversion, replication, recombination, and repair, to cell wall/membrane/envelope biogenesis and archaella production. History events reconstruction shows that these genes, acquired by putative common ancestors, may determine the evolutionary and functional divergences of "E-plasma", which is much more developed than other representatives of the order Thermoplasmatales. In addition, the ancestral hereditary reconstruction strongly indicates the placement of Thermogymnomonas acidicola close to the root of the Thermoplasmatales. CONCLUSIONS: This study has analysed the metagenome-assembled genome of "E-plasma", which denotes the basis of their predominance in Parys Mountain environmental microbiome, their global ubiquity, and points into the right direction of further cultivation attempts. The results suggest distinct evolutionary trajectories of organisms comprising the order Thermoplasmatales, which is important for the understanding of their evolution and lifestyle.

Increasing concentration of pure micro- and macro-LDPE and PP plastic negatively affect crop biomass, nutrient cycling, and microbial biomass.

Over the last 50 years, the intense use of agricultural plastic in the form of mulch films has led to an accumulation of plastic in soil, creating a legacy of plastic in agricultural fields. Plastic often contains additives, however it is still largely unknown how these compounds affect soil properties, potentially influencing or masking effects of the plastic itself. Therefore, the aim of this study was to investigate the effects of pure plastics of varying sizes and concentrations, to improve our understanding of plastic-only interactions within soil-plant mesocosms. Maize (Zea mays L.) was grown over eight weeks following the addition of micro and macro low-density polyethylene and polypropylene at increasing concentrations (equivalent to 1, 10, 25, and 50 years mulch film use) and the effects of plastic on key soil and plant properties were measured. We found the effect of both macro and microplastic on soil and plant health is negligible in the short-term (1 to <10 years). However, ≥ 10 years of plastic application for both plastic types and sizes resulted in a clear negative effect on plant growth and microbial biomass. This study provides vital insight into the effect of both macro and microplastics on soil and plant properties.

Enzyme adaptation to habitat thermal legacy shapes the thermal plasticity of marine microbiomes.

Microbial communities respond to temperature with physiological adaptation and compositional turnover. Whether thermal selection of enzymes explains marine microbiome plasticity in response to temperature remains unresolved. By quantifying the thermal behaviour of seven functionally-independent enzyme classes (esterase, extradiol dioxygenase, phosphatase, beta-galactosidase, nuclease, transaminase, and aldo-keto reductase) in native proteomes of marine sediment microbiomes from the Irish Sea to the southern Red Sea, we record a significant effect of the mean annual temperature (MAT) on enzyme response in all cases. Activity and stability profiles of 228 esterases and 5 extradiol dioxygenases from sediment and seawater across 70 locations worldwide validate this thermal pattern. Modelling the esterase phase transition temperature as a measure of structural flexibility confirms the observed relationship with MAT. Furthermore, when considering temperature variability in sites with non-significantly different MATs, the broadest range of enzyme thermal behaviour and the highest growth plasticity of the enriched heterotrophic bacteria occur in samples with the widest annual thermal variability. These results indicate that temperature-driven enzyme selection shapes microbiome thermal plasticity and that thermal variability finely tunes such processes and should be considered alongside MAT in forecasting microbial community thermal response.

Thermophilic Carboxylesterases from Hydrothermal Vents of the Volcanic Island of Ischia Active on Synthetic and Biobased Polymers and Mycotoxins.

