High-throughput genetics enables identification of nutrient utilization and accessory energy metabolism genes in a model methanogen.

Archaea are widespread in the environment and play fundamental roles in diverse ecosystems; however, characterization of their unique biology requires advanced tools. This is particularly challenging when characterizing gene function. Here, we generate randomly barcoded transposon libraries in the model methanogenic archaeon Methanococcus maripaludis and use high-throughput growth methods to conduct fitness assays (RB-TnSeq) across over 100 unique growth conditions. Using our approach, we identified new genes involved in nutrient utilization and response to oxidative stress. We identified novel genes for the usage of diverse nitrogen sources in M. maripaludis including a putative regulator of alanine deamination and molybdate transporters important for nitrogen fixation. Furthermore, leveraging the fitness data, we inferred that M. maripaludis can utilize additional nitrogen sources including ʟ-glutamine, ᴅ-glucuronamide, and adenosine. Under autotrophic growth conditions, we identified a gene encoding a domain of unknown function (DUF166) that is important for fitness and hypothesize that it has an accessory role in carbon dioxide assimilation. Finally, comparing fitness costs of oxygen versus sulfite stress, we identified a previously uncharacterized class of dissimilatory sulfite reductase-like proteins (Dsr-LP; group IIId) that is important during growth in the presence of sulfite. When overexpressed, Dsr-LP conferred sulfite resistance and enabled use of sulfite as the sole sulfur source. The high-throughput approach employed here allowed for generation of a large-scale data set that can be used as a resource to further understand gene function and metabolism in the archaeal domain.IMPORTANCEArchaea are widespread in the environment, yet basic aspects of their biology remain underexplored. To address this, we apply randomly barcoded transposon libraries (RB-TnSeq) to the model archaeon Methanococcus maripaludis. RB-TnSeq coupled with high-throughput growth assays across over 100 unique conditions identified roles for previously uncharacterized genes, including several encoding proteins with domains of unknown function (DUFs). We also expand on our understanding of carbon and nitrogen metabolism and characterize a group IIId dissimilatory sulfite reductase-like protein as a functional sulfite reductase. This data set encompasses a wide range of additional conditions including stress, nitrogen fixation, amino acid supplementation, and autotrophy, thus providing an extensive data set for the archaeal community to mine for characterizing additional genes of unknown function.

Origin of biogeographically distinct ecotypes during laboratory evolution.

Resource partitioning is central to the incredible productivity of microbial communities, including gigatons in annual methane emissions through syntrophic interactions. Previous work revealed how a sulfate reducer (Desulfovibrio vulgaris, Dv) and a methanogen (Methanococcus maripaludis, Mm) underwent evolutionary diversification in a planktonic context, improving stability, cooperativity, and productivity within 300-1000 generations. Here, we show that mutations in just 15 Dv and 7 Mm genes within a minimal assemblage of this evolved community gave rise to co-existing ecotypes that were spatially enriched within a few days of culturing in a fluidized bed reactor. The spatially segregated communities partitioned resources in the simulated subsurface environment, with greater lactate utilization by attached Dv but partial utilization of resulting H2 by low affinity hydrogenases of Mm in the same phase. The unutilized H2 was scavenged by high affinity hydrogenases of planktonic Mm, producing copious amounts of methane. Our findings show how a few mutations can drive resource partitioning amongst niche-differentiated ecotypes, whose interplay synergistically improves productivity of the entire mutualistic community.

Proteomic and transcriptomic analysis of selenium utilization in Methanococcus maripaludis.

