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The division and cell wall (dcw) gene cluster in Bacteria comprises 17 genes encoding key steps in peptidoglycan synthesis and cytokinesis. To understand the origin and evolution of this cluster, we analysed its presence in over 1,000 bacterial genomes. We show that the dcw gene cluster is strikingly conserved in both gene content and gene order across all Bacteria and has undergone only a few rearrangements in some phyla, potentially linked to cell envelope specificities, but not directly to cell shape. A large concatenation of the 12 most conserved dcw cluster genes produced a robust tree of Bacteria that is largely consistent with recent phylogenies based on frequently used markers. Moreover, evolutionary divergence analyses show that the dcw gene cluster offers advantages in defining high-rank taxonomic boundaries and indicate at least two main phyla in the Candidate Phyla Radiation (CPR) matching a sharp dichotomy in dcw gene cluster arrangement. Our results place the origin of the dcw gene cluster in the Last Bacterial Common Ancestor and show that it has evolved vertically for billions of years, similar to major cellular machineries such as the ribosome. The strong phylogenetic signal, combined with conserved genomic synteny at large evolutionary distances, makes the dcw gene cluster a robust alternative set of markers to resolve the ever-growing tree of Bacteria.

Compared with the extensive knowledge on cell division in model eukaryotes and bacteria, little is known about how archaea divide. Interestingly, both endosomal sorting complex required for transport (ESCRT)-based and FtsZ-based cell division systems are found in members of the Archaea. In the past couple of years, several studies have started to shed light on FtsZ-based cell division processes in members of the Euryarchaeota. In this review we highlight recent findings in this emerging field of research. We present current knowledge of the cell division machinery of halophiles which relies on two FtsZ proteins, and we compare it with that of methanobacteria, which relies on only one FtsZ. Finally, we discuss how these differences relate to the distinct cell envelopes of these two archaeal model systems.

Dividing cells into two daughter cells is a complicated process that in bacteria and eukaryotes requires many proteins to work together. For archaea that divide via an FtsZ-based mechanism, only three proteins of the cell division machinery could so far be identified. These are two tubulin homologs, FtsZ1, FtsZ2 and the membrane anchor of FtsZ2, SepF. Here, we investigate additional archaeal cell division proteins that were identified by immunoprecipitation of SepF. These proteins comprise a single PRC-barrel domain and strictly co-occur with FtsZ. Two out of three PRC-barrel domain containing proteins found inHaloferax volcanii, CdpB1 and CdpB2 localize to the site of cell division in a SepF-dependent manner. Moreover, depletions and deletions cause severe cell division defects, generating drastically enlarged cells. Fluorescence microscopy of tagged FtsZ1, FtsZ2 and SepF in CdpB1/2 deletion strains revealed that the divisome is unusually disordered and not organized into a distinct ring-like structure at the cell centre. Biochemical analysis of CdpB homologs from different archaeal species showed that SepF interacts directly with CdpB1, which in turn binds to CdpB2, forming a tripartite complex. A crystal structure of CdpB1 and B2 recapitulated these interactions and suggested how these proteins might form filaments, possibly aligning SepF and therefore the FtsZ2 ring during cell division. In summary, we demonstrate that PRC domain proteins play essential roles in FtsZ based cell division in archaea.

