Localized microbially induced inflammation influences distant healthy tissues in the human oral cavity.

Variation in human immune response to the same bacterial or viral pathogen is well established in the literature. Variation in immune response to microbial challenge has also been observed within the human oral cavity. Our recent study focused on characterizing observed variations in microbially induced gingival inflammation-resulting in three distinct clinical Inflammatory Responder Types (IRTs): High-IRT, Low-IRT, and Slow-IRT. Here, we applied a high-resolution temporal multiomic analysis during microbially induced inflammation in order to characterize the effects of localized oral inflammation on distant healthy tissues in young healthy adults. Our results highlight a nonlocalized subclinical effect with alterations in proinflammatory host mediators and an ecological shift toward dysbiosis within the subgingival microbiome in an IRT-dependent manner-despite maintained oral hygiene. Our results provide mechanistic insight into how healthy tissues within humans are influenced by distant localized inflammation and may ultimately become susceptible to disease.

Transcriptome of Epibiont Saccharibacteria Nanosynbacter lyticus Strain TM7x During the Establishment of Symbiosis.

Saccharibacteria Nanosynbacter lyticus strain TM7x is a member of the broadly distributed candidate phylum radiation. These bacteria have ultrasmall cell sizes, have reduced genomes, and live as epibionts on the surfaces of other bacteria. The mechanisms by which they establish and maintain this relationship are not yet fully understood. The transcriptomes of the epibiont TM7x and its host bacteria Schaalia odontolytica strain XH001 were captured across the establishment of symbiosis during both the initial interaction and stable symbiosis. The results showed a dynamic interaction with large shifts in gene expression for both species between the initial encounter and stable symbiosis, notably in transporter genes. During stable symbiosis, the host XH001 showed higher gene expression for peptidoglycan biosynthesis, mannosylation, cell cycle and stress-related genes, whereas it showed lower expression of chromosomal partitioning genes. This was consistent with the elongated cell shape seen in XH001 infected with TM7x and our discovery that infection resulted in thickened cell walls. Within TM7x, increased pili, type IV effector genes, and arginine catabolism/biosynthesis gene expression during stable symbiosis implied a key role for these functions in the interaction. Consistent with its survival and persistence in the human microbiome as an obligate epibiont with reduced de novo biosynthetic capacities, TM7x also showed higher levels of energy production and peptidoglycan biosynthesis, but lower expression of stress-related genes, during stable symbiosis. These results imply that TM7x and its host bacteria keep a delicate balance in order to sustain an episymbiotic lifestyle. IMPORTANCE Nanosynbacter lyticus type strain TM7x is the first cultivated member of the Saccharibacteria and the candidate phyla radiation (CPR). It was discovered to be ultrasmall in cell size with a highly reduced genome that establishes an obligate epibiotic relationship with its host bacterium. The CPR is a large, monophyletic radiation of bacteria with reduced genomes that includes Saccharibacteria. The vast majority of the CPR have yet to be cultivated, and our insights into these unique organisms to date have been derived from only a few Saccharibacteria species. Being obligate parasites, it is unknown how these ultrasmall Saccharibacteria, which are missing many de novo biosynthetic pathways, are maintained at a high prevalence within the human microbiome as well as in the environment.

Klebsiella and Providencia emerge as lone survivors following long-term starvation of oral microbiota.