Hydrothermal vents are geographically widespread and host microorganisms with robust enzymes useful in various industrial applications. We examined microbial communities and carboxylesterases of two terrestrial hydrothermal vents of the volcanic island of Ischia (Italy) predominantly composed of Firmicutes, Proteobacteria, and Bacteroidota. High-temperature enrichment cultures with the polyester plastics polyhydroxybutyrate and polylactic acid (PLA) resulted in an increase of Thermus and Geobacillus species and to some extent Fontimonas and Schleiferia species. The screening at 37 to 70°C of metagenomic fosmid libraries from above enrichment cultures identified three hydrolases (IS10, IS11, and IS12), all derived from yet-uncultured Chloroflexota and showing low sequence identity (33 to 56%) to characterized enzymes. Enzymes expressed in Escherichia coli exhibited maximal esterase activity at 70 to 90°C, with IS11 showing the highest thermostability (90% activity after 20-min incubation at 80°C). IS10 and IS12 were highly substrate promiscuous and hydrolyzed all 51 monoester substrates tested. Enzymes were active with PLA, polyethylene terephthalate model substrate, and mycotoxin T-2 (IS12). IS10 and IS12 had a classical α/ß-hydrolase core domain with a serine hydrolase catalytic triad (Ser155, His280, and Asp250) in their hydrophobic active sites. The crystal structure of IS11 resolved at 2.92 Å revealed the presence of a N-terminal ß-lactamase-like domain and C-terminal lipocalin domain. The catalytic cleft of IS11 included catalytic Ser68, Lys71, Tyr160, and Asn162, whereas the lipocalin domain enclosed the catalytic cleft like a lid and contributed to substrate binding. Our study identified novel thermotolerant carboxylesterases with a broad substrate range, including polyesters and mycotoxins, for potential applications in biotechnology. IMPORTANCE High-temperature-active microbial enzymes are important biocatalysts for many industrial applications, including recycling of synthetic and biobased polyesters increasingly used in textiles, fibers, coatings and adhesives. Here, we identified three novel thermotolerant carboxylesterases (IS10, IS11, and IS12) from high-temperature enrichment cultures from Ischia hydrothermal vents and incubated with biobased polymers. The identified metagenomic enzymes originated from uncultured Chloroflexota and showed low sequence similarity to known carboxylesterases. Active sites of IS10 and IS12 had the largest effective volumes among the characterized prokaryotic carboxylesterases and exhibited high substrate promiscuity, including hydrolysis of polyesters and mycotoxin T-2 (IS12). Though less promiscuous than IS10 and IS12, IS11 had a higher thermostability with a high temperature optimum (80 to 90°C) for activity and hydrolyzed polyesters, and its crystal structure revealed an unusual lipocalin domain likely involved in substrate binding. The polyesterase activity of these enzymes makes them attractive candidates for further optimization and potential application in plastics recycling.

Microbial Diversity of a Disused Copper Mine Site (Parys Mountain, UK), Dominated by Intensive Eukaryotic Filamentous Growth.

The Parys Mountain copper mine (Wales, UK) contains a wide range of discrete environmental microniches with various physicochemical conditions that shape microbial community composition. Our aim was to assess the microbial community in the sediments and overlying water column in an acidic mine drainage (AMD) site containing abundant filamentous biogenic growth via application of a combination of chemical analysis and taxonomic profiling using 16S rRNA gene amplicon sequencing. Our results were then compared to previously studied sites at Parys Mt. Overall, the sediment microbiome showed a dominance of bacteria over archaea, particularly those belonging to Proteobacteria (genera Acidiphilium and Acidisphaera), Acidobacteriota (subgroup 1), Chloroflexota (AD3 cluster), Nitrospirota (Leptospirillum) and the uncultured Planctomycetota/CPIa-3 termite group. Archaea were only present in the sediment in small quantities, being represented by the Terrestrial Miscellaneous Euryarchaeota Group (TMEG), Thermoplasmatales and Ca. Micrarchaeota (Ca. Micracaldota). Bacteria, mostly of the genera Acidiphilium and Leptospirillum, also dominated within the filamentous streamers while archaea were largely absent. This study found pH and dissolved solutes to be the most important parameters correlating with relative proportions of bacteria to archaea in an AMD environment and revealed the abundance patterns of native acidophilic prokaryotes inhabiting Parys Mt sites and their niche specificities.

Calm and Frenzy: marine obligate hydrocarbonoclastic bacteria sustain ocean wellness.

According to current estimates, the annual volume of crude oil entering the ocean due to both anthropogenic activities and naturally occurring seepages reaches approximately 8.3 million metric tons. Huge discharges from accidents have caused large-scale environmental disasters with extensive damage to the marine ecosystem. The natural clean-up of petroleum spills in marine environments is carried out primarily by naturally occurring obligate hydrocarbonoclastic bacteria (OHCB). The natural hosts of OHCB include a range of marine primary producers, unicellular photosynthetic eukaryotes and cyanobacteria, which have been documented as both, suppliers of hydrocarbon-like compounds that fuel the 'cryptic' hydrocarbon cycle and as a source of isolation of new OHCB. A very new body of evidence suggests that OHCB are not only the active early stage colonizers of plastics and hence the important component of the ocean's 'plastisphere' but also encode an array of enzymes experimentally proven to act on petrochemical and bio-based polymers.