Mobile genetic elements (MGEs), especially viruses, have a major impact on microbial communities. Methanogenic archaea play key environmental and economical roles, being the main producers of methane -a potent greenhouse gas and an energy source. They are widespread in diverse anoxic artificial and natural environments, including animal gut microbiomes. However, their viruses remain vastly unknown. Here, we carried out a global investigation of MGEs in 3436 genomes and metagenome-assembled genomes covering all known diversity of methanogens and using a newly assembled CRISPR database consisting of 60,000 spacers of methanogens, the most extensive collection to date. We obtained 248 high-quality (pro)viral and 63 plasmid sequences assigned to hosts belonging to nine main orders of methanogenic archaea, including the first MGEs of Methanonatronarchaeales, Methanocellales and Methanoliparales archaea. We found novel CRISPR arrays in ‘Ca. Methanomassiliicoccus intestinalis’ and ‘Ca. Methanomethylophilus’ genomes with spacers targeting small ssDNA viruses of the Smacoviridae, supporting and extending the hypothesis of an interaction between smacoviruses and gut associated Methanomassiliicoccales. Gene network analysis shows that methanogens encompass a unique and interconnected MGE repertoire, including novel viral families belonging to head-tailed Caudoviricetes, but also icosahedral and archaeal-specific pleomorphic, spherical, and spindle (pro)viruses. We reveal well-delineated modules for virus-host interaction, genome replication and virion assembly, and a rich repertoire of defense and counter-defense systems suggesting a highly dynamic and complex network of interactions between methanogens and their MGEs. We also identify potential conjugation systems composed of VirB4, VirB5 and VirB6 proteins encoded on plasmids and (pro)viruses of Methanosarcinales, the first report in Euryarchaeota. We identified 15 new families of viruses infecting Methanobacteriales, the most prominent archaea in the gut microbiome. These encode a large repertoire of protein domains for recognizing and cleaving pseudomurein for viral entry and egress, suggesting convergent adaptation of bacterial and archaeal viruses to the presence of a cell wall. Finally, we highlight an enrichment of glycan-binding domains (immunoglobulin-like (Ig-like)/Flg_new) and diversity-generating retroelements (DGRs) in viruses from gut-associated methanogens, suggesting a role in adaptation to host environments and remarkable convergence with phages infecting gut-associated bacteria. Our work represents an important step toward the characterization of the vast repertoire of MGEs associated with methanogens, including a better understanding of their role in regulating their communities globally and the development of much-needed genetic tools.

International audience; Recent data support the hypothesis that Gram-positive bacteria (monoderms) arose from Gram-negative ones (diderms) through loss of the outer membrane (OM), but how this happened remains unknown. As tethering of the OM is essential for cell envelope stability in diderm bacteria, its destabilization may have been involved in this transition. In the present study, we present an in-depth analysis of the four known main OM-tethering systems across the Tree of Bacteria (ToB). We show that the presence of such systems follows the ToB with a bimodal distribution matching the deepest phylogenetic divergence between Terrabacteria and Gracilicutes. Whereas the lipoprotein peptidoglycan-associated lipoprotein (Pal) is restricted to the Gracilicutes, along with a more sporadic occurrence of OmpA, and Braun's lipoprotein is present only in a subclade of Gammaproteobacteria, diderm Terrabacteria display, as the main system, the OmpM protein. We propose an evolutionary scenario whereby OmpM represents a simple, ancestral OM-tethering system that was later replaced by one based on Pal after the emergence of the Lol machinery to deliver lipoproteins to the OM, with OmpA as a possible transition state. We speculate that the existence of only one main OM-tethering system in the Terrabacteria would have allowed the multiple OM losses specifically inferred in this clade through OmpM perturbation, and we provide experimental support for this hypothesis by inactivating all four ompM gene copies in the genetically tractable diderm Firmicute Veillonella parvula. High-resolution imaging and tomogram reconstructions reveal a non-lethal phenotype in which vast portions of the OM detach from the cells, forming huge vesicles with an inflated periplasm shared by multiple dividing cells. Together, our results highlight an ancient shift of OM-tethering systems in bacterial evolution and suggest a mechanism for OM loss and the multiple emergences of the monoderm phenotype from diderm ancestors.

The human gut microbiome plays an important role in health, but its archaeal diversity remains largely unexplored. In the present study, we report the analysis of 1,167 nonredundant archaeal genomes (608 high-quality genomes) recovered from human gastrointestinal tract, sampled across 24 countries and rural and urban populations. We identified previously undescribed taxa including 3 genera, 15 species and 52 strains. Based on distinct genomic features, we justify the split of the Methanobrevibacter smithii clade into two separate species, with one represented by the previously undescribed ‘Candidatus Methanobrevibacter intestini’. Patterns derived from 28,581 protein clusters showed significant associations with sociodemographic characteristics such as age groups and lifestyle. We additionally show that archaea are characterized by specific genomic and functional adaptations to the host and carry a complex virome. Our work expands our current understanding of the human archaeome and provides a large genome catalogue for future analyses to decipher its impact on human physiology., Recovery of 1,167 nonredundant archaeal genomes from the human gut microbiomes reveals previously undescribed genera, associations with sociodemographic factors and the presence of an archaeal virome.