It is well-understood that many bacteria have evolved to survive catastrophic events using a variety of mechanisms, which include expression of stress-response genes, quiescence, necrotrophy, and metabolic advantages obtained through mutation. However, the dynamics of individuals leveraging these abilities to gain a competitive advantage in an ecologically complex setting remain unstudied. In this study, we observed the saliva microbiome throughout the ecological perturbation of long-term starvation, allowing only the species best equipped to access and use the limited resources to survive. During the first several days, the community underwent a death phase that resulted in a ∼50-100-fold reduction in the number of viable cells. Interestingly, after this death phase, only three species, Klebsiella pneumoniae, Klebsiella oxytoca, and Providencia alcalifaciens, all members of the family Enterobacteriaceae, appeared to be transcriptionally active and recoverable. Klebsiella are significant human pathogens, frequently resistant to multiple antibiotics, and recently, ectopic colonization of the gut by oral Klebsiella was documented to induce dysbiosis and inflammation. MetaOmics analyses provided several leads for further investigation regarding the ecological success of the Enterobacteriaceae. The isolates accumulated single nucleotide polymorphisms in known growth advantage in stationary phase alleles and produced natural products closely resembling antimicrobial cyclic depsipeptides. The results presented in this study suggest that pathogenic Enterobacteriaceae persist much longer than their more benign neighbors in the salivary microbiome when faced with starvation. This is particularly significant, given that hospital surfaces contaminated with oral fluids, especially sinks and drains, are well-established sources of outbreaks of drug-resistant Enterobacteriaceae.

Ultrasmall epibiont Nanosynbacter lyticus strain TM7x and host bacteria transcriptional activity after initial host parasitism.

Background: Oral Saccharibacteria Nanosynbacter lyticus strain TM7× lives as an ultrasmall epibiont on the surface of its host, Schaalia odontolytica strain XH001. Establishing this interaction is a poorly understood multi-step process. The recovery phase marks a shift in the TM7×/host interaction, switching from the early killing phase, with extensive host cell death, to a stable symbiosis phase where the host and epibiont can grow together. Results: Transcriptomes of TM7× and host, XH001, were captured during the recovery phase and compared to uninfected host and the early host/epibiont interaction (initial encounter). XH001 showed increased expression for rhamnose cell wall components and for the precursor to peptidoglycan while TM7× showed increases in the peptidoglycan pathway. Transporter expression was generally increased for both organisms during recovery compared to the initial encounter, though, XH001 showed lower amino acid transporter expression. Consistent with host parasitism, XH001 showed increased expression of various stress-related genes during recovery while TM7× showed reduced stress. TM7× displayed higher expression of type IV pili, consistent with increased attachment to new hosts. Conclusion: As TM7× is a member of the broadly distributed Candidate Phyla Radiation with small genomes lacking numerous biosynthetic pathways, this study provides further insights into how these epibionts interact and modulate their host bacteria.

Specific host metabolite and gut microbiome alterations are associated with bone loss during spaceflight.

Understanding the axis of the human microbiome and physiological homeostasis is an essential task in managing deep-space-travel-associated health risks. The NASA-led Rodent Research 5 mission enabled an ancillary investigation of the gut microbiome, varying exposure to microgravity (flight) relative to ground controls in the context of previously shown bone mineral density (BMD) loss that was observed in these flight groups. We demonstrate elevated abundance of Lactobacillus murinus and Dorea sp. during microgravity exposure relative to ground control through whole-genome sequencing and 16S rRNA analyses. Specific functionally assigned gene clusters of L. murinus and Dorea sp. capable of producing metabolites, lactic acid, leucine/isoleucine, and glutathione are enriched. These metabolites are elevated in the microgravity-exposed host serum as shown by liquid chromatography-tandem mass spectrometry (LC-MS/MS) metabolomic analysis. Along with BMD loss, ELISA reveals increases in osteocalcin and reductions in tartrate-resistant acid phosphatase 5b signifying additional loss of bone homeostasis in flight.

Insights into the virulence of oral biofilms: discoveries from proteomics.

This review covers developments in the study of polymicrobial communities, biofilms and selected areas of host response relevant to dental plaque and related areas of oral biology. The emphasis is on recent studies in which proteomic methods, particularly those using mass spectrometry as a readout, have played a major role in the investigation. The last 5-10 years have seen a transition of such methods from the periphery of oral biology to the mainstream, as in other areas of biomedical science. For reasons of focus and space, the authors do not discuss biomarker studies relevant to improved diagnostics for oral health, as this literature is rather substantial in its own right and deserves a separate treatment. Here, global gene regulation studies of plaque-component organisms, biofilm formation, multispecies interactions and host-microbe interactions are discussed. Several aspects of proteomics methodology that are relevant to the studies of multispecies systems are commented upon.