Structure and evolutionary trace-assisted screening of a residue swapping the substrate ambiguity and chiral specificity in an esterase.

Our understanding of enzymes with high substrate ambiguity remains limited because their large active sites allow substrate docking freedom to an extent that seems incompatible with stereospecificity. One possibility is that some of these enzymes evolved a set of evolutionarily fitted sequence positions that stringently allow switching substrate ambiguity and chiral specificity. To explore this hypothesis, we targeted for mutation a serine ester hydrolase (EH3) that exhibits an impressive 71-substrate repertoire but is not stereospecific (e.e. 50%). We used structural actions and the computational evolutionary trace method to explore specificity-swapping sequence positions and hypothesized that position I244 was critical. Driven by evolutionary action analysis, this position was substituted to leucine, which together with isoleucine appears to be the amino acid most commonly present in the closest homologous sequences (max. identity, ca. 67.1%), and to phenylalanine, which appears in distant homologues. While the I244L mutation did not have any functional consequences, the I244F mutation allowed the esterase to maintain a remarkable 53-substrate range while gaining stereospecificity properties (e.e. 99.99%). These data support the possibility that some enzymes evolve sequence positions that control the substrate scope and stereospecificity. Such residues, which can be evolutionarily screened, may serve as starting points for further designing substrate-ambiguous, yet chiral-specific, enzymes that are greatly appreciated in biotechnology and synthetic chemistry.

Corrigendum: High Representation of Archaea Across All Depths in Oxic and Low-pH Sediment Layers Underlying an Acidic Stream.

[This corrects the article DOI: 10.3389/fmicb.2020.576520.].

Utilization of low-molecular-weight organic compounds by the filterable fraction of a lotic microbiome.

Filterable microorganisms participate in dissolved organic carbon (DOC) cycling in freshwater systems, however their exact functional role remains unknown. We determined the taxonomic identity and community dynamics of prokaryotic microbiomes in the 0.22 µm-filtered fraction and unfiltered freshwater from the Conwy River (North Wales, UK) in microcosms and, using targeted metabolomics and 14C-labelling, examined their role in the utilization of amino acids, organic acids and sugars spiked at environmentally-relevant (nanomolar) concentrations. To identify changes in community structure, we used 16S rRNA amplicon and shotgun sequencing. Unlike the unfiltered water samples where the consumption of DOC was rapid, the filtered fraction showed a 3-day lag phase before the consumption started. Analysis of functional categories of clusters of orthologous groups of proteins (COGs) showed that COGs associated with energy production increased in number in both fractions with substrate addition. The filtered fraction utilized low-molecular-weight (LMW) DOC at much slower rates than the whole community. Addition of nanomolar concentrations of LMW DOC did not measurably influence the composition of the microbial community nor the rate of consumption across all substrate types in either fraction. We conclude that due to their low activity, filterable microorganisms play a minor role in LMW DOC processing within a short residence time of lotic freshwater systems.

High Representation of Archaea Across All Depths in Oxic and Low-pH Sediment Layers Underlying an Acidic Stream.

Parys Mountain or Mynydd Parys (Isle of Anglesey, United Kingdom) is a mine-impacted environment, which accommodates a variety of acidophilic organisms. Our previous research of water and sediments from one of the surface acidic streams showed a high proportion of archaea in the total microbial community. To understand the spatial distribution of archaea, we sampled cores (0-20 cm) of sediment and conducted chemical analyses and taxonomic profiling of microbiomes using 16S rRNA gene amplicon sequencing in different core layers. The taxonomic affiliation of sequencing reads indicated that archaea represented between 6.2 and 54% of the microbial community at all sediment depths. Majority of archaea were associated with the order Thermoplasmatales, with the most abundant group of sequences being clustered closely with the phylotype B_DKE, followed by "E-plasma," "A-plasma," other yet uncultured Thermoplasmatales with Ferroplasma and Cuniculiplasma spp. represented in minor proportions. Thermoplasmatales were found at all depths and in the whole range of chemical conditions with their abundance correlating with sediment Fe, As, Cr, and Mn contents. The bacterial microbiome component was largely composed in all layers of sediment by members of the phyla Proteobacteria, Actinobacteria, Nitrospirae, Firmicutes, uncultured Chloroflexi (AD3 group), and Acidobacteria. This study has revealed a high abundance of Thermoplasmatales in acid mine drainage-affected sediment layers and pointed at these organisms being the main contributors to carbon, and probably to iron and sulfur cycles in this ecosystem.