DNA in cells is associated with proteins that constrain its structure and affect DNA-templated processes including transcription and replication. HU and histones are the main constituents of chromatin in bacteria and eukaryotes, respectively, with few exceptions. Archaea, in contrast, have diverse repertoires of nucleoid-associated proteins (NAPs). To analyse the evolutionary and ecological drivers of this diversity, we combined a phylogenomic survey of known and predicted NAPs with quantitative proteomic data. We identify the Diaforarchaea as a hotbed of NAP gain and loss, and experimentally validate candidate NAPs in two members of this clade, Thermoplasma volcanium and Methanomassiliicoccus luminyensis. Proteomic analysis across a diverse sample of 19 archaea revealed that NAP investment varies from 5% of total protein. This variation is predicted by growth temperature. We propose that high levels of chromatinization have evolved as a mechanism to prevent uncontrolled helix denaturation at higher temperatures, with implications for the origin of chromatin in both archaea and eukaryotes.

International audience; Iron–sulfur (Fe–S) clusters are ubiquitous cofactors essential for life. It is largely thought that the emergence of oxygenic photosynthesis and progressive oxygenation of the atmosphere led to the origin of multiprotein machineries (ISC, NIF and SUF) assisting Fe–S cluster synthesis in the presence of oxidative stress and shortage of bioavailable iron. However, previous analyses have left unclear the origin and evolution of these systems. Here, we combine exhaustive homology searches with genomic context analysis and phylogeny to precisely identify Fe–S cluster biogenesis systems in over 10,000 archaeal and bacterial genomes. We highlight the existence of two additional and clearly distinct ‘minimal’ Fe–S cluster assembly machineries, MIS (minimal iron–sulfur) and SMS (SUF-like minimal system), which we infer in the last universal common ancestor (LUCA) and we experimentally validate SMS as a bona fide Fe–S cluster biogenesis system. These ancestral systems were kept in archaea whereas they went through stepwise complexification in bacteria to incorporate additional functions for higher Fe–S cluster synthesis efficiency leading to SUF, ISC and NIF. Horizontal gene transfers and losses then shaped the current distribution of these systems, driving ecological adaptations such as the emergence of aerobic lifestyles in archaea. Our results show that dedicated machineries were in place early in evolution to assist Fe–S cluster biogenesis and that their origin is not directly linked to Earth oxygenation.

International audience; Methane is one of the most important greenhouse gases on Earth and holds an important place in the global carbon cycle. Archaea are the only organisms that use methanogenesis to produce energy and rely on the methyl–coenzyme M reductase complex (Mcr). Over the last decade, new results have significantly reshaped our view of the diversity of methane-related pathways in the Archaea. Many new lineages that synthesize or use methane have been identified across the whole archaeal tree, leading to a greatly expanded diversity of substrates and mechanisms. In this review, we present the state of the art of these advances and how they challenge established scenarios of the origin and evolution of methanogenesis, and we discuss the potential trajectories that may have led to this strikingly wide range of metabolisms.

Significance Chromatin in eukaryotes is built around histone–DNA complexes, which act as platforms for the integration of regulatory information. Different layers of information are integrated in a combinatorial fashion, for example by exchanging core histones for variants with different properties. We show that histone variants also exist in archaea. In particular, we identify unique archaeal variants that act as capstones, preventing extension of histone–DNA complexes. Importantly, we show that some archaeal histone variants are ancient and have been maintained as distinct units for hundreds of millions of years. Our work suggests that complex combinatorial chromatin that uses histones as its building blocks exists outside eukaryotes and that the ancestor of eukaryotes might have already had complex chromatin., Nucleosomes in eukaryotes act as platforms for the dynamic integration of epigenetic information. Posttranslational modifications are reversibly added or removed and core histones exchanged for paralogous variants, in concert with changing demands on transcription and genome accessibility. Histones are also common in archaea. Their role in genome regulation, however, and the capacity of individual paralogs to assemble into histone–DNA complexes with distinct properties remain poorly understood. Here, we combine structural modeling with phylogenetic analysis to shed light on archaeal histone paralogs, their evolutionary history, and capacity to generate combinatorial chromatin states through hetero-oligomeric assembly. Focusing on the human commensal Methanosphaera stadtmanae as a model archaeal system, we show that the heteromeric complexes that can be assembled from its seven histone paralogs vary substantially in DNA binding affinity and tetramer stability. Using molecular dynamics simulations, we go on to identify unique paralogs in M. stadtmanae and Methanobrevibacter smithii that are characterized by unstable interfaces between dimers. We propose that these paralogs act as capstones that prevent stable tetramer formation and extension into longer oligomers characteristic of model archaeal histones. Importantly, we provide evidence from phylogeny and genome architecture that these capstones, as well as other paralogs in the Methanobacteriales, have been maintained for hundreds of millions of years following ancient duplication events. Taken together, our findings indicate that at least some archaeal histone paralogs have evolved to play distinct and conserved functional roles, reminiscent of eukaryotic histone variants. We conclude that combinatorially complex histone-based chromatin is not restricted to eukaryotes and likely predates their emergence.