Proteomics of Streptococcus gordonii within a model developing oral microbial community.

BACKGROUND: Streptococcus gordonii is one of several species that can initiate the formation of oral biofilms that develop into the complex multispecies microbial communities referred to as dental plaque. It is in the context of dental plaque that periodontal pathogens such as Porphyromonas gingivalis cause disease. We have previously reported a whole cell quantitative proteomics investigation of P. gingivalis in a model dental plaque community of S. gordonii, P. gingivalis, and Fusobacterium nucleatum. Here we report the adaptation of S. gordonii to the same model. RESULTS: 1122 S. gordonii proteins were detected in S. gordonii control samples, 915 in communities with F. nucleatum, 849 with P. gingivalis, and 649 with all three organisms. Quantitative comparisons showed extensive proteome changes in association with F. nucleatum or P. gingivalis individually or both P. gingivalis and F. nucleatum together. The changes were species specific, though the P. gingivalis interaction may be dominant, indicated by large differences between the proteomes with F. nucleatum or P. gingivalis but limited changes between communities with P. gingivalis or both P. gingivalis and F. nucleatum. The results were inspected manually and an ontology analysis conducted using DAVID. Extensive changes were seen in nutrition pathways with increases in energy metabolism and changes in the resulting byproducts, while the acid and sugar repressed PTS (phosphoenolpyruvate dependent phosphotransferase system) sugar transport systems showed decreases. These results were seen across all the multispecies samples, though with different profiles according to the partner species. F. nucleatum association decreased proteins for the metabolic end products acetate and ethanol but increased lactate, the primary source of acidity from streptococcal cultures. P. gingivalis containing samples had a reduction in levels of proteins for ethanol and formate but increased proteins for both acetate and lactate production. The communities also showed increases in exopolysaccharide synthesis, amino acid biosynthesis, and oxidative stress protection and decreases in adhesion and transporter proteins. CONCLUSION: This study showed that S. gordonii demonstrates species specific responses during interactions with F. nucleatum or P. gingivalis. Extensive changes were seen in energy metabolism and byproduct production implicating nutrient transfer as an important community interaction.

Strain-Level Variation and Diverse Host Bacterial Responses in Episymbiotic Saccharibacteria.

Saccharibacteria (TM7), which are obligate episymbionts growing on the surface of host bacteria, may play an important role in oral disease, such as periodontitis (1, 2). As TM7 is a newly cultured lineage of bacteria, its research is limited by the small number of isolated representatives relative to the number of TM7 genomes assembled from culture-independent studies (3-5). A comprehensive view of both TM7 taxa and TM7 strain-level variations remains opaque. In this study, we expanded our previously developed TM7 baiting method into using many host bacteria in parallel, which allowed us to obtain 37 TM7 strains from the human oral cavity. These strains were further classified into low-enrichment (LE, n = 24) and high-enrichment (HE, n = 13) groups based on their proficiency at propagating on host bacteria. Of the 13 HE strains, 10 belong to "Candidatus Nanosynbacter sp." strain HMT-352 (human microbial taxon) (6), enabling us to explore both the phenotypic and genomic strain variations within a single TM7 species. We show that TM7 HMT-352 strains exhibit a diverse host range and varied growth dynamics during the establishment of their episymbiotic relationship with host bacteria. Furthermore, despite HMT-352 strains sharing a majority of their genes, we identified several gene clusters that may play a pivotal role in host affinity. More importantly, our comparative analyses also provide TM7 gene candidates associated with strain-level phenotypic variation that may be important for episymbiotic interactions with host bacteria. IMPORTANCE Candidate phylum radiation (CPR) bacteria comprise a poorly understood phylum that is estimated to encompass ∼26% of all diversity of domain bacteria. Among CPR bacteria, the Saccharibacteria lineage (TM7) is of particular interest, as it is found in high abundance in the mammal microbiome and has been associated with oral disease. While many CPR genomes, TM7 included, have been acquired through culture-independent methods, only a small number of representatives have been isolated. Such isolated representatives, however, shed light on the physiology, pathogenesis, and episymbiotic interactions of TM7. Combined with genomic analyses, experiments involving isolated representatives can distinguish phylogenetic to phenotypic discrepancies and better identify genes of importance. In this study, we utilized multiple host bacteria in parallel to isolate TM7 bacteria and examined strain-level variation in TM7 to reveal key genes that may drive TM7-host interactions. Our findings accentuate that broad phylogenetic characterization of CPR is the next step in understanding these bacteria.

