Rich Boden

Stable-isotope probing implicates Methylophaga spp and novel Gammaproteobacteria in marine methanol and methylamine metabolism

The metabolism of one-carbon (C1) compounds in the marine environment affects global warming, seawater ecology and atmospheric chemistry. Despite their global significance, marine microorganisms that consume C1 compounds in situ remain poorly characterized. Stable-isotope probing (SIP) is an ideal tool for linking the function and phylogeny of methylotrophic organisms by the metabolism and incorporation of stable-isotope-labelled substrates into nucleic acids. By combining DNA-SIP and time-series sampling, we characterized the organisms involved in the assimilation of methanol and methylamine in coastal sea water (Plymouth, UK). Labelled nucleic acids were analysed by denaturing gradient gel electrophoresis (DGGE) and clone libraries of 16S rRNA genes. In addition, we characterized the functional gene complement of labelled nucleic acids with an improved primer set targeting methanol dehydrogenase (mxaF) and newly designed primers for methylamine dehydrogenase (mauA). Predominant DGGE phylotypes, 16S rRNA, methanol and methylamine dehydrogenase gene sequences, and cultured isolates all implicated Methylophaga spp, moderately halophilic marine methylotrophs, in the consumption of both methanol and methylamine. Additionally, an mxaF sequence obtained from DNA extracted from sea water clustered with those detected in 13C-DNA, suggesting a predominance of Methylophaga spp among marine methylotrophs. Unexpectedly, most predominant 16S rRNA and functional gene sequences from 13C-DNA were clustered in distinct substrate-specific clades, with 16S rRNA genes clustering with sequences from the Gammaproteobacteria. These clades have no cultured representatives and reveal an ecological adaptation of particular uncultured methylotrophs to specific C1 compounds in the coastal marine environment.

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.

Microbial degradation of dimethylsulphide and related C1-sulphur compounds: organisms and pathways controlling fluxes of sulphur in the biosphere

Dimethylsulphide (DMS) plays a major role in the global sulphur cycle. It has important implications for atmospheric chemistry, climate regulation, and sulphur transport from the marine to the atmospheric and terrestrial environments. In addition, DMS acts as an info-chemical for a wide range of organisms ranging from micro-organisms to mammals. Micro-organisms that cycle DMS are widely distributed in a range of environments, for instance, oxic and anoxic marine, freshwater and terrestrial habitats. Despite the importance of DMS that has been unearthed by many studies since the early 1970s, the understanding of the biochemistry, genetics, and ecology of DMS-degrading micro-organisms is still limited. This review examines current knowledge on the microbial cycling of DMS and points out areas for future research that should shed more light on the role of organisms degrading DMS and related compounds in the biosphere.

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

ABSTRACT Marine microorganisms that consume one-carbon (C1) 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 C1 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 13C-labeled methanol, monomethylamine, dimethylamine, methyl bromide, and dimethyl sulfide to identify microbial populations involved in the turnover of C1 compounds, using DNA stable isotope probing. The [13C]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 13C-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 C1 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 13C-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.

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 13C-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.

Complete Genome Sequence of the Aerobic Marine Methanotroph Methylomonas methanica MC09

Methylomonas methanica MC09 is a mesophilic, halotolerant, aerobic, methanotrophic member of the Gammaproteobacteria, isolated from coastal seawater. Here we present the complete genome sequence of this strain, the first available from an aerobic marine methanotroph.

Novel methylotrophic bacteria isolated from the River Thames (London, UK)

Enrichment and elective culture for methylotrophs from sediment of the River Thames in central London yielded a diversity of pure cultures representing several genera of Gram-negative and Gram-positive bacteria, which were mainly of organisms not generally regarded as typically methylotrophic. Substrates leading to successful isolations included methanol, monomethylamine, dimethylamine, trimethylamine, methanesulfonate and dimethylsulfone. Several isolates were studied in detail and shown by their biochemical and morphological properties and 16S rRNA gene sequencing to be Sphingomonas melonis strain ET35, Mycobacterium fluoranthenivorans strain DSQ3, Rhodococcus erythropolis strain DSQ4, Brevibacterium casei strain MSQ5, Klebsiella oxytoca strains MMA/F and MMA/1, Pseudomonas mendocina strain TSQ4, and Flavobacterium sp. strains MSA/1 and MMA/2. The results show that facultative methylotrophy is present across a wide range of Bacteria, suggesting that turnover of diverse C(1)-compounds is of much greater microbiological and environmental significance than is generally thought. The origins of the genes encoding the enzymes of methylotrophy in diverse heterotrophs need further study, and could further our understanding of the phylogeny and antiquity of methylotrophic systems.

