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Transformation, the uptake of DNA directly from the environment, is a major driver of gene flow in microbial populations. In bacteria, DNA uptake requires a nuclease that processes dsDNA to ssDNA, which is subsequently transferred into the cell and incorporated into the genome. However, the process of DNA uptake in archaea is still unknown. Previously, we cataloged genes essential to natural transformation in Methanococcus maripaludis, but few homologs of bacterial transformation‐associated genes were identified. Here, we characterize one gene, MMJJ_16440 (named here as ecnA), to be an extracellular nuclease. We show that EcnA is Ca2+‐activated, present on the cell surface, and essential for transformation. While EcnA can degrade several forms of DNA, the highest activity was observed with ssDNA as a substrate. Activity was also observed with circular dsDNA, suggesting that EcnA is an endonuclease. This is the first biochemical characterization of a transformation‐associated protein in a member of the archaeal domain and suggests that both archaeal and bacterial transformation initiate in an analogous fashion. [ABSTRACT FROM AUTHOR]

Methanogenic archaea are the only organisms that produce CH4 as part of their energy-generating metabolism. They are ubiquitous in oxidant-depleted, anoxic environments such as aquatic sediments, anaerobic digesters, inundated agricultural fields, the rumen of cattle, and the hindgut of termites, where they catalyze the terminal reactions in the degradation of organic matter. Methanogenesis is the only metabolism that is restricted to members of the domain Archaea. Here, we discuss the importance of model organisms in the history of methanogen research, including their role in the discovery of the archaea and in the biochemical and genetic characterization of methanogenesis. We also discuss outstanding questions in the field and newly emerging model systems that will expand our understanding of this uniquely archaeal metabolism. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Methanogenesis is an anaerobic metabolism responsible for the generation of >90% of the methane formed on Earth today, with important implications for fuels production and global warming. Although methanogenic Archaea have been cultured for over 70 years, key insights regarding electron flow and energy conservation in methanogenesis have only recently emerged. Fundamental differences between two metabolic types of methanogenesis, hydrogenotrophic and methylotrophic, are now understood, with implications for metabolic versatility and the potential for engineering of methanogens to utilize new substrates. The development of model species with genetic and bioinformatic tools has advanced the field and holds potential for further characterizing and engineering of methanogenesis. Our understanding of a related pathway, anaerobic methane oxidation, is in its infancy. [ABSTRACT FROM AUTHOR]

Flavin-based electron bifurcation has recently been characterized as an essential energy conservation mechanism that is utilized by hydrogenotrophic methanogenic Archaea to generate low-potential electrons in an ATP-independent manner. Electron bifurcation likely takes place at the flavin associated with the α subunit of heterodisulfide reductase (HdrA). In Methanococcus maripaludis the electrons for this reaction come from either formate or H2 via formate dehydrogenase (Fdh) or Hdr-associated hydrogenase (Vhu). However, how these enzymes bind to HdrA to deliver electrons is unknown. Here, we present evidence that the δ subunit of hydrogenase (VhuD) is central to the interaction of both enzymes with HdrA. When Ai. maripaludis is grown under conditions where both Fdh and Vhu are expressed, these enzymes compete for binding to VhuD, which in turn binds to HdrA. Under these conditions, both enzymes are fully functional and are bound to VhuD in substoichiometric quantities. We also show that Fdh copurifies specifically with VhuD in the absence of other hydrogenase subunits. Surprisingly, in the absence of Vhu, growth on hydrogen still occurs; we show that this involves F420-reducing hydrogenase. The data presented here represent an initial characterization of specific protein interactions centered on Hdr in a hydrogenotrophic methanogen that utilizes multiple electron donors for growth. [ABSTRACT FROM AUTHOR]

The H2-dependent methylene-tetrahydromethanopterin dehydrogenase ( Hmd), also known as the [ Fe]-hydrogenase, is found only in methanogens without cytochromes. In contrast to the binuclear metal centers of the [ Ni Fe]- and [ Fe Fe]-hydrogenases, the [ Fe]-hydrogenase contains only a single Fe atom, which is coordinated by a novel guanylylpyridinol cofactor in the active site. The biosynthesis of the cofactor is not well understood and the responsible genes are unknown. However, seven genes ( hmd co-occurring genes, hcg) encoding proteins of unknown function are always associated with the hmd gene. In the model methanogen Methanococcus maripaludis, we used a genetic background in which a deletion of hmd had a distinct growth phenotype, and made null-mutations in each hcg gene as well as in a gene encoding the Hmd paralog Hmd II, which is hypothesized to function as a scaffold for cofactor synthesis. Deletions in all seven hcg genes resulted in the same growth phenotype as a deletion in hmd, suggesting they are required for Hmd function. In all cases, genetic complementation of the mutation restored the wild-type phenotype. A deletion in hmdII had no effect. [ABSTRACT FROM AUTHOR]

