One-carbon Compounds Research Articles

Sensing and signaling for peroxisome autophagic degradation (pexophagy) in yeasts

yeast cells, similarly to cells of other eukaryotic organisms, possess intracellular organelles, including that of peroxisomes also known as microbodies . Enzymes of oxidative metabolism, mainly hydrogen peroxide generating oxidases, catalase, some enzymes of glyoxylic cycle and enzymes involved in catabolism of unusual carbon sources (n-alkanes, methanol) are located in peroxisomes. Especially important role is played by peroxisomes in methylotrophic yeasts, unique eukaryotic organisms capable to utilize one-carbon compound, methanol. Active proliferation and biogenesis of peroxisomes occur on methanol, so these organelles can occupy between 30 and 80% of cellular volume. After shift of methanol-grown cells into media with multicarbon substrates, such as glucose or ethanol, an excess of peroxisomes degrades in the specific process known as autophagic degradation of peroxisomes or pexophagy. There are 36 AuTophaGy related genes, known as ATG genes, which products are also involved in pexophagy. At the same time, not much is known on mechanisms of glucose and ethanol sensing and signaling which initia te pexophagy process. Proteins Pfk1(α-subunit of phosphofructokinase), Slt2 (mitogen-activating protein kinase) Gpr1 and Gpa2 (components of GPCr system) and Snf3 and Ggt2 (highand low-affinity glucose sensors) were found to be involved in signaling of glucose-induced pexophagy in Saccharomyces cerevisiae. In the methylotrophic yeast Pichia pastoris, glucose sensing protein Gss1 was found to be important for glucose-induced pexophagy. Very few is known on mechanisms of ethanol sensing and signaling during pexophagy which is an important problem for future studies.

SIGN EPISTASIS LIMITS EVOLUTIONARY TRADE-OFFS AT THE CONFLUENCE OF SINGLE- AND MULTI-CARBON METABOLISM INMETHYLOBACTERIUM EXTORQUENSAM1

Adaptation of one set of traits is often accompanied by attenuation of traits important in other selective environments, leading to fitness trade-offs. The mechanisms that either promote or prevent the emergence of trade-offs remain largely unknown, and are difficult to discern in most systems. Here, we investigate the basis of trade-offs that emerged during experimental evolution of Methylobacterium extorquens AM1 to distinct growth substrates. After 1500 generations of adaptation to a multi-carbon substrate, succinate (S), many lineages had lost the ability to use one-carbon compounds such as methanol (M), generating a mixture of M(+) and M(-) evolved phenotypes. We show that trade-offs in M(-) strains consistently arise via antagonistic pleiotropy through recurrent selection for loss-of-function mutations to ftfL (formate-tetrahydrofolate ligase), which improved growth on S while simultaneously eliminating growth on M. But if loss of FtfL was beneficial, why were M trade-offs not found in all populations? We discovered that eliminating FtfL was not universally beneficial on S, as it was neutral or even deleterious in certain evolved lineages that remained M(+) . This suggests that sign epistasis with earlier arising mutations prevented the emergence of mutations that drove trade-offs through antagonistic pleiotropy, limiting the evolution of metabolic specialists in some populations.

Fluorescence-Based Bacterial Bioreporter for Specific Detection of Methyl Halide Emissions in the Environment

Methyl halides are volatile one-carbon compounds responsible for substantial depletion of stratospheric ozone. Among them, chloromethane (CH3Cl) is the most abundant halogenated hydrocarbon in the atmosphere. Global budgets of methyl halides in the environment are still poorly understood due to uncertainties in their natural sources, mainly from vegetation, and their sinks, which include chloromethane-degrading bacteria. A bacterial bioreporter for the detection of methyl halides was developed on the basis of detailed knowledge of the physiology and genetics of Methylobacterium extorquens CM4, an aerobic alphaproteobacterium which utilizes chloromethane as the sole source of carbon and energy. A plasmid construct with the promoter region of the chloromethane dehalogenase gene cmuA fused to a promotorless yellow fluorescent protein gene cassette resulted in specific methyl halide-dependent fluorescence when introduced into M. extorquens CM4. The bacterial whole-cell bioreporter allowed detection of methyl halides at femtomolar levels and quantification at concentrations above 10 pM (approximately 240 ppt). As shown for the model chloromethane-producing plant Arabidopsis thaliana in particular, the bioreporter may provide an attractive alternative to analytical chemical methods to screen for natural sources of methyl halide emissions.

