Purification of the formate-tetrahydrofolate ligase from Methylobacterium extorquens AM1 and demonstration of its requirement for methylotrophic growth

The serine cycle methylotroph Methylobacterium extorquens AM1 contains two pterin-dependent pathways for C(1) transfers, the tetrahydrofolate (H(4)F) pathway and the tetrahydromethanopterin (H(4)MPT) pathway, and both are required for growth on C(1) compounds. With the exception of formate-tetrahydrofolate ligase (FtfL, alternatively termed formyl-H(4)F synthetase), all of the genes encoding the enzymes comprising these two pathways have been identified, and the corresponding gene products have been purified and characterized. We present here the purification and characterization of FtfL from M. extorquens AM1 and the confirmation that this enzyme is encoded by an ftfL homolog identified previously through transposon mutagenesis. Phenotypic analyses of the ftfL mutant strain demonstrated that FtfL activity is required for growth on C(1) compounds. Unlike mutants defective for the H(4)MPT pathway, the ftfL mutant strain does not exhibit phenotypes indicative of defective formaldehyde oxidation. Furthermore, the ftfL mutant strain remained competent for wild-type conversion of [(14)C]methanol to [(14)C]CO(2). Collectively, these data confirm our previous presumptions that the H(4)F pathway is not the key formaldehyde oxidation pathway in M. extorquens AM1. Rather, our data suggest an alternative model for the role of the H(4)F pathway in this organism in which it functions to convert formate to methylene H(4)F for assimilatory metabolism.     

Formaldehyde-detoxifying role of the tetrahydromethanopterin-linked pathway in Methylobacterium extorquens AM1

The facultative methylotroph Methylobacterium extorquens AM1 possesses two pterin-dependent pathways for C(1) transfer between formaldehyde and formate, the tetrahydrofolate (H(4)F)-linked pathway and the tetrahydromethanopterin (H(4)MPT)-linked pathway. Both pathways are required for growth on C(1) substrates; however, mutants defective for the H(4)MPT pathway reveal a unique phenotype of being inhibited by methanol during growth on multicarbon compounds such as succinate. It has been previously proposed that this methanol-sensitive phenotype is due to the inability to effectively detoxify formaldehyde produced from methanol. Here we present a comparative physiological characterization of four mutants defective in the H(4)MPT pathway and place them into three different phenotypic classes that are concordant with the biochemical roles of the respective enzymes. We demonstrate that the analogous H(4)F pathway present in M. extorquens AM1 cannot fulfill the formaldehyde detoxification function, while a heterologously expressed pathway linked to glutathione and NAD(+) can successfully substitute for the H(4)MPT pathway. Additionally, null mutants were generated in genes previously thought to be essential, indicating that the H(4)MPT pathway is not absolutely required during growth on multicarbon compounds. These results define the role of the H(4)MPT pathway as the primary formaldehyde oxidation and detoxification pathway in M. extorquens AM1.     

Formate as the main branch point for methylotrophic metabolism in Methylobacterium extorquens AM1

In serine cycle methylotrophs, methylene tetrahydrofolate (H4F) is the entry point of reduced one-carbon compounds into the serine cycle for carbon assimilation during methylotrophic metabolism. In these bacteria, two routes are possible for generating methylene H4F from formaldehyde during methylotrophic growth: one involving the reaction of formaldehyde with H4F to generate methylene H4F and the other involving conversion of formaldehyde to formate via methylene tetrahydromethanopterin-dependent enzymes and conversion of formate to methylene H4F via H4F-dependent enzymes. Evidence has suggested that the direct condensation reaction is the main source of methylene H4F during methylotrophic metabolism. However, mutants lacking enzymes that interconvert methylene H4F and formate are unable to grow on methanol, suggesting that this route for methylene H4F synthesis should have a significant role in biomass production during methylotrophic metabolism. This problem was investigated in Methylobacterium extorquens AM1. Evidence was obtained suggesting that the existing deuterium assay might overestimate the flux through the direct condensation reaction. To test this possibility, it was shown that only minor assimilation into biomass occurred in mutants lacking the methylene H4F synthesis pathway through formate. These results suggested that the methylene H4F synthesis pathway through formate dominates assimilatory flux. A revised kinetic model was used to validate this possibility, showing that physiologically plausible parameters in this model can account for the metabolic fluxes observed in vivo. These results all support the suggestion that formate, not formaldehyde, is the main branch point for methylotrophic metabolism in M. extorquens AM1.     