Methylophaga lonarensis sp. nov., a moderately haloalkaliphilic methylotroph isolated from the soda lake sediments of a meteorite impact crater

A moderately haloalkaliphilic methylotrophic bacterium possessing the ribulose monophosphate pathway for carbon assimilation, designated MPL(T), was isolated from Lonar Lake sediment microcosms that were oxidizing methane for two weeks. The isolate utilized methanol and was an aerobic, Gram-negative, asporogenous, motile, short rod that multiplied by binary fission. The isolate required NaHCO(3) or NaCl for growth and, although not auxotrophic for vitamin B(12), had enhanced growth with vitamin B(12). Optimal growth occurred with 0.5-2% (w/v) NaCl, at 28-30 °C and at pH 9.0-10.0. The cellular fatty acid profile consisted primarily of straight-chain saturated C(16:0) and unsaturated C(16:1)ω7c and C(18:1)ω7c. The major ubiquinone was Q-8. The dominant phospholipids were phosphatidylethanolamine, phosphatidylglycerol and diphosphatidylglycerol. Cells accumulated ectoine as the main compatible solute. The DNA G+C content was 50.0 mol%. The isolate exhibited 94.0-95.4% 16S rRNA gene sequence similarity with the type strains of methylotrophs belonging to the genus Methylophaga and 31% DNA-DNA relatedness with the reference strain, Methylophaga alcalica VKM B-2251(T). It is proposed that strain MPL(T) represents a novel species, Methylophaga lonarensis sp. nov. (type strain MPL(T)=VKM B-2684(T)=MCC 1002(T)).

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.

Purification and Characterization of Dimethylsulfide Monooxygenase from Hyphomicrobium sulfonivorans

Dimethylsulfide (DMS) is a volatile organosulfur compound which has been implicated in the biogeochemical cycling of sulfur and in climate control. Microbial degradation is a major sink for DMS. DMS metabolism in some bacteria involves its oxidation by a DMS monooxygenase in the first step of the degradation pathway; however, this enzyme has remained uncharacterized until now. We have purified a DMS monooxygenase from Hyphomicrobium sulfonivorans, which was previously isolated from garden soil. The enzyme is a member of the flavin-linked monooxygenases of the luciferase family and is most closely related to nitrilotriacetate monooxygenases. It consists of two subunits: DmoA, a 53-kDa FMNH₂-dependent monooxygenase, and DmoB, a 19-kDa NAD(P)H-dependent flavin oxidoreductase. Enzyme kinetics were investigated with a range of substrates and inhibitors. The enzyme had a K(m) of 17.2 (± 0.48) μM for DMS (k(cat) = 5.45 s⁻¹) and a V(max) of 1.25 (± 0.01) μmol NADH oxidized min⁻¹ (mg protein⁻¹). It was inhibited by umbelliferone, 8-anilinonaphthalenesulfonate, a range of metal-chelating agents, and Hg²(+), Cd²(+), and Pb²(+) ions. The purified enzyme had no activity with the substrates of related enzymes, including alkanesulfonates, aldehydes, nitrilotriacetate, or dibenzothiophenesulfone. The gene encoding the 53-kDa enzyme subunit has been cloned and matched to the enzyme subunit by mass spectrometry. DMS monooxygenase represents a new class of FMNH₂-dependent monooxygenases, based on its specificity for dimethylsulfide and the molecular phylogeny of its predicted amino acid sequence. The gene encoding the large subunit of DMS monooxygenase is colocated with genes encoding putative flavin reductases, homologues of enzymes of inorganic and organic sulfur compound metabolism, and enzymes involved in riboflavin synthesis.