The unique metabolism of SAR11 aquatic bacteria

The deeply branching clade of abundant, globally distributed aquatic α-Proteobacteria known as "SAR11", are adapted to nutrient-poor environments such as the surface waters of the open ocean. Unknown prior to 1990, uncultured until 2002, members of the SAR11 clade can now be cultured in artificial, defined media to densities three orders of magnitude higher than in unamended natural media. Cultivation in natural and defined media has confirmed genomic and metagenomic predictions such as an inability to reduce sulfate to sulfide, a requirement for pyruvate, an ability to oxidize a wide variety of methylated and one-carbon compounds for energy, and an unusual form of conditional glycine auxotrophy. Here we describe the metabolism of the SAR11 type strain Candidatus "Pelagibacter ubique" str. HTCC1062, as revealed by genome-assisted studies of laboratory cultures. We also describe the discovery of SAR11 and field studies that have been done on natural populations.

One-carbon metabolism: An aging-cancer crossroad for the gerosuppressant metformin

The gerosuppressant metformin operates as an efficient inhibitor of the mTOR/S6K1 gerogenic pathway due to its ability to ultimately activate the energy-sensor AMPK. If an aging-related decline in the AMPK sensitivity to cellular stress is a crucial event for mTOR-driven aging and aging-related diseases, including cancer, unraveling new proximal causes through which AMPK activation endows its gerosuppressive effects may offer not only a better understanding of metformin function but also the likely possibility of repositioning our existing gerosuppressant drugs. Here we provide our perspective on recent findings suggesting that de novo biosynthesis of purine nucleotides, which is based on the metabolism of one-carbon compounds, is a new target for metformin's actions at the crossroads of aging and cancer.

Draft Genome Sequence of the Methane-Oxidizing Bacterium Methylococcus capsulatus (Texas)

Methanotrophic bacteria perform major roles in global carbon cycles via their unique enzymatic activities that enable the oxidation of one-carbon compounds, most notably methane. Here we describe the annotated draft genome sequence of the aerobic methanotroph Methylococcus capsulatus (Texas), a type strain originally isolated from sewer sludge.

Regulation of the Histidine Utilization (Hut) System in Bacteria

The ability to degrade the amino acid histidine to ammonia, glutamate, and a one-carbon compound (formate or formamide) is a property that is widely distributed among bacteria. The four or five enzymatic steps of the pathway are highly conserved, and the chemistry of the reactions displays several unusual features, including the rearrangement of a portion of the histidase polypeptide chain to yield an unusual imidazole structure at the active site and the use of a tightly bound NAD molecule as an electrophile rather than a redox-active element in urocanase. Given the importance of this amino acid, it is not surprising that the degradation of histidine is tightly regulated. The study of that regulation led to three central paradigms in bacterial regulation: catabolite repression by glucose and other carbon sources, nitrogen regulation and two-component regulators in general, and autoregulation of bacterial regulators. This review focuses on three groups of organisms for which studies are most complete: the enteric bacteria, for which the regulation is best understood; the pseudomonads, for which the chemistry is best characterized; and Bacillus subtilis, for which the regulatory mechanisms are very different from those of the Gram-negative bacteria. The Hut pathway is fundamentally a catabolic pathway that allows cells to use histidine as a source of carbon, energy, and nitrogen, but other roles for the pathway are also considered briefly here.

Protease inhibitors: Silver bullets for chronic hepatitis C infection?

Recent trials evaluated the safety and efficacy of two protease inhibitors, boceprevir (Victrelis) and telaprevir (Incivek), added to standard care with pegylated interferon and ribavirin, in patients with chronic hepatitis C virus (HCV) infection. These drugs open the door for triple therapy and other new therapies involving combinations of other direct-acting antiviral agents to become the new standard of care for this population.

