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Abstract Background Bacterial protein biosynthesis usually depends on a formylated methionyl start tRNA but Staphylococcus aureus is viable in the absence of Fmt, the tRNAMet formyl transferase. fmt mutants exhibit reduced growth rates indicating that the function of certain proteins depends on formylated N-termini but it has remained unclear, which cellular processes are abrogated by the lack of formylation. Results In order to elucidate how global metabolic processes are affected by the absence of formylated proteins the exometabolome of an S. aureus fmt mutant was compared with that of the parental strain and the transcription of corresponding enzymes was analyzed to identify possible regulatory changes. The mutant consumed glucose and other carbon sources slower than the wild type. While the turnover of several metabolites remained unaltered fmt inactivation led to increases pyruvate release and, concomitantly, reduced pyruvate dehydrogenase activity. In parallel, the release of the pyruvate-derived metabolites lactate, acetoin, and alanine was reduced. The anaerobic degradation of arginine was also reduced in the fmt mutant compared to the wild-type strain. Moreover, the lack of formylated proteins caused increased susceptibility to the antibiotics trimethoprim and sulamethoxazole suggesting that folic acid-dependant pathways were perturbed in the mutant. Conclusions These data indicate that formylated proteins are crucial for specific bacterial metabolic processes and they may help to understand why it has remained important during bacterial evolution to initiate protein biosynthesis with a formylated tRNAMet.

Abstract Background Pseudoalteromonas haloplanktis is a cold-adapted γ-proteobacterium isolated from Antarctic sea ice. It is characterized by remarkably high growth rates at low temperatures. P. haloplanktis is one of the model organisms of cold-adapted bacteria and has been suggested as an alternative host for the soluble overproduction of heterologous proteins which tend to form inclusion bodies in established expression hosts. Despite the progress in establishing P. haloplanktis as an alternative expression host the cell densities obtained with this organism, which is unable to use glucose as a carbon source, are still low. Here we present the first fed-batch cultivation strategy for this auspicious alternative expression host. Results The key for the fed-batch cultivation of P. haloplanktis was the replacement of peptone by casamino acids, which have a much higher solubility and allow a better growth control. In contrast to the peptone medium, on which P. haloplanktis showed different growth phases, on a casamino acids-containing, phosphate-buffered medium P. haloplanktis grew exponentially with a constant growth rate until the stationary phase. A fed-batch process was established by feeding of casamino acids with a constant rate resulting in a cell dry weight of about 11 g l-1 (OD540 = 28) which is a twofold increase of the highest densities which have been obtained with P. haloplanktis so far and an eightfold increase of the density obtained in standard shake flask cultures. The cell density was limited in the fed-batch cultivation by the relatively low solubility of casamino acids (about 100 g l-1), which was proven by pulse addition of casamino acid powder which increased the cell density to about 20 g l-1 (OD540 = 55). Conclusion The growth of P. haloplanktis to higher cell densities on complex medium is possible. A first fed-batch fermentation strategy could be established which is feasible to be used in lab-scale or for industrial purposes. The substrate concentration of the feeding solution was found to influence the maximal biomass yield considerably. The bottleneck for growing P. haloplanktis to high cell densities still remains the availability of a highly concentrated substrate and the reduction of the substrate complexity. However, our results indicate glutamic acid as a major carbon source, which provides a good basis for further improvement of the fed-batch process.

Brown macroalgae synthesize large amounts of fucoidans, sulfated fucose-containing polysaccharides, in the ocean. Fucoidans are of importance for their recently discovered contribution to marine carbon dioxide sequestration and due to their potential applications in biotechnology and biomedicine. However, fucoidans have high intra- and intermolecular diversity that challenges assignment of structure to biological function and the development of applications. Fucoidan-active enzymes may be used to simplify this diversity by producing defined oligosaccharides more applicable for structural refinement, characterization, and structure to function assignment for example via bioassays. In this study, we combined MALDI mass spectrometry with biocatalysis to show that the endo-fucoidanases P5AFcnA and Wv323 can produce defined oligosaccharide structures directly from unrefined macroalgal biomass. P5AFcnA released oligosaccharides from seven commercial fucoidan extracts in addition to unrefined biomass of three macroalgae species indicating a broadly applicable approach reproducible across 10 species. Both MALDI-TOF/TOF and AP-MALDI-Orbitrap systems were used, demonstrating that the approach is not instrument-specific and exploiting their combined high-throughput and high-resolution capabilities. Overall, the combination of MALDI-MS and endo-fucoidanase assays offers high-throughput evaluation of fucoidan samples and also enables extraction of defined oligosaccharides of known structure from unrefined seaweed biomass., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

microbeMASST, a taxonomically informed mass spectrometry (MS) search tool, tackles limited microbial metabolite annotation in untargeted metabolomics experiments. Leveraging a curated database of >60,000 microbial monocultures, users can search known and unknown MS/MS spectra and link them to their respective microbial producers via MS/MS fragmentation patterns. Identification of microbe-derived metabolites and relative producers without a priori knowledge will vastly enhance the understanding of microorganisms' role in ecology and human health., (© 2024. The Author(s).)

