A dynamic, ring-forming MucB / RseB-like protein influences spore shape in Bacillus subtilis.

How organisms develop into specific shapes is a central question in biology. The maintenance of bacterial shape is connected to the assembly and remodelling of the cell envelope. In endospore-forming bacteria, the pre-spore compartment (the forespore) undergoes morphological changes that result in a spore of defined shape, with a complex, multi-layered cell envelope. However, the mechanisms that govern spore shape remain poorly understood. Here, using a combination of fluorescence microscopy, quantitative image analysis, molecular genetics and transmission electron microscopy, we show that SsdC (formerly YdcC), a poorly-characterized new member of the MucB / RseB family of proteins that bind lipopolysaccharide in diderm bacteria, influences spore shape in the monoderm Bacillus subtilis. Sporulating cells lacking SsdC fail to adopt the typical oblong shape of wild-type forespores and are instead rounder. 2D and 3D-fluorescence microscopy suggest that SsdC forms a discontinuous, dynamic ring-like structure in the peripheral membrane of the mother cell, near the mother cell proximal pole of the forespore. A synthetic sporulation screen identified genetic relationships between ssdC and genes involved in the assembly of the spore coat. Phenotypic characterization of these mutants revealed that spore shape, and SsdC localization, depend on the coat basement layer proteins SpoVM and SpoIVA, the encasement protein SpoVID and the inner coat protein SafA. Importantly, we found that the ΔssdC mutant produces spores with an abnormal-looking cortex, and abolishing cortex synthesis in the mutant largely suppresses its shape defects. Thus, SsdC appears to play a role in the proper assembly of the spore cortex, through connections to the spore coat. Collectively, our data suggest functional diversification of the MucB / RseB protein domain between diderm and monoderm bacteria and identify SsdC as an important factor in spore shape development.

Localization and functional characterization of the alternative oxidase in Naegleria.

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.

One or two membranes? Diderm Firmicutes challenge the Gram-positive/Gram-negative divide.

How, when and why the transition between cell envelopes with one membrane (Gram-positives or monoderms) and two (Gram-negative or diderms) occurred in Bacteria is a key unanswered question in evolutionary biology. Different hypotheses have been put forward, suggesting that either the monoderm or the diderm phenotype is ancestral. The existence of diderm members in the classically monoderm Firmicutes challenges the Gram-positive/Gram-negative divide and provides a great opportunity to tackle the issue. In this review, we present current knowledge on the diversity of bacterial cell envelopes, including these atypical Firmicutes. We discuss how phylogenomic analysis supports the hypothesis that the diderm cell envelope architecture is an ancestral character in the Firmicutes, and that the monoderm phenotype in this phylum arose multiple times independently by loss of the outer membrane. Given the overwhelming distribution of diderm phenotypes with respect to monoderm ones, this scenario likely extends to the ancestor of all bacteria. Finally, we discuss the recent development of genetic tools for Veillonella parvula, a diderm Firmicute member of the human microbiome, which indicates it as an emerging new experimental model to investigate fundamental aspects of the diderm/monoderm transition.

Autotransporters Drive Biofilm Formation and Autoaggregation in the Diderm Firmicute Veillonella parvula.

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.IMPORTANCEVeillonella 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 phytopathogenic nature of Dickeya aquatica 174/2 and the dynamic early evolution of Dickeya pathogenicity.

Dickeya is a genus of phytopathogenic enterobacterales causing soft rot in a variety of plants (e.g. potato, chicory, maize). Among the species affiliated to this genus, Dickeya aquatica, described in 2014, remained particularly mysterious because it had no known host. Furthermore, while D. aquatica was proposed to represent a deep-branching species among Dickeya genus, its precise phylogenetic position remained elusive. Here, we report the complete genome sequence of the D. aquatica type strain 174/2. We demonstrate the affinity of D. aquatica strain 174/2 for acidic fruits such as tomato and cucumber and show that exposure of this bacterium to acidic pH induces twitching motility. An in-depth phylogenomic analysis of all available Dickeya proteomes pinpoints D. aquatica as the second deepest branching lineage within this genus and reclassifies two lineages that likely correspond to new genomospecies (gs.): Dickeya gs. poaceaephila (Dickeya sp NCPPB 569) and Dickeya gs. undicola (Dickeya sp 2B12), together with a new putative genus, tentatively named Prodigiosinella. Finally, from comparative analyses of Dickeya proteomes, we infer the complex evolutionary history of this genus, paving the way to study the adaptive patterns and processes of Dickeya to different environmental niches and hosts. In particular, we hypothesize that the lack of xylanases and xylose degradation pathways in D. aquatica could reflect adaptation to aquatic charophyte hosts which, in contrast to land plants, do not contain xyloglucans.

