Loss-of-function mutations are main drivers of adaptations during short-term evolution.

We noticed that during short-term experimental evolution and carcinogenesis, mutations causing gene inactivation (i.e., nonsense mutations or frameshifts) are frequent. Our meta-analysis of 65 experiments using modified dN/dS statistics indicated that nonsense mutations are adaptive in different experimental conditions and we empirically confirmed this prediction. Using yeast S. cerevisiae as a model we show that fixed or highly frequent gene loss-of-function mutations are almost exclusively adaptive in the majority of experiments.

Apoptotic Factors Are Evolutionarily Conserved Since Mitochondrial Domestication.

The mechanisms initiating apoptotic programmed cell death in diverse eukaryotes are very similar. Basically, the mitochondrial permeability transition activates apoptotic proteases, DNases, and flavoproteins such as apoptosis-inducing factors (AIFs). According to the hypothesis of the endosymbiotic origin of apoptosis, these mechanisms evolved during mitochondrial domestication. Various phylogenetic analyses, including ours, have suggested that apoptotic factors were eubacterial protomitochondrial toxins used for killing protoeukaryotic hosts. Here, we tested whether the function of yeast Saccharomyces cerevisiae apoptotic proteases (metacaspases Mca1 and Nma111), DNase Nuc1, and flavoprotein Ndi1 can be substituted with orthologs from remotely related eukaryotes such as plants, protists, and eubacteria. We found that orthologs of remotely related eukaryotic and even eubacterial proteins can initiate apoptosis in yeast when triggered by chemical stresses. This observation suggests that apoptotic mechanisms have been maintained since mitochondrial domestication, which occurred approximately 1,800 Mya. Additionally, it supports the hypothesis that some of these apoptotic factors could be modified eubacterial toxins.

Improvement of native structure-based peptides as efficient inhibitors of protein-protein interactions of SARS-CoV-2 spike protein and human ACE2.

New pathogens responsible for novel human disease outbreaks in the last two decades are mainly the respiratory system viruses. Not different was the last pandemic episode, caused by infection of a severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). One of the extensively explored targets, in the recent scientific literature, as a possible way for rapid development of COVID-19 specific drug(s) is the interaction between the receptor-binding domain of the virus' spike (S) glycoprotein and human receptor angiotensin-converting enzyme 2 (hACE2). This protein-protein recognition process is involved in the early stages of the SARS-CoV-2 life cycle leading to the host cell membrane penetration. Thus, disrupting this interaction may block or significantly reduce the infection caused by the novel pathogen. Previously we have designed (by in silico structure-based analysis) three very short peptides having sequences inspirited by hACE2 native fragments, which effectively bind to the SARS-CoV-2 S protein and block its interaction with the human receptor. In continuation of the above mentioned studies, here we presented an application of molecular modeling approach resulting in improved binding affinity of the previously proposed ligand and its enhanced ability to inhibit meaningful host-virus protein-protein interaction. The new optimized hexapeptide binds to the virus protein with affinity one magnitude higher than the initial ligand and, as a very short peptide, has also great potential for further drug development. The peptide-based strategy is rapid and cost-effective for developing and optimizing efficient protein-protein interactions disruptors and may be successfully applied to discover antiviral candidates against other future emerging human viral infections.

The Anti-Inflammatory Effect of Cabbage Leaves Explained by the Influence of bol-miRNA172a on FAN Expression.

Recently, the possibility of cross-kingdom gene expression regulation by miRNAs from other species ("xenomiRs"), specifically from plants, has acquired scientific meaning. Based on the one of oldest methods for dealing with inflammation via the use of cabbage leaf compresses, we investigated the effects of Brassica oleracea derived miR172a on the potential human target gene encoding FAN (Factor Associated with Neutral Sphingomyelinase Activation) protein. In vitro experiments showed a decrease in FAN protein levels in both human and mouse cells transfected with bol-miRNA172a. As the FAN protein mediates inflammatory responses, the potential of miR172a to mitigate the inflammatory process was tested in a mouse model of rheumatoid arthritis. Animal studies showed the decreased oedema of inflamed paws in mouse with rheumatoid arthritis model induced after treatment with miR172a.

Genetic interaction network has a very limited impact on the evolutionary trajectories in continuous culture-grown populations of yeast.