Label-free molecular imaging techniques such as matrix-assisted laser desorption ionization mass spectrometry imaging (MALDI-MSI) enable the direct and simultaneous mapping of hundreds of different metabolites in thin sections of biological tissues. However, in host-microbe interactions it remains challenging to localize microbes and to assign metabolites to the host versus members of the microbiome. We therefore developed a correlative imaging approach combining MALDI-MSI with fluorescence in situ hybridization (FISH) on the same section to identify and localize microbial cells. Here, we detail metaFISH as a robust and easy method for assigning the spatial distribution of metabolites to microbiome members based on imaging of nucleic acid probes, down to single-cell resolution. We describe the steps required for tissue preparation, on-tissue hybridization, fluorescence microscopy, data integration into a correlative image dataset, matrix application and MSI data acquisition. Using metaFISH, we map hundreds of metabolites and several microbial species to the micrometer scale on a single tissue section. For example, intra- and extracellular bacteria, host cells and their associated metabolites can be localized in animal tissues, revealing their complex metabolic interactions. We explain how we identify low-abundance bacterial infection sites as regions of interest for high-resolution MSI analysis, guiding the user to a trade-off between metabolite signal intensities and fluorescence signals. MetaFISH is suitable for a broad range of users from environmental microbiologists to clinical scientists. The protocol requires ~2 work days., (© 2023. Springer Nature Limited.)

Sterols are vital for nearly all eukaryotes. Their distribution differs in plants and animals, with phytosterols commonly found in plants whereas most animals are dominated by cholesterol. We show that sitosterol, a common sterol of plants, is the most abundant sterol in gutless marine annelids. Using multiomics, metabolite imaging, heterologous gene expression, and enzyme assays, we show that these animals synthesize sitosterol de novo using a noncanonical C-24 sterol methyltransferase (C 24 -SMT). This enzyme is essential for sitosterol synthesis in plants, but not known from most bilaterian animals. Our phylogenetic analyses revealed that C 24 -SMTs are present in representatives of at least five animal phyla, indicating that the synthesis of sterols common to plants is more widespread in animals than currently known.

Neuropeptides are important regulators of animal physiology and behavior. Hitherto the gold standard for the localization of neuropeptides have been immunohistochemical methods that require the synthesis of antibody panels, while another limiting factor has been the brain's opacity for subsequent in situ light or fluorescence microscopy. To address these limitations, we explored the integration of high-resolution mass spectrometry imaging (MSI) with microtomography for a multiplexed mapping of neuropeptides in two evolutionary distant ant species, Atta sexdens and Lasius niger . For analyzing the spatial distribution of chemically diverse peptide molecules across the brain in each species, the acquisition of serial mass spectrometry images was essential. As a result, we have comparatively mapped the three-dimensional (3D) distributions of eight conserved neuropeptides throughout the brain microanatomy. We demonstrate that integrating the 3D MSI data into high-resolution anatomy models can be critical for studying organs with high plasticity such as brains of social insects. Several peptides, like the tachykinin-related peptides (TK) 1 and 4, were widely distributed in many brain areas of both ant species, whereas others, for instance myosuppressin, were restricted to specific regions only. Also, we detected differences at the species level; many peptides were identified in the optic lobe of L. niger , but only one peptide (ITG-like) was found in this region in A. sexdens . Building upon MS imaging studies on neuropeptides in invertebrate model systems, our approach leverages correlative MSI and computed microtomography for investigating fundamental neurobiological processes by visualizing the unbiased 3D neurochemistry in its complex anatomic environment., (© The Author(s) 2023. Published by Oxford University Press on behalf of National Academy of Sciences.)

Brown algae annually convert gigatons of carbon dioxide into carbohydrates, including the complex extracellular matrix polysaccharide fucoidan. Due to its persistence in the environment, fucoidan is potentially a pathway for marine carbon sequestration. Rates of fucoidan secretion by brown algae remain unknown due to the challenge of identifying and quantifying complex polysaccharides in seawater. We adapted the techniques of anion exchange chromatography, enzyme-linked immunosorbent assay, and biocatalytic enzyme-based assay for detection and quantification of fucoidan. We found the brown alga Fucus vesiculosus at the Baltic Sea coast of south-west Finland to secrete 0.3% of their biomass as fucoidan per day. Dissolved fucoidan concentrations in seawater adjacent to algae reached up to 0.48 mg L -1 . Fucoidan accumulated during incubations of F. vesiculosus , significantly more in light than in darkness. Maximum estimation by acid hydrolysis indicated fucoidan secretion at a rate of 28 to 40 mg C kg -1 h -1 , accounting for 44 to 50% of all exuded dissolved organic carbon. Composed only of carbon, oxygen, hydrogen, and sulfur, fucoidan secretion does not consume nutrients enabling carbon sequestration independent of algal growth. Extrapolated over a year, the algae sequester more carbon into secreted fucoidan than their biomass. The global utility of fucoidan secretion is an alternative pathway for carbon dioxide removal by brown algae without the need to harvest or bury algal biomass.