International audience; The alternative oxidase (AOX) is a protein involved in supporting enzymatic reactions of the Krebs cycle in instances when the canonical (cytochrome-mediated) respiratory chain has been inhibited, while allowing for the maintenance of cell growth and necessary metabolic processes for survival. Among eukaryotes, alternative oxidases have dispersed distribution and are found in plants, fungi, and protists, including Naegleria ssp. Naegleria species are free-living unicellular amoeboflagellates and include the pathogenic species of N. fowleri, the so-called "brain-eating amoeba." Using a multidisciplinary approach, we aimed to understand the evolution, localization, and function of AOX and the role that plays in Naegleria's biology. Our analyses suggest that AOX was present in last common ancestor of the genus and structure prediction showed that all functional residues are also present in Naegleria species. Using cellular and biochemical techniques, we also functionally characterize N. gruberi's AOX in its mitochondria, and we demonstrate that its inactivation affects its proliferation. Consequently, we discuss the benefits of the presence of this protein in Naegleria species, along with its potential pathogenicity role in N. fowleri. We predict that our findings will spearhead new explorations to understand the cell biology, metabolism, and evolution of Naegleria and other free-living relatives.

Iron-sulfur (Fe-S) clusters are ancient and ubiquitous protein cofactors and play irreplaceable roles in many metabolic and regulatory processes. Fe-S clusters are built and distributed to Fe-S enzymes by dedicated protein networks. The core components of these networks are widely conserved and highly versatile. However, Fe-S proteins and enzymes are often inactive outside their native host species. We sought to systematically investigate the compatibility of Fe-S networks with non-native Fe-S enzymes. By using collections of Fe-S enzyme orthologs representative of the entire range of prokaryotic diversity, we uncovered a striking correlation between phylogenetic distance and probability of functional expression. Moreover, coexpression of a heterologous Fe-S biogenesis pathway increases the phylogenetic range of orthologs that can be supported by the foreign host. We also find that Fe-S enzymes that require specific electron carrier proteins are rarely functionally expressed unless their taxon-specific reducing partners are identified and co-expressed. We demonstrate how these principles can be applied to improve the activity of a radical S-adenosyl methionine(rSAM) enzyme from a Streptomyces antibiotic biosynthesis pathway in Escherichia coli. Our results clarify how oxygen sensitivity and incompatibilities with foreign Fe-S and electron transfer networks each impede heterologous activity. In particular, identifying compatible electron transfer proteins and heterologous Fe-S biogenesis pathways may prove essential for engineering functional Fe-S enzyme-dependent pathways.

Archaea are active members of the gut microbiome, but a thorough analysis of their diversity and abundance in a wide range of animals is lacking. Here, we examined both quantitatively and qualitatively the gut archaeome of 269 species from invertebrates to primates. Archaea are present across many animals and mostly represented by four genera and one family of methanogens, but also members of Thaumarchaeota. Five major events of adaptation to the gut in the Archaea were identified. Host phylogeny, diet, and intestinal tract physiology are key factors shaping the structure and abundance of the archaeome. The abundance of methanogens is positively correlated with diet fibre content in mammals and hydrogenotrophic methyl-reducing methanogenesis (the main methanogenesis pathway in many animals) is linked to diet and methyl compounds-producing bacteria. Our results provide unprecedented insights on the intestinal archaeome and pave the way for further studies on their role in this environment.