Extreme halophilic archaea derive from two distinct methanogen Class II lineages.

Phylogenetic analyses of conserved core genes have disentangled most of the ancient relationships in Archaea. However, some groups remain debated, like the DPANN, a deep-branching super-phylum composed of nanosized archaea with reduced genomes. Among these, the Nanohaloarchaea require high-salt concentrations for growth. Their discovery in 2012 was significant because they represent, together with Halobacteria (a Class belonging to Euryarchaeota), the only two described lineages of extreme halophilic archaea. The phylogenetic position of Nanohaloarchaea is highly debated, being alternatively proposed as the sister-lineage of Halobacteria or a member of the DPANN super-phylum. Pinpointing the phylogenetic position of extreme halophilic archaea is important to improve our knowledge of the deep evolutionary history of Archaea and the molecular adaptive processes and evolutionary paths that allowed their emergence. Using comparative genomic approaches, we identified 258 markers carrying a reliable phylogenetic signal. By combining strategies limiting the impact of biases on phylogenetic inference, we showed that Nanohaloarchaea and Halobacteria represent two independent lines that derived from two distinct but related methanogen Class II lineages. This implies that adaptation to high salinity emerged twice independently in Archaea and indicates that emergence of Nanohaloarchaea within DPANN in previous studies is likely the consequence of a tree reconstruction artifact, challenging the existence of this super-phylum.

Structure of the rare archaeal biosphere and seasonal dynamics of active ecotypes in surface coastal waters.

Marine Archaea are important players among microbial plankton and significantly contribute to biogeochemical cycles, but details regarding their community structure and long-term seasonal activity and dynamics remain largely unexplored. In this study, we monitored the interannual archaeal community composition of abundant and rare biospheres in northwestern Mediterranean Sea surface waters by pyrosequencing 16S rDNA and rRNA. A detailed analysis of the rare biosphere structure showed that the rare archaeal community was composed of three distinct fractions. One contained the rare Archaea that became abundant at different times within the same ecosystem; these cells were typically not dormant, and we hypothesize that they represent a local seed bank that is specific and essential for ecosystem functioning through cycling seasonal environmental conditions. The second fraction contained cells that were uncommon in public databases and not active, consisting of aliens to the studied ecosystem and representing a nonlocal seed bank of potential colonizers. The third fraction contained Archaea that were always rare but actively growing; their affiliation and seasonal dynamics were similar to the abundant microbes and could not be considered a seed bank. We also showed that the major archaeal groups, Thaumarchaeota marine group I and Euryarchaeota group II.B in winter and Euryarchaeota group II.A in summer, contained different ecotypes with varying activities. Our findings suggest that archaeal diversity could be associated with distinct metabolisms or life strategies, and that the rare archaeal biosphere is composed of a complex assortment of organisms with distinct histories that affect their potential for growth.

Temporal Dynamics of Active Prokaryotic Nitrifiers and Archaeal Communities from River to Sea.

To test if different niches for potential nitrifiers exist in estuarine systems, we assessed by pyrosequencing the diversity of archaeal gene transcript markers for taxonomy (16S ribosomal RNA (rRNA)) during an entire year along a salinity gradient in surface waters of the Charente estuary (Atlantic coast, France). We further investigated the potential for estuarine prokaryotes to oxidize ammonia and hydrolyze urea by quantifying thaumarchaeal amoA and ureC and bacterial amoA transcripts. Our results showed a succession of different nitrifiers from river to sea with bacterial amoA transcripts dominating in the freshwater station while archaeal transcripts were predominant in the marine station. The 16S rRNA sequence analysis revealed that Thaumarchaeota marine group I (MGI) were the most abundant overall but other archaeal groups like Methanosaeta were also potentially active in winter (December-March) and Euryarchaeota marine group II (MGII) were dominant in seawater in summer (April-August). Each station also contained different Thaumarchaeota MGI phylogenetic clusters, and the clusters' microdiversity was associated to specific environmental conditions suggesting the presence of ecotypes adapted to distinct ecological niches. The amoA and ureC transcript dynamics further indicated that some of the Thaumarchaeota MGI subclusters were involved in ammonia oxidation through the hydrolysis of urea. Our findings show that ammonia-oxidizing Archaea and Bacteria were adapted to contrasted conditions and that the Thaumarchaeota MGI diversity probably corresponds to distinct metabolisms or life strategies.