Hydrogenotrophic methanogenic Archaea are defined by an H2 requirement for growth. Despite this requirement, many hydrogenotrophs are also capable of growth with formate as an electron donor for methanogenesis. While certain responses of these organisms to hydrogen availability have been characterized, responses to formate starvation have not been reported. Here we report that during continuous culture of Methanococcus maripaludis under defined nutrient conditions, growth yields relative to methane production decreased markedly with either H2 excess or formate excess. Analysis of the growth yields of several mutants suggests that this phenomenon occurs independently of the storage of intracellular carbon or a transcriptional response to methanogenesis. Using microarray analysis, we found that the expression of genes encoding coenzyme F420-dependent steps of methanogenesis, including one of two formate dehydrogenases, increased with H2 starvation but with formate occurred at high levels regardless of limitation or excess. One gene, encoding H2-dependent methylene-tetrahydromethanopterin dehydrogenase, decreased in expression with either H2 limitation or formate limitation. Expression of genes for the second formate dehydrogenase, molybdenum-dependent formylmethanofuran dehydrogenase, and molybdenum transport increased specifically with formate limitation. Of the two formate dehydrogenases, only the first could support growth on formate in batch culture where formate was in excess. [ABSTRACT FROM AUTHOR]

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 (DflaK) 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. [ABSTRACT FROM AUTHOR]

Hydrogenotrophic methanogens produce CH4 using H2 as an electron donor to reduce CO2. In the absence of H2, many are able to use formate or alcohols as alternate electron donors. Methanogens from the order Methanomicrobiales are capable of growth with H2, but many lack genes encoding hydrogenases that are typically found in other hydrogenotrophic methanogens. In an effort to better understand electron flow in methanogens from the Methanomicrobiales, we undertook a genetic and biochemical study of heterodisulfide reductase (Hdr) in Methanoculleus thermophilus. Hdr catalyzes an essential reaction by coupling the first and last steps of methanogenesis through flavin-based electron bifurcation. Hdr from M. thermophilus copurified with formate dehydrogenase (Fdh) and only displayed activity when formate was supplied as an electron donor. We found no evidence of an Hdr-associated hydrogenase, and H2 could not function as an electron donor, even with Hdr purified from cells grown on H2. We found that cells catalyze a formate hydrogenlyase activity that is likely essential for generating the formate needed for the Hdr reaction. Together, these results highlight the importance of formate as an electron donor for methanogenesis and suggest the ability to use formate is closely integrated into the methanogenic pathway in organisms from the order Methanomicrobiales. IMPORTANCE Methanogens from the order Methanomicrobiales are thought to prefer H2 as an electron donor for growth. They are ubiquitous in anaerobic environments, such as in wastewater treatment facilities, anaerobic digesters, and the rumen, where they catalyze the terminal steps in the breakdown of organic matter. However, despite their importance, the metabolism of these organisms remains understudied. Using a genetic and biochemical approach, we show that formate metabolism is closely integrated into methanogenesis in Methanoculleus thermophilus. This is due to a requirement for formate as the electron donor to heterodisulfide reductase (Hdr), an enzyme responsible for catalyzing essential reactions in methanogenesis by linking the initial CO2 fixing step to the exergonic terminal reaction of the pathway. These results suggest that hydrogen is not necessarily the preferred electron donor for all hydrogenotrophic methanogens and provide insight into the metabolism of methanogens from the order Methanomicrobiales. [ABSTRACT FROM AUTHOR]