Archaea are common constituents of the gut microbiome of humans, ruminants, and termites but little is known about their diversity and abundance in other animals. Here, we analyse sequencing and quantification data of archaeal and bacterial 16S rRNA genes from 250 species of animals covering a large taxonomic spectrum. We detect the presence of archaea in 175 animal species belonging to invertebrates, fish, amphibians, birds, reptiles and mammals. We identify five dominant gut lineages, corresponding to Methanobrevibacter, Methanosphaera, Methanocorpusculum, Methanimicrococcus and “Ca. Methanomethylophilaceae”. Some archaeal clades, notably within Methanobrevibacter, are associated to certain hosts, suggesting specific adaptations. The non-methanogenic lineage Nitrososphaeraceae (Thaumarchaeota) is frequently present in animal samples, although at low abundance, but may have also adapted to the gut environment. Host phylogeny, diet type, fibre content, and intestinal tract physiology are major drivers of the diversity and abundance of the archaeome in mammals. The overall abundance of archaea is more influenced by these factors than that of bacteria. Methanogens reducing methyl-compounds with H2 can represent an important fraction of the overall methanogens in many animals. Together with CO2-reducing methanogens, they are influenced by diet and composition of gut bacteria. Our results provide key elements toward our understanding of the ecology of archaea in the gut, an emerging and important field of investigation.

Across the tree of life, DNA in living cells is associated with proteins that coat chromosomes, constrain their structure and influence DNA-templated processes such as transcription and replication. In bacteria and eukaryotes, HU and histones, respectively, are the principal constituents of chromatin, with few exceptions. Archaea, in contrast, have more diverse repertoires of nucleoid-associated proteins (NAPs). The evolutionary and ecological drivers behind this diversity are poorly understood. Here, we combine a systematic phylogenomic survey of known and predicted NAPs with quantitative protein abundance data to shed light on the forces governing the evolution of archaeal chromatin. Our survey highlights the Diaforarchaea as a hotbed of NAP innovation and turnover. Loss of histones and Alba in the ancestor of this clade was followed by multiple lineage-specific horizontal acquisitions of DNA-binding proteins from other prokaryotes. Intriguingly, we find that one family of Diaforarchaea, the Methanomethylophilaceae, lacks any known NAPs. Comparative analysis of quantitative proteomics data across a panel of 19 archaea revealed that investment in NAP production varies over two orders of magnitude, from 5% of total protein. Integrating genomic and ecological data, we demonstrate that growth temperature is an excellent predictor of relative NAP investment across archaea. Our results suggest that high levels of chromatinization have evolved as a mechanism to prevent uncontrolled helix opening and runaway denaturation – rather than, for example, to globally orchestrate gene expression – with implications for the origin of chromatin in both archaea and eukaryotes.

Recent data support the hypothesis that Gram-positive bacteria (monoderms) arose from Gram-negatives (diderms) through loss of the outer membrane (OM). However how this happened remains unknown. Considering that tethering of the OM is essential for cell envelope stability in diderm bacteria we hypothesize that its destabilization may have been involved in OM loss. Here, we present an in-depth analysis of the four main OM tethering systems across all Bacteria. We show that their distribution strikingly follows the bacterial phylogeny with a bimodal distribution matching the deepest phylogenetic cleavage between Terrabacteria (a clade encompassing Cyanobacteria, Deinococcus/Thermus, Firmicutes, etc.) and Gracilicutes (a clade encompassing Proteobacteria, Bacteroidetes, Spirochaetes, etc.). Diderm Terrabacteria display as the main system OmpM, a porin that attaches non-covalently to modified peptidoglycan or to secondary cell wall polymers. In contrast, the lipoprotein Pal is restricted to the Gracilicutes along with a more sporadic occurrence of OmpA. While Braun’s lipoprotein Lpp is largely considered as the textbook example of OM attachment, it is actually present only in a subclade of Gammaproteobacteria. We propose an evolutionary scenario whereby the last common bacterial ancestor used a system based on OmpM, which was later replaced by one based on the lipoprotein Pal concomitantly to the emergence of the Lol machinery to address lipoproteins to the OM, with OmpA as a possible transition state. We speculate that the existence of only one main OM tethering system in the Terrabacteria would have allowed the multiple emergences of the monoderm phenotype specifically observed in this clade through OmpM perturbation. We test this hypothesis by inactivating all four ompM gene copies in the genetically tractable diderm Firmicute Veillonella parvula. The resulting mutant is severely affected in growth and displays high sensitivity to OM stress. High resolution imaging and tomogram reconstructions reveal a dramatic - yet non-lethal - phenotype, in which vast portions of the OM detach, producing large vesicles surrounding multiple monoderm-like cells sharing a common periplasm. Complementation by a single OmpM rescues the phenotype to a normal cell envelope. Together, our results highlight an ancient shift in bacterial evolution involving OM tethering systems. They suggest a possible mechanism for OM loss and a high flexibility of the cell envelope in diderm Firmicutes, making them ideal models to further refine our understanding of the mechanisms involved in bacterial OM stability, and opening the way to recapitulate the monoderm/diderm transition in the laboratory.