Proteins containing photosynthetic reaction centre domains modulate FtsZ-based archaeal cell division.

Cell division in all domains of life requires the orchestration of many proteins, but in Archaea most of the machinery remains poorly characterized. Here we investigate the FtsZ-based cell division mechanism in Haloferax volcanii and find proteins containing photosynthetic reaction centre (PRC) barrel domains that play an essential role in archaeal cell division. We rename these proteins cell division protein B 1 (CdpB1) and CdpB2. Depletions and deletions in their respective genes cause severe cell division defects, generating drastically enlarged cells. Fluorescence microscopy of tagged FtsZ1, FtsZ2 and SepF in CdpB1 and CdpB2 mutant strains revealed an unusually disordered divisome that is not organized into a distinct ring-like structure. Biochemical analysis shows that SepF forms a tripartite complex with CdpB1/2 and crystal structures suggest that these two proteins might form filaments, possibly aligning SepF and the FtsZ2 ring during cell division. Overall our results indicate that PRC-domain proteins play essential roles in FtsZ-based cell division in Archaea.

Borg extrachromosomal elements of methane-oxidizing archaea have conserved and expressed genetic repertoires.

Borgs are huge extrachromosomal elements (ECE) of anaerobic methane-consuming "Candidatus Methanoperedens" archaea. Here, we used nanopore sequencing to validate published complete genomes curated from short reads and to reconstruct new genomes. 13 complete and four near-complete linear genomes share 40 genes that define a largely syntenous genome backbone. We use these conserved genes to identify new Borgs from peatland soil and to delineate Borg phylogeny, revealing two major clades. Remarkably, Borg genes encoding nanowire-like electron-transferring cytochromes and cell surface proteins are more highly expressed than those of host Methanoperedens, indicating that Borgs augment the Methanoperedens activity in situ. We reconstructed the first complete 4.00 Mbp genome for a Methanoperedens that is inferred to be a Borg host and predicted its methylation motifs, which differ from pervasive TC and CC methylation motifs of the Borgs. Thus, methylation may enable Methanoperedens to distinguish their genomes from those of Borgs. Very high Borg to Methanoperedens ratios and structural predictions suggest that Borgs may be capable of encapsulation. The findings clearly define Borgs as a distinct class of ECE with shared genomic signatures, establish their diversification from a common ancestor with genetic inheritance, and raise the possibility of periodic existence outside of host cells.

Short-term dynamics of diversity patterns: evidence of continual reassembly within lacustrine small eukaryotes.

The short-term variation in the community structure of freshwater small eukaryotes (0.2-5 µm) was investigated in a mesotrophic lake every 2-3 days over one summer by coupling three molecular methods: 454 amplicon pyrosequencing, qPCR and TSA-FISH. The pyrosequencing approach unveiled a much more extensive small-eukaryotic diversity (991 OTUs) than has been described previously. The vast majority of the diversity described was represented by rare OTUs (≤ 0.01% of reads) belonging primarily to Cryptomycota, Dikarya and photosynthetic organisms, which were never detected as abundant in any of the samples. The small eukaryote community was characterized by a continual and important reassembly. These rearrangements involved the 20 'core taxa' (≥ 1% of reads), and, were essentially due to a handful of OTUs that were detected in intermediate abundance (0.01-1% of reads) and sporadically in dominant taxa. Putative bacterivorous (Ciliophora and Cercozoa) as well as parasitic and saprotrophic taxa (Perkinsozoa and Cryptomycota) were involved in these changes of diversity. A putative infection of microalgae by a lacustrine perkinsozoan was also reported for the first time in this study. Open questions regarding both the patterns that govern the rapid small eukaryote reassemblies and the possible biogeography of these organisms arise from this study.

Analyses of cell wall synthesis in Clostridioides difficile reveal a diversification in cell division mechanisms in endospore-forming bacteria.