Methanococcus maripaludis utilizes selenocysteine- (Sec-) containing proteins (selenoproteins), mostly active in the organism's primary energy metabolism, methanogenesis. During selenium depletion, M. maripaludis employs a set of enzymes containing cysteine (Cys) instead of Sec. The genes coding for these Sec-/Cys-containing isoforms were the only genes known of which expression is influenced by the selenium status of the cell. Using proteomics and transcriptomics, approx. 7% and 12%, respectively, of all genes/proteins were found differentially expressed/synthesized in response to the selenium supply. Some of the genes identified involve methanogenesis, nitrogenase functions, and putative transporters. An increase of transcript abundance for putative transporters under selenium depletion indicated the organism's effort to tap into alternative sources of selenium. M. maripaludis is known to utilize selenite and dimethylselenide as selenium sources. To expand this list, a selenium-responsive reporter strain was assessed with nine other, environmentally relevant selenium species. While the effect of some was very similar to that of selenite, others were effectively utilized at lower concentrations. Conversely, selenate and seleno-amino acids were only utilized at unphysiologically high concentrations and two compounds were not utilized at all. To address the role of the selenium-regulated putative transporters, M. maripaludis mutant strains lacking one or two of the putative transporters were tested for the capability to utilize the different selenium species. Of the five putative transporters analyzed by loss-of-function mutagenesis, none appeared to be absolutely required for utilizing any of the selenium species tested, indicating they have redundant and/or overlapping specificities or are not dedicated selenium transporters. IMPORTANCE: While selenium metabolism in microorganisms has been studied intensively in the past, global gene expression approaches have not been employed so far. Furthermore, the use of different selenium sources, widely environmentally interconvertible via biotic and abiotic processes, was also not extensively studied before. Methanococcus maripaludis JJ is ideally suited for such analyses, thanks to its known selenium usage and available genetic tools. Thus, an overall view on the selenium regulon of M. maripaludis was obtained via transcriptomic and proteomic analyses, which inspired further experimentation. This led to demonstrating the use of selenium sources M. maripaludis was previously not known to employ. Also, an attempt-although so far unsuccessful-was made to pinpoint potential selenium transporter genes, in order to deepen our understanding of trace element utilization in this important model organism.

Microbial electrosynthesis of single cell protein and methane by coupling fast-growing Methanococcus maripaludis with microbial electrolysis cells.

Single cell protein (SCP) is a promising alternative protein source, as its production bypasses the disadvantages of animal protein production in industrial agriculture. Coupling a fast-growing hydrogen consuming organism with microbial electrolysis cells (MECs) could be a viable method for SCP production. In this study, a fast-growing and protein-rich methanogen, Methanococcus maripaludis was selected as the primary SCP source. The inoculation of M. maripaludis in MECs triggered cell synthesis with methane production. The doubling time measured was 11.2 h and the specific growth rate was 0.062 1/h. The highest SCP production rate was 13.7 mg/L/h. In the dried biomass, the weight of protein was over 60 %. Amino acid profiling of the harvested biomass demonstrated high abundance of essential amino acids. The electron flux analysis indicated that 31.3 % electrons in the electrochemical systems were directed into SCP synthesis. These results illustrated the potential for SCP production by coupling a fast-growing methanogen with MECs.

Functionally redundant formate dehydrogenases enable formate-dependent growth in Methanococcus maripaludis.

Methanogens are essential for the complete remineralization of organic matter in anoxic environments. Most cultured methanogens are hydrogenotrophic, using H2 as an electron donor to reduce CO2 to CH4, but in the absence of H2 many can also use formate. Formate dehydrogenase (Fdh) is essential for formate oxidation, where it transfers electrons for the reduction of coenzyme F420 or to a flavin-based electron bifurcating reaction catalyzed by heterodisulfide reductase (Hdr), the terminal reaction of methanogenesis. Furthermore, methanogens that use formate encode at least two isoforms of Fdh in their genomes, but how these different isoforms participate in methanogenesis is unknown. Using Methanococcus maripaludis, we undertook a biochemical characterization of both Fdh isoforms involved in methanogenesis. Both Fdh1 and Fdh2 interacted with Hdr to catalyze the flavin-based electron bifurcating reaction, and both reduced F420 at similar rates. F420 reduction preceded flavin-based electron bifurcation activity for both enzymes. In a Δfdh1 mutant background, a suppressor mutation was required for Fdh2 activity. Genome sequencing revealed that this mutation resulted in the loss of a specific molybdopterin transferase (moeA), allowing for Fdh2-dependent growth, and the metal content of the proteins suggested that isoforms are dependent on either molybdenum or tungsten for activity. These data suggest that both isoforms of Fdh are functionally redundant, but their activities in vivo may be limited by gene regulation or metal availability under different growth conditions. Together these results expand our understanding of formate oxidation and the role of Fdh in methanogenesis.