Tuning the Properties of Natural Promiscuous Enzymes by Engineering Their Nano-environment.

Owing to their outstanding catalytic properties, enzymes represent powerful tools for carrying out a wide range of (bio)chemical transformations with high proficiency. In this context, enzymes with high biocatalytic promiscuity are somewhat neglected. Here, we demonstrate that a meticulous modification of a synthetic shell that surrounds an immobilized enzyme possessing broad substrate specificity allows the resulting nanobiocatalyst to be endowed with enantioselective properties while maintaining a high level of substrate promiscuity. Our results show that control of the enzyme nano-environment enables tuning of both substrate specificity and enantioselectivity. Further, we demonstrate that our strategy of enzyme supramolecular engineering allows the enzyme to be endowed with markedly enhanced stability in an organic solvent (i.e., acetonitrile). The versatility of the method was assessed with two additional substrate-promiscuous and structurally different enzymes, for which improvements in enantioselectivity and stability were confirmed. We expect this method to promote the use of supramolecularly engineered promiscuous enzymes in industrially relevant biocatalytic processes.

Hydrocarbon-Degrading Bacteria Alcanivorax and Marinobacter Associated With Microalgae Pavlova lutheri and Nannochloropsis oculata.

Marine hydrocarbon-degrading bacteria play an important role in natural petroleum biodegradation processes and were initially associated with man-made oil spills or natural seeps. There is no full clarity though on what, in the absence of petroleum, their natural niches are. Few studies pointed at some marine microalgae that produce oleophilic compounds (alkanes, long-chain fatty acids, and alcohols) as potential natural hosts of these bacteria. We established Dansk crude oil-based enrichment cultures with photobioreactor-grown marine microalgae cultures Pavlova lutheri and Nannochloropsis oculata and analyzed the microbial succession using cultivation and SSU (16S) rRNA amplicon sequencing. We found that petroleum enforced a strong selection for members of Alpha- and Gamma-proteobacteria in both enrichment cultures with the prevalence of Alcanivorax and Marinobacter spp., well-known hydrocarbonoclastic bacteria. In total, 48 non-redundant bacterial strains were isolated and identified to represent genera Alcanivorax, Marinobacter, Thalassospira, Hyphomonas, Halomonas, Marinovum, Roseovarius, and Oleibacter, which were abundant in sequencing reads in both crude oil enrichments. Our assessment of public databases demonstrated some overlaps of geographical sites of isolation of Nannochloropsis and Pavlova with places of molecular detection and isolation of Alcanivorax and Marinobacter spp. Our study suggests that these globally important hydrocarbon-degrading bacteria are associated with P. lutheri and N. oculata.

Proteome Cold-Shock Response in the Extremely Acidophilic Archaeon, Cuniculiplasma divulgatum.