BACKGROUND: The impact of genetic interaction networks on evolution is a fundamental issue. Previous studies have demonstrated that the topology of the network is determined by the properties of the cellular machinery. Functionally related genes frequently interact with one another, and they establish modules, e.g., modules of protein complexes and biochemical pathways. In this study, we experimentally tested the hypothesis that compensatory evolutionary modifications, such as mutations and transcriptional changes, occur frequently in genes from perturbed modules of interacting genes. RESULTS: Using Saccharomyces cerevisiae haploid deletion mutants as a model, we investigated two modules lacking COG7 or NUP133, which are evolutionarily conserved genes with many interactions. We performed laboratory evolution experiments with these strains in two genetic backgrounds (with or without additional deletion of MSH2), subjecting them to continuous culture in a non-limiting minimal medium. Next, the evolved yeast populations were characterized through whole-genome sequencing and transcriptome analyses. No obvious compensatory changes resulting from inactivation of genes already included in modules were identified. The supposedly compensatory inactivation of genes in the evolved strains was only rarely observed to be in accordance with the established fitness effect of the genetic interaction network. In fact, a substantial majority of the gene inactivations were predicted to be neutral in the experimental conditions used to determine the interaction network. Similarly, transcriptome changes during continuous culture mostly signified adaptation to growth conditions rather than compensation of the absence of the COG7, NUP133 or MSH2 genes. However, we noticed that for genes whose inactivation was deleterious an upregulation of transcription was more common than downregulation. CONCLUSIONS: Our findings demonstrate that the genetic interactions and the modular structure of the network described by others have very limited effects on the evolutionary trajectory following gene deletion of module elements in our experimental conditions and has no significant impact on short-term compensatory evolution. However, we observed likely compensatory evolution in functionally related (albeit non-interacting) genes.

Metabolic Potential, Ecology and Presence of Associated Bacteria Is Reflected in Genomic Diversity of Mucoromycotina.

Mucoromycotina are often considered mainly in pathogenic context but their biology remains understudied. We describe the genomes of six Mucoromycotina fungi representing distant saprotrophic lineages within the subphylum (i.e., Umbelopsidales and Mucorales). We selected two Umbelopsis isolates from soil (i.e., U. isabellina, U. vinacea), two soil-derived Mucor isolates (i.e., M. circinatus, M. plumbeus), and two Mucorales representatives with extended proteolytic activity (i.e., Thamnidium elegans and Mucor saturninus). We complement computational genome annotation with experimental characteristics of their digestive capabilities, cell wall carbohydrate composition, and extensive total lipid profiles. These traits inferred from genome composition, e.g., in terms of identified encoded enzymes, are in accordance with experimental results. Finally, we link the presence of associated bacteria with observed characteristics. Thamnidium elegans genome harbors an additional, complete genome of an associated bacterium classified to Paenibacillus sp. This fungus displays multiple altered traits compared to the remaining isolates, regardless of their evolutionary distance. For instance, it has expanded carbon assimilation capabilities, e.g., efficiently degrades carboxylic acids, and has a higher diacylglycerol:triacylglycerol ratio and skewed phospholipid composition which suggests a more rigid cellular membrane. The bacterium can complement the host enzymatic capabilities, alter the fungal metabolism, cell membrane composition but does not change the composition of the cell wall of the fungus. Comparison of early-diverging Umbelopsidales with evolutionary younger Mucorales points at several subtle differences particularly in their carbon source preferences and encoded carbohydrate repertoire. Nevertheless, all tested Mucoromycotina share features including the ability to produce 18:3 gamma-linoleic acid, use TAG as the storage lipid and have fucose as a cell wall component.

Slow Adaptive Response of Budding Yeast Cells to Stable Conditions of Continuous Culture Can Occur without Genome Modifications.

Continuous cultures assure the invariability of environmental conditions and the metabolic state of cultured microorganisms, whereas batch-cultured cells undergo constant changes in nutrients availability. For that reason, continuous culture is sometimes employed in the whole transcriptome, whole proteome, or whole metabolome studies. However, the typical method for establishing uniform growth of a cell population, i.e., by limited chemostat, results in the enrichment of the cell population gene pool with mutations adaptive for starvation conditions. These adaptive changes can skew the results of large-scale studies. It is commonly assumed that these adaptations reflect changes in the genome, and this assumption has been confirmed experimentally in rare cases. Here we show that in a population of budding yeast cells grown for over 200 generations in continuous culture in non-limiting minimal medium and therefore not subject to selection pressure, remodeling of transcriptome occurs, but not as a result of the accumulation of adaptive mutations. The observed changes indicate a shift in the metabolic balance towards catabolism, a decrease in ribosome biogenesis, a decrease in general stress alertness, reorganization of the cell wall, and transactions occurring at the cell periphery. These adaptive changes signify the acquisition of a new lifestyle in a stable nonstressful environment. The absence of underlying adaptive mutations suggests these changes may be regulated by another mechanism.