A robust genetic toolbox for fine-tuning gene expression in the CO2-Fixing methanogenic archaeon Methanococcus maripaludis.

Libraries of well-characterized genetic elements for fine-tuning gene expression are essential for biological and biotechnological research and applications. The fast-growing and genetically tractable methanogen, Methanococcus maripaludis, is a promising host organism for biotechnological conversion of carbon dioxide and renewable hydrogen into fuels and value-added products, as well as fundamental biological studies of archaea. However, the lack of molecular tools for gene expression has hindered its application as a workhorse to fine-tune gene and metabolic pathway expressions. In this study, we developed a genetic toolbox, including libraries of promoters, ribosome binding sites (RBS), and neutral sites for chromosomal integration, to facilitate precise gene expression in M. maripaludis. We generated a promoter library consisting of 81 constitutive promoters with expression strengths spanning a ∼104-fold dynamic range. Importantly, we identified a base composition rule for strong archaeal promoters and successfully remodeled weak promoters, enhancing their activities by up to 120-fold. We also established an RBS library containing 42 diverse RBS sequences with translation strengths covering a ∼100-fold dynamic range. Additionally, we identified eight neutral sites and developed a one-step, Cas9-based marker-less knock-in approach for chromosomal integration. We successfully applied the characterized promoter and RBS elements to significantly improve recombinant protein expression by 41-fold and modulate essential gene expression to generate corresponding physiological changes in M. maripaludis. Therefore, this work establishes a solid foundation for utilizing this autotrophic methanogen as an ideal workhorse for archaeal biology and biotechnological studies and applications.

Random transposon mutagenesis identifies genes essential for transformation in Methanococcus maripaludis.

Natural transformation, the process whereby a cell acquires DNA directly from the environment, is an important driver of evolution in microbial populations, yet the mechanism of DNA uptake is only characterized in bacteria. To expand our understanding of natural transformation in archaea, we undertook a genetic approach to identify a catalog of genes necessary for transformation in Methanococcus maripaludis. Using an optimized method to generate random transposon mutants, we screened 6144 mutant strains for defects in natural transformation and identified 25 transformation-associated candidate genes. Among these are genes encoding components of the type IV-like pilus, transcription/translation associated genes, genes encoding putative membrane bound transport proteins, and genes of unknown function. Interestingly, similar genes were identified regardless of whether replicating or integrating plasmids were provided as a substrate for transformation. Using allelic replacement mutagenesis, we confirmed that several genes identified in these screens are essential for transformation. Finally, we identified a homolog of a membrane bound substrate transporter in Methanoculleus thermophilus and verified its importance for transformation using allelic replacement mutagenesis, suggesting a conserved mechanism for DNA transfer in multiple archaea. These data represent an initial characterization of the genes important for transformation which will inform efforts to understand gene flow in natural populations. Additionally, knowledge of the genes necessary for natural transformation may assist in identifying signatures of transformation machinery in archaeal genomes and aid the establishment of new model genetic systems for studying archaea.

Proteomic Analysis of Methanococcus voltae Grown in the Presence of Mineral and Nonmineral Sources of Iron and Sulfur.

Iron sulfur (Fe-S) proteins are essential and ubiquitous across all domains of life, yet the mechanisms underpinning assimilation of iron (Fe) and sulfur (S) and biogenesis of Fe-S clusters are poorly understood. This is particularly true for anaerobic methanogenic archaea, which are known to employ more Fe-S proteins than other prokaryotes. Here, we utilized a deep proteomics analysis of Methanococcus voltae A3 cultured in the presence of either synthetic pyrite (FeS2) or aqueous forms of ferrous iron and sulfide to elucidate physiological responses to growth on mineral or nonmineral sources of Fe and S. The liquid chromatography-mass spectrometry (LCMS) shotgun proteomics analysis included 77% of the predicted proteome. Through a comparative analysis of intra- and extracellular proteomes, candidate proteins associated with FeS2 reductive dissolution, Fe and S acquisition, and the subsequent transport, trafficking, and storage of Fe and S were identified. The proteomic response shows a large and balanced change, suggesting that M. voltae makes physiological adjustments involving a range of biochemical processes based on the available nutrient source. Among the proteins differentially regulated were members of core methanogenesis, oxidoreductases, membrane proteins putatively involved in transport, Fe-S binding ferredoxin and radical S-adenosylmethionine proteins, ribosomal proteins, and intracellular proteins involved in Fe-S cluster assembly and storage. This work improves our understanding of ancient biogeochemical processes and can support efforts in biomining of minerals. IMPORTANCE Clusters of iron and sulfur are key components of the active sites of enzymes that facilitate microbial conversion of light or electrical energy into chemical bonds. The proteins responsible for transporting iron and sulfur into cells and assembling these elements into metal clusters are not well understood. Using a microorganism that has an unusually high demand for iron and sulfur, we conducted a global investigation of cellular proteins and how they change based on the mineral forms of iron and sulfur. Understanding this process will answer questions about life on early earth and has application in biomining and sustainable sources of energy.