The archaeon Cuniculiplasma divulgatum is ubiquitous in acidic environments with low-to-moderate temperatures. However, molecular mechanisms underlying its ability to thrive at lower temperatures remain unexplored. Using mass spectrometry (MS)-based proteomics, we analysed the effect of short-term (3 h) exposure to cold. The C. divulgatum genome encodes 2016 protein-coding genes, from which 819 proteins were identified in the cells grown under optimal conditions. In line with the peptidolytic lifestyle of C. divulgatum, its intracellular proteome revealed the abundance of proteases, ABC transporters and cytochrome C oxidase. From 747 quantifiable polypeptides, the levels of 582 proteins showed no change after the cold shock, whereas 104 proteins were upregulated suggesting that they might be contributing to cold adaptation. The highest increase in expression appeared in low-abundance (0.001-0.005 fmol%) proteins for polypeptides' hydrolysis (metal-dependent hydrolase), oxidation of amino acids (FAD-dependent oxidoreductase), pyrimidine biosynthesis (aspartate carbamoyltransferase regulatory chain proteins), citrate cycle (2-oxoacid ferredoxin oxidoreductase) and ATP production (V type ATP synthase). Importantly, the cold shock induced a substantial increase (6% and 9%) in expression of the most-abundant proteins, thermosome beta subunit and glutamate dehydrogenase. This study has outlined potential mechanisms of environmental fitness of Cuniculiplasma spp. allowing them to colonise acidic settings at low/moderate temperatures.

Functional microbiome deficits associated with ageing: Chronological age threshold.

Composition of the gut microbiota changes during ageing, but questions remain about whether age is also associated with deficits in microbiome function and whether these changes occur sharply or progressively. The ability to define these deficits in populations of different ages may help determine a chronological age threshold at which deficits occur and subsequently identify innovative dietary strategies for active and healthy ageing. Here, active gut microbiota and associated metabolic functions were evaluated using shotgun proteomics in three well-defined age groups consisting of 30 healthy volunteers, namely, ten infants, ten adults and ten elderly individuals. Samples from each volunteer at intervals of up to 6 months (n = 83 samples) were used for validation. Ageing gradually increases the diversity of gut bacteria that actively synthesize proteins, that is by 1.4-fold from infants to elderly individuals. An analysis of functional deficits consistently identifies a relationship between tryptophan and indole metabolism and ageing (p < 2.8e-8 ). Indeed, the synthesis of proteins involved in tryptophan and indole production and the faecal concentrations of these metabolites are directly correlated (r2  > .987) and progressively decrease with age (r2  > .948). An age threshold for a 50% decrease is observed ca. 11-31 years old, and a greater than 90% reduction is observed from the ages of 34-54 years. Based on recent investigations linking tryptophan with abundance of indole and other "healthy" longevity molecules and on the results from this small cohort study, dietary interventions aimed at manipulating tryptophan deficits since a relatively "young" age of 34 and, particularly, in the elderly are recommended.

Microbial Communities of Polymetallic Deposits' Acidic Ecosystems of Continental Climatic Zone With High Temperature Contrasts.

Acid mine drainage (AMD) systems are globally widespread and are an important source of metal pollution in riverine and coastal systems. Microbial AMD communities have been extensively studied for their ability to thrive under extremely acidic conditions and for their immense contribution to the dissolution of metal ores. However, little is known on microbial inhabitants of AMD systems subjected to extremely contrasting continental seasonal temperature patterns as opposed to maritime climate zones, experiencing much weaker annual temperature variations. Here, we investigated three types of AMD sites in Eastern Transbaikalia (Russia). In this region, all surface water bodies undergo a deep and long (up to 6 months) freezing, with seasonal temperatures varying between -33 and +24°C, which starkly contrasts the common well-studied AMD environments. We sampled acidic pit lake (Sherlovaya Gora site) located in the area of a polymetallic deposit, acidic drainage water from Bugdaya gold-molybdenum-tungsten deposit and Ulan-Bulak natural acidic spring. These systems showed the abundance of bacteria-derived reads mostly affiliated with Actinobacteria, Acidobacteria, Alpha- and Gammaproteobacteria, chloroplasts, Chloroflexi, Bacteroidetes, and Firmicutes. Furthermore, candidate taxa "Ca. Saccharibacteria" (previously known as TM7), "Ca. Parcubacteria" (OD1) and WPS-2 were represented in substantial quantities (10-20%). Heterotrophy and iron redox cycling can be considered as central processes of carbon and energy flow for majority of detected bacterial taxa. Archaea were detected in low numbers, with Terrestrial Miscellaneous Euryarchaeal Group (TMEG), to be most abundant (3%) in acidic spring Ulan-Bulak. Composition of these communities was found to be typical in comparison to other AMD sites; however, certain groups (as Ignavibacteriae) could be specifically associated with this area. This study provides insight into the microbial diversity patterns in acidic ecosystems formed in areas of polymetallic deposits in extreme continental climate zone with contrasting temperature parameters.

Diversity of "Ca. Micrarchaeota" in Two Distinct Types of Acidic Environments and Their Associations with Thermoplasmatales.

"Candidatus Micrarchaeota" are widely distributed in acidic environments; however, their cultivability and our understanding of their interactions with potential hosts are very limited. Their habitats were so far attributed with acidic sites, soils, peats, freshwater systems, and hypersaline mats. Using cultivation and culture-independent approaches (16S rRNA gene clonal libraries, high-throughput amplicon sequencing of V3-V4 region of 16S rRNA genes), we surveyed the occurrence of these archaea in geothermal areas on Kamchatka Peninsula and Kunashir Island and assessed their taxonomic diversity in relation with another type of low-pH environment, acid mine drainage stream (Wales, UK). We detected "Ca. Micrarchaeota" in thermophilic heterotrophic enrichment cultures of Kunashir and Kamchatka that appeared as two different phylotypes, namely "Ca. Mancarchaeum acidiphilum"-, and ARMAN-2-related, alongside their potential hosts, Cuniculiplasma spp. and other Thermoplasmatales archaea without defined taxonomic position. These clusters of "Ca. Micrarchaeota" together with three other groups were also present in mesophilic acid mine drainage community. Present work expands our knowledge on the diversity of "Ca. Micrarchaeota" in thermophilic and mesophilic acidic environments, suggests cultivability patterns of acidophilic archaea and establishes potential links between low-abundance species of thermophilic "Ca. Micrarchaeota" and certain Thermoplasmatales, such as Cuniculiplasma spp. in situ.

Archaea dominate the microbial community in an ecosystem with low-to-moderate temperature and extreme acidity.

BACKGROUND: The current view suggests that in low-temperature acidic environments, archaea are significantly less abundant than bacteria. Thus, this study of the microbiome of Parys Mountain (Anglesey, UK) sheds light on the generality of this current assumption. Parys Mountain is a historically important copper mine and its acid mine drainage (AMD) water streams are characterised by constant moderate temperatures (8-18 °C), extremely low pH (1.7) and high concentrations of soluble iron and other metal cations. RESULTS: Metagenomic and SSU rRNA amplicon sequencing of DNA from Parys Mountain revealed a significant proportion of archaea affiliated with Euryarchaeota, which accounted for ca. 67% of the community. Within this phylum, potentially new clades of Thermoplasmata were overrepresented (58%), with the most predominant group being "E-plasma", alongside low-abundant Cuniculiplasmataceae, 'Ca. Micrarchaeota' and 'Terrestrial Miscellaneous Euryarchaeal Group' (TMEG) archaea, which were phylogenetically close to Methanomassilicoccales and clustered with counterparts from acidic/moderately acidic settings. In the sediment, archaea and Thermoplasmata contributed the highest numbers in V3-V4 amplicon reads, in contrast with the water body community, where Proteobacteria, Nitrospirae, Acidobacteria and Actinobacteria outnumbered archaea. Cultivation efforts revealed the abundance of archaeal sequences closely related to Cuniculiplasma divulgatum in an enrichment culture established from the filterable fraction of the water sample. Enrichment cultures with unfiltered samples showed the presence of Ferrimicrobium acidiphilum, C. divulgatum, 'Ca. Mancarchaeum acidiphilum Mia14', 'Ca. Micrarchaeota'-related and diverse minor (< 2%) bacterial metagenomic reads. CONCLUSION: Contrary to expectation, our study showed a high abundance of archaea in this extremely acidic mine-impacted environment. Further, archaeal populations were dominated by one particular group, suggesting that they are functionally important. The prevalence of archaea over bacteria in these microbiomes and their spatial distribution patterns represents a novel and important advance in our understanding of acidophile ecology. We also demonstrated a procedure for the specific enrichment of cell wall-deficient members of the archaeal component of this community, although the large fraction of archaeal taxa remained unculturable. Lastly, we identified a separate clustering of globally occurring acidophilic members of TMEG that collectively belong to a distinct order within Thermoplasmata with yet unclear functional roles in the ecosystem.