International audience; Single-stranded (ss) DNA-binding proteins are found in all three domains of life where they play vital roles in nearly all aspects of DNA metabolism by binding to and stabilizing exposed ssDNA and acting as platforms onto which DNA-processing activities can assemble. The ssDNA-binding factors SSB and RPA are extremely well conserved across bacteria and eukaryotes, respectively, and comprise one or more OB-fold ssDNA-binding domains. In the third domain of life, the archaea, multiple types of ssDNA-binding protein are found with a variety of domain architectures and subunit compositions, with OB-fold ssDNA-binding domains being a characteristic of most, but not all. This chapter summarizes current knowledge of the distribution, structure, and biological function of the archaeal ssDNA-binding factors, highlighting key features shared between clades and those that distinguish the proteins of different clades from one another. The likely cellular functions of the proteins are discussed and gaps in current knowledge identified.

Single-stranded (ss) DNA-binding proteins are found in all three domains of life where they play vital roles in nearly all aspects of DNA metabolism by binding to and stabilizing exposed ssDNA and acting as platforms onto which DNA-processing activities can assemble. The ssDNA-binding factors SSB and RPA are extremely well conserved across bacteria and eukaryotes, respectively, and comprise one or more OB-fold ssDNA-binding domains. In the third domain of life, the archaea, multiple types of ssDNA-binding protein are found with a variety of domain architectures and subunit compositions, with OB-fold ssDNA-binding domains being a characteristic of most, but not all. This chapter summarizes current knowledge of the distribution, structure, and biological function of the archaeal ssDNA-binding factors, highlighting key features shared between clades and those that distinguish the proteins of different clades from one another. The likely cellular functions of the proteins are discussed and gaps in current knowledge identified.

The cell cycle is a fundamental process that has been extensively studied in bacteria. However, many of its components and their interactions with machineries involved in other cellular processes are poorly understood. Furthermore, most knowledge relies on the study of a few models, but the real diversity of the cell division apparatus and its evolution are largely unknown. Here, we present a massive in-silico analysis of cell division and associated processes in around 1,000 genomes of the Firmicutes, a major bacterial phylum encompassing models (i.e. Bacillus subtilis, Streptococcus pneumoniae, and Staphylococcus aureus), as well as many important pathogens. We analyzed over 160 proteins by using an original approach combining phylogenetic reconciliation, phylogenetic profiles, and gene cluster survey. Our results reveal the presence of substantial differences among clades and pinpoints a number of evolutionary hotspots. In particular, the emergence of Bacilli coincides with an expansion of the gene repertoires involved in cell wall synthesis and remodeling. We also highlight major genomic rearrangements at the emergence of Streptococcaceae. We establish a functional network in Firmicutes that allows identifying new functional links inside one same process such as between FtsW (peptidoglycan polymerase) and a previously undescribed Penicilin-Binding Protein or between different processes, such as replication and cell wall synthesis. Finally, we identify new candidates involved in sporulation and cell wall synthesis. Our results provide a previously undescribed view on the diversity of the bacterial cell cycle, testable hypotheses for further experimental studies, and a methodological framework for the analysis of any other biological system.

Many of the most promising applications of synthetic biology, including engineering of microbes for renewable chemical production, relies upon the ability of genetically-tractable hosts to express heterologous enzymes from foreign species. While countless methods for facilitating heterologous enzyme expression have been developed, comparable tools for facilitating heterologous enzyme activity are generally lacking. Such tools are needed to fully exploit the biosynthetic potential of the natural world. Here, using the model bacterium Escherichia coli, we investigate why iron-sulphur (Fe-S) enzymes are often inactive when heterologously expressed. By applying a simple growth complementation assay with collections of Fe-S enzyme orthologs from a wide range of prokaryotic diversity, we uncover a striking correlation between phylogenetic distance and probability of functional expression. Moreover, co-expression of a heterologous Fe-S biogenesis pathway increases the phylogenetic range of orthologs that can be functionally expressed. On the other hand, we find that heterologous Fe-S enzymes that require specific electron carrier proteins within their natural host are rarely functionally expressed unless their specific reducing partners are identified and co-expressed. We demonstrate in vitro that such selectivity in part derives from a need for low-potential electron donors. Our results clarify how phylogenetic distance and electron transfer biochemistry each separately impact functional heterologous expression and provide insight into how these barriers can be overcome for successful microbial engineering involving Fe-S enzymes.

Correction to: Nature Microbiology https://doi.org/10.1038/s41564-020-0733-x , published online 8 June 2020. In the version of this Consensus Statement originally published, Pablo Yarza was mistakenly not included in the author list. Also, in Supplementary Table 1, Alexander Jaffe was missing from the list of endorsees. These errors have now been corrected and the updated Supplementary Table 1 is available online.

Phylogenomic analysis supports a diderm ancestor of the Firmicutes and points to an early origin of two-membraned cells in Bacteria and the derived nature of the Gram-positive envelope following multiple outer membrane losses. The transition between cell envelopes with one membrane (Gram-positive or monoderm) and those with two membranes (Gram-negative or diderm) is a fundamental open question in the evolution of Bacteria. Evidence of the presence of two independent diderm lineages, the Halanaerobiales and the Negativicutes, within the classically monoderm Firmicutes has blurred the monoderm/diderm divide and specifically anticipated that other members with an outer membrane (OM) might exist in this phylum. Here, by screening 1,639 genomes of uncultured Firmicutes for signatures of an OM, we highlight a third and deep branching diderm clade, the Limnochordia, strengthening the hypothesis of a diderm ancestor and the occurrence of independent transitions leading to the monoderm phenotype. Phyletic patterns of over 176,000 protein families constituting the Firmicutes pan-proteome identify those that strongly correlate with the diderm phenotype and suggest the existence of new potential players in OM biogenesis. In contrast, we find practically no largely conserved core of monoderms, a fact possibly linked to different ways of adapting to repeated OM losses. Phylogenetic analysis of a concatenation of main OM components totalling nearly 2,000 amino acid positions illustrates the common origin and vertical evolution of most diderm bacterial envelopes. Finally, mapping the presence/absence of OM markers onto the tree of Bacteria shows the overwhelming presence of diderm phyla and the non-monophyly of monoderm ones, pointing to an early origin of two-membraned cells and the derived nature of the Gram-positive envelope following multiple OM losses.

The Archaea present profound differences compared to Bacteria in fundamental molecular and cellular processes. While most Archaea divide by binary fission using an FtsZ-based system similar to Bacteria, they lack the majority of the components forming the complex bacterial divisome. Moreover, how FtsZ precisely functions and interacts with other proteins to assemble the archaeal division machinery remains largely unknown. Notably, among the multiple bacterial factors that tether FtsZ to the membrane during cell constriction, Archaea only possess SepF-like homologues, but their function has not been demonstrated. Here, we combine structural, cellular, and evolutionary approaches to demonstrate that SepF is the FtsZ anchor in the human-associated archaeon Methanobrevibacter smithii. 3D super-resolution microscopy of immunolabeled cells shows that M. smithii SepF co-localizes with FtsZ at the division plane. We also show that M. smithii SepF binds both to membranes and FtsZ, inducing filament bundling. High-resolution crystal structures of archaeal SepF alone and in complex with FtsZCTD reveal that SepF forms a dimer with a specific homodimerization interface. This drives a strikingly different binding mode from what is observed in Bacteria. Finally, analysis of the distribution and phylogeny of SepF and FtsZ indicates that these proteins date back to the Last Universal Common Ancestor (LUCA) and that Archaea may have retained features of an ancestral minimal cell division system, while Bacteria likely diverged to accommodate the emergence of the complex machinery required to coordinate cytokinesis with the rigid peptidoglycan cell wall and the appearance of additional FtsZ tethers. Our results contribute key insights into the largely understudied mechanisms of archaeal cell division, and pave the way for a better understanding of the processes underlying the divide between the two prokaryotic domains.