Growth rate-dependent coordination of catabolism and anabolism in the archaeon Methanococcus maripaludis under phosphate limitation.

Catabolic and anabolic processes are finely coordinated in microorganisms to provide optimized fitness under varying environmental conditions. Understanding this coordination and the resulting physiological traits reveals fundamental strategies of microbial acclimation. Here, we characterized the system-level physiology of Methanococcus maripaludis, a niche-specialized methanogenic archaeon, at different dilution rates ranging from 0.09 to 0.003 h-1 in chemostat experiments under phosphate (i.e., anabolic) limitation. Phosphate was supplied as the limiting nutrient, while formate was supplied in excess as the catabolic substrate and carbon source. We observed a decoupling of catabolism and anabolism resulting in lower biomass yield relative to catabolically limited cells at the same dilution rates. In addition, the mass abundance of several coarse-grained proteome sectors (i.e., combined abundance of proteins grouped based on their function) exhibited a linear relationship with growth rate, mostly ribosomes and their biogenesis. Accordingly, cellular RNA content also correlated with growth rate. Although the methanogenesis proteome sector was invariant, the metabolic capacity for methanogenesis, measured as methane production rates immediately after transfer to batch culture, correlated with growth rate suggesting translationally independent regulation that allows cells to only increase catabolic activity under growth-permissible conditions. These observations are in stark contrast to the physiology of M. maripaludis under formate (i.e., catabolic) limitation, where cells keep an invariant proteome including ribosomal content and a high methanogenesis capacity across a wide range of growth rates. Our findings reveal that M. maripaludis employs fundamentally different strategies to coordinate global physiology during anabolic phosphate and catabolic formate limitation.

The Fluorescence-Activating and Absorption-Shifting Tag (FAST) Enables Live-Cell Fluorescence Imaging of Methanococcus maripaludis.

Live-cell fluorescence imaging of methanogenic archaea has been limited due to the strictly anoxic conditions required for growth and issues with autofluorescence associated with electron carriers in central metabolism. Here, we show that the fluorescence-activating and absorption-shifting tag (FAST) complexed with the fluorogenic ligand 4-hydroxy-3-methylbenzylidene-rhodanine (HMBR) overcomes these issues and displays robust fluorescence in Methanococcus maripaludis. We also describe a mechanism to visualize cells under anoxic conditions using a fluorescence microscope. Derivatives of FAST were successfully applied for protein abundance analysis, subcellular localization analysis, and determination of protein-protein interactions. FAST fusions to both formate dehydrogenase (Fdh) and F420-reducing hydrogenase (Fru) displayed increased fluorescence in cells grown on formate-containing medium, consistent with previous studies suggesting the increased abundance of these proteins in the absence of H2. Additionally, FAST fusions to both Fru and the ATPase associated with the archaellum (FlaI) showed a membrane localization in single cells observed using anoxic fluorescence microscopy. Finally, a split reporter translationally fused to the alpha and beta subunits of Fdh reconstituted a functionally fluorescent molecule in vivo via bimolecular fluorescence complementation. Together, these observations demonstrate the utility of FAST as a tool for studying members of the methanogenic archaea. IMPORTANCE Methanogenic archaea are important members of anaerobic microbial communities where they catalyze essential reactions in the degradation of organic matter. Developing additional tools for studying the cell biology of these organisms is essential to understanding them at a mechanistic level. Here, we show that FAST, in combination with the fluorogenic ligand HMBR, can be used to monitor protein dynamics in live cells of M. maripaludis. The application of FAST holds promise for future studies focused on the metabolism and physiology of methanogenic archaea.