Draft Genome Sequences of Nine "Candidatus Nanosynbacter sp. HMT-352" Strains Cultured from the Human Oral Cavity.

Here, we report draft genome sequences for nine strains of "Candidatus Nanosynbacter sp. HMT-352." These strains and their sequences were used to interrogate strain-level variations in host range, gene content, and growth dynamics among the phylum "Candidatus Saccharibacteria."

An integrated proteomics/transcriptomics approach points to oxygen as the main electron sink for methanol metabolism in Methylotenera mobilis.

Methylotenera species, unlike their close relatives in the genera Methylophilus, Methylobacillus, and Methylovorus, neither exhibit the activity of methanol dehydrogenase nor possess mxaFI genes encoding this enzyme, yet they are able to grow on methanol. In this work, we integrated a genome-wide proteomics approach, shotgun proteomics, and a genome-wide transcriptomics approach, shotgun transcriptome sequencing (RNA-seq), of Methylotenera mobilis JLW8 to identify genes and enzymes potentially involved in methanol oxidation, with special attention to alternative nitrogen sources, to address the question of whether nitrate could play a role as an electron acceptor in place of oxygen. Both proteomics and transcriptomics identified a limited number of genes and enzymes specifically responding to methanol. This set includes genes involved in oxidative stress response systems, a number of oxidoreductases, including XoxF-type alcohol dehydrogenases, a type II secretion system, and proteins without a predicted function. Nitrate stimulated expression of some genes in assimilatory nitrate reduction and denitrification pathways, while ammonium downregulated some of the nitrogen metabolism genes. However, none of these genes appeared to respond to methanol, which suggests that oxygen may be the main electron sink during growth on methanol. This study identifies initial targets for future focused physiological studies, including mutant analysis, which will provide further details into this novel process.

Overlapping repressor binding sites regulate expression of the Methanococcus maripaludis glnK(1) operon.

The euryarchaeal transcriptional repressor NrpR regulates a variety of nitrogen assimilation genes by 2-oxoglutarate-reversible binding to conserved palindromic operators. The number and positioning of these operators varies among promoter regions of regulated genes, suggesting NrpR can bind in different patterns. Particularly intriguing is the contrast between the nif and glnK(1) promoter regions of Methanococcus maripaludis, where two operators are present but with different configurations. Here we study NrpR binding and regulation at the glnK(1) promoter, where the two operator sequences overlap and occur on opposite faces of the double helix. We find that both operators function in binding, with a dimer of NrpR binding simultaneously to each overlapping operator. We show in vivo that the first operator plays a primary role in regulation and the second operator plays an enhancing role. This is the first demonstration of overlapping operators functioning in Archaea.

Expressed genome of Methylobacillus flagellatus as defined through comprehensive proteomics and new insights into methylotrophy.