Phenazines are a class of bacterially produced redox-active metabolites that are found in natural, industrial, and clinical environments. In Pseudomonas spp., phenazine-1-carboxylic acid (PCA)--the precursor of all phenazine metabolites--facilitates nutrient acquisition, biofilm formation, and competition with other organisms. While the removal of phenazines negatively impacts these activities, little is known about the genes or enzymes responsible for phenazine degradation by other organisms. Here, we report that the first step of PCA degradation by Mycobacterium fortuitum is catalyzed by a phenazine-degrading decarboxylase (PhdA). PhdA is related to members of the UbiD protein family that rely on a prenylated flavin mononucleotide cofactor for activity. The gene for PhdB, the enzyme responsible for cofactor synthesis, is present in a putative operon with the gene encoding PhdA in a region of the M. fortuitum genome that is essential for PCA degradation. PhdA and PhdB are present in all known PCA-degrading organisms from the Actinobacteria. M. fortuitum can also catabolize other Pseudomonas-derived phenazines such as phenazine-1-carboxamide, 1-hydroxyphenazine, and pyocyanin. On the basis of our previous work and the current characterization of PhdA, we propose that degradation converges on a common intermediate: dihydroxyphenazine. An understanding of the genes responsible for degradation will enable targeted studies of phenazine degraders in diverse environments. [ABSTRACT FROM AUTHOR]

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 flavinbased 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. [ABSTRACT FROM AUTHOR]

The complete remineralization of organic matter in anoxic environments relies on communities of microorganisms that ferment organic acids and alcohols to CH4. This is accomplished through syntrophic association of H2 or formate producing bacteria and methanogenic archaea, where exchange of these intermediates enables growth of both organisms. While these communities are essential to Earth's carbon cycle, our understanding of the dynamics of H2 or formate exchanged is limited. Here, we establish a model partnership between Syntrophotalea carbinolica and Methanococcus maripaludis. Through sequencing a transposon mutant library of M. maripaludis grown with ethanol oxidizing S. carbinolica, we found that genes encoding the F420-dependent formate dehydrogenase (Fdh) and F420-dependent methylene-tetrahydromethanopterin dehydrogenase (Mtd) are important for growth. Competitive growth of M. maripaludis mutants defective in either H2 or formate metabolism verified that, across multiple substrates, interspecies formate exchange was dominant in these communities. Agitation of these cultures to facilitate diffusive loss of H2 to the culture headspace resulted in an even greater competitive advantage for M. maripaludis strains capable of oxidizing formate. Finally, we verified that an M. maripaludis Dmtd mutant had a defect during syntrophic growth. Together, these results highlight the importance of formate exchange for the growth of methanogens under syntrophic conditions. [ABSTRACT FROM AUTHOR]

Naturally competent organisms are capable of DNA uptake directly from the environment through the process of transformation. Despite the importance of transformation to microbial evolution, DNA uptake remains poorly characterized outside of the bacterial domain. Here, we identify the pilus as a necessary component of the transformation machinery in archaea. We describe two naturally competent organisms, Methanococcus maripaludis and Methanoculleus thermophilus. In M. maripaludis, replicative vectors were transferred with an average efficiency of 2.4 × 10³ transformants μg-1 DNA. In M. thermophilus, integrative vectors were transferred with an average efficiency of 2.7 × 10³ transformants μg-1 DNA. Additionally, natural transformation of M. thermophilus could be used to introduce chromosomal mutations. To our knowledge, this is the first demonstration of a method to introduce targeted mutations in a member of the order Methanomicrobiales. For both organisms, mutants lacking structural components of the type IV-like pilus filament were defective for DNA uptake, demonstrating the importance of pili for natural transformation. Interestingly, competence could be induced in a noncompetent strain of M. maripaludis by expressing pilin genes from a replicative vector. These results expand the known natural competence pili to include examples from the archaeal domain and highlight the importance of pili for DNA uptake in diverse microbial organisms. [ABSTRACT FROM AUTHOR]

Methanogens catalyze the critical methane-producing step (called methanogenesis) in the anaerobic decomposition of organic matter. Here, we present the first predictive model of global gene regulation of methanogenesis in a hydrogenotrophic methanogen, Methanococcus maripaludis. We generated a comprehensive list of genes (protein-coding and noncoding) for M. maripaludis through integrated analysis of the transcriptome structure and a newly constructed Peptide Atlas. The environment and gene- regulatory influence network (EGRIN) model of the strain was constructed from a compendium of transcriptome data that was collected over 58 different steady-state and time-course experiments that were performed in chemostats or batch cultures under a spectrum of environmental perturbations that modulated methanogenesis. Analyses of the EGRIN model have revealed novel components of methanogenesis that included at least three additional protein-coding genes of previously unknown function as well as one noncoding RNA. We discovered that at least five regulatory mechanisms act in a combinatorial scheme to intercoordinate key steps of methanogenesis with different processes such as motility, ATP biosynthesis, and carbon assimilation. Through a combination of genetic and environmental perturbation experiments we have validated the EGRIN-predicted role of two novel transcription factors in the regulation of phosphate-dependent repression of formate dehydrogenase"a key enzyme in the methanogenesis pathway. The EGRIN model demonstrates regulatory affiliations within methanogenesis as well as between methanogenesis and other cellular functions. [ABSTRACT FROM AUTHOR]