Efficient CRISPR/Cas12a-Based Genome-Editing Toolbox for Metabolic Engineering in Methanococcus maripaludis.

The rapid-growing and genetically tractable methanogen Methanococcus maripaludis is a promising host organism for the biotechnological conversion of carbon dioxide and renewable hydrogen to fuels and value-added products. Expansion of its product scope through metabolic engineering necessitates reliable and efficient genetic tools, particularly for genome edits that affect the primary metabolism and cell growth. Here, we have designed a genome-editing toolbox by utilizing Cas12a from Lachnospiraceae bacterium ND2006 (LbCas12a) in combination with the homology-directed repair machinery endogenously present in M. maripaludis. This toolbox can delete target genes with a success rate of up to 95%, despite the hyperpolyploidy of M. maripaludis. For the purpose of demonstrating a large deletion, the M. maripaludis flagellum operon (∼8.9 kbp) was replaced by the Escherichia coli ß-glucuronidase gene. To facilitate metabolic engineering and flux balancing in M. maripaludis, the relative strength of 15 different promoters was quantified in the presence of two common growth substrates, either formate or carbon dioxide and hydrogen. This CRISPR/LbCas12a toolbox can be regarded as a reliable and quick method for genome editing in a methanogen.

CryoEM reveals the stochastic nature of individual ATP binding events in a group II chaperonin.

Chaperonins are homo- or hetero-oligomeric complexes that use ATP binding and hydrolysis to facilitate protein folding. ATP hydrolysis exhibits both positive and negative cooperativity. The mechanism by which chaperonins coordinate ATP utilization in their multiple subunits remains unclear. Here we use cryoEM to study ATP binding in the homo-oligomeric archaeal chaperonin from Methanococcus maripaludis (MmCpn), consisting of two stacked rings composed of eight identical subunits each. Using a series of image classification steps, we obtained different structural snapshots of individual chaperonins undergoing the nucleotide binding process. We identified nucleotide-bound and free states of individual subunits in each chaperonin, allowing us to determine the ATP occupancy state of each MmCpn particle. We observe distinctive tertiary and quaternary structures reflecting variations in nucleotide occupancy and subunit conformations in each chaperonin complex. Detailed analysis of the nucleotide distribution in each MmCpn complex indicates that individual ATP binding events occur in a statistically random manner for MmCpn, both within and across the rings. Our findings illustrate the power of cryoEM to characterize a biochemical property of multi-subunit ligand binding cooperativity at the individual particle level.

Examining Pathways of Iron and Sulfur Acquisition, Trafficking, Deployment, and Storage in Mineral-Grown Methanogen Cells.

Methanogens have a high demand for iron (Fe) and sulfur (S); however, little is known of how they acquire, deploy, and store these elements and how this, in turn, affects their physiology. Methanogens were recently shown to reduce pyrite (FeS2), generating aqueous iron sulfide (FeSaq) clusters that are likely assimilated as a source of Fe and S. Here, we compared the phenotypes of Methanococcus voltae grown with FeS2 or ferrous iron [Fe(II)] and sulfide (HS-). FeS2-grown cells are 33% smaller yet have 193% more Fe than Fe(II)/HS--grown cells. Whole-cell electron paramagnetic resonance revealed similar distributions of paramagnetic Fe, although FeS2-grown cells showed a broad spectral feature attributed to intracellular thioferrate-like nanoparticles. Differential proteomic analyses showed similar expression of core methanogenesis enzymes, indicating that Fe and S source does not substantively alter the energy metabolism of cells. However, a homolog of the Fe(II) transporter FeoB and its putative transcriptional regulator DtxR were up-expressed in FeS2-grown cells, suggesting that cells sense Fe(II) limitation. Two homologs of IssA, a protein putatively involved in coordinating thioferrate nanoparticles, were also up-expressed in FeS2-grown cells. We interpret these data to indicate that, in FeS2-grown cells, DtxR cannot sense Fe(II) and therefore cannot downregulate FeoB. We suggest this is due to the transport of Fe(II) complexed with sulfide (FeSaq), leading to excess Fe that is sequestered by IssA as a thioferrate-like species. This model provides a framework for the design of targeted experiments aimed at further characterizing Fe acquisition and homeostasis in M. voltae and other methanogens. IMPORTANCE FeS2 is the most abundant sulfide mineral in the Earth's crust and is common in environments inhabited by methanogenic archaea. FeS2 can be reduced by methanogens, yielding aqueous FeSaq clusters that are thought to be a source of Fe and S. Here, we show that growth of Methanococcus voltae on FeS2 results in smaller cell size and higher Fe content per cell, with Fe likely stored intracellularly as thioferrate-like nanoparticles. Fe(II) transporters and storage proteins were upregulated in FeS2-grown cells. These responses are interpreted to result from cells incorrectly sensing Fe(II) limitation due to assimilation of Fe(II) as FeSaq. These findings have implications for our understanding of how Fe/S availability influences methanogen physiology and the biogeochemical cycling of these elements.

Study of Fe-S Cluster Proteins in Methanococcus maripaludis , a Model Archaeal Organism.

Iron-sulfur (Fe-S) clusters are among the oldest and most versatile cofactors present in all domains of life. Many bacterial and eukaryotic Fe-S proteins have been well-characterized, whereas the archaeal ones are less studied. Fe-S proteins are particularly abundant and play essential roles in methanogenic archaea. Methanococcus maripaludis is a model methanogen with available genetic tools. Here, we describe the techniques for anaerobic cultivation of M. maripaludis with formate, liposome-mediated transformation, expression and anoxic affinity purification of Fe-S proteins, Fe-S cluster reconstitution, and analysis of Fe-S proteins by UV-visible absorption spectroscopy.

The Oligosaccharyltransferase AglB Supports Surface-Associated Growth and Iron Oxidation in Methanococcus maripaludis.

Most microbial organisms grow as surface-attached communities known as biofilms. However, the mechanisms whereby methanogenic archaea grow attached to surfaces have remained understudied. Here, we show that the oligosaccharyltransferase AglB is essential for growth of Methanococcus maripaludis strain JJ on glass or metal surfaces. AglB glycosylates several cellular structures, such as pili, archaella, and the cell surface layer (S-layer). We show that the S-layer of strain JJ, but not strain S2, is a glycoprotein, that only strain JJ was capable of growth on surfaces, and that deletion of aglB blocked S-layer glycosylation and abolished surface-associated growth. A strain JJ mutant lacking structural components of the type IV-like pilus did not have a growth defect under any conditions tested, while a mutant lacking the preflagellin peptidase (ΔflaK) was defective for surface growth only when formate was provided as the sole electron donor. Finally, for strains that are capable of Fe0 oxidation, we show that deletion of aglB decreases the rate of anaerobic Fe0 oxidation, presumably due to decreased association of biomass with the Fe0 surface. Together, these data provide an initial characterization of surface-associated growth in a member of the methanogenic archaea. IMPORTANCE Methanogenic archaea are responsible for producing the majority of methane on Earth and catalyze the terminal reactions in the degradation of organic matter in anoxic environments. Methanogens often grow as biofilms associated with surfaces or partner organisms; however, the molecular details of surface-associated growth remain uncharacterized. We have found evidence that glycosylation of the cell surface layer is essential for growth of M. maripaludis on surfaces and can enhance rates of anaerobic iron corrosion. These results provide insight into the physiology of surface-associated methanogenic organisms and highlight the importance of surface association for anaerobic iron corrosion.

An alternative resource allocation strategy in the chemolithoautotrophic archaeon Methanococcus maripaludis.

Most microorganisms in nature spend the majority of time in a state of slow or zero growth and slow metabolism under limited energy or nutrient flux rather than growing at maximum rates. Yet, most of our knowledge has been derived from studies on fast-growing bacteria. Here, we systematically characterized the physiology of the methanogenic archaeon Methanococcus maripaludis during slow growth. M. maripaludis was grown in continuous culture under energy (formate)-limiting conditions at different dilution rates ranging from 0.09 to 0.002 h-1, the latter corresponding to 1% of its maximum growth rate under laboratory conditions (0.23 h-1). While the specific rate of methanogenesis correlated with growth rate as expected, the fraction of cellular energy used for maintenance increased and the maintenance energy per biomass decreased at slower growth. Notably, proteome allocation between catabolic and anabolic pathways was invariant with growth rate. Unexpectedly, cells maintained their maximum methanogenesis capacity over a wide range of growth rates, except for the lowest rates tested. Cell size, cellular DNA, RNA, and protein content as well as ribosome numbers also were largely invariant with growth rate. A reduced protein synthesis rate during slow growth was achieved by a reduction in ribosome activity rather than via the number of cellular ribosomes. Our data revealed a resource allocation strategy of a methanogenic archaeon during energy limitation that is fundamentally different from commonly studied versatile chemoheterotrophic bacteria such as E. coli.

Synergistic epistasis enhances the co-operativity of mutualistic interspecies interactions.

Early evolution of mutualism is characterized by big and predictable adaptive changes, including the specialization of interacting partners, such as through deleterious mutations in genes not required for metabolic cross-feeding. We sought to investigate whether these early mutations improve cooperativity by manifesting in synergistic epistasis between genomes of the mutually interacting species. Specifically, we have characterized evolutionary trajectories of syntrophic interactions of Desulfovibrio vulgaris (Dv) with Methanococcus maripaludis (Mm) by longitudinally monitoring mutations accumulated over 1000 generations of nine independently evolved communities with analysis of the genotypic structure of one community down to the single-cell level. We discovered extensive parallelism across communities despite considerable variance in their evolutionary trajectories and the perseverance within many evolution lines of a rare lineage of Dv that retained sulfate-respiration (SR+) capability, which is not required for metabolic cross-feeding. An in-depth investigation revealed that synergistic epistasis across pairings of Dv and Mm genotypes had enhanced cooperativity within SR- and SR+ assemblages, enabling their coexistence within the same community. Thus, our findings demonstrate that cooperativity of a mutualism can improve through synergistic epistasis between genomes of the interacting species, enabling the coexistence of mutualistic assemblages of generalists and their specialized variants.

Structural plasticity of a designer protein sheds light on ß-propeller protein evolution.

ß-propeller proteins are common in nature, where they are observed to adopt 4- to 10-fold internal rotational pseudo-symmetry. This size diversity can be explained by the evolutionary process of gene duplication and fusion. In this study, we investigated a distorted ß-propeller protein, an apparent intermediate between two symmetries. From this template, we created a perfectly symmetric 9-bladed ß-propeller named Cake, using computational design and ancestral sequence reconstruction. The designed repeat sequence was found to be capable of generating both 8-fold and 9-fold propellers which are highly stable. Cake variants with 2-10 identical copies of the repeat sequence were characterised by X-ray crystallography and in solution. They were found to be highly stable, and to self-assemble into 8- or 9-fold symmetrical propellers. These findings show that the ß-propeller fold allows sufficient structural plasticity to permit a given blade to assemble different forms, a transition from even to odd changes in blade number, and provide a potential explanation for the wide diversity of repeat numbers observed in natural propeller proteins. DATABASE: Structural data are available in Protein Data Bank database under the accession numbers 6TJB, 6TJC, 6TJD, 6TJE, 6TJF, 6TJG, 6TJH and 6TJI.

Fine-Tuning Protein Self-Organization by Orthogonal Chemo-Optogenetic Tools.

A universal gain-of-function approach for the spatiotemporal control of protein activity is highly desirable when reconstituting biological modules in vitro. Here we used orthogonal translation with a photocaged amino acid to map and elucidate molecular mechanisms in the self-organization of the prokaryotic filamentous cell-division protein (FtsZ) that is highly relevant for the assembly of the division ring in bacteria. We masked a tyrosine residue of FtsZ by site-specific incorporation of a photocaged tyrosine analogue. While the mutant still shows self-assembly into filaments, dynamic self-organization into ring patterns can no longer be observed. UV-mediated uncaging revealed that tyrosine 222 is essential for the regulation of the protein's GTPase activity, self-organization, and treadmilling dynamics. Thus, the light-mediated assembly of functional protein modules appears to be a promising minimal-regulation strategy for building up molecular complexity towards a minimal cell.