Distribution of Tetrahydromethanopterin-Dependent Enzymes in Methylotrophic Bacteria and Phylogeny of Methenyl Tetrahydromethanopterin Cyclohydrolases

The methylotrophic proteobacterium Methylobacterium extorquens AM1 possesses tetrahydromethanopterin (H(4)MPT)-dependent enzymes, which are otherwise specific to methanogenic and sulfate-reducing archaea and which have been suggested to be involved in formaldehyde oxidation to CO(2) in M. extorquens AM1. The distribution of H(4)MPT-dependent enzyme activities in cell extracts of methylotrophic bacteria from 13 different genera are reported. H(4)MPT-dependent activities were detected in all of the methylotrophic and methanotrophic proteobacteria tested that assimilate formaldehyde by the serine or ribulose monophosphate pathway. H(4)MPT-dependent activities were also found in autotrophic Xanthobacter strains. However, no H(4)MPT-dependent enzyme activities could be detected in other autotrophic alpha-proteobacteria or in gram-positive methylotrophic bacteria. Genes encoding methenyl H(4)MPT cyclohydrolase (mch genes) were cloned and sequenced from several proteobacteria. Bacterial and archaeal Mch sequences have roughly 35% amino acid identity and form distinct groups in phylogenetic analysis.     

Oxidation of C1-compounds in Pseudomonas C.

Extracts of Pseudomonas C grown on methanol as a sole carbon and energy source contain a methanol dehydrogenase activity which can be coupled to phenazine methosulfate. This enzyme catalyzes two reactions namely the conversion of methanol to formaldehyde (phenazine methosulfate coupled) and the oxidation of formaldehyde to formate (2,6-dichloroindophenol-coupled). Activities of glutathione-dependent formaldehyde dehydrogenase (NAD+) and formate dehydrogenase (NAD+) were also detected in the extracts. The addition of D-ribulose 5-phosphate to the reaction mixtures caused a marked increase in the formaldehyde-dependent reduction of NAD+ or NADP+. In addition, the oxidation of [14C]formaldehyde to CO2, by extracts of Pseudomonas C, increased when D-ribulose 5-phosphate was present in the assay mixtures. The amount of radioactivity found in CO2, was 6;8-times higher when extracts of methanol-grown Pseudomonas C were incubated for a short period of time with [1-14C]glucose 6-phosphate than with [U-14C]glucose 6-phosphate. These data, and the presence of high specific activities of hexulose phosphate synthase, phosphoglucoisomerase, glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase indicate that in methanol-grown Pseudomonas C, formaldehyde carbon is oxidized to CO2 both via a cyclic pathway which includes the enzymes mentioned and via formate as an oxidation intermediate, with the former predominant.     

Proteome analyses of hydrogen-producing hyperthermophilic archaeon Thermococcus onnurineus NA1 in different one-carbon substrate culture conditions

Thermococcus onnurineus NA1, a sulfur-reducing hyperthermophilic archaeon, is capable of H(2)-producing growth, considered to be hydrogenogenic carboxydotrophy. Utilization of formate as a sole energy source has been well studied in T. onnurineus NA1. However, whether formate can be used as its carbon source remains unknown. To obtain a global view of the metabolic characteristics of H(2)-producing growth, a quantitative proteome analysis of T. onnurineus NA1 grown on formate, CO, and starch was performed by combining one-dimensional SDS-PAGE with nano UPLC-MS(E). A total of 587 proteins corresponding to 29.7% of the encoding genes were identified, and the major metabolic pathways (especially energy metabolism) were characterized at the protein level. Expression of glycolytic enzymes was common but more highly induced in starch-grown cells. In contrast, enzymes involved in key steps of the gluconeogenesis and pentose phosphate pathways were strongly up-regulated in formate-grown cells, suggesting that formate could be utilized as a carbon source by T. onnurineus NA1. In accordance with the genomic analysis, comprehensive proteomic analysis also revealed a number of hydrogenase clusters apparently associated with formate metabolism. On the other hand, CODH and CO-induced hydrogenases belonging to the Hyg4-II cluster, as well as sulfhydrogenase-I and Mbx, were prominently expressed during CO culture. Our data suggest that CO can be utilized as a sole energy source for H(2) production via an electron transport mechanism and that CO(2) produced from catabolism or CO oxidation by CODH and CO-induced hydrogenases may subsequently be assimilated into the organic carbon. Overall, proteomic comparison of formate- and CO-grown cells with starch-grown cells revealed that a single carbon compound, such as formate and CO, can be utilized as an efficient substrate to provide cellular carbon and/or energy by T. onnurineus NA1.     

Tetrahydromethanopterin, a carbon carrier in methanogenesis

Evidence obtained by 13C NMR spectroscopy indicates that tetrahydromethanopterin (H4MPT) serves as a carbon carrier for C1 units at the methine, methylene, and methyl levels of oxidation. All three derivatives of H4MPT served as substrates for methanogenesis by cell extracts under a hydrogen atmosphere; in each instance, methane evolved at a rate comparable to that obtained when 2-(methylthio)ethanesulfonic acid was used as the substrate. Each C1 derivative of H4MPT stimulated the reduction of CO2 as efficiently as 2-(methylthio)ethanesulfonic acid. High resolution fast atom bombardment mass spectrometry indicated that the product of the spontaneous reaction of formaldehyde with H4MPT was methylene-H4MPT, with the molecular formula C31H45N6O16P. 13C NMR spectroscopy of hexamethylenetetramine, a model compound, suggested that the methylene group in methylene-H4MPT was bound to two nitrogen atoms. Molecular formulas of C31H44N6O16P and C31H47N6O16P were assigned to methenyl-H4MPT+, and methyl-H4MPT, by high resolution fast atom bombardment mass spectrometry. 1H NMR spectroscopy of methyl-H4MPT indicated that the methyl group was bound to a nitrogen atom. Sensitivity of each derivative to oxygen was noted. Apparent extinction coefficients of H4MPT and its derivatives were recorded. Evidence for the enzymatic synthesis of methylene-H4MPT from methenyl-H4MPT+ is presented.     

Studies on lung tumours. III. Oxidative metabolism of dimethylnitrosamine by rodent and human lung tissue.

The oxidative metabolism of the carcinogen dimethylnitrosamine (DMN) was studied in mouse, rat, hamster and human respiratory tissue. [14C]DMN was purified by Dowex-1-bisulfite column chromatography to remove a contaminant (probably [14C]formaldehyde) interfering with the enzyme assay. Since formaldehyde and methyl carbonium ions - yielding methanol with water - are considered to be the primary products of DMN metabolism, tissue slices were assayed for the production of [14C]CO2 from 14C-labelled methanol, formaldehyde, formate, and DMN. Oxidation of formaldehyde to formate was not, but oxidation of formate to CO2 was very much rate-limiting. This rate-limiting step was circumvented by introducing quantitative chemical oxidation of formate to CO2 by mercury(II)chloride following the enzymic reaction. Since oxidation of methanol to CO2 proved to be insignificant, production of CO2 from DMN by lung tissue enzymes and HgCl2 may serve as a parameter for N-demethylating activity and the production of the suspected carcinogenically active methyl carbonium ions. The DMN-N-demethylating activities of lung tissue slices of two mouse strains with widely different susceptibilities to formation of lung adenomas by DMN differed significantly, but the difference seemed too small to explain the divergence in tumourigenic response. The enzymatic activities decreased in hamster bronchus, hamster trachea, hamster lung, GRS/A mouse lung, C3Hf/A mouse lung, human lung, Sprague-Dawley rat lung, in that order. The reported resistance of the hamster respiratory system to tumour induction by DMN may therefore not be due to poor DMN-N-demethylating capacity.     

Metabolism of methylamine in the tea plant (Thea sinensis L.)

1. The metabolism of methylamine in excised shoot tips of tea was studied with micromolar amounts of [(14)C]methylamine. Of the [(14)C]methylamine supplied 57% was utilized by tea shoots during the 10h experimental period. 2. The main products of [(14)C]methylamine metabolism in tea shoots were serine, gamma-glutamylmethylamide, theobromine, caffeine and CO(2). There was also incorporation of the label into glutamate, aspartate, RNA purine nucleotides and S-adenosylmethionine. 3. The formation of methylamine from gamma-glutamylmethylamide was confirmed by feeding tea shoots with gamma-glutamyl[(14)C]methylamide. The products of gamma-glutamyl[(14)C]methylamide metabolism in tea plants were serine, theobromine, caffeine, glutamate and aspartate. 4. The results indicate that the oxidation of methylamine to formaldehyde is the first step of methylamine utilization. Labelled formaldehyde released by the metabolism of methylamine leads to the incorporation of the label into metabolites on the C(1) pathways of this compound. It is also suggested that formaldehyde is further oxidized via formate to CO(2). 5. The role of gamma-glutamylmethylamide in methylamine metabolism in tea plants is discussed. 6. Results support the view that theobromine is the immediate precursor of caffeine.     

How an enzyme binds the C1 carrier tetrahydromethanopterin. Structure of the tetrahydromethanopterin-dependent formaldehyde-activating enzyme (Fae) from Methylobacterium extorquens AM1

Tetrahydromethanopterin (H4 MPT) is a tetrahydrofolate analogue involved as a C1 carrier in the metabolism of various groups of microorganisms. How H4MPT is bound to the respective C1 unit converting enzymes remained elusive. We describe here the structure of the homopentameric formaldehyde-activating enzyme (Fae) from Methylobacterium extorquens AM1 established at 2.0 angstrom without and at 1.9 angstrom with methylene-H4MPT bound. Methylene-H4MPT is bound in an "S"-shaped conformation into the cleft formed between two adjacent subunits. Coenzyme binding is accompanied by side chain rearrangements up to 5 angstrom and leads to a rigidification of the C-terminal arm, a formation of a new hydrophobic cluster, and an inversion of the amide side chain of Gln88. Methylene-H4MPT in Fae shows a characteristic kink between the tetrahydropyrazine and the imidazolidine rings of 70 degrees that is more pronounced than that reported for free methylene-H4MPT in solution (50 degrees). Fae is an essential enzyme for energy metabolism and formaldehyde detoxification of this bacterium and catalyzes the formation of methylene-H4MPT from H4MPT and formaldehyde. The molecular mechanism ofthis reaction involving His22 as acid catalyst is discussed.     

Inhibition of CO2 production from aminopyrine or methanol by cyanamide or crotonaldehyde and the role of mitochondrial aldehyde dehydrogenase in formaldehyde oxidation

Previous results have shown that cyanamide or crotonaldehyde are effective inhibitors of the oxidation of formaldehyde by the low-Km mitochondrial aldehyde dehydrogenase, but do not affect the activity of the glutathione-dependent formaldehyde dehydrogenase. These compounds were used to evaluate the enzyme pathways responsible for the oxidation of formaldehyde generated during the metabolism of aminopyrine or methanol by isolated hepatocytes. Both cyanamide and crotonaldehyde inhibited the production of 14CO2 from 14C-labeled aminopyrine by 30-40%. These agents caused an accumulation of formaldehyde which was identical to the loss in CO2 production, indicating that the inhibition of CO2 production reflected an inhibition of formaldehyde oxidation. The oxidation of methanol was stimulated by the addition of glyoxylic acid, which increases the rate of H2O2 generation. Crotonaldehyde inhibited CO2 production from methanol, but caused a corresponding increase in formaldehyde accumulation. The partial sensitivity of CO2 production to inhibition by cyanamide or crotonaldehyde suggests that both the mitochondrial aldehyde dehydrogenase and formaldehyde dehydrogenase contribute towards the metabolism of formaldehyde which is generated from mixed-function oxidase activity or from methanol, just as both enzyme systems contribute towards the metabolism of exogenously added formaldehyde.     

Formaldehyde metabolism by Escherichia coli. In vivo carbon, deuterium, and two-dimensional NMR observations of multiple detoxifying pathways

13C NMR has been used to demonstrate the metabolism of dilute solutions of labeled formaldehyde by Escherichia coli to methanol, formate, carbon dioxide, and several other unidentified metabolites which contain labeled CH2 groups. Aeration of bacterial suspensions within the spectrometer dramatically increased the rate of oxidation to formate and carbon dioxide. Deoxygenation with nitrogen gas virtually abolished all metabolism, as did the exposure of bacteria to very high formaldehyde concentrations. Deuterium NMR of whole cells in deuterium-depleted water further demonstrated the conversion of formaldehyde-d2 to methanol-d2, ruling out a formaldehyde dismutase as an important species. Two-dimensional proton-carbon chemical shift correlation was used to reveal the chemical shifts of the protons attached to 13C labels in metabolites. The results indicate that formaldehyde is efficiently detoxified by the bacterial cell through a route or routes which do not appear to involve tetrahydrofolate. This detoxification may be in competition with the lethal antibacterial processes associated with formaldehyde.     