Current models of bacterial cell division assume that the core synthases of the multiprotein divisome complex, FtsW-FtsI, are the primary drivers of septal peptidoglycan (PG) synthesis. These enzymes are typically encoded in the highly conserved division and cell wall (dcw) cluster and are considered to be universally essential for cell division. Here, we combine bioinformatics analyses with functional characterization in the pathogen Clostridioides difficile to show that dcw-encoded PG synthases have undergone a surprising specialization in the sole endospore-forming phylum, Firmicutes, to fulfill sporulation-specific roles. We describe a novel role for these enzymes in synthesizing septal PG during the sporulation-specific mode of cell division in C. difficile. Although these enzymes are directly regulated by canonical divisome components during this process, dcw-encoded PG synthases and their divisome regulators are unexpectedly dispensable for cell division during normal growth. Instead, C. difficile uses its sole bifunctional class A penicillin-binding protein (aPBP) to drive cell division, revealing a previously unreported role for this class of PG synthases as the core divisome enzyme. Collectively, our findings reveal how the emergence of endosporulation in the Firmicutes phylum was a key driver for the functional repurposing of an otherwise universally conserved cellular process such as cell division. Moreover, they indicate that C. difficile, and likely other clostridia, assemble a divisome that differs markedly from previously studied bacteria, thus representing an attractive, unique target for therapeutic purposes.

Diversification of division mechanisms in endospore-forming bacteria revealed by analyses of peptidoglycan synthesis in Clostridioides difficile.

The bacterial enzymes FtsW and FtsI, encoded in the highly conserved dcw gene cluster, are considered to be universally essential for the synthesis of septal peptidoglycan (PG) during cell division. Here, we show that the pathogen Clostridioides difficile lacks a canonical FtsW/FtsI pair, and its dcw-encoded PG synthases have undergone a specialization to fulfill sporulation-specific roles, including synthesizing septal PG during the sporulation-specific mode of cell division. Although these enzymes are directly regulated by canonical divisome components during this process, dcw-encoded PG synthases and their divisome regulators are dispensable for cell division during normal growth. Instead, C. difficile uses a bifunctional class A penicillin-binding protein as the core divisome PG synthase, revealing a previously unreported role for this class of enzymes. Our findings support that the emergence of endosporulation in the Firmicutes phylum facilitated the functional repurposing of cell division factors. Moreover, they indicate that C. difficile, and likely other clostridia, assemble a distinct divisome that therefore may represent a unique target for therapeutic interventions.

The Mla system of diderm Firmicute Veillonella parvula reveals an ancestral transenvelope bridge for phospholipid trafficking.

E. coli and most other diderm bacteria (those with two membranes) have an inner membrane enriched in glycerophospholipids (GPLs) and an asymmetric outer membrane (OM) containing GPLs in its inner leaflet and primarily lipopolysaccharides in its outer leaflet. In E. coli, this lipid asymmetry is maintained by the Mla system which consists of six proteins: the OM lipoprotein MlaA extracts GPLs from the outer leaflet, and the periplasmic chaperone MlaC transfers them across the periplasm to the inner membrane complex MlaBDEF. However, GPL trafficking still remains poorly understood, and has only been studied in a handful of model species. Here, we investigate GPL trafficking in Veillonella parvula, a diderm Firmicute with an Mla system that lacks MlaA and MlaC, but contains an elongated MlaD. V. parvula mla mutants display phenotypes characteristic of disrupted lipid asymmetry which can be suppressed by mutations in tamB, supporting that these two systems have opposite GPL trafficking functions across diverse bacterial lineages. Structural modelling and subcellular localisation assays suggest that V. parvula MlaD forms a transenvelope bridge, comprising a typical inner membrane-localised MCE domain and, in addition, an outer membrane ß-barrel. Phylogenomic analyses indicate that this elongated MlaD type is widely distributed across diderm bacteria and likely forms part of the ancestral functional core of the Mla system, which would be composed of MlaEFD only.

Ancient origin and constrained evolution of the division and cell wall gene cluster in Bacteria.

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.

An ancient divide in outer membrane tethering systems in bacteria suggests a mechanism for the diderm-to-monoderm transition.

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.

Genetic Screens Identify Additional Genes Implicated in Envelope Remodeling during the Engulfment Stage of Bacillus subtilis Sporulation.