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 14C-labeled compounds to 14CO2 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 14C-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 CO2 in the upper ocean.

Methylorosula polaris gen. nov., sp. nov., an aerobic, facultatively methylotrophic psychrotolerant bacterium from tundra wetland soil

Three strains of Gram-negative, aerobic, motile bacteria with bipolar flagella were isolated from acidic tundra wetland soils near the city of Vorkuta and from the Chukotka and Yugorsky Peninsulas and designated strains V-022(T), Ch-022 and Ju-022. The cells were rod-shaped, 0.5-0.6 µm in width and 1.3-4.5 µm in length and reproduced by irregular fission. These bacteria were facultative methylotrophs that used methanol, methylamines and a wide range of other sources of carbon and energy such as sugars and polysaccharides, ethanol and amino acids. The isolates used the Calvin-Benson pathway for the assimilation of one-carbon compounds and were unable to fix nitrogen. The new strains were moderately acidophilic and psychrotolerant, capable of growth over a pH range of 4.0 to 7.8, with optimum growth at pH 5.5-6.0. Growth occurred between 4 and 30 °C (optimum 20-25 °C). The principal phospholipid fatty acid was C(18:1)ω7c. The DNA G+C content of strain V-022(T) was 65.2 mol%. Analysis of the 16S rRNA gene sequences revealed that all three isolates V-022(T), Ch-022 and Yu-022 exhibited almost identical 16S rRNA gene sequences (99.9% gene sequence similarity) and formed a new lineage within the class Alphaproteobacteria. The name Methylorosula polaris is suggested to accommodate this new genus and novel species with strain V-022(T) (=DSM 22001(T)=VKM V-2485(T)) as the type strain of the type species.

Draft Genome Sequence of the Chemolithoheterotrophic, Halophilic Methylotroph Methylophaga thiooxydans DMS010

Methylophaga thiooxydans is a mesophilic, obligately halophilic bacterium that is capable of methylotrophic growth on a range of one-carbon compounds as well as chemolithoheterotrophic growth at the expense of thiosulfate. Here we present the draft genome sequence of Methylophaga thiooxydans DMS010 (DSM 22068(T), VKM B2586(T)), the type strain of the species, which has allowed prediction of the genes involved in one-carbon metabolism, nitrogen metabolism, and other aspects of central metabolism.

Facultative methylotrophs from the human oral cavity and methylotrophy in strains of Gordonia, Leifsonia, and Microbacterium

We show that bacteria with methylotrophic potential are ubiquitous in the human mouth microbiota. Numerous strains of Actinobacteria (Brevibacterium, Gordonia, Leifsonia, Microbacterium, Micrococcus, Rhodococcus) and Proteobacteria (Achromobacter, Klebsiella, Methylobacterium, Pseudomonas, Ralstonia) were isolated, and one strain of each of the eleven genera was studied in detail. These strains expressed enzymes associated with methylotrophic metabolism (methanol, methylamine, and formate dehydrogenases), and the assimilation of one-carbon compounds by the serine pathway (hydroxypyruvate reductase). Methylotrophic growth of the strains was enhanced by the addition of glass beads to cultures, suggesting that they may naturally occur in biofilms in the mouth. This is the first report of Gordonia, Leifsonia, and Rhodococcus being present in the mouth and of the unequivocal demonstration for the first time of the methylotrophic potential of strains of Gordonia, Leifsonia, and Microbacterium.

Transcriptional response of hepatic largemouth bass (Micropterus salmoides) mRNA upon exposure to environmental contaminants

Microarrays enable gene transcript expression changes in near-whole genomes to be assessed in response to environmental stimuli. We utilized oligonucleotide microarrays and subsequent gene set enrichment analysis (GSEA) to assess patterns of gene expression changes in male largemouth bass (Micropterus salmoides) hepatic tissues after a 96 h exposure to common environmental contaminants. Fish were exposed to atrazine, cadmium chloride, PCB 126, phenanthrene and toxaphene via intraperitoneal injection with target body burdens of 3.0, 0.00067, 2.5, 50 and 100 µg g(-1), respectively. This was conducted in an effort to identify potential biomarkers of exposure. The expressions of 4, 126, 118, 137 and 58 mRNA transcripts were significantly (P ≤ 0.001, fold change ≥2×) affected by exposure to atrazine, cadmium chloride, PCB 126, phenanthrene and toxaphene exposures, respectively. GSEA revealed that none, four, five, five and three biological function gene ontology categories were significantly influenced by exposure to these chemicals, respectively. We observed that cadmium chloride elicited ethanol metabolism responses, and along with PCB 126 and phenanthrene affected transcripts associated with protein biosynthesis. PCB 126, phenanthrene and toxaphene also influenced one-carbon compound metabolism while PCB 126 and phenanthrene affected mRNA transcription and mRNA export from the nucleus and may have induced an antiestrogenic response. Atrazine was found to alter the expression of few hepatic transcripts. This work has highlighted several biological processes of interest that may be helpful in the development of gene transcript biomarkers of chemical exposure in fish.

Environmental proteomics of microbial plankton in a highly productive coastal upwelling system

Metaproteomics is one of a suite of new approaches providing insights into the activities of microorganisms in natural environments. Proteins, the final products of gene expression, indicate cellular priorities, taking into account both transcriptional and posttranscriptional control mechanisms that control adaptive responses. Here, we report the proteomic composition of the < 1.2 μm fraction of a microbial community from Oregon coast summer surface waters, detected with two-dimensional liquid chromatography coupled with electrospray tandem mass spectrometry. Spectra corresponding to proteins involved in protein folding and biosynthesis, transport, and viral capsid structure were the most frequently detected. A total of 36% of all the detected proteins were best matches to the SAR11 clade, and other abundant coastal microbial clades were also well represented, including the Roseobacter clade (17%), oligotrophic marine gammaproteobacteria group (6%), OM43 clade (1%). Viral origins were attributed to 2.5% of proteins. In contrast to oligotrophic waters, phosphate transporters were not highly detected in this nutrient-rich system. However, transporters for amino acids, taurine, polyamines and glutamine synthetase were among the most highly detected proteins, supporting predictions that carbon and nitrogen are more limiting than phosphate in this environment. Intriguingly, one of the highly detected proteins was methanol dehydrogenase originating from the OM43 clade, providing further support for recent reports that the metabolism of one-carbon compounds by these streamlined methylotrophs might be an important feature of coastal ocean biogeochemistry.

Methanol Assimilation in Methylobacterium extorquens AM1: Demonstration of All Enzymes and Their Regulation

Background Methylobacterium extorquens AM1 is an aerobic facultative methylotrophic α-proteobacterium that can use reduced one-carbon compounds such as methanol, but also multi-carbon substrates like acetate (C2) or succinate (C4) as sole carbon and energy source. The organism has gained interest as future biotechnological production platform based on methanol as feedstock.Methodology/Principal FindingsWe present a comprehensive study of all postulated enzymes for the assimilation of methanol and their regulation in response to the carbon source. Formaldehyde, which is derived from methanol oxidation, is assimilated via the serine cycle, which starts with glyoxylate and forms acetyl-CoA. Acetyl-CoA is assimilated via the proposed ethylmalonyl-CoA pathway, which thereby regenerates glyoxylate. To further the understanding of the central carbon metabolism we identified and quantified all enzymes of the pathways involved in methanol assimilation. We observed a strict differential regulation of their activity level depending on whether C1, C2 or C4 compounds are used. The enzymes, which are specifically required for the utilization of the individual substrates, were several-fold up-regulated and those not required were down-regulated. The enzymes of the ethylmalonyl-CoA pathway showed specific activities, which were higher than the calculated minimal values that can account for the observed growth rate. Yet, some enzymes of the serine cycle, notably its first and last enzymes serine hydroxymethyl transferase and malate thiokinase, exhibit much lower values and probably are rate limiting during methylotrophic growth. We identified the natural C1 carrying coenzyme as tetrahydropteroyl-tetraglutamate rather than tetrahydrofolate.Conclusion/SignificanceThis study provides the first complete picture of the enzymes required for methanol assimilation, the regulation of their activity levels in response to the growth substrate, and the identification of potential growth limiting steps.