In recent years, techniques have been developed and perfected for high-throughput identification of proteins and their accurate partial sequencing by shotgun nano-liquid chromatography-tandem mass spectrometry (nano-LC-MS/MS), making it feasible to assess global protein expression profiles in organisms with sequenced genomes. We implemented comprehensive proteomics to assess the expressed portion of the genome of Methylobacillus flagellatus during methylotrophic growth. We detected a total of 1,671 proteins (64% of the inferred proteome), including all the predicted essential proteins. Nonrandom patterns observed with the nondetectable proteins appeared to correspond to silent genomic islands, as inferred through functional profiling and genome localization. The protein contents in methylamine- and methanol-grown cells showed a significant overlap, confirming the commonality of methylotrophic metabolism downstream of the primary oxidation reactions. The new insights into methylotrophy include detection of proteins for the N-methylglutamate methylamine oxidation pathway that appears to be auxiliary and detection of two alternative enzymes for both the 6-phosphogluconate dehydrogenase reaction (GndA and GndB) and the formate dehydrogenase reaction (FDH1 and FDH4). Mutant analysis revealed that GndA and FDH4 are crucial for the organism's fitness, while GndB and FDH1 are auxiliary.

Complete Genome Sequence of Strain BB001, a Novel Epibiont Bacterium from the Candidate Phylum Saccharibacteria (TM7).

Strain BB001 is cultivated from the human oral cavity on its basibiont bacterial host Actinomyces sp. It is an ultrasmall bacterium with a reduced genome that grows obligately on its bacterial host. BB001 is the first member of human microbiome taxon 957.

Acquisition and Adaptation of Ultra-small Parasitic Reduced Genome Bacteria to Mammalian Hosts.

The first cultivated representative of the enigmatic phylum Saccharibacteria (formerly TM7) was isolated from humans and revealed an ultra-small cell size (200-300 nm), a reduced genome with limited biosynthetic capabilities, and a unique parasitic lifestyle. TM7x was the only cultivated member of the candidate phyla radiation (CPR), estimated to encompass 26% of the domain Bacteria. Here we report on divergent genomes from major lineages across the Saccharibacteria phylum in humans and mammals, as well as from ancient dental calculus. These lineages are present at high prevalence within hosts. Direct imaging reveals that all groups are ultra-small in size, likely feeding off commensal bacteria. Analyses suggest that multiple acquisition events in the past led to the current wide diversity, with convergent evolution of key functions allowing Saccharibacteria from the environment to adapt to mammals. Ultra-small, parasitic CPR bacteria represent a relatively unexplored paradigm of prokaryotic interactions within mammalian microbiomes.

Pathway analysis for intracellular Porphyromonas gingivalis using a strain ATCC 33277 specific database.

BACKGROUND: Porphyromonas gingivalis is a Gram-negative intracellular pathogen associated with periodontal disease. We have previously reported on whole-cell quantitative proteomic analyses to investigate the differential expression of virulence factors as the organism transitions from an extracellular to intracellular lifestyle. The original results with the invasive strain P. gingivalis ATCC 33277 were obtained using the genome sequence available at the time, strain W83 [GenBank: AE015924]. We present here a re-processed dataset using the recently published genome annotation specific for strain ATCC 33277 [GenBank: AP009380] and an analysis of differential abundance based on metabolic pathways rather than individual proteins. RESULTS: Qualitative detection was observed for 1266 proteins using the strain ATCC 33277 annotation for 18 hour internalized P. gingivalis within human gingival epithelial cells and controls exposed to gingival cell culture medium, an improvement of 7% over the W83 annotation. Internalized cells showed increased abundance of proteins in the energy pathway from asparagine/aspartate amino acids to ATP. The pathway producing one short chain fatty acid, propionate, showed increased abundance, while that of another, butyrate, trended towards decreased abundance. The translational machinery, including ribosomal proteins and tRNA synthetases, showed a significant increase in protein relative abundance, as did proteins responsible for transcription. CONCLUSION: Use of the ATCC 33277 specific genome annotation resulted in improved proteome coverage with respect to the number of proteins observed both qualitatively in terms of protein identifications and quantitatively in terms of the number of calculated abundance ratios. Pathway analysis showed a significant increase in overall protein synthetic and transcriptional machinery in the absence of significant growth. These results suggest that the interior of host cells provides a more energy rich environment compared to the extracellular milieu. Shifts in the production of cytotoxic fatty acids by intracellular P. gingivalis may play a role in virulence. Moreover, despite extensive genomic re-arrangements between strains W83 and 33277, there is sufficient sequence similarity at the peptide level for proteomic abundance trends to be largely accurate when using the heterologous strain annotated genome as the reference for database searching.