Plasmidome of an environmental Acinetobacter lwoffii strain originating from a former gold and arsenic mine.

Emerging important Acinetobacter strains commonly accommodate a plethora of mobile elements including plasmids of different size. Plasmids, apart from encoding modules enabling their self-replication and/or transmission, can carry a diverse number of genes, allowing the host cell to survive in an environment that would otherwise be lethal or restrictive for growth. The present study characterizes the plasmidome generated from an arsenic-resistant strain named ZS207, classified as Acinetobacter lwoffii. Sequencing effort revealed the presence of nine plasmids in the size between 4.3 and 38.4 kb as well as one 186.6 kb megaplasmid. All plasmids, except the megaplasmid, do apparently not confer distinguishing phenotypic features. In contrast, the megaplasmid carries arsenic and heavy metals resistance regions similar to those found in permafrost A. lwoffii strains. In-depth in silico analyses have shown a significant similarity between the regions from these plasmids, especially concerning multiple transposable elements, transfer and mobilization genes, and toxin-antitoxin systems. Since ars genes encode proteins of major significance in terms of potential use in bioremediation, arsenic resistance level of ZS207 was determined and the functionality of selected ars genes was examined. Additionally, we checked the functionality of plasmid-encoded toxin-antitoxin systems and their impact on the formation of persister cells.

Evaluation of keratin biomaterial containing silver nanoparticles as a potential wound dressing in full-thickness skin wound model in diabetic mice.

Keratin is a cytoskeletal scaffolding protein essential for wound healing and tissue recovery. The aim of the study was to evaluate the potential role of insoluble fur keratin-derived powder containing silver nanoparticles (FKDP-AgNP) in the allogenic full-thickness surgical skin wound model in diabetic mice. The scanning electron microscopy image evidenced that the keratin surface is covered by a single layer of silver nanoparticles. Data obtained from dynamic light scattering and micellar electrokinetic chromatography showed three fractions of silver nanoparticles with an average diameter of 130, 22.5, and 5 nm. Microbiologic results revealed that the designed insoluble FKDP-AgNP dressing to some extent inhibit the growth of Escherichia coli and Staphylococcus aureus. In vitro assays showed that the FKDP-AgNP dressing did not inhibit fibroblast growth or induce hemolysis. In vivo studies using a diabetic mice model confirmed biocompatible properties of the insoluble keratin dressings. FKDP-AgNP significantly accelerated wound closure and epithelization at Days 5 and 8 (p < .05) when compared with controls. Histological examination of the inflammatory response documented that FKDP-AgNP-treated wounds contained predominantly macrophages, whereas their untreated variants showed mixed cell infiltrates rich in neutrophils. Wound inflammatory response based on macrophages favors tissue remodeling and healing. In conclusion, the investigated FKDP-AgNP dressing consisting of an insoluble fraction of keratin, which is biocompatible, significantly accelerated wound healing in a diabetic mouse model.

Activity and Identification of Methanotrophic Bacteria in Arable and No-Tillage Soils from Lublin Region (Poland).

Methanotrophic bacteria are able to use methane (CH4) as a sole carbon and energy source. Photochemical oxidation of methane takes place in the stratosphere, whereas in the troposphere, this process is carried out by methanotrophic bacteria. On the one hand, it is known that the efficiency of biological CH4 oxidation is dependent on the mode of land use but, on the other hand, the knowledge of this impact on methanotrophic activity (MTA) is still limited. Thus, the aim of the study was to determine the CH4 oxidation ability of methanotrophic bacteria inhabiting selected arable and no-tillage soils from the Lublin region (Albic Luvisol, Brunic Arenosol, Haplic Chernozem, Calcaric Cambisol) and to identify bacteria involved in this process. MTA was determined based on incubation of soils in air with addition of methane at the concentrations of 0.002, 0.5, 1, 5, and 10%. The experiment was conducted in a temperature range of 10-30 °C. Methanotrophs in soils were identified by next-generation sequencing (NGS). MTA was confirmed in all investigated soils (in the entire range of the tested methane concentrations and temperatures, except for the arable Albic Luvisol). Importantly, the MTA values in the no-tillage soil were nearly two-fold higher than in the cultivated soils. Statistical analysis indicated a significant influence of land use, type of soil, temperature, and especially methane concentration (p < 0.05) on MTA. Metagenomic analysis confirmed the presence of methanotrophs from the genus Methylocystis (Alphaproteobacteria) in the studied soils (except for the arable Albic Luvisol). Our results also proved the ability of methanotrophic bacteria to oxidize methane although they constituted only up to 0.1% of the total bacterial community.

An Apoptotic and Endosymbiotic Explanation of the Warburg and the Inverse Warburg Hypotheses.

Otto Warburg, a Nobel prize winner, observed that cancer cells typically "switch" from aerobic to anaerobic respiration. He hypothesized that mitochondrial damage induces neoplastic transformation. In contrast, pathological aging is observed mainly in neuron cells in neurodegenerative diseases. Oxidative respiration is particularly active in neurons. There is inverse comorbidity between cancer and neurodegenerative diseases. This led to the creation of the "inverse Warburg hypothesis", according to which excessive mitochondrial activity induces pathological aging. The findings of our studies suggest that both the Warburg effect and the "inverse Warburg hypothesis" can be elucidated by the activation or suppression of apoptosis through oxidative respiration. The key outcome of our phylogenetic studies was the discovery that apoptosis and apoptosis-like cell death evolved due to an evolutionary "arms race" conducted between "prey" protomitochondrion and "predator" primitive eukaryotes. The ancestral protomitochondrial machinery produces and releases toxic mitochondrial proteins. Extant apoptotic factors evolved from these toxins. Our experiments indicate that the mitochondrial machinery is directly involved in adaptation to aerobic conditions. Additionally, our hypothesis is supported by the fact that different apoptotic factors are directly involved in respiration.

Development of a novel keratin dressing which accelerates full-thickness skin wound healing in diabetic mice: In vitro and in vivo studies.

Impaired wound healing is a major medical problem in diabetes. The objective of this study was to determine the possible application of an insoluble fraction of fur-derived keratin biomaterial as a wound dressing in a full thickness surgical skin wound model in mice ( n = 20) with iatrogenically induced diabetes. The obtained keratin dressing was examined in vitro and in vivo. In vitro study showed the keratin dressing is tissue biocompatible and non-toxic for murine fibroblasts. Antimicrobial examination revealed the keratin dressing inhibited the growth of S. aureus and E. coli. In vivo studies showed the obtained dressing significantly ( p < 0.05) accelerated healing during the first week after surgery compared to control wounds. Keratin dressings were incorporated naturally into granulation and regenerating tissue without any visible signs of inflammatory response, which was confirmed by clinical and histopathological analysis. It is one of the first studies to show application of insoluble keratin proteins and its properties as a wound dressing. The obtained keratin dressing accelerated wound healing in mice with iatrogenically induced diabetes. Therefore, it can be considered as a safe and efficient wound dressing. Although future studies are needed to explain the molecular mechanism behind fur-derived keratin effect during the multilayer wound healing process, our findings may open the way for a new class of insoluble fur keratin dressings in chronic difficult to heal wounds treatment.

Ancestral State Reconstruction of the Apoptosis Machinery in the Common Ancestor of Eukaryotes.

Apoptotic cell death is a type of eukaryotic cell death. In animals, it regulates development, is involved in cancer suppression, and causes cell death during pathological aging of neuronal cells in neurodegenerative diseases such as Alzheimer's. Mitochondrial apoptotic-like cell death, a form of primordial apoptosis, also occurs in unicellular organisms. Here, we ask the question why the apoptosis machinery has been acquired and maintained in unicellular organisms and attempt to answer it by performing ancestral state reconstruction. We found indications of an ancient evolutionary arms race between protomitochondria and host cells, leading to the establishment of the currently existing apoptotic pathways. According to this reconstruction, the ancestral protomitochondrial apoptosis machinery contained both caspases and metacaspases, four types of apoptosis induction factors (AIFs), both fungal and animal OMI/HTR proteases, and various apoptotic DNases. This leads to the prediction that in extant unicellular eukaryotes, the apoptotic factors are involved in mitochondrial respiration and their activity is needed exclusively in aerobic conditions. We test this prediction experimentally using yeast and find that a loss of the main apoptotic factors is beneficial under anaerobic conditions yet deleterious under aerobic conditions in the absence of lethal stimuli. We also point out potential medical implications of these findings.