Active methylotrophs in the sediments of Lonar Lake, a saline and alkaline ecosystem formed by meteor impact

Lonar Lake is a unique saline and alkaline ecosystem formed by meteor impact in the Deccan basalts in India around 52,000 years ago. To investigate the role of methylotrophy in the cycling of carbon in this unusual environment, stable-isotope probing (SIP) was carried out using the one-carbon compounds methane, methanol and methylamine. Denaturing gradient gel electrophoresis fingerprinting analyses performed with heavy (13)C-labelled DNA retrieved from sediment microcosms confirmed the enrichment and labelling of active methylotrophic communities. Clone libraries were constructed using PCR primers targeting 16S rRNA genes and functional genes. Methylomicrobium, Methylophaga and Bacillus spp. were identified as the predominant active methylotrophs in methane, methanol and methylamine SIP microcosms, respectively. Absence of mauA gene amplification in the methylamine SIP heavy fraction also indicated that methylamine metabolism in Lonar Lake sediments may not be mediated by the methylamine dehydrogenase enzyme pathway. Many gene sequences retrieved in this study were not affiliated with extant methanotrophs or methylotrophs. These sequences may represent hitherto uncharacterized novel methylotrophs or heterotrophic organisms that may have been cross-feeding on methylotrophic metabolites or biomass. This study represents an essential first step towards understanding the relevance of methylotrophy in the soda lake sediments of an unusual impact crater structure.

The microbial methane cycle

This special issue highlights several recent discoveries in the microbial methane cycle, including the diversity and activity of methanotrophic bacteria in special habitats, distribution and contribution of the newly discovered Verrucomicrobia, metabolism of methane and related one-carbon compounds such as methanol and methylamine in freshwater and marine environments, methanol and methane-dependent nitrate reduction, the relationships of methane cycle microorganisms with plants and animals, and the environmental factors that regulate microbial processes of the methane cycle. These articles also highlight the plethora of new organisms and metabolism relating to the methane cycle that have been discovered in recent years and outline the many questions in the methane cycle that still need to be addressed. It is clear that despite a tremendous amount of research on the biology of the methane cycle, the microbes involved in catalysing methane production and consumption harbour many secrets that need to be disclosed in order for us to fully understand how the biogeochemical methane cycle is regulated in the environment, and for us to make future predictions about the global sources and sinks of methane and how anthropogenic changes impact on the cycling of this important greenhouse gas.

Life without light: microbial diversity and evidence of sulfur- and ammonium-based chemolithotrophy in Movile Cave

Microbial diversity in Movile Cave (Romania) was studied using bacterial and archaeal 16S rRNA gene sequence and functional gene analyses, including ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO), soxB (sulfate thioesterase/thiohydrolase) and amoA (ammonia monooxygenase). Sulfur oxidizers from both Gammaproteobacteria and Betaproteobacteria were detected in 16S rRNA, soxB and RuBisCO gene libraries. DNA-based stable-isotope probing analyses using 13C-bicarbonate showed that Thiobacillus spp. were most active in assimilating CO2 and also implied that ammonia and nitrite oxidizers were active during incubations. Nitrosomonas spp. were detected in both 16S rRNA and amoA gene libraries from the 'heavy' DNA and sequences related to nitrite-oxidizing bacteria Nitrospira and Candidatus 'Nitrotoga' were also detected in the 'heavy' DNA, which suggests that ammonia/nitrite oxidation may be another major primary production process in this unique ecosystem. A significant number of sequences associated with known methylotrophs from the Betaproteobacteria were obtained, including Methylotenera, Methylophilus and Methylovorus, supporting the view that cycling of one-carbon compounds may be an important process within Movile Cave. Other sequences detected in the bacterial 16S rRNA clone library included Verrucomicrobia, Firmicutes, Bacteroidetes, alphaproteobacterial Rhodobacterales and gammaproteobacterial Xanthomonadales. Archaeal 16S rRNA sequences retrieved were restricted within two groups, namely the Deep-sea Hydrothermal Vent Euryarchaeota group and the Miscellaneous Crenarchaeotic group. No sequences related to known sulfur-oxidizing archaea, ammonia-oxidizing archaea, methanogens or anaerobic methane-oxidizing archaea were detected in this clone library. The results provided molecular biological evidence to support the hypothesis that Movile Cave is driven by chemolithoautotrophy, mainly through sulfur oxidation by sulfur-oxidizing bacteria and reveal that ammonia- and nitrite-oxidizing bacteria may also be major primary producers in Movile Cave.