Proteomics of Porphyromonas gingivalis within a model oral microbial community.

BACKGROUND: Porphyromonas gingivalis is a periodontal pathogen that resides in a complex multispecies microbial biofilm community known as dental plaque. Confocal laser scanning microscopy showed that P. gingivalis can assemble into communities in vitro with Streptococcus gordonii and Fusobacterium nucleatum, common constituents of dental plaque. Whole cell quantitative proteomics, along with mutant construction and analysis, were conducted to investigate how P. gingivalis adapts to this three species community. RESULTS: 1156 P. gingivalis proteins were detected qualitatively during comparison of the three species model community with P. gingivalis incubated alone under the same conditions. Integration of spectral counting and summed signal intensity analyses of the dataset showed that 403 proteins were down-regulated and 89 proteins up-regulated. The proteomics results were inspected manually and an ontology analysis conducted using DAVID. Significant decreases were seen in proteins involved in cell shape and the formation of the cell envelope, as well as thiamine, cobalamin, and pyrimidine synthesis and DNA repair. An overall increase was seen in proteins involved in protein synthesis. HmuR, a TonB dependent outer membrane receptor, was up-regulated in the community and an hmuR deficient mutant was deficient in three species community formation, but was unimpaired in its ability to form mono- or dual-species biofilms. CONCLUSION: Collectively, these results indicate that P. gingivalis can assemble into a heterotypic community with F. nucleatum and S. gordonii, and that a community lifestyle provides physiologic support for P. gingivalis. Proteins such as HmuR, that are up-regulated, can be necessary for community structure.

Quantitative proteomics of nutrient limitation in the hydrogenotrophic methanogen Methanococcus maripaludis.

BACKGROUND: Methanogenic Archaea play key metabolic roles in anaerobic ecosystems, where they use H2 and other substrates to produce methane. Methanococcus maripaludis is a model for studies of the global response to nutrient limitations. RESULTS: We used high-coverage quantitative proteomics to determine the response of M. maripaludis to growth-limiting levels of H2, nitrogen, and phosphate. Six to ten percent of the proteome changed significantly with each nutrient limitation. H2 limitation increased the abundance of a wide variety of proteins involved in methanogenesis. However, one protein involved in methanogenesis decreased: a low-affinity [Fe] hydrogenase, which may dominate over a higher-affinity mechanism when H2 is abundant. Nitrogen limitation increased known nitrogen assimilation proteins. In addition, the increased abundance of molybdate transport proteins suggested they function for nitrogen fixation. An apparent regulon governed by the euryarchaeal nitrogen regulator NrpR is discussed. Phosphate limitation increased the abundance of three different sets of proteins, suggesting that all three function in phosphate transport. CONCLUSION: The global proteomic response of M. maripaludis to each nutrient limitation suggests a wider response than previously appreciated. The results give new insight into the function of several proteins, as well as providing information that should contribute to the formulation of a regulatory network model.

Roles of coenzyme F420-reducing hydrogenases and hydrogen- and F420-dependent methylenetetrahydromethanopterin dehydrogenases in reduction of F420 and production of hydrogen during methanogenesis.

Reduced coenzyme F420 (F420H2) is an essential intermediate in methanogenesis from CO2. During methanogenesis from H2 and CO2, F420H2 is provided by the action of F420-reducing hydrogenases. However, an alternative pathway has been proposed, where H2-dependent methylenetetrahydromethanopterin dehydrogenase (Hmd) and F420H2-dependent methylenetetrahydromethanopterin dehydrogenase (Mtd) together reduce F420 with H2. Here we report the construction of mutants of Methanococcus maripaludis that are defective in each putative pathway. Their analysis demonstrates that either pathway supports growth on H2 and CO2. Furthermore, we show that during growth on formate instead of H2, where F420H2 is a direct product of formate oxidation, H2 production occurs. H2 presumably arises from the oxidation of F420H2, and the analysis of the mutants during growth on formate suggests that this too can occur by either pathway. We designate the alternative pathway for the interconversion of H2 and F420H2 the Hmd-Mtd cycle.

Global responses of Methanococcus maripaludis to specific nutrient limitations and growth rate.

Continuous culture, transcriptome arrays, and measurements of cellular amino acid pools and tRNA charging levels were used to determine the response of Methanococcus maripaludis to leucine limitation. For comparison, the responses to phosphate and H2 limitations were measured as well. In addition, the effect of growth rate was determined. Leucine limitation resulted in a broad response. tRNA(Leu) charging decreased, but only small increases in mRNA were seen for amino acid biosynthesis genes. However, the cellular levels of free isoleucine and valine showed significant increases, indicating a coordinate regulation of branched-chain amino acids at a post-mRNA level. Leucine limitation also resulted in increased mRNA abundance for ribosomal protein genes, increased rRNA abundance, and decreased mRNA abundance for genes of methanogenesis. In contrast, phosphate limitation induced a specific response, a marked increase in mRNA levels for a phosphate transporter. Some mRNA levels responded to more than one factor; for example, transcripts for flagellum synthesis genes decreased under conditions of leucine limitation and increased under H2 limitation. Increased growth rate resulted in increased mRNA levels for ribosomal protein genes, increased rRNA abundance, and increased mRNA for a gene encoding an S-layer protein.

Formate-dependent H2 production by the mesophilic methanogen Methanococcus maripaludis.

Methanococcus maripaludis, an H(2)- and formate-utilizing methanogen, produced H(2) at high rates from formate. The rates and kinetics of H(2) production depended upon the growth conditions, and H(2) availability during growth was a major factor. Specific activities of resting cells grown with formate or H(2) were 0.4 to 1.4 U mg(-1) (dry weight). H(2) production in formate-grown cells followed Michaelis-Menten kinetics, and the concentration of formate required for half-maximal activity (K(f)) was 3.6 mM. In contrast, in H(2)-grown cells this process followed sigmoidal kinetics, and the K(f) was 9 mM. A key enzyme for formate-dependent H(2) production was formate dehydrogenase, Fdh. H(2) production and growth were severely reduced in a mutant containing a deletion of the gene encoding the Fdh1 isozyme, indicating that it was the primary Fdh. In contrast, a mutant containing a deletion of the gene encoding the Fdh2 isozyme possessed near-wild-type activities, indicating that this isozyme did not play a major role. H(2) production by a mutant containing a deletion of the coenzyme F(420)-reducing hydrogenase Fru was also severely reduced, suggesting that the major pathway of H(2) production comprised Fdh1 and Fru. Because a Deltafru-Deltafrc mutant retained 10% of the wild-type activity, an additional pathway is present. Mutants possessing deletions of the gene encoding the F(420)-dependent methylene-H(4)MTP dehydrogenase (Mtd) or the H(2)-forming methylene-H(4)MTP dehydrogenase (Hmd) also possessed reduced activity, which suggested that this second pathway was comprised of Fdh1-Mtd-Hmd. In contrast to H(2) production, the cellular rates of methanogenesis were unaffected in these mutants, which suggested that the observed H(2) production was not a direct intermediate of methanogenesis. In conclusion, high rates of formate-dependent H(2) production demonstrated the potential of M. maripaludis for the microbial production of H(2) from formate.