Biosynthesis and replication, from A to Z Four nucleobases. adenine (A), cytosine (C), guanine (G), and thymine (T), are usually thought to be invariable in DNA. In bacterial viruses, however, each of the DNA bases have variations that help them to escape degradation by bacterial restriction enzymes. In the genome of cyanophage S-2L, A is completely replaced by diaminopurine (Z), which forms three hydrogen bonds with T and thus creates non–Watson-Crick base pairing in the DNA of this virus (see the Perspective by Grome and Isaacs). Zhou et al. and Sleiman et al. determined the biochemical pathway that produces Z, which revealed more Z genomes in viruses hosted in bacteria distributed widely in the environment and phylogeny. Pezo et al. identified a DNA polymerase that incorporates Z into DNA while rejecting A. These findings enrich our understanding of biodiversity and expand the genetic palette for synthetic biology. Science , this issue p. 512 , 516 , 520 ; see also p. 460

Veillonella parvula is an anaerobic commensal and opportunistic pathogen whose ability to adhere to surfaces or other bacteria and form biofilms is critical for it to inhabit complex human microbial communities such as the gut and oral microbiota. Although the adhesive capacity of V. parvula has been previously described, very little is known about the underlying molecular mechanisms due to a lack of genetically amenable Veillonella strains. In this study, we took advantage of a naturally transformable V. parvula isolate and newly adapted genetic tools to identify surface-exposed adhesins called autotransporters as the main molecular determinants of adhesion in this bacterium. This work therefore provides new insights on an important aspect of the V. parvula lifestyle, opening new possibilities for mechanistic studies of the contribution of biofilm formation to the biology of this major commensal of the oral-digestive tract., The Negativicutes are a clade of the Firmicutes that have retained the ancestral diderm character and possess an outer membrane. One of the best studied Negativicutes, Veillonella parvula, is an anaerobic commensal and opportunistic pathogen inhabiting complex human microbial communities, including the gut and the dental plaque microbiota. Whereas the adhesion and biofilm capacities of V. parvula are expected to be crucial for its maintenance and development in these environments, studies of V. parvula adhesion have been hindered by the lack of efficient genetic tools to perform functional analyses in this bacterium. Here, we took advantage of a recently described naturally transformable V. parvula isolate, SKV38, and adapted tools developed for the closely related Clostridia spp. to perform random transposon and targeted mutagenesis to identify V. parvula genes involved in biofilm formation. We show that type V secreted autotransporters, typically found in diderm bacteria, are the main determinants of V. parvula autoaggregation and biofilm formation and compete with each other for binding either to cells or to surfaces, with strong consequences for V. parvula biofilm formation capacity. The identified trimeric autotransporters have an original structure compared to classical autotransporters identified in Proteobacteria, with an additional C-terminal domain. We also show that inactivation of the gene coding for a poorly characterized metal-dependent phosphohydrolase HD domain protein conserved in the Firmicutes and their closely related diderm phyla inhibits autotransporter-mediated biofilm formation. This study paves the way for further molecular characterization of V. parvula interactions with other bacteria and the host within complex microbiota environments. IMPORTANCE Veillonella parvula is an anaerobic commensal and opportunistic pathogen whose ability to adhere to surfaces or other bacteria and form biofilms is critical for it to inhabit complex human microbial communities such as the gut and oral microbiota. Although the adhesive capacity of V. parvula has been previously described, very little is known about the underlying molecular mechanisms due to a lack of genetically amenable Veillonella strains. In this study, we took advantage of a naturally transformable V. parvula isolate and newly adapted genetic tools to identify surface-exposed adhesins called autotransporters as the main molecular determinants of adhesion in this bacterium. This work therefore provides new insights on an important aspect of the V. parvula lifestyle, opening new possibilities for mechanistic studies of the contribution of biofilm formation to the biology of this major commensal of the oral-digestive tract.