Taxonomic and chemical assessment of exceptionally abundant rock mine biofilm.

BACKGROUND: An exceptionally thick biofilm covers walls of ancient gold and arsenic Zloty Stok mine (Poland) in the apparent absence of organic sources of energy. METHODS AND RESULTS: We have characterized this microbial community using culture-dependent and independent methods. We sequenced amplicons of the 16S rRNA gene obtained using generic primers and additional primers targeted at Archaea and Actinobacteria separately. Also, we have cultured numerous isolates from the biofilm on different media under aerobic and anaerobic conditions. We discovered very high biodiversity, and no single taxonomic group was dominant. The majority of almost 4,000 OTUs were classified above genus level indicating presence of novel species. Elemental analysis, performed using SEM-EDS and X-ray, of biofilm samples showed that carbon, sulphur and oxygen were not evenly distributed in the biofilm and that their presence is highly correlated. However, the distribution of arsenic and iron was more flat, and numerous intrusions of elemental silver and platinum were noted, indicating that microorganisms play a key role in releasing these elements from the rock. CONCLUSIONS: Altogether, the picture obtained throughout this study shows a very rich, complex and interdependent system of rock biofilm. The chemical heterogeneity of biofilm is a likely explanation as to why this oligotrophic environment is capable of supporting such high microbial diversity.

Microbial biodiversity of meadows under different modes of land use: catabolic and genetic fingerprinting.

The main goal of the study was to find differences in the bacterial community structure resulting from different ways of meadow management in order to get the first insight into microbial biodiversity in meadow samples. The next generation sequencing technique (454-pyrosequencing) was accompanied with the community level physiological profiling (CLPP) method in order to acquire combined knowledge of both genetic and catabolic bacterial fingerprinting of two studied meadows (hayland and pasture). Soil samples (FAO: Mollic Gleysol) were taken in April 2015 from the surface layer (0-20 cm). Significant differences of the bacterial community structure between the two analyzed meadows resulted from different land mode were evidenced by pyrosequencing and CLPP techniques. It was found that Alpha- and Gammaproteobacteria dominated in the hayland, whereas Delta- and Betaproteobacteria prevailed in the pasture. Additionally, the hayland displayed lower Firmicutes diversity than the pasture. Predominant bacterial taxa: Acidobacteria, together with Chloroflexi and Bacteroidetes seemed to be insensitive to the mode of land use, because their abundance remained at a similar level in the both studied meadows. The CLPP analysis confirmed much faster degradation of the carbon sources by microorganisms from the hayland rather than from the pasture. Amino acids were the most favoured carbon source groups utilized by microorganisms in contrast to carbohydrates, which were utilized to the lowest extent. The study clearly proved that the consequences of even moderate anthropogenic management are always changes in bacterial community structure and their metabolic activity. Bacterial taxa that are sensitive and resistant on modes of land use were determined.

Metagenomic Analysis of Some Potential Nitrogen-Fixing Bacteria in Arable Soils at Different Formation Processes.

The main goal of the study was to determine the diversity of the potential nitrogen-fixing (PNF) bacteria inhabiting agricultural (A) soils versus wastelands serving as controls (C). The soils were classified into three groups based on the formation process: autogenic soils (Albic Luvisols, Brunic Arenosols, Haplic Phaeozem) formed on loess material, hydrogenic soils (Mollic Gleysols, Eutric Fluvisol, Eutric Histosol) formed under the effect of stagnant water and lithogenic soils (Rendzina Leptosols) formed on limestone. In order to determine the preferable conditions for PNF bacteria, the relationships between the soil chemical features and bacterial operational taxonomic units (OTUs) were tested. Additionally, the nitrogen content and fertilisation requirement of the lithogenic (LG), autogenic (AG) and hydrogenic (HG) soils were discussed. The composition of the bacterial communities was analysed with the next-generation sequencing (NGS) by the Ion Torrent™ technology. The sequences were clustered into OTU based on a 99 % similarity threshold. The arable soils tested were distinctly dominated by ß-Proteobacteria representatives of PNF bacteria belonging to the genus Burkholderia. Bacteria from the α-Proteobacteria class and Devosia genus were subdominants. A free-living Cyanobacteria population dominated in A rather than in C soils. We have found that both soil agricultural management and soil formation processes are the most conducive factors for PNF bacteria, as a majority of these microorganisms inhabit the AG group of soils, whilst the LG soils with the lowest abundance of PNF bacteria revealed the need for additional mineral fertilisation. Our studies have also indicated that there are close relationships between soil classification with respect to soil formation processes and PNF bacteria preference for occupation of soil niches.

Detection of Plant miRNAs Abundance in Human Breast Milk.

Breast milk is a natural food and important component of infant nutrition. Apart from the alimentary substances, breast milk contains many important bioactive compounds, including endogenous microRNA molecules (miRNAs). These regulatory molecules were identified in various mammalian biological fluids and were shown to be mostly packed in exosomes. Recently, it was revealed that plant food-derived miRNAs are stably present in human blood and regulate the expression of specific human genes. Since then, the scientific community has focused its efforts on contradicting or confirming this discovery. With the same intention, qRT-PCR experiments were performed to evaluate the presence of five plant food-derived miRNAs (miR166a, miR156a, miR157a, miR172a and miR168a) in breast milk (whole milk and exosomes) from healthy volunteers. In whole milk samples, all examined miRNAs were identified, while only two of these miRNAs were confirmed to be present in exosomes. The plant miRNA concentration in the samples ranged from 4 to 700 fM. Complementary bioinformatics analysis suggests that the evaluated plant miRNAs may potentially influence several crucial biological pathways in the infant organism.

Mapping Protein-Protein Interactions of the Resistance-Related Bacterial Zeta Toxin-Epsilon Antitoxin Complex (ε2ζ2) with High Affinity Peptide Ligands Using Fluorescence Polarization.

Toxin-antitoxin systems constitute a native survival strategy of pathogenic bacteria and thus are potential targets of antibiotic drugs. Here, we target the Zeta-Epsilon toxin-antitoxin system, which is responsible for the stable maintenance of certain multiresistance plasmids in Gram-positive bacteria. Peptide ligands were designed on the basis of the ε2ζ2 complex. Three α helices of Zeta forming the protein-protein interaction (PPI) site were selected and peptides were designed conserving the residues interacting with Epsilon antitoxin while substituting residues binding intramolecularly to other parts of Zeta. Designed peptides were synthesized with an N-terminal fluoresceinyl-carboxy-residue for binding assays and provided active ligands, which were used to define the hot spots of the ε2ζ2 complex. Further shortening and modification of the binding peptides provided ligands with affinities <100 nM, allowing us to determine the most relevant PPIs and implement a robust competition binding assay.

Noteworthy Facts about a Methane-Producing Microbial Community Processing Acidic Effluent from Sugar Beet Molasses Fermentation.

Anaerobic digestion is a complex process involving hydrolysis, acidogenesis, acetogenesis and methanogenesis. The separation of the hydrogen-yielding (dark fermentation) and methane-yielding steps under controlled conditions permits the production of hydrogen and methane from biomass. The characterization of microbial communities developed in bioreactors is crucial for the understanding and optimization of fermentation processes. Previously we developed an effective system for hydrogen production based on long-term continuous microbial cultures grown on sugar beet molasses. Here, the acidic effluent from molasses fermentation was used as the substrate for methanogenesis in an upflow anaerobic sludge blanket bioreactor. This study focused on the molecular analysis of the methane-yielding community processing the non-gaseous products of molasses fermentation. The substrate for methanogenesis produces conditions that favor the hydrogenotrophic pathway of methane synthesis. Methane production results from syntrophic metabolism whose key process is hydrogen transfer between bacteria and methanogenic Archaea. High-throughput 454 pyrosequencing of total DNA isolated from the methanogenic microbial community and bioinformatic sequence analysis revealed that the domain Bacteria was dominated by Firmicutes (mainly Clostridia), Bacteroidetes, δ- and γ-Proteobacteria, Cloacimonetes and Spirochaetes. In the domain Archaea, the order Methanomicrobiales was predominant, with Methanoculleus as the most abundant genus. The second and third most abundant members of the Archaeal community were representatives of the Methanomassiliicoccales and the Methanosarcinales. Analysis of the methanogenic sludge by scanning electron microscopy with Energy Dispersive X-ray Spectroscopy and X-ray diffraction showed that it was composed of small highly heterogeneous mineral-rich granules. Mineral components of methanogenic granules probably modulate syntrophic metabolism and methanogenic pathways. A rough functional analysis from shotgun data of the metagenome demonstrated that our knowledge of methanogenesis is poor and/or the enzymes responsible for methane production are highly effective, since despite reasonably good sequencing coverage, the details of the functional potential of the microbial community appeared to be incomplete.