Demonstration of the ethylmalonyl-CoA pathway by using 13 C metabolomics

The assimilation of one-carbon (C1) compounds, such as methanol, by serine cycle methylotrophs requires the continuous regeneration of glyoxylate. Instead of the glyoxylate cycle, this process is achieved by a not yet established pathway where CoA thioesters are known to play a key role. We applied state-of-the-art metabolomics and (13)C metabolomics strategies to demonstrate how glyoxylate is generated during methylotrophic growth in the isocitrate lyase-negative methylotroph Methylobacterium extorquens AM1. High-resolution mass spectrometry showed the presence of CoA thioesters specific to the recently proposed ethylmalonyl-CoA pathway. The operation of this pathway was demonstrated by short-term (13)C-labeling experiments, which allowed determination of the sequence of reactions from the order of label incorporation into the different CoA derivatives. Analysis of (13)C positional enrichment in glycine by NMR was consistent with the predicted labeling pattern as a result of the operation of the ethylmalonyl-CoA pathway and the unique operation of the latter for glyoxylate generation during growth on methanol. The results also revealed that 2 molecules of glyoxylate were regenerated in this process. This work provides a complete pathway for methanol assimilation in the model methylotroph M. extorquens AM1 and represents an important step toward the determination of the overall topology of its metabolic network. The operation of the ethylmalonyl-CoA pathway in M. extorquens AM1 has major implications for the physiology of these methylotrophs and their role in nature, and it also provides a common ground for C1 and C2 compound assimilation in isocitrate lyase-negative bacteria.

Substrate-Specific Clades of Active Marine Methylotrophs Associated with a Phytoplankton Bloom in a Temperate Coastal Environment

Marine microorganisms that consume one-carbon (C(1)) compounds are poorly described, despite their impact on global climate via an influence on aquatic and atmospheric chemistry. This study investigated marine bacterial communities involved in the metabolism of C(1) compounds. These communities were of relevance to surface seawater and atmospheric chemistry in the context of a bloom that was dominated by phytoplankton known to produce dimethylsulfoniopropionate. In addition to using 16S rRNA gene fingerprinting and clone libraries to characterize samples taken from a bloom transect in July 2006, seawater samples from the phytoplankton bloom were incubated with (13)C-labeled methanol, monomethylamine, dimethylamine, methyl bromide, and dimethyl sulfide to identify microbial populations involved in the turnover of C(1) compounds, using DNA stable isotope probing. The [(13)C]DNA samples from a single time point were characterized and compared using denaturing gradient gel electrophoresis (DGGE), fingerprint cluster analysis, and 16S rRNA gene clone library analysis. Bacterial community DGGE fingerprints from (13)C-labeled DNA were distinct from those obtained with the DNA of the nonlabeled community DNA and suggested some overlap in substrate utilization between active methylotroph populations growing on different C(1) substrates. Active methylotrophs were affiliated with Methylophaga spp. and several clades of undescribed Gammaproteobacteria that utilized methanol, methylamines (both monomethylamine and dimethylamine), and dimethyl sulfide. rRNA gene sequences corresponding to populations assimilating (13)C-labeled methyl bromide and other substrates were associated with members of the Alphaproteobacteria (e.g., the family Rhodobacteraceae), the Cytophaga-Flexibacter-Bacteroides group, and unknown taxa. This study expands the known diversity of marine methylotrophs in surface seawater and provides a comprehensive data set for focused cultivation and metagenomic analyses in the future.