Seagrasses are among the most efficient sinks of carbon dioxide on Earth. While carbon sequestration in terrestrial plants is linked to the microorganisms living in their soils, the interactions of seagrasses with their rhizospheres are poorly understood. Here, we show that the seagrass, Posidonia oceanica excretes sugars, mainly sucrose, into its rhizosphere. These sugars accumulate to µM concentrations-nearly 80 times higher than previously observed in marine environments. This finding is unexpected as sugars are readily consumed by microorganisms. Our experiments indicated that under low oxygen conditions, phenolic compounds from P. oceanica inhibited microbial consumption of sucrose. Analyses of the rhizosphere community revealed that many microbes had the genes for degrading sucrose but these were only expressed by a few taxa that also expressed genes for degrading phenolics. Given that we observed high sucrose concentrations underneath three other species of marine plants, we predict that the presence of plant-produced phenolics under low oxygen conditions allows the accumulation of labile molecules across aquatic rhizospheres., (© 2022. The Author(s).)

Eukaryotes may experience oxygen deprivation under both physiological and pathological conditions. Because oxygen shortage leads to a reduction in cellular energy production, all eukaryotes studied so far conserve energy by suppressing their metabolism. However, the molecular physiology of animals that naturally and repeatedly experience anoxia is underexplored. One such animal is the marine nematode Laxus oneistus. It thrives, invariably coated by its sulfur-oxidizing symbiont Candidatus Thiosymbion oneisti, in anoxic sulfidic or hypoxic sand. Here, transcriptomics and proteomics showed that, whether in anoxia or not, L. oneistus mostly expressed genes involved in ubiquitination, energy generation, oxidative stress response, immune response, development, and translation. Importantly, ubiquitination genes were also highly expressed when the nematode was subjected to anoxic sulfidic conditions, together with genes involved in autophagy, detoxification and ribosome biogenesis. We hypothesize that these degradation pathways were induced to recycle damaged cellular components (mitochondria) and misfolded proteins into nutrients. Remarkably, when L. oneistus was subjected to anoxic sulfidic conditions, lectin and mucin genes were also upregulated, potentially to promote the attachment of its thiotrophic symbiont. Furthermore, the nematode appeared to survive oxygen deprivation by using an alternative electron carrier (rhodoquinone) and acceptor (fumarate), to rewire the electron transfer chain. On the other hand, under hypoxia, genes involved in costly processes (e.g., amino acid biosynthesis, development, feeding, mating) were upregulated, together with the worm's Toll-like innate immunity pathway and several immune effectors (e.g., bactericidal/permeability-increasing proteins, fungicides). In conclusion, we hypothesize that, in anoxic sulfidic sand, L. oneistus upregulates degradation processes, rewires the oxidative phosphorylation and reinforces its coat of bacterial sulfur-oxidizers. In upper sand layers, instead, it appears to produce broad-range antimicrobials and to exploit oxygen for biosynthesis and development., (© 2022. The Author(s).)

Marine coastlines colonized by seagrasses are a net source of methane to the atmosphere. However, methane emissions from these environments are still poorly constrained, and the underlying processes and responsible microorganisms remain largely unknown. Here, we investigated methane turnover in seagrass meadows of Posidonia oceanica in the Mediterranean Sea. The underlying sediments exhibited median net fluxes of methane into the water column of ca. 106 µmol CH 4 ⋅ m -2 ⋅ d -1 Our data show that this methane production was sustained by methylated compounds produced by the plant, rather than by fermentation of buried organic carbon. Interestingly, methane production was maintained long after the living plant died off, likely due to the persistence of methylated compounds, such as choline, betaines, and dimethylsulfoniopropionate, in detached plant leaves and rhizomes. We recovered multiple mcrA gene sequences, encoding for methyl-coenzyme M reductase (Mcr), the key methanogenic enzyme, from the seagrass sediments. Most retrieved mcrA gene sequences were affiliated with a clade of divergent Mcr and belonged to the uncultured Candidatus Helarchaeota of the Asgard superphylum, suggesting a possible involvement of these divergent Mcr in methane metabolism. Taken together, our findings identify the mechanisms controlling methane emissions from these important blue carbon ecosystems., Competing Interests: The authors declare no competing interest., (Copyright © 2022 the Author(s). Published by PNAS.)

Our understanding of metabolic interactions between small symbiotic animals and bacteria or parasitic eukaryotes that reside within their bodies is extremely limited. This gap in knowledge originates from a methodological challenge, namely to connect histological changes in host tissues induced by beneficial and parasitic (micro)organisms to the underlying metabolites. We addressed this challenge and developed chemo-histo-tomography (CHEMHIST), a culture-independent approach to connect anatomic structure and metabolic function in millimeter-sized symbiotic animals. CHEMHIST combines chemical imaging of metