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Reporting in Nature Communications, Huo and colleagues provide three-dimensional structures of a bacterial immune defense system called Gabija. This work builds on recently published structural and functional studies and contributes strong evidence that protein assembly formation is essential for antiviral function.

RNA-guided endonucleases form the crux of diverse biological processes and technologies, including adaptive immunity, transposition, and genome editing. Some of these enzymes are components of insertion sequences (IS) in the IS200/IS605 and IS607 transposon families. Both IS families encode a TnpA transposase and a TnpB nuclease, an RNA-guided enzyme ancestral to CRISPR-Cas12s. In eukaryotes, TnpB homologs occur as two distinct types, Fanzor1s and Fanzor2s. We analyzed the evolutionary relationships between prokaryotic TnpBs and eukaryotic Fanzors, which revealed that both Fanzor1s and Fanzor2s stem from a single lineage of IS607 TnpBs with unusual active site arrangement. The widespread nature of Fanzors implies that the properties of this particular lineage of IS607 TnpBs were particularly suited to adaptation in eukaryotes. Biochemical analysis of an IS607 TnpB and Fanzor1s revealed common strategies employed by TnpBs and Fanzors to co-evolve with their cognate transposases. Collectively, our results provide a new model of sequential evolution from IS607 TnpBs to Fanzor2s, and Fanzor2s to Fanzor1s that details how genes of prokaryotic origin evolve to give rise to new protein families in eukaryotes.

In the ongoing debates about eukaryogenesis-the series of evolutionary events leading to the emergence of the eukaryotic cell from prokaryotic ancestors-members of the Asgard archaea play a key part as the closest archaeal relatives of eukaryotes1. However, the nature and phylogenetic identity of the last common ancestor of Asgard archaea and eukaryotes remain unresolved2-4. Here we analyse distinct phylogenetic marker datasets of an expanded genomic sampling of Asgard archaea and evaluate competing evolutionary scenarios using state-of-the-art phylogenomic approaches. We find that eukaryotes are placed, with high confidence, as a well-nested clade within Asgard archaea and as a sister lineage to Hodarchaeales, a newly proposed order within Heimdallarchaeia. Using sophisticated gene tree and species tree reconciliation approaches, we show that analogous to the evolution of eukaryotic genomes, genome evolution in Asgard archaea involved significantly more gene duplication and fewer gene loss events compared with other archaea. Finally, we infer that the last common ancestor of Asgard archaea was probably a thermophilic chemolithotroph and that the lineage from which eukaryotes evolved adapted to mesophilic conditions and acquired the genetic potential to support a heterotrophic lifestyle. Our work provides key insights into the prokaryote-to-eukaryote transition and a platform for better understanding the emergence of cellular complexity in eukaryotic cells.

Ice-binding proteins (IBPs) are a group of ecologically and biotechnologically relevant enzymes produced by psychrophilic organisms. Although putative IBPs containing the domain of unknown function (DUF) 3494 have been identified in many taxa of polar microbes, our knowledge of their genetic and structural diversity in natural microbial communities is limited. Here, we used samples from sea ice and sea water collected in the central Arctic Ocean as part of the MOSAiC expedition for metagenome sequencing and the subsequent analyses of metagenome-assembled genomes (MAGs). By linking structurally diverse IBPs to particular environments and potential functions, we reveal that IBP sequences are enriched in interior ice, have diverse genomic contexts and cluster taxonomically. Their diverse protein structures may be a consequence of domain shuffling, leading to variable combinations of protein domains in IBPs and probably reflecting the functional versatility required to thrive in the extreme and variable environment of the central Arctic Ocean.

The transcriptional regulatory network in prokaryotes controls global gene expression mostly through transcription factors (TFs), which are DNA-binding proteins. Chromatin immunoprecipitation (ChIP) with DNA sequencing methods can identify TF binding sites across the genome, providing a bottom-up, mechanistic understanding of how gene expression is regulated. ChIP provides indispensable evidence toward the goal of acquiring a comprehensive understanding of cellular adaptation and regulation, including condition-specificity. ChIP-derived data's importance and labor-intensiveness motivate its broad dissemination and reuse, which is currently an unmet need in the prokaryotic domain. To fill this gap, we present proChIPdb (prochipdb.org), an information-rich, interactive web database. This website collects public ChIP-seq/-exo data across several prokaryotes and presents them in dashboards that include curated binding sites, nucleotide-resolution genome viewers, and summary plots such as motif enrichment sequence logos. Users can search for TFs of interest or their target genes, download all data, dashboards, and visuals, and follow external links to understand regulons through biological databases and the literature. This initial release of proChIPdb covers diverse organisms, including most major TFs of Escherichia coli, and can be expanded to support regulon discovery across the prokaryotic domain.

We advance an account that grounds cognition, specifically decision-making, in an activity all organisms as autonomous systems must perform to keep themselves viable-controlling their production mechanisms. Production mechanisms, as we characterize them, perform activities such as procuring resources from their environment, putting these resources to use to construct and repair the organism's body and moving through the environment. Given the variable nature of the environment and the continual degradation of the organism, these production mechanisms must be regulated by control mechanisms that select when a production is required and how it should be carried out. To operate on production mechanisms, control mechanisms need to procure information through measurement processes and evaluate possible actions. They are making decisions. In all organisms, these decisions are made by multiple different control mechanisms that are organized not hierarchically but heterarchically. In many cases, they employ internal models of features of the environment with which the organism must deal. Cognition, in the form of decision-making, is thus fundamental to living systems which must control their production mechanisms. This article is part of the theme issue 'Basal cognition: conceptual tools and the view from the single cell'.

The archaeal tailed viruses (arTV), evolutionarily related to tailed double-stranded DNA (dsDNA) bacteriophages of the class Caudoviricetes, represent the most common isolates infecting halophilic archaea. Only a handful of these viruses have been genomically characterized, limiting our appreciation of their ecological impacts and evolution. Here, we present 37 new genomes of haloarchaeal tailed virus isolates, more than doubling the current number of sequenced arTVs. Analysis of all 63 available complete genomes of arTVs, which we propose to classify into 14 new families and 3 orders, suggests ancient divergence of archaeal and bacterial tailed viruses and points to an extensive sharing of genes involved in DNA metabolism and counterdefense mechanisms, illuminating common strategies of virus-host interactions with tailed bacteriophages. Coupling of the comparative genomics with the host range analysis on a broad panel of haloarchaeal species uncovered 4 distinct groups of viral tail fiber adhesins controlling the host range expansion. The survey of metagenomes using viral hallmark genes suggests that the global architecture of the arTV community is shaped through recurrent transfers between different biomes, including hypersaline, marine, and anoxic environments.

The assembly of single-amplified genomes (SAGs) and metagenome-assembled genomes (MAGs) has led to a surge in genome-based discoveries of members affiliated with Archaea and Bacteria, bringing with it a need to develop guidelines for nomenclature of uncultivated microorganisms. The International Code of Nomenclature of Prokaryotes (ICNP) only recognizes cultures as 'type material', thereby preventing the naming of uncultivated organisms. In this Consensus Statement, we propose two potential paths to solve this nomenclatural conundrum. One option is the adoption of previously proposed modifications to the ICNP to recognize DNA sequences as acceptable type material; the other option creates a nomenclatural code for uncultivated Archaea and Bacteria that could eventually be merged with the ICNP in the future. Regardless of the path taken, we believe that action is needed now within the scientific community to develop consistent rules for nomenclature of uncultivated taxa in order to provide clarity and stability, and to effectively communicate microbial diversity.

The assembly of single-amplified genomes (SAGs) and metagenome-assembled genomes (MAGs) has led to a surge in genome-based discoveries of members affiliated with Archaea and Bacteria, bringing with it a need to develop guidelines for nomenclature of uncultivated microorganisms. The International Code of Nomenclature of Prokaryotes (ICNP) only recognizes cultures as 'type material', thereby preventing the naming of uncultivated organisms. In this Consensus Statement, we propose two potential paths to solve this nomenclatural conundrum. One option is the adoption of previously proposed modifications to the ICNP to recognize DNA sequences as acceptable type material; the other option creates a nomenclatural code for uncultivated Archaea and Bacteria that could eventually be merged with the ICNP in the future. Regardless of the path taken, we believe that action is needed now within the scientific community to develop consistent rules for nomenclature of uncultivated taxa in order to provide clarity and stability, and to effectively communicate microbial diversity.

Genome-scale models (GEMs) of bacterial strains' metabolism have been formulated and used over the past 20 years. Recently, with the number of genome sequences exponentially increasing, multi-strain GEMs have proved valuable to define the properties of a species. Here, through four major stages, we extend the original Protocol used to generate a GEM for a single strain to enable multi-strain GEMs: (i) obtain or generate a high-quality model of a reference strain; (ii) compare the genome sequence between a reference strain and target strains to generate a homology matrix; (iii) generate draft strain-specific models from the homology matrix; and (iv) manually curate draft models. These multi-strain GEMs can be used to study pan-metabolic capabilities and strain-specific differences across a species, thus providing insights into its range of lifestyles. Unlike the original Protocol, this procedure is scalable and can be partly automated with the Supplementary Jupyter notebook Tutorial. This Protocol Extension joins the ranks of other comparable methods for generating models such as CarveMe and KBase. This extension of the original Protocol takes on the order of weeks to multiple months to complete depending on the availability of a suitable reference model.

Bacterial autotrophs often rely on CO2 concentrating mechanisms (CCMs) to assimilate carbon. Although many CCM proteins have been identified, a systematic screen of the components of CCMs is lacking. Here, we performed a genome-wide barcoded transposon screen to identify essential and CCM-related genes in the γ-proteobacterium Halothiobacillus neapolitanus. Screening revealed that the CCM comprises at least 17 and probably no more than 25 genes, most of which are encoded in 3 operons. Two of these operons (DAB1 and DAB2) contain a two-gene locus that encodes a domain of unknown function (Pfam: PF10070) and a putative cation transporter (Pfam: PF00361). Physiological and biochemical assays demonstrated that these proteins-which we name DabA and DabB, for DABs accumulate bicarbonate-assemble into a heterodimeric complex, which contains a putative β-carbonic anhydrase-like active site and functions as an energy-coupled inorganic carbon (Ci) pump. Interestingly, DAB operons are found in a diverse range of bacteria and archaea. We demonstrate that functional DABs are present in the human pathogens Bacillus anthracis and Vibrio cholerae. On the basis of these results, we propose that DABs constitute a class of energized Ci pumps and play a critical role in the metabolism of Ci throughout prokaryotic phyla.

Microbiomes from every environment contain a myriad of uncultivated archaeal and bacterial viruses, but studying these viruses is hampered by the lack of a universal, scalable taxonomic framework. We present vConTACT v.2.0, a network-based application utilizing whole genome gene-sharing profiles for virus taxonomy that integrates distance-based hierarchical clustering and confidence scores for all taxonomic predictions. We report near-identical (96%) replication of existing genus-level viral taxonomy assignments from the International Committee on Taxonomy of Viruses for National Center for Biotechnology Information virus RefSeq. Application of vConTACT v.2.0 to 1,364 previously unclassified viruses deposited in virus RefSeq as reference genomes produced automatic, high-confidence genus assignments for 820 of the 1,364. We applied vConTACT v.2.0 to analyze 15,280 Global Ocean Virome genome fragments and were able to provide taxonomic assignments for 31% of these data, which shows that our algorithm is scalable to very large metagenomic datasets. Our taxonomy tool can be automated and applied to metagenomes from any environment for virus classification.

Microbiomes from every environment contain a myriad of uncultivated archaeal and bacterial viruses, but studying these viruses is hampered by the lack of a universal, scalable taxonomic framework. We present vConTACT v.2.0, a network-based application utilizing whole genome gene-sharing profiles for virus taxonomy that integrates distance-based hierarchical clustering and confidence scores for all taxonomic predictions. We report near-identical (96%) replication of existing genus-level viral taxonomy assignments from the International Committee on Taxonomy of Viruses for National Center for Biotechnology Information virus RefSeq. Application of vConTACT v.2.0 to 1,364 previously unclassified viruses deposited in virus RefSeq as reference genomes produced automatic, high-confidence genus assignments for 820 of the 1,364. We applied vConTACT v.2.0 to analyze 15,280 Global Ocean Virome genome fragments and were able to provide taxonomic assignments for 31% of these data, which shows that our algorithm is scalable to very large metagenomic datasets. Our taxonomy tool can be automated and applied to metagenomes from any environment for virus classification.

Understanding the complex interactions of protein posttranslational modifications (PTMs) represents a major challenge in metabolic engineering, synthetic biology, and the biomedical sciences. Here, we present a workflow that integrates multiplex automated genome editing (MAGE), genome-scale metabolic modeling, and atomistic molecular dynamics to study the effects of PTMs on metabolic enzymes and microbial fitness. This workflow incorporates complementary approaches across scientific disciplines; provides molecular insight into how PTMs influence cellular fitness during nutrient shifts; and demonstrates how mechanistic details of PTMs can be explored at different biological scales. As a proof of concept, we present a global analysis of PTMs on enzymes in the metabolic network of Escherichia coli Based on our workflow results, we conduct a more detailed, mechanistic analysis of the PTMs in three proteins: enolase, serine hydroxymethyltransferase, and transaldolase. Application of this workflow identified the roles of specific PTMs in observed experimental phenomena and demonstrated how individual PTMs regulate enzymes, pathways, and, ultimately, cell phenotypes.

Genomic information is essential for taxonomic, phylogenetic, and functional studies to comprehensively decipher the characteristics of microorganisms, to explore microbiomes through metagenomics, and to answer fundamental questions of nature and human life. However, large gaps remain in the available genomic sequencing information published for bacterial and archaeal species, and the gaps are even larger for fungal type strains. The Global Catalogue of Microorganisms (GCM) leads an internationally coordinated effort to sequence type strains and close gaps in the genomic maps of microorganisms. Hence, the GCM aims to promote research by deep-mining genomic data.

BackgroundThe majority of eukaryotic genomes are unfinished due to the algorithmic challenges of assembling them. A variety of assembly and scaffolding tools are available, but it is not always obvious which tool or parameters to use for a specific genome size and complexity. It is, therefore, common practice to produce multiple assemblies using different assemblers and parameters, then select the best one for public release. A more compelling approach would allow one to merge multiple assemblies with the intent of producing a higher quality consensus assembly, which is the objective of assembly reconciliation.ResultsSeveral assembly reconciliation tools have been proposed in the literature, but their strengths and weaknesses have never been compared on a common dataset. We fill this need with this work, in which we report on an extensive comparative evaluation of several tools. Specifically, we evaluate contiguity, correctness, coverage, and the duplication ratio of the merged assembly compared to the individual assemblies provided as input.ConclusionsNone of the tools we tested consistently improved the quality of the input GAGE and synthetic assemblies. Our experiments show an increase in contiguity in the consensus assembly when the original assemblies already have high quality. In terms of correctness, the quality of the results depends on the specific tool, as well as on the quality and the ranking of the input assemblies. In general, the number of misassemblies ranges from being comparable to the best of the input assembly to being comparable to the worst of the input assembly.

BackgroundBacteriophage-prokaryote dynamics and interaction are believed to be important in governing microbiome composition and ecosystem functions, yet our limited knowledge of the spatial and temporal variation in phage and prokaryotic community compositions precludes accurate assessment of their roles and impacts. Anaerobic digesters are ideal model systems to examine phage-host interaction, owing to easy access, stable operation, nutrient-rich environment, and consequently enormous numbers of phages and prokaryotic cells.ResultsEquipped with high-throughput, cutting-edge environmental genomics techniques, we examined phage and prokaryotic community composition of four anaerobic digesters in full-scale wastewater treatment plants across China. Despite the relatively stable process performance in biogas production, phage and prokaryotic groups fluctuated monthly over a year of study period, showing significant correlations between those two groups at the α- and β-diversity levels. Strikingly, phages explained 40.6% of total variations of the prokaryotic community composition, much higher than the explanatory power by abiotic factors (14.5%). Consequently, phages were significantly (P

Over the past 3.8 billion years, the maximum size of life has increased by approximately 18 orders of magnitude. Much of this increase is associated with two major evolutionary innovations: the evolution of eukaryotes from prokaryotic cells approximately 1.9 billion years ago (Ga), and multicellular life diversifying from unicellular ancestors approximately 0.6 Ga. However, the quantitative relationship between organismal size and structural complexity remains poorly documented. We assessed this relationship using a comprehensive dataset that includes organismal size and level of biological complexity for 11 172 extant genera. We find that the distributions of sizes within complexity levels are unimodal, whereas the aggregate distribution is multimodal. Moreover, both the mean size and the range of size occupied increases with each additional level of complexity. Increases in size range are non-symmetric: the maximum organismal size increases more than the minimum. The majority of the observed increase in organismal size over the history of life on the Earth is accounted for by two discrete jumps in complexity rather than evolutionary trends within levels of complexity. Our results provide quantitative support for an evolutionary expansion away from a minimal size constraint and suggest a fundamental rescaling of the constraints on minimal and maximal size as biological complexity increases.

BackgroundBacteriophage-prokaryote dynamics and interaction are believed to be important in governing microbiome composition and ecosystem functions, yet our limited knowledge of the spatial and temporal variation in phage and prokaryotic community compositions precludes accurate assessment of their roles and impacts. Anaerobic digesters are ideal model systems to examine phage-host interaction, owing to easy access, stable operation, nutrient-rich environment, and consequently enormous numbers of phages and prokaryotic cells.ResultsEquipped with high-throughput, cutting-edge environmental genomics techniques, we examined phage and prokaryotic community composition of four anaerobic digesters in full-scale wastewater treatment plants across China. Despite the relatively stable process performance in biogas production, phage and prokaryotic groups fluctuated monthly over a year of study period, showing significant correlations between those two groups at the α- and β-diversity levels. Strikingly, phages explained 40.6% of total variations of the prokaryotic community composition, much higher than the explanatory power by abiotic factors (14.5%). Consequently, phages were significantly (P

BackgroundThe majority of eukaryotic genomes are unfinished due to the algorithmic challenges of assembling them. A variety of assembly and scaffolding tools are available, but it is not always obvious which tool or parameters to use for a specific genome size and complexity. It is, therefore, common practice to produce multiple assemblies using different assemblers and parameters, then select the best one for public release. A more compelling approach would allow one to merge multiple assemblies with the intent of producing a higher quality consensus assembly, which is the objective of assembly reconciliation.ResultsSeveral assembly reconciliation tools have been proposed in the literature, but their strengths and weaknesses have never been compared on a common dataset. We fill this need with this work, in which we report on an extensive comparative evaluation of several tools. Specifically, we evaluate contiguity, correctness, coverage, and the duplication ratio of the merged assembly compared to the individual assemblies provided as input.ConclusionsNone of the tools we tested consistently improved the quality of the input GAGE and synthetic assemblies. Our experiments show an increase in contiguity in the consensus assembly when the original assemblies already have high quality. In terms of correctness, the quality of the results depends on the specific tool, as well as on the quality and the ranking of the input assemblies. In general, the number of misassemblies ranges from being comparable to the best of the input assembly to being comparable to the worst of the input assembly.

The elucidation of the genetic code remains among the most influential discoveries in biology. While innumerable studies have validated the general universality of the code and its value in predicting and analyzing protein coding sequences, established and emerging work has also suggested that full genome decryption may benefit from a greater consideration of a codon's neighborhood within an mRNA than has been broadly applied. This Review examines the evidence for context cues in translation, with a focus on several recent studies that reveal broad roles for mRNA context in programming translation start sites, the rate of translation elongation, and stop codon identity.

The elucidation of the genetic code remains among the most influential discoveries in biology. While innumerable studies have validated the general universality of the code and its value in predicting and analyzing protein coding sequences, established and emerging work has also suggested that full genome decryption may benefit from a greater consideration of a codon's neighborhood within an mRNA than has been broadly applied. This Review examines the evidence for context cues in translation, with a focus on several recent studies that reveal broad roles for mRNA context in programming translation start sites, the rate of translation elongation, and stop codon identity.

Whole-cell biocatalysts have proven a tractable path toward sustainable production of bulk and fine chemicals. Yet the screening of libraries of cellular designs to identify best-performing biocatalysts is most often a low-throughput endeavor. For this reason, the development of biosensors enabling real-time monitoring of production has attracted attention. Here we applied systematic engineering of multiple parameters to search for a general biosensor design in the budding yeast Saccharomyces cerevisiae based on small-molecule binding transcriptional activators from the prokaryote superfamily of LysR-type transcriptional regulators (LTTRs). We identified a design supporting LTTR-dependent activation of reporter gene expression in the presence of cognate small-molecule inducers. As proof of principle, we applied the biosensors for in vivo screening of cells producing naringenin or cis,cis-muconic acid at different levels, and found that reporter gene output correlated with production. The transplantation of prokaryotic transcriptional activators into the eukaryotic chassis illustrates the potential of a hitherto untapped biosensor resource useful for biotechnological applications.

Whole-cell biocatalysts have proven a tractable path toward sustainable production of bulk and fine chemicals. Yet the screening of libraries of cellular designs to identify best-performing biocatalysts is most often a low-throughput endeavor. For this reason, the development of biosensors enabling real-time monitoring of production has attracted attention. Here we applied systematic engineering of multiple parameters to search for a general biosensor design in the budding yeast Saccharomyces cerevisiae based on small-molecule binding transcriptional activators from the prokaryote superfamily of LysR-type transcriptional regulators (LTTRs). We identified a design supporting LTTR-dependent activation of reporter gene expression in the presence of cognate small-molecule inducers. As proof of principle, we applied the biosensors for in vivo screening of cells producing naringenin or cis,cis-muconic acid at different levels, and found that reporter gene output correlated with production. The transplantation of prokaryotic transcriptional activators into the eukaryotic chassis illustrates the potential of a hitherto untapped biosensor resource useful for biotechnological applications.

DNA methylation acts in concert with restriction enzymes to protect the integrity of prokaryotic genomes. Studies in a limited number of organisms suggest that methylation also contributes to prokaryotic genome regulation, but the prevalence and properties of such non-restriction-associated methylation systems remain poorly understood. Here, we used single molecule, real-time sequencing to map DNA modifications including m6A, m4C, and m5C across the genomes of 230 diverse bacterial and archaeal species. We observed DNA methylation in nearly all (93%) organisms examined, and identified a total of 834 distinct reproducibly methylated motifs. This data enabled annotation of the DNA binding specificities of 620 DNA Methyltransferases (MTases), doubling known specificities for previously hard to study Type I, IIG and III MTases, and revealing their extraordinary diversity. Strikingly, 48% of organisms harbor active Type II MTases with no apparent cognate restriction enzyme. These active 'orphan' MTases are present in diverse bacterial and archaeal phyla and show motif specificities and methylation patterns consistent with functions in gene regulation and DNA replication. Our results reveal the pervasive presence of DNA methylation throughout the prokaryotic kingdoms, as well as the diversity of sequence specificities and potential functions of DNA methylation systems.

There is only limited understanding of the impact of high p(CO2) on soil biomes. We have studied a floodplain wetland where long-term emanations of temperate volcanic CO2 (mofettes) are associated with accumulation of carbon from the Earth's mantle. With an integrated approach using isotope geochemistry, soil activity measurements and multi-omics analyses, we demonstrate that high (nearly pure) CO2 concentrations have strongly affected pathways of carbon production and decomposition and therefore carbon turnover. In particular, a promotion of dark CO2 fixation significantly increased the input of geogenic carbon in the mofette when compared to a reference wetland soil exposed to normal levels of CO2. Radiocarbon analysis revealed that high quantities of mofette soil carbon originated from the assimilation of geogenic CO2 (up to 67%) via plant primary production and subsurface CO2 fixation. However, the preservation and accumulation of almost undegraded organic material appeared to be facilitated by the permanent exclusion of meso- to macroscopic eukaryotes and associated physical and/or ecological traits rather than an impaired biochemical potential for soil organic matter decomposition. Our study shows how CO2-induced changes in diversity and functions of the soil community can foster an unusual biogeochemical profile.

There is only limited understanding of the impact of high p(CO2) on soil biomes. We have studied a floodplain wetland where long-term emanations of temperate volcanic CO2 (mofettes) are associated with accumulation of carbon from the Earth's mantle. With an integrated approach using isotope geochemistry, soil activity measurements and multi-omics analyses, we demonstrate that high (nearly pure) CO2 concentrations have strongly affected pathways of carbon production and decomposition and therefore carbon turnover. In particular, a promotion of dark CO2 fixation significantly increased the input of geogenic carbon in the mofette when compared to a reference wetland soil exposed to normal levels of CO2. Radiocarbon analysis revealed that high quantities of mofette soil carbon originated from the assimilation of geogenic CO2 (up to 67%) via plant primary production and subsurface CO2 fixation. However, the preservation and accumulation of almost undegraded organic material appeared to be facilitated by the permanent exclusion of meso- to macroscopic eukaryotes and associated physical and/or ecological traits rather than an impaired biochemical potential for soil organic matter decomposition. Our study shows how CO2-induced changes in diversity and functions of the soil community can foster an unusual biogeochemical profile.

DNA methylation acts in concert with restriction enzymes to protect the integrity of prokaryotic genomes. Studies in a limited number of organisms suggest that methylation also contributes to prokaryotic genome regulation, but the prevalence and properties of such non-restriction-associated methylation systems remain poorly understood. Here, we used single molecule, real-time sequencing to map DNA modifications including m6A, m4C, and m5C across the genomes of 230 diverse bacterial and archaeal species. We observed DNA methylation in nearly all (93%) organisms examined, and identified a total of 834 distinct reproducibly methylated motifs. This data enabled annotation of the DNA binding specificities of 620 DNA Methyltransferases (MTases), doubling known specificities for previously hard to study Type I, IIG and III MTases, and revealing their extraordinary diversity. Strikingly, 48% of organisms harbor active Type II MTases with no apparent cognate restriction enzyme. These active 'orphan' MTases are present in diverse bacterial and archaeal phyla and show motif specificities and methylation patterns consistent with functions in gene regulation and DNA replication. Our results reveal the pervasive presence of DNA methylation throughout the prokaryotic kingdoms, as well as the diversity of sequence specificities and potential functions of DNA methylation systems.

BackgroundRecent studies posit a reciprocal dependency between the microbiomes associated with humans and indoor environments. However, none of these metagenome surveys has considered the viability of constituent microorganisms when inferring impact on human health.ResultsReported here are the results of a viability-linked metagenomics assay, which (1) unveil a remarkably complex community profile for bacteria, fungi, and viruses and (2) bolster the detection of underrepresented taxa by eliminating biases resulting from extraneous DNA. This approach enabled, for the first time ever, the elucidation of viral genomes from a cleanroom environment. Upon comparing the viable biomes and distribution of phylotypes within a cleanroom and adjoining (uncontrolled) gowning enclosure, the rigorous cleaning and stringent control countermeasures of the former were observed to select for a greater presence of anaerobes and spore-forming microflora. Sequence abundance and correlation analyses suggest that the viable indoor microbiome is influenced by both the human microbiome and the surrounding ecosystem(s).ConclusionsThe findings of this investigation constitute the literature's first ever account of the indoor metagenome derived from DNA originating solely from the potential viable microbial population. Results presented in this study should prove valuable to the conceptualization and experimental design of future studies on indoor microbiomes aimed at inferring impact on human health.

BackgroundRecent studies posit a reciprocal dependency between the microbiomes associated with humans and indoor environments. However, none of these metagenome surveys has considered the viability of constituent microorganisms when inferring impact on human health.ResultsReported here are the results of a viability-linked metagenomics assay, which (1) unveil a remarkably complex community profile for bacteria, fungi, and viruses and (2) bolster the detection of underrepresented taxa by eliminating biases resulting from extraneous DNA. This approach enabled, for the first time ever, the elucidation of viral genomes from a cleanroom environment. Upon comparing the viable biomes and distribution of phylotypes within a cleanroom and adjoining (uncontrolled) gowning enclosure, the rigorous cleaning and stringent control countermeasures of the former were observed to select for a greater presence of anaerobes and spore-forming microflora. Sequence abundance and correlation analyses suggest that the viable indoor microbiome is influenced by both the human microbiome and the surrounding ecosystem(s).ConclusionsThe findings of this investigation constitute the literature's first ever account of the indoor metagenome derived from DNA originating solely from the potential viable microbial population. Results presented in this study should prove valuable to the conceptualization and experimental design of future studies on indoor microbiomes aimed at inferring impact on human health.

A bacterial lipoxygenase (LOX) shows a deuterium kinetic isotope effect (KIE) that is similar in magnitude and temperature dependence to the very large KIE of eukaryotic LOXs. This occurs despite the evolutionary distance, an ~25% smaller catalytic domain, and an increase in Ea of ~11 kcal/mol. Site-specific mutagenesis leads to a protein variant with an Ea similar to that of the prototypic plant LOX, providing possible insight into the origin of evolutionary differences. These findings, which extend the phenomenon of hydrogen tunneling to a prokaryotic LOX, are discussed in the context of a role for protein size and/or flexibility in enzymatic hydrogen tunneling.

A bacterial lipoxygenase (LOX) shows a deuterium kinetic isotope effect (KIE) that is similar in magnitude and temperature dependence to the very large KIE of eukaryotic LOXs. This occurs despite the evolutionary distance, an ~25% smaller catalytic domain, and an increase in Ea of ~11 kcal/mol. Site-specific mutagenesis leads to a protein variant with an Ea similar to that of the prototypic plant LOX, providing possible insight into the origin of evolutionary differences. These findings, which extend the phenomenon of hydrogen tunneling to a prokaryotic LOX, are discussed in the context of a role for protein size and/or flexibility in enzymatic hydrogen tunneling.

Permeases of the prokaryotic phosphoenolpyruvate-sugar phosphotransferase system (PTS) catalyse sugar transport coupled to sugar phosphorylation. The lipid composition of a membrane determines the activities of these enzyme/transporters as well as the degree of coupling of phosphorylation to transport. We have investigated mechanisms of PTS permease biogenesis and identified cytoplasmic (soluble) forms of these integral membrane proteins. We found that the catalytic activities of the soluble forms differ from those of the membrane-embedded forms. Transport via the latter is much more sensitive to lipid composition than to phosphorylation, and some of these enzymes are much more sensitive to the lipid environment than others. While the membrane-embedded PTS permeases are always dimeric, the cytoplasmic forms are micellar, either monomeric or dimeric. Scattered published evidence suggests that other integral membrane proteins also exist in cytoplasmic micellar forms. The possible functions of cytoplasmic PTS permeases in biogenesis, intracellular sugar phosphorylation and permease storage are discussed.

Permeases of the prokaryotic phosphoenolpyruvate-sugar phosphotransferase system (PTS) catalyse sugar transport coupled to sugar phosphorylation. The lipid composition of a membrane determines the activities of these enzyme/transporters as well as the degree of coupling of phosphorylation to transport. We have investigated mechanisms of PTS permease biogenesis and identified cytoplasmic (soluble) forms of these integral membrane proteins. We found that the catalytic activities of the soluble forms differ from those of the membrane-embedded forms. Transport via the latter is much more sensitive to lipid composition than to phosphorylation, and some of these enzymes are much more sensitive to the lipid environment than others. While the membrane-embedded PTS permeases are always dimeric, the cytoplasmic forms are micellar, either monomeric or dimeric. Scattered published evidence suggests that other integral membrane proteins also exist in cytoplasmic micellar forms. The possible functions of cytoplasmic PTS permeases in biogenesis, intracellular sugar phosphorylation and permease storage are discussed.

Posttranslational modification is an evolutionarily conserved mechanism for regulating protein activity, binding affinity, and stability. Compared with established posttranslational modifications such as phosphorylation or ubiquitination, posttranslational modification by protons within physiological pH ranges is a less recognized mechanism for regulating protein function. By changing the charge of amino acid side chains, posttranslational modification by protons can drive dynamic changes in protein conformation and function. Addition and removal of a proton is rapid and reversible and, in contrast to most other posttranslational modifications, does not require an enzyme. Signaling specificity is achieved by only a minority of sites in proteins titrating within the physiological pH range. Here, we examine the structural mechanisms and functional consequences of proton posttranslational modification of pH-sensing proteins regulating different cellular processes.

Posttranslational modification is an evolutionarily conserved mechanism for regulating protein activity, binding affinity, and stability. Compared with established posttranslational modifications such as phosphorylation or ubiquitination, posttranslational modification by protons within physiological pH ranges is a less recognized mechanism for regulating protein function. By changing the charge of amino acid side chains, posttranslational modification by protons can drive dynamic changes in protein conformation and function. Addition and removal of a proton is rapid and reversible and, in contrast to most other posttranslational modifications, does not require an enzyme. Signaling specificity is achieved by only a minority of sites in proteins titrating within the physiological pH range. Here, we examine the structural mechanisms and functional consequences of proton posttranslational modification of pH-sensing proteins regulating different cellular processes.

MVP, a Methanococcus jannaschii voltage-gated potassium channel, was cloned and shown to operate in eukaryotic and prokaryotic cells. Like pacemaker channels, MVP opens on hyperpolarization using S4 voltage sensors like those in classical channels activated by depolarization. The MVP S4 span resembles classical sensors in sequence, charge, topology and movement, traveling inward on hyperpolarization and outward on depolarization (via canaliculi in the protein that bring the extracellular and internal solutions into proximity across a short barrier). Thus, MVP opens with sensors inward indicating a reversal of S4 position and pore state compared to classical channels. Homologous channels in mammals and plants are expected to function similarly.

MVP, a Methanococcus jannaschii voltage-gated potassium channel, was cloned and shown to operate in eukaryotic and prokaryotic cells. Like pacemaker channels, MVP opens on hyperpolarization using S4 voltage sensors like those in classical channels activated by depolarization. The MVP S4 span resembles classical sensors in sequence, charge, topology and movement, traveling inward on hyperpolarization and outward on depolarization (via canaliculi in the protein that bring the extracellular and internal solutions into proximity across a short barrier). Thus, MVP opens with sensors inward indicating a reversal of S4 position and pore state compared to classical channels. Homologous channels in mammals and plants are expected to function similarly.