PrimerEvalPy: a tool for in-silico evaluation of primers for targeting the microbiome.

BACKGROUND: The selection of primer pairs in sequencing-based research can greatly influence the results, highlighting the need for a tool capable of analysing their performance in-silico prior to the sequencing process. We therefore propose PrimerEvalPy, a Python-based package designed to test the performance of any primer or primer pair against any sequencing database. The package calculates a coverage metric and returns the amplicon sequences found, along with information such as their average start and end positions. It also allows the analysis of coverage for different taxonomic levels. RESULTS: As a case study, PrimerEvalPy was used to test the most commonly used primers in the literature against two oral 16S rRNA gene databases containing bacteria and archaea. The results showed that the most commonly used primer pairs in the oral cavity did not match those with the highest coverage. The best performing primer pairs were found for the detection of oral bacteria and archaea. CONCLUSIONS: This demonstrates the importance of a coverage analysis tool such as PrimerEvalPy to find the best primer pairs for specific niches. The software is available under the MIT licence at https://gitlab.citius.usc.es/lara.vazquez/PrimerEvalPy .

Interpreting an Archaean paleoenvironment through 3D imagery of microbialites.

While stromatolites, and to a lesser extent thrombolites, have been extensively studied in order to unravel Precambrian (>539 Ma) biological evolution, studies of clastic-dominated microbially induced sedimentary structures (MISS) are relatively scarce. The lack of a consolidated record of clastic microbialites creates questions about how much (and what) information on depositional and taphonomic settings can be gleaned from these fossils. We used µCT scanning, a non-destructive X-ray-based 3D imaging method, to reconstruct morphologies of ancient MISS and mat textures in two previously described coastal Archaean samples from the ~3.48 Ga Dresser Formation, Pilbara, Western Australia. The aim of this study was to test the ability of µCT scanning to visualize and make 3D measurements that can be used to interpret the biotic-environmental interactions. Fossil MISS including mat laminae with carpet-like textures in one sample and mat rip-up chips in the second sample were investigated. Compiled δ13C and δ34S analyses of specimens from the Dresser Fm. are consistent with a taxonomically diverse community that could be capable of forming such MISS. 3D measurements of fossil microbial mat chips indicate significant biostabilization and suggest formation in flow velocities >25 cm s-1. Given the stratigraphic location of these chips in a low-flow lagoonal layer, we conclude that these chips formed due to tidal influence, as these assumed velocities are consistent with recent modeling of Archaean tides. The success of µCT scanning in documenting these microbialite features validates this technique both as a first step analysis for rare samples prior to the use of more destructive techniques and as a valuable tool for gaining insight into microbialite taphonomy.

AnnoView enables large-scale analysis, comparison, and visualization of microbial gene neighborhoods.

The analysis and comparison of gene neighborhoods is a powerful approach for exploring microbial genome structure, function, and evolution. Although numerous tools exist for genome visualization and comparison, genome exploration across large genomic databases or user-generated datasets remains a challenge. Here, we introduce AnnoView, a web server designed for interactive exploration of gene neighborhoods across the bacterial and archaeal tree of life. Our server offers users the ability to identify, compare, and visualize gene neighborhoods of interest from 30 238 bacterial genomes and 1672 archaeal genomes, through integration with the comprehensive Genome Taxonomy Database and AnnoTree databases. Identified gene neighborhoods can be visualized using pre-computed functional annotations from different sources such as KEGG, Pfam and TIGRFAM, or clustered based on similarity. Alternatively, users can upload and explore their own custom genomic datasets in GBK, GFF or CSV format, or use AnnoView as a genome browser for relatively small genomes (e.g. viruses and plasmids). Ultimately, we anticipate that AnnoView will catalyze biological discovery by enabling user-friendly search, comparison, and visualization of genomic data. AnnoView is available at http://annoview.uwaterloo.ca.

Analysis of nearly 3000 archaeal genomes from terrestrial geothermal springs sheds light on interconnected biogeochemical processes.

Terrestrial geothermal springs are physicochemically diverse and host abundant populations of Archaea. However, the diversity, functionality, and geological influences of these Archaea are not well understood. Here we explore the genomic diversity of Archaea in 152 metagenomes from 48 geothermal springs in Tengchong, China, collected from 2016 to 2021. Our dataset is comprised of 2949 archaeal metagenome-assembled genomes spanning 12 phyla and 392 newly identified species, which increases the known species diversity of Archaea by ~48.6%. The structures and potential functions of the archaeal communities are strongly influenced by temperature and pH, with high-temperature acidic and alkaline springs favoring archaeal abundance over Bacteria. Genome-resolved metagenomics and metatranscriptomics provide insights into the potential ecological niches of these Archaea and their potential roles in carbon, sulfur, nitrogen, and hydrogen metabolism. Furthermore, our findings illustrate the interplay of competition and cooperation among Archaea in biogeochemical cycles, possibly arising from overlapping functional niches and metabolic handoffs. Taken together, our study expands the genomic diversity of Archaea inhabiting geothermal springs and provides a foundation for more incisive study of biogeochemical processes mediated by Archaea in geothermal ecosystems.

Extremophiles and their expanding biotechnological applications.

Microbial life is not restricted to any particular setting. Over the past several decades, it has been evident that microbial populations can exist in a wide range of environments, including those with extremes in temperature, pressure, salinity, and pH. Bacteria and Archaea are the two most reported types of microbes that can sustain in extreme environments, such as hot springs, ice caves, acid drainage, and salt marshes. Some can even grow in toxic waste, organic solvents, and heavy metals. These microbes are called extremophiles. There exist certain microorganisms that are found capable of thriving in two or more extreme physiological conditions simultaneously, and are regarded as polyextremophiles. Extremophiles possess several physiological and molecular adaptations including production of extremolytes, ice nucleating proteins, pigments, extremozymes and exopolysaccharides. These metabolites are used in many biotechnological industries for making biofuels, developing new medicines, food additives, cryoprotective agents etc. Further, the study of extremophiles holds great significance in astrobiology. The current review summarizes the diversity of microorganisms inhabiting challenging environments and the biotechnological and therapeutic applications of the active metabolites obtained as a response to stress conditions. Bioprospection of extremophiles provides a progressive direction with significant enhancement in economy. Moreover, the introduction to omics approach including whole genome sequencing, single cell genomics, proteomics, metagenomics etc., has made it possible to find many unique microbial communities that could be otherwise difficult to cultivate using traditional methods. These findings might be capable enough to state that discovery of extremophiles can bring evolution to biotechnology.

Dynamic evolution of the mTHF gene family associated with primary metabolism across life.

BACKGROUND: The folate cycle of one-carbon (C1) metabolism, which plays a central role in the biosynthesis of nucleotides and amino acids, demonstrates the significance of metabolic adaptation. We investigated the evolutionary history of the methylenetetrahydrofolate dehydrogenase (mTHF) gene family, one of the main drivers of the folate cycle, across life. RESULTS: Through comparative genomic and phylogenetic analyses, we found that several lineages of Archaea lacked domains vital for folate cycle function such as the mTHF catalytic and NAD(P)-binding domains of FolD. Within eukaryotes, the mTHF gene family diversified rapidly. For example, several duplications have been observed in lineages including the Amoebozoa, Opisthokonta, and Viridiplantae. In a common ancestor of Opisthokonta, FolD and FTHFS underwent fusion giving rise to the gene MTHFD1, possessing the domains of both genes. CONCLUSIONS: Our evolutionary reconstruction of the mTHF gene family associated with a primary metabolic pathway reveals dynamic evolution, including gene birth-and-death, gene fusion, and potential horizontal gene transfer events and/or amino acid convergence.

Regulatory sequence-based discovery of anti-defense genes in archaeal viruses.

In silico identification of viral anti-CRISPR proteins (Acrs) has relied largely on the guilt-by-association method using known Acrs or anti-CRISPR associated proteins (Acas) as the bait. However, the low number and limited spread of the characterized archaeal Acrs and Aca hinders our ability to identify Acrs using guilt-by-association. Here, based on the observation that the few characterized archaeal Acrs and Aca are transcribed immediately post viral infection, we hypothesize that these genes, and many other unidentified anti-defense genes (ADG), are under the control of conserved regulatory sequences including a strong promoter, which can be used to predict anti-defense genes in archaeal viruses. Using this consensus sequence based method, we identify 354 potential ADGs in 57 archaeal viruses and 6 metagenome-assembled genomes. Experimental validation identified a CRISPR subtype I-A inhibitor and the first virally encoded inhibitor of an archaeal toxin-antitoxin based immune system. We also identify regulatory proteins potentially akin to Acas that can facilitate further identification of ADGs combined with the guilt-by-association approach. These results demonstrate the potential of regulatory sequence analysis for extensive identification of ADGs in viruses of archaea and bacteria.

Differences in archaeal diversity and potential ecological functions between saline and hypersaline lakes on Qinghai-Tibet Plateau were driven by multiple environmental and non-environmental factors beyond the salinity.

BACKGROUND: Saline lakes are home to various archaea that play special and crucial roles in the global biogeochemical cycle. The Qinghai-Tibet Plateau hosts a large number of lakes with diverse salinity ranging from 0.1 to over 400 g/L, harboring complex and diverse archaea. To the best of our knowledge, the formation mechanisms and potential ecological roles of archaea in Qinghai-Tibetan Plateau saline lakes remain largely unknown. RESULTS: Using High-throughput Illumina sequencing, we uncovered the vastly distinct archaea communities between two typical saline lakes with significant salinity differences on the Qinghai Tibet Plateau (Qinghai saline lake and Chaka hypersaline lake) and suggested archaea played different important roles in methanogenesis-related and nitrate reduction-related functions of these two lakes, respectively. Rather than the individual effect of salinity, the composite effect of salinity with diverse environmental parameters (e.g., temperature, chlorophyll a, total nitrogen, and total phosphorus) dominated the explanation of the variations in archaeal community structure in different habitats. Based on the network analysis, we further found the correlations between dominant archaeal OTUs were tight but significantly different between the two habitats, implying that archaeal interactions may also largely determine the shape of archaeal communities. CONCLUSION: The present study improved our understanding of the structure and function of archaea in different saline lakes on the Qinghai-Tibet Plateau and provided a new perspective on the mechanisms underlying shaping their communities.

Origin and modern microbial ecology of secondary mineral deposits in Lehman Caves, Great Basin National Park, NV, USA.

Lehman Caves is an extensively decorated high desert cave that represents one of the main tourist attractions in Great Basin National Park, Nevada. Although traditionally considered a water table cave, recent studies identified abundant speleogenetic features consistent with a hypogenic and, potentially, sulfuric acid origin. Here, we characterized white mineral deposits in the Gypsum Annex (GA) passage to determine whether these secondary deposits represent biogenic minerals formed during sulfuric acid corrosion and explored microbial communities associated with these and other mineral deposits throughout the cave. Powder X-ray diffraction (pXRD), scanning electron microscopy with electron dispersive spectroscopy (SEM-EDS), and electron microprobe analyses (EPMA) showed that, while most white mineral deposits from the GA contain gypsum, they also contain abundant calcite, silica, and other phases. Gypsum and carbonate-associated sulfate isotopic values of these deposits are variable, with δ34SV-CDT between +9.7‰ and +26.1‰, and do not reflect depleted values typically associated with replacement gypsum formed during sulfuric acid speleogenesis. Petrographic observations show that the sulfates likely co-precipitated with carbonate and SiO2 phases. Taken together, these data suggest that the deposits resulted from later-stage meteoric events and not during an initial episode of sulfuric acid speleogenesis. Most sedimentary and mineral deposits in Lehman Caves have very low microbial biomass, with the exception of select areas along the main tour route that have been impacted by tourist traffic. High-throughput 16S rRNA gene amplicon sequencing showed that microbial communities in GA sediments are distinct from those in other parts of the cave. The microbial communities that inhabit these oligotrophic secondary mineral deposits include OTUs related to known ammonia-oxidizing Nitrosococcales and Thaumarchaeota, as well as common soil taxa such as Acidobacteriota and Proteobacteria. This study reveals microbial and mineralogical diversity in a previously understudied cave and expands our understanding of the geomicrobiology of desert hypogene cave systems.

Colonization characteristics and dynamic transition of archaea communities on polyethylene and polypropylene microplastics in the sediments of mangrove ecosystems.

Microplastics are a growing concern in mangrove ecosystems; however, their effects on archaeal communities and related ecological processes remain unclear. We conducted in situ biofilm-enrichment experiments to investigate the ecological influence of polyethylene (PE) and polypropylene microplastics on archaeal communities in the sediments of mangrove ecosystems. The archaeal community present on microplastics was distinct from that of the surrounding sediments at an early stage but became increasingly similar over time. Bathyarchaeota, Thaumarchaeota, Euryarchaeota, and Asgardaeota were the most abundant phyla. Methanolobus, an archaeal biomarker, was enriched in PE biofilms, and significantly controlled by homogeneous selection in the plastisphere, indicating an increased potential risk of methane emission. The dominant archaeal assembly process in the sediments was deterministic (58.85%-70.47%), while that of the PE biofilm changed from stochastic to deterministic during the experiment. The network of PE plastispheres showed less complexity and competitive links, and higher modularity and stability than that of sediments. Functional prediction showed an increase in aerobic ammonia oxidation during the experiment, whereas methanogenesis and chemoheterotrophy were significantly higher in the plastisphere. This study provides novel insights into the impact of microplastic pollution on archaeal communities and their mediating ecological functions in mangrove ecosystems.

Tracking investigation of archaeal composition and methanogenesis function from parental to offspring pigs.

Archaea play a crucial role in microbial systems, including driving biochemical reactions and affecting host health by producing methane through hydrogen. The study of swine gut archaea has a positive significance in reducing methane emissions and improving feed utilization efficiency. However, the development and functional changes of archaea in the pig intestines have been overlooked for a long time. In this study, 54 fecal samples were collected from 36 parental pigs (18 boars and 18 pregnant/lactating sows), and 108 fecal samples from 18 offspring pigs during lactation, nursery, growing, and finishing stages were tracked and collected for metagenomic sequencing. We obtained 14 archaeal non-redundant metagenome-assembled genomes (MAGs). These archaea were classified as Methanobacteriota and Thermoplasmatota at the phylum level, and Methanobrevibacter, Methanosphaera, MX-02, and UBA71 at the genus level, involving hydrogenotrophic, methylotrophic, and acetoclastic pathways. The hydrogenotrophic pathway dominated the methanogenesis function, and the vast majority of archaea participated in it. Dietary changes profoundly affected the archaeal composition and methanogenesis function in pigs. The abundance of hydrogen-producing bacteria in parental pigs fed high-fiber diets was higher than that in offspring pigs fed low-fiber diets. The methanogenesis function was positively correlated with fiber decomposition functions and negatively correlated with the starch decomposition function. Increased abundance of sulfate reductase and fumarate reductase, as well as decreased acetate/propionate ratio, indicated that the upregulation of alternative hydrogen uptake pathways competing with methanogens may be the reason for the reduced methanogenesis function. These findings contribute to providing information and direction in the pig industry for the development of strategies to reduce methane emissions, improve feed efficiency, and maintain intestinal health.

Light Control in Microbial Systems.

Light is a key environmental component influencing many biological processes, particularly in prokaryotes such as archaea and bacteria. Light control techniques have revolutionized precise manipulation at molecular and cellular levels in recent years. Bacteria, with adaptability and genetic tractability, are promising candidates for light control studies. This review investigates the mechanisms underlying light activation in bacteria and discusses recent advancements focusing on light control methods and techniques for controlling bacteria. We delve into the mechanisms by which bacteria sense and transduce light signals, including engineered photoreceptors and light-sensitive actuators, and various strategies employed to modulate gene expression, protein function, and bacterial motility. Furthermore, we highlight recent developments in light-integrated methods of controlling microbial responses, such as upconversion nanoparticles and optical tweezers, which can enhance the spatial and temporal control of bacteria and open new horizons for biomedical applications.

New insights into the coal-associated methane architect: the ancient archaebacteria.

Exploration and marketable exploitation of coalbed methane (CBM) as cleaner fuel has been started globally. In addition, incidence of methane in coal basins is an imperative fraction of global carbon cycle. Significantly, subsurface coal ecosystem contains methane forming archaea. There is a rising attention in optimizing microbial coal gasification to exploit the abundant or inexpensive coal reserves worldwide. Therefore, it is essential to understand the coalbeds in geo-microbial perspective. Current review provides an in-depth analysis of recent advances in our understanding of how methanoarchaea are distributed in coal deposits globally. Specially, we highlight the findings on coal-associated methanoarchaeal existence, abundance, diversity, metabolic activity, and biogeography in diverse coal basins worldwide. Growing evidences indicates that we have arrived an exciting era of archaeal research. Moreover, gasification of coal into methane by utilizing microbial methanogenesis is a considerable way to mitigate the energy crisis for the rising world population.

Soil prokaryotic and fungal biome structures associated with crop disease status across the Japan Archipelago.

Archaea, bacteria, and fungi in the soil are increasingly recognized as determinants of agricultural productivity and sustainability. A crucial step for exploring soil microbiomes with important ecosystem functions is to perform statistical analyses on the potential relationship between microbiome structure and functions based on comparisons of hundreds or thousands of environmental samples collected across broad geographic ranges. In this study, we integrated agricultural field metadata with microbial community analyses by targeting 2,903 bulk soil samples collected along a latitudinal gradient from cool-temperate to subtropical regions in Japan (26.1-42.8 °N). The data involving 632 archaeal, 26,868 bacterial, and 4,889 fungal operational taxonomic units detected across the fields of 19 crop plant species allowed us to conduct statistical analyses (permutational analyses of variance, generalized linear mixed models, randomization analyses, and network analyses) on the relationship among edaphic factors, microbiome compositions, and crop disease prevalence. We then examined whether the diverse microbes form species sets varying in potential ecological impacts on crop plants. A network analysis suggested that the observed prokaryotes and fungi were classified into several species sets (network modules), which differed substantially in association with crop disease prevalence. Within the network of microbe-to-microbe coexistence, ecologically diverse microbes, such as an ammonium-oxidizing archaeon, an antibiotics-producing bacterium, and a potentially mycoparasitic fungus, were inferred to play key roles in shifts between crop-disease-promotive and crop-disease-suppressive states of soil microbiomes. The bird's-eye view of soil microbiome structure will provide a basis for designing and managing agroecosystems with high disease-suppressive functions.IMPORTANCEUnderstanding how microbiome structure and functions are organized in soil ecosystems is one of the major challenges in both basic ecology and applied microbiology. Given the ongoing worldwide degradation of agroecosystems, building frameworks for exploring structural diversity and functional profiles of soil microbiomes is an essential task. Our study provides an overview of cropland microbiome states in light of potential crop-disease-suppressive functions. The large data set allowed us to explore highly functional species sets that may be stably managed in agroecosystems. Furthermore, an analysis of network architecture highlighted species that are potentially used to cause shifts from disease-prevalent states of agroecosystems to disease-suppressive states. By extending the approach of comparative analyses toward broader geographic ranges and diverse agricultural practices, agroecosystem with maximized biological functions will be further explored.

Removal of phosphoglycolate in hyperthermophilic archaea.

Many organisms that utilize the Calvin-Benson-Bassham (CBB) cycle for autotrophic growth harbor metabolic pathways to remove and/or salvage 2-phosphoglycolate, the product of the oxygenase activity of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco). It has been presumed that the occurrence of 2-phosphoglycolate salvage is linked to the CBB cycle, and in particular, the C2 pathway to the CBB cycle and oxygenic photosynthesis. Here, we examined 2-phosphoglycolate salvage in the hyperthermophilic archaeon Thermococcus kodakarensis, an obligate anaerobe that harbors a Rubisco that functions in the pentose bisphosphate pathway. T. kodakarensis harbors enzymes that have the potential to convert 2-phosphoglycolate to glycine and serine, and their genes were identified by biochemical and/or genetic analyses. 2-phosphoglycolate phosphatase activity increased 1.6-fold when cells were grown under microaerobic conditions compared to anaerobic conditions. Among two candidates, TK1734 encoded a phosphatase specific for 2-phosphoglycolate, and the enzyme was responsible for 80% of the 2-phosphoglycolate phosphatase activity in T. kodakarensis cells. The TK1734 disruption strain displayed growth impairment under microaerobic conditions, which was relieved upon addition of sodium sulfide. In addition, glycolate was detected in the medium when T. kodakarensis was grown under microaerobic conditions. The results suggest that T. kodakarensis removes 2-phosphoglycolate via a phosphatase reaction followed by secretion of glycolate to the medium. As the Rubisco in T. kodakarensis functions in the pentose bisphosphate pathway and not in the CBB cycle, mechanisms to remove 2-phosphoglycolate in this archaeon emerged independent of the CBB cycle.

Bacterioruberin: Biosynthesis, Antioxidant Activity, and Therapeutic Applications in Cancer and Immune Pathologies.

Halophilic archaea, also termed haloarchaea, are a group of moderate and extreme halophilic microorganisms that constitute the major microbial populations in hypersaline environments. In these ecosystems, mainly aquatic, haloarchaea are constantly exposed to ionic and oxidative stress due to saturated salt concentrations and high incidences of UV radiation (mainly in summer). To survive under these harsh conditions, haloarchaea have developed molecular adaptations including hyperpigmentation. Regarding pigmentation, haloarchaeal species mainly synthesise the rare C50 carotenoid called bacterioruberin (BR) and its derivatives, monoanhydrobacterioruberin and bisanhydrobacterioruberin. Due to their colours and extraordinary antioxidant properties, BR and its derivatives have been the aim of research in several research groups all over the world during the last decade. This review aims to summarise the most relevant characteristics of BR and its derivatives as well as describe their reported antitumoral, immunomodulatory, and antioxidant biological activities. Based on their biological activities, these carotenoids can be considered promising natural biomolecules that could be used as tools to design new strategies and/or pharmaceutical formulas to fight against cancer, promote immunomodulation, or preserve skin health, among other potential uses.

A vast repertoire of secondary metabolites potentially influences community dynamics and biogeochemical processes in cold seeps.

In deep-sea cold seeps, microbial communities thrive on the geological seepage of hydrocarbons and inorganic compounds, differing from photosynthetically driven ecosystems. However, their biosynthetic capabilities remain largely unexplored. Here, we analyzed 81 metagenomes, 33 metatranscriptomes, and 7 metabolomes derived from nine different cold seep areas to investigate their secondary metabolites. Cold seep microbiomes encode diverse and abundant biosynthetic gene clusters (BGCs). Most BGCs are affiliated with understudied bacteria and archaea, including key mediators of methane and sulfur cycling. The BGCs encode diverse antimicrobial compounds that potentially shape community dynamics and various metabolites predicted to influence biogeochemical cycling. BGCs from key players are widely distributed and highly expressed, with their abundance and expression levels varying with sediment depth. Sediment metabolomics reveals unique natural products, highlighting uncharted chemical potential and confirming BGC activity in these sediments. Overall, these results demonstrate that cold seep sediments serve as a reservoir of hidden natural products and sheds light on microbial adaptation in chemosynthetically driven ecosystems.

Microbial Diversity and Open Questions about the Deep Tree of Life.

In this perspective, we explore the transformative impact and inherent limitations of metagenomics and single-cell genomics on our understanding of microbial diversity and their integration into the Tree of Life. We delve into the key challenges associated with incorporating new microbial lineages into the Tree of Life through advanced phylogenomic approaches. Additionally, we shed light on enduring debates surrounding various aspects of the microbial Tree of Life, focusing on recent advances in some of its deepest nodes, such as the roots of bacteria, archaea, and eukaryotes. We also bring forth current limitations in genome recovery and phylogenomic methodology, as well as new avenues of research to uncover additional key microbial lineages and resolve the shape of the Tree of Life.