During bacterial endospore formation, the developing spore is internalized into the mother cell through a phagocytic-like process called engulfment, which involves synthesis and hydrolysis of peptidoglycan. Engulfment peptidoglycan hydrolysis requires the widely conserved and well-characterized DMP complex, composed of SpoIID, SpoIIM, and SpoIIP. In contrast, although peptidoglycan synthesis has been implicated in engulfment, the protein players involved are less well defined. The widely conserved SpoIIIAH-SpoIIQ interaction is also required for engulfment efficiency, functioning like a ratchet to promote membrane migration around the forespore. Here, we screened for additional factors required for engulfment using transposon sequencing in Bacillus subtilis mutants with mild engulfment defects. We discovered that YrvJ, a peptidoglycan hydrolase, and the MurA paralog MurAB, involved in peptidoglycan precursor synthesis, are required for efficient engulfment. Cytological analyses suggest that both factors are important for engulfment when the DMP complex is compromised and that MurAB is additionally required when the SpoIIIAH-SpoIIQ ratchet is abolished. Interestingly, despite the importance of MurAB for sporulation in B. subtilis, phylogenetic analyses of MurA paralogs indicate that there is no correlation between sporulation and the number of MurA paralogs and further reveal the existence of a third MurA paralog, MurAC, within the Firmicutes. Collectively, our studies identify two new factors that are required for efficient envelop remodeling during sporulation and highlight the importance of peptidoglycan precursor synthesis for efficient engulfment in B. subtilis and likely other endospore-forming bacteria. IMPORTANCE In bacteria, cell envelope remodeling is critical for cell growth and division. This is also the case during the development of bacteria into highly resistant endospores (spores), known as sporulation. During sporulation, the developing spore becomes internalized inside the mother cell through a phagocytic-like process called engulfment, which is essential to form the cell envelope of the spore. Engulfment involves both the synthesis and hydrolysis of peptidoglycan and the stabilization of migrating membranes around the developing spore. Importantly, although peptidoglycan synthesis has been implicated during engulfment, the specific genes that contribute to this molecular element of engulfment have remained unclear. Our study identifies two new factors that are required for efficient envelope remodeling during engulfment and emphasizes the importance of peptidoglycan precursor synthesis for efficient engulfment in the model organism Bacillus subtilis and likely other endospore-forming bacteria. Finally, our work highlights the power of synthetic screens to reveal additional genes that contribute to essential processes during sporulation.

Single-Stranded DNA-Binding Proteins in the Archaea.

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.

Comparative genomic analysis of Methanimicrococcus blatticola provides insights into host adaptation in archaea and the evolution of methanogenesis.

Other than the Methanobacteriales and Methanomassiliicoccales, the characteristics of archaea that inhabit the animal microbiome are largely unknown. Methanimicrococcus blatticola, a member of the Methanosarcinales, currently reunites two unique features within this order: it is a colonizer of the animal digestive tract and can only reduce methyl compounds with H2 for methanogenesis, a increasingly recognized metabolism in the archaea and whose origin remains debated. To understand the origin of these characteristics, we have carried out a large-scale comparative genomic analysis. We infer the loss of more than a thousand genes in M. blatticola, by far the largest genome reduction across all Methanosarcinales. These include numerous elements for sensing the environment and adapting to more stable gut conditions, as well as a significant remodeling of the cell surface components likely involved in host and gut microbiota interactions. Several of these modifications parallel those previously observed in phylogenetically distant archaea and bacteria from the animal microbiome, suggesting large-scale convergent mechanisms of adaptation to the gut. Strikingly, M. blatticola has lost almost all genes coding for the H4MPT methyl branch of the Wood-Ljungdahl pathway (to the exception of mer), a phenomenon never reported before in any member of Class I or Class II methanogens. The loss of this pathway illustrates one of the evolutionary processes that may have led to the emergence of methyl-reducing hydrogenotrophic methanogens, possibly linked to the colonization of organic-rich environments (including the animal gut) where both methyl compounds and hydrogen are abundant.

A third purine biosynthetic pathway encoded by aminoadenine-based viral DNA genomes.

Cells have two purine pathways that synthesize adenine and guanine ribonucleotides from phosphoribose via inosylate. A chemical hybrid between adenine and guanine, 2-aminoadenine (Z), replaces adenine in the DNA of the cyanobacterial virus S-2L. We show that S-2L and Vibrio phage PhiVC8 encode a third purine pathway catalyzed by PurZ, a distant paralog of succinoadenylate synthase (PurA), the enzyme condensing aspartate and inosylate in the adenine pathway. PurZ condenses aspartate with deoxyguanylate into dSMP (N6-succino-2-amino-2'-deoxyadenylate), which undergoes defumarylation and phosphorylation to give dZTP (2-amino-2'-deoxyadenosine-5'-triphosphate), a substrate for the phage DNA polymerase. Crystallography and phylogenetics analyses indicate a close relationship between phage PurZ and archaeal PurA enzymes. Our work elucidates the biocatalytic innovation that remodeled a DNA building block beyond canonical molecular biology.