Transformation, the uptake of DNA directly from the environment, is a major driver of gene flow in microbial populations. In bacteria, DNA uptake requires a nuclease that processes dsDNA to ssDNA, which is subsequently transferred into the cell and incorporated into the genome. However, the process of DNA uptake in archaea is still unknown. Previously, we cataloged genes essential to natural transformation in Methanococcus maripaludis, but few homologs of bacterial transformation-associated genes were identified. Here, we characterize one gene, MMJJ_16440 (named here as ecnA), to be an extracellular nuclease. We show that EcnA is Ca 2+ -activated, present on the cell surface, and essential for transformation. While EcnA can degrade several forms of DNA, the highest activity was observed with ssDNA as a substrate. Activity was also observed with circular dsDNA, suggesting that EcnA is an endonuclease. This is the first biochemical characterization of a transformation-associated protein in a member of the archaeal domain and suggests that both archaeal and bacterial transformation initiate in an analogous fashion., (© 2024 The Author(s). Molecular Microbiology published by John Wiley & Sons Ltd.)

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. maripaludi s 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., Competing Interests: The authors declare no conflict of interest.

Methanocaldococcus sp. strain FS406-22, a hyperthermophilic methanogen, fixes nitrogen with a minimal set of known nif genes. Only four structural nif genes, nifH , nifD , nifK , and nifE, are present in a cluster, and a nifB homolog is present elsewhere in the genome. nifN , essential for the final synthesis of the iron-molybdenum cofactor of nitrogenase in well-characterized diazotrophs, is absent from FS406-22. In addition, FS406-22 encodes four novel hypothetical proteins, and a ferredoxin, in the nif cluster. Here, we develop a set of genetic tools for FS406-22 and test the functionality of genes in the nif cluster by making markerless in-frame deletion mutations. Deletion of the gene for one hypothetical protein, designated Hp4, delayed the initiation of diazotrophic growth and decreased the growth rate, an effect we confirmed by genetic complementation. NifE also appeared to play a role in diazotrophic growth, and the encoding of Hp4 and NifE in a single operon suggested they may work together in some way in the synthesis of the nitrogenase cofactor. No role could be discerned for any of the other hypothetical proteins, nor for the ferredoxin, despite the presence of these genes in a variety of related organisms. Possible pathways and evolutionary scenarios for the synthesis of the nitrogenase cofactor in an organism that lacks nifN are discussed. IMPORTANCE Methanocaldococcus has been considered a model genus, but genetic tools have not been forthcoming until recently. Here, we develop and illustrate the utility of positive selection with either of two selective agents (simvastatin and neomycin), negative selection, generation of markerless in-frame deletion mutations, and genetic complementation. These genetic tools should be useful for a variety of related species. We address the question of the minimal set of nif genes, which has implications for how nitrogen fixation evolved.

We report the complete genome sequence of Methanospirillum hungatei strain GP1 (DSM 1101). Strain GP1 oxidizes H 2 , formate, and secondary alcohols as the substrates for methanogenesis. Members of the genus are model organisms used to study syntrophic growth with bacterial partners, but secondary alcohol metabolism remains poorly studied.