Fundamentals of methanogenic pathways that are key to the biomethanation of complex biomass

The conversion of biomass to CH4 (biomethanation) involves an anaerobic microbial food chain composed of at least three metabolic groups of which the first two decompose the complex biomass primarily to acetate, formate, and H2. The thermodynamics of these conversions are unfavorable requiring a symbiosis with the CH4-producing group (methanogens) that metabolize the decomposition products to favorable concentrations. The methanogens produce CH4 by two major pathways, conversion of the methyl group of acetate and reduction of CO2 coupled to the oxidation of formate or H2. This review covers recent advances in the fundamental understanding of both methanogenic pathways with the view of stimulating research towards improving the rate and reliability of the overall biomethanation process.     

Biochemistry of methanogenesis.

Methane is a product of the energy-yielding pathways of the largest and most phylogenetically diverse group in the Archaea. These organisms have evolved three pathways that entail a novel and remarkable biochemistry. All of the pathways have in common a reduction of the methyl group of methyl-coenzyme M (CH3-S-CoM) to CH4. Seminal studies on the CO2-reduction pathway have revealed new cofactors and enzymes that catalyze the reduction of CO2 to the methyl level (CH3-S-CoM) with electrons from H2 or formate. Most of the methane produced in nature originates from the methyl group of acetate. CO dehydrogenase is a key enzyme catalyzing the decarbonylation of acetyl-CoA; the resulting methyl group is transferred to CH3-S-CoM, followed by reduction to methane using electrons derived from oxidation of the carbonyl group to CO2 by the CO dehydrogenase. Some organisms transfer the methyl group of methanol and methylamines to CH3-S-CoM; electrons for reduction of CH3-S-CoM to CH4 are provided by the oxidation of methyl groups to CO2.     

Tetrahydromethanopterin-dependent methanogenesis from non-physiological C1 donors in Methanobacterium thermoautotrophicum.

Methanogenesis from the non-physiological C1 donors thioproline, thiazolidine, hexamethylenetetramine, formaldehyde (HCHO), and HOCH2-S-coenzyme M (CoM) was catalyzed by cell extracts of Methanobacterium thermoautotrophicum under a hydrogen atmosphere. Tetrahydromethanopterin (H4MPT) and HS-CoM were required in the reaction mixture. The non-physiological compounds were found to be in chemical equilibrium with HCHO, which has been shown to react spontaneously with H4MPT to form methylene-H4MPT, an intermediate of the methanogenic pathway at the formaldehyde level of oxidation. Highfield (360 MHZ) 1H and 13C nuclear magnetic resonance studies performed on the interaction between HCHO and HS-CoM showed that these compounds are in equilibrium with HOCH2-S-CoM and that the equilibrium is pH dependent. When methanogenesis from the non-physiological donors was followed under a nitrogen atmosphere, the C1 moiety from each compound underwent a disproportionation, forming methenyl-H4MPT+ and methane. The compounds tested served as substrates for the enzymatic synthesis of methenyl-H4MPT+.     

Energetics of methanogenesis studied in vesicular systems

Methanogenesis is restricted to a group of prokaryotic microorganisms which thrive in strictly anaerobic habitats where they play an indispensable role in the anaerobic food chain. Methanogenic bacteria possess a number of unique cofactors and coenzymes that play an important role in their specialized metabolism. Methanogenesis from a number of simple substrates such as H2 + CO2, formate, methanol, methylamines, and acetate is associated with the generation of transmembrane electrochemical gradients of protons and sodium ions which serve as driving force for a number of processes such as the synthesis of ATP via an ATP synthase, reverse electron transfer, and solute uptake. Several unique reactions of the methanogenic pathways have been identified that are involved in energy transduction. Their role and importance for the methanogenic metabolism are described.     

Methanogenic pathways in Methanosphaera stadtmanae

Methanosphaera stadtmanae reduces methanol to CH4 in a similar way as Methanosarcina barkeri. Low activities of 5,10-methylenetetrahydromethanopterin dehydrogenase (MTDH) and reductase (MTR) were found. From studies on formaldehyde oxidation and reduction it was concluded that most likely the inability to reduce CO2 to CH4 was due to the lack of an active or the presence of an inactive CO2 reductase system and methyltetrahydromethanopterin (methyl-H4MPT): coenzyme M methyltransferase. Methanofuran was not detected, while the presence of a pterin, analogous to H4MPT, could be substantiated from its degradation products in boiled extracts.     

Purine biosynthesis in the domain Archaea without folates or modified folates.

The established pathway for the last two steps in purine biosynthesis, the conversion of 5-aminoimidazole-4-carboxamide ribonucleotide (ZMP) to IMP, is known to utilize 10-formyl-tetrahydrofolate as the required C1 donor cofactor. The biosynthetic conversion of ZMP to IMP in three members of the domain Archaea, Methanobacterium thermoautotrophicum deltaH, M. thermoautotrophicum Marburg, and Sulfolobus solfataricus, however, has been demonstrated to occur with only formate and ATP serving as cofactors. Thus, in these archaea, which use methanopterin (MPT) or another modified folate in place of folate as the C1 carrier coenzyme, neither folate nor a modified folate serves as a cofactor for this biosynthetic transformation. It is concluded that archaea, which function with modified folates such as MPT, are able to carry out purine biosynthesis without the involvement of folates or modified folates.     

Single-carbon catabolism in acetogens: analysis of carbon flow in Acetobacterium woodii and Butyribacterium methylotrophicum by fermentation and 13C nuclear magnetic resonance measurement.

The catabolism of methanol, formate, or carbon monoxide to acetate or butyrate or both was examined in two acetogenic bacteria. Butyribacterium methylotrophicum simultaneously transformed methanol and formate mainly to butyrate with concomitant H2 and CO2 production and consumption. In contrast, methanol plus CO was primarily converted to acetate, and only slight amounts of CO2 were produced. In vivo 13C nuclear magnetic resonance analysis of [13C]methanol transformation by B. methylotrophicum indicated that methanol was predominantly incorporated into the methyl of acetate. 13CO2 was produced and then consumed, and butyrate was formed from the condensation of two acetate precursors. The analysis of the position of acetate labeled by a given 13C single-carbon substrate when B. methylotrophicum or Acetobacterium woodii was grown in the presence of a second one-carbon substrate indicated two trends: when methanol was consumed, CO, CO2, or formate predominantly labeled the acetate carboxyl; when CO was consumed, CO2 and formate were principally funneled into the acetate methyl group, and CO remained a better carboxyl precursor. These data suggest a model of acetate synthesis via the combined operation of two readily reversible single-carbon pathways which are linked by CO2.     

One carbon metabolism in SAR11 pelagic marine bacteria

The SAR11 Alphaproteobacteria are the most abundant heterotrophs in the oceans and are believed to play a major role in mineralizing marine dissolved organic carbon. Their genomes are among the smallest known for free-living heterotrophic cells, raising questions about how they successfully utilize complex organic matter with a limited metabolic repertoire. Here we show that conserved genes in SAR11 subgroup Ia (Candidatus Pelagibacter ubique) genomes encode pathways for the oxidation of a variety of one-carbon compounds and methyl functional groups from methylated compounds. These pathways were predicted to produce energy by tetrahydrofolate (THF)-mediated oxidation, but not to support the net assimilation of biomass from C1 compounds. Measurements of cellular ATP content and the oxidation of (14)C-labeled compounds to (14)CO(2) indicated that methanol, formaldehyde, methylamine, and methyl groups from glycine betaine (GBT), trimethylamine (TMA), trimethylamine N-oxide (TMAO), and dimethylsulfoniopropionate (DMSP) were oxidized by axenic cultures of the SAR11 strain Ca. P. ubique HTCC1062. Analyses of metagenomic data showed that genes for C1 metabolism occur at a high frequency in natural SAR11 populations. In short term incubations, natural communities of Sargasso Sea microbial plankton expressed a potential for the oxidation of (14)C-labeled formate, formaldehyde, methanol and TMAO that was similar to cultured SAR11 cells and, like cultured SAR11 cells, incorporated a much larger percentage of pyruvate and glucose (27-35%) than of C1 compounds (2-6%) into biomass. Collectively, these genomic, cellular and environmental data show a surprising capacity for demethylation and C1 oxidation in SAR11 cultures and in natural microbial communities dominated by SAR11, and support the conclusion that C1 oxidation might be a significant conduit by which dissolved organic carbon is recycled to CO(2) in the upper ocean.