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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.

[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.

Metagenomic studies are revolutionizing our understanding of microbes in the biosphere. They have uncovered numerous proteins of unknown function in tens of essentially unstudied lineages that lack cultivated representatives. Notably, few of these microorganisms have been visualized, and even fewer have been described ultra-structurally in their essentially intact, physiologically relevant states. Here, we present cryogenic transmission electron microscope (cryo-TEM) 2D images and 3D tomographic datasets for archaeal species from natural acid mine drainage (AMD) microbial communities. Ultrastructural findings indicate the importance of microbial interconnectedness via a range of mechanisms, including direct cytoplasmic bridges and pervasive pili. The data also suggest a variety of biological structures associated with cell-cell interfaces that lack explanation. Some may play roles in inter-species interactions. Interdependences amongst the archaea may have confounded prior isolation efforts. Overall, the findings underline knowledge gaps related to archaeal cell components and highlight the likely importance of co-evolution in shaping microbial lineages.

Metagenomic studies are revolutionizing our understanding of microbes in the biosphere. They have uncovered numerous proteins of unknown function in tens of essentially unstudied lineages that lack cultivated representatives. Notably, few of these microorganisms have been visualized, and even fewer have been described ultra-structurally in their essentially intact, physiologically relevant states. Here, we present cryogenic transmission electron microscope (cryo-TEM) 2D images and 3D tomographic datasets for archaeal species from natural acid mine drainage (AMD) microbial communities. Ultrastructural findings indicate the importance of microbial interconnectedness via a range of mechanisms, including direct cytoplasmic bridges and pervasive pili. The data also suggest a variety of biological structures associated with cell-cell interfaces that lack explanation. Some may play roles in inter-species interactions. Interdependences amongst the archaea may have confounded prior isolation efforts. Overall, the findings underline knowledge gaps related to archaeal cell components and highlight the likely importance of co-evolution in shaping microbial lineages.

Some bacteria and archaea have evolved metabolic strategies that enable them to live in environments contaminated by toxic metals. In fact, many bacteria and archaea take advantage of the redox sensitivity of these very same metals to gain energy via anaerobic respiration. Here, metagenomic techniques were developed and applied along with conventional physiological and ecological methods to elucidate multiple modes of adaptation of bacteria and archaea in metal-contaminated acid mine drainage and aquifer environments contaminated by mine tailings. These approaches provided insight into how these organisms survive and thrive in these environments and how they differentiate themselves from each other.Many of the microbial species in acid mine drainage and mine tailings-contaminated aquifer environments are difficult to culture in the laboratory. Thus, a focus of the research was metabolic analysis of these organisms via analysis of genes and genomes recovered from microbial communities and isolates. Many of the genes are novel, and likely required for specific environmental adaptation, but they are difficult or impossible to functionally characterize based on conventional homology methods. A new method was developed to deal with the challenge of identifying poorly annotated or hypothetical genes of importance in adaptation to metal-contaminated environments. This probabilistic approach is based on conserved gene order between the genomes of interest with distant relatives. The annotation method was used in conjunction with traditional comparative genomics to differentiate a group of co-occurring archaea based on their genetic metabolic potential. These microorganisms grow in biofilm communities in an acid mine drainage system within the Richmond Mine, near Redding in Northern California, USA. Microbial biofilms growing at the air-solution interface were sampled from solutions with pH values of < 1.2, temperatures of up to 48 °C, and mM concentrations of zinc, coppe

Some bacteria and archaea have evolved metabolic strategies that enable them to live in environments contaminated by toxic metals. In fact, many bacteria and archaea take advantage of the redox sensitivity of these very same metals to gain energy via anaerobic respiration. Here, metagenomic techniques were developed and applied along with conventional physiological and ecological methods to elucidate multiple modes of adaptation of bacteria and archaea in metal-contaminated acid mine drainage and aquifer environments contaminated by mine tailings. These approaches provided insight into how these organisms survive and thrive in these environments and how they differentiate themselves from each other.Many of the microbial species in acid mine drainage and mine tailings-contaminated aquifer environments are difficult to culture in the laboratory. Thus, a focus of the research was metabolic analysis of these organisms via analysis of genes and genomes recovered from microbial communities and isolates. Many of the genes are novel, and likely required for specific environmental adaptation, but they are difficult or impossible to functionally characterize based on conventional homology methods. A new method was developed to deal with the challenge of identifying poorly annotated or hypothetical genes of importance in adaptation to metal-contaminated environments. This probabilistic approach is based on conserved gene order between the genomes of interest with distant relatives. The annotation method was used in conjunction with traditional comparative genomics to differentiate a group of co-occurring archaea based on their genetic metabolic potential. These microorganisms grow in biofilm communities in an acid mine drainage system within the Richmond Mine, near Redding in Northern California, USA. Microbial biofilms growing at the air-solution interface were sampled from solutions with pH values of < 1.2, temperatures of up to 48 °C, and mM concentrations of zinc, coppe