Hydrogenotrophic methanogens produce CH 4 using H 2 as an electron donor to reduce CO 2 In the absence of H 2 , many are able to use formate or alcohols as alternate electron donors. Methanogens from the order Methanomicrobiales are capable of growth with H 2 , but many lack genes encoding hydrogenases that are typically found in other hydrogenotrophic methanogens. In an effort to better understand electron flow in methanogens from the Methanomicrobiales , we undertook a genetic and biochemical study of heterodisulfide reductase (Hdr) in Methanoculleus thermophilus Hdr catalyzes an essential reaction by coupling the first and last steps of methanogenesis through flavin-based electron bifurcation. Hdr from M. thermophilus copurified with formate dehydrogenase (Fdh) and only displayed activity when formate was supplied as an electron donor. We found no evidence of an Hdr-associated hydrogenase, and H 2 could not function as an electron donor, even with Hdr purified from cells grown on H 2 We found that cells catalyze a formate hydrogenlyase activity that is likely essential for generating the formate needed for the Hdr reaction. Together, these results highlight the importance of formate as an electron donor for methanogenesis and suggest the ability to use formate is closely integrated into the methanogenic pathway in organisms from the order Methanomicrobiales IMPORTANCE Methanogens from the order Methanomicrobiales are thought to prefer H 2 as an electron donor for growth. They are ubiquitous in anaerobic environments, such as in wastewater treatment facilities, anaerobic digesters, and the rumen, where they catalyze the terminal steps in the breakdown of organic matter. However, despite their importance, the metabolism of these organisms remains understudied. Using a genetic and biochemical approach, we show that formate metabolism is closely integrated into methanogenesis in Methanoculleus thermophilus This is due to a requirement for formate as the electron donor to heterodisulfide reductase (Hdr), an enzyme responsible for catalyzing essential reactions in methanogenesis by linking the initial CO 2 fixing step to the exergonic terminal reaction of the pathway. These results suggest that hydrogen is not necessarily the preferred electron donor for all hydrogenotrophic methanogens and provide insight into the metabolism of methanogens from the order Methanomicrobiales ., (Copyright © 2021 American Society for Microbiology.)

Unlabelled: Diverse bacteria, including several Pseudomonas species, produce a class of redox-active metabolites called phenazines that impact different cell types in nature and disease. Phenazines can affect microbial communities in both positive and negative ways, where their presence is correlated with decreased species richness and diversity. However, little is known about how the concentration of phenazines is modulated in situ and what this may mean for the fitness of members of the community. Through culturing of phenazine-degrading mycobacteria, genome sequencing, comparative genomics, and molecular analysis, we identified several conserved genes that are important for the degradation of three Pseudomonas-derived phenazines: phenazine-1-carboxylic acid (PCA), phenazine-1-carboxamide (PCN), and pyocyanin (PYO). PCA can be used as the sole carbon source for growth by these organisms. Deletion of several genes in Mycobacterium fortuitum abolishes the degradation phenotype, and expression of two genes in a heterologous host confers the ability to degrade PCN and PYO. In cocultures with phenazine producers, phenazine degraders alter the abundance of different phenazine types. Not only does degradation support mycobacterial catabolism, but also it provides protection to bacteria that would otherwise be inhibited by the toxicity of PYO. Collectively, these results serve as a reminder that microbial metabolites can be actively modified and degraded and that these turnover processes must be considered when the fate and impact of such compounds in any environment are being assessed., Importance: Phenazine production by Pseudomonas spp. can shape microbial communities in a variety of environments ranging from the cystic fibrosis lung to the rhizosphere of dryland crops. For example, in the rhizosphere, phenazines can protect plants from infection by pathogenic fungi. The redox activity of phenazines underpins their antibiotic activity, as well as providing pseudomonads with important physiological benefits. Our discovery that soil mycobacteria can catabolize phenazines and thereby protect other organisms against phenazine toxicity suggests that phenazine degradation may influence turnover in situ. The identification of genes involved in the degradation of phenazines opens the door to monitoring turnover in diverse environments, an essential process to consider when one is attempting to understand or control communities influenced by phenazines., (Copyright © 2015 Costa et al.)

Flavin-based electron bifurcation has recently been characterized as an essential energy conservation mechanism that is utilized by hydrogenotrophic methanogenic Archaea to generate low-potential electrons in an ATP-independent manner. Electron bifurcation likely takes place at the flavin associated with the α subunit of heterodisulfide reductase (HdrA). In Methanococcus maripaludis the electrons for this reaction come from either formate or H2 via formate dehydrogenase (Fdh) or Hdr-associated hydrogenase (Vhu). However, how these enzymes bind to HdrA to deliver electrons is unknown. Here, we present evidence that the δ subunit of hydrogenase (VhuD) is central to the interaction of both enzymes with HdrA. When M. maripaludis is grown under conditions where both Fdh and Vhu are expressed, these enzymes compete for binding to VhuD, which in turn binds to HdrA. Under these conditions, both enzymes are fully functional and are bound to VhuD in substoichiometric quantities. We also show that Fdh copurifies specifically with VhuD in the absence of other hydrogenase subunits. Surprisingly, in the absence of Vhu, growth on hydrogen still occurs; we show that this involves F420-reducing hydrogenase. The data presented here represent an initial characterization of specific protein interactions centered on Hdr in a hydrogenotrophic methanogen that utilizes multiple electron donors for growth.

Methanogens catalyze the critical methane-producing step (called methanogenesis) in the anaerobic decomposition of organic matter. Here, we present the first predictive model of global gene regulation of methanogenesis in a hydrogenotrophic methanogen, Methanococcus maripaludis. We generated a comprehensive list of genes (protein-coding and noncoding) for M. maripaludis through integrated analysis of the transcriptome structure and a newly constructed Peptide Atlas. The environment and gene-regulatory influence network (EGRIN) model of the strain was constructed from a compendium of transcriptome data that was collected over 58 different steady-state and time-course experiments that were performed in chemostats or batch cultures under a spectrum of environmental perturbations that modulated methanogenesis. Analyses of the EGRIN model have revealed novel components of methanogenesis that included at least three additional protein-coding genes of previously unknown function as well as one noncoding RNA. We discovered that at least five regulatory mechanisms act in a combinatorial scheme to intercoordinate key steps of methanogenesis with different processes such as motility, ATP biosynthesis, and carbon assimilation. Through a combination of genetic and environmental perturbation experiments we have validated the EGRIN-predicted role of two novel transcription factors in the regulation of phosphate-dependent repression of formate dehydrogenase-a key enzyme in the methanogenesis pathway. The EGRIN model demonstrates regulatory affiliations within methanogenesis as well as between methanogenesis and other cellular functions.

Hydrogenotrophic methanogenic Archaea are defined by an H2 requirement for growth. Despite this requirement, many hydrogenotrophs are also capable of growth with formate as an electron donor for methanogenesis. While certain responses of these organisms to hydrogen availability have been characterized, responses to formate starvation have not been reported. Here we report that during continuous culture of Methanococcus maripaludis under defined nutrient conditions, growth yields relative to methane production decreased markedly with either H2 excess or formate excess. Analysis of the growth yields of several mutants suggests that this phenomenon occurs independently of the storage of intracellular carbon or a transcriptional response to methanogenesis. Using microarray analysis, we found that the expression of genes encoding coenzyme F420-dependent steps of methanogenesis, including one of two formate dehydrogenases, increased with H2 starvation but with formate occurred at high levels regardless of limitation or excess. One gene, encoding H2-dependent methylene-tetrahydromethanopterin dehydrogenase, decreased in expression with either H2 limitation or formate limitation. Expression of genes for the second formate dehydrogenase, molybdenum-dependent formylmethanofuran dehydrogenase, and molybdenum transport increased specifically with formate limitation. Of the two formate dehydrogenases, only the first could support growth on formate in batch culture where formate was in excess.

Unlabelled: Hydrogenotrophic methanogenic Archaea require reduced ferredoxin as an anaplerotic source of electrons for methanogenesis. H(2) oxidation by the hydrogenase Eha provides these electrons, consistent with an H(2) requirement for growth. Here we report the identification of alternative pathways of ferredoxin reduction in Methanococcus maripaludis that operate independently of Eha to stimulate methanogenesis. A suppressor mutation that increased expression of the glycolytic enzyme glyceraldehyde-3-phosphate:ferredoxin oxidoreductase resulted in a strain capable of H(2)-independent ferredoxin reduction and growth with formate as the sole electron donor. In this background, it was possible to eliminate all seven hydrogenases of M. maripaludis. Alternatively, carbon monoxide oxidation by carbon monoxide dehydrogenase could also generate reduced ferredoxin that feeds into methanogenesis. In either case, the reduced ferredoxin generated was inefficient at stimulating methanogenesis, resulting in a slow growth phenotype. As methanogenesis is limited by the availability of reduced ferredoxin under these conditions, other electron donors, such as reduced coenzyme F(420), should be abundant. Indeed, when F(420)-reducing hydrogenase was reintroduced into the hydrogenase-free mutant, the equilibrium of H(2) production via an F(420)-dependent formate:H(2) lyase activity shifted markedly toward H(2) compared to the wild type., Importance: Hydrogenotrophic methanogens are thought to require H(2) as a substrate for growth and methanogenesis. Here we show alternative pathways in methanogenic metabolism that alleviate this H(2) requirement and demonstrate, for the first time, a hydrogenotrophic methanogen that is capable of growth in the complete absence of H(2). The demonstration of alternative pathways in methanogenic metabolism suggests that this important group of organisms is metabolically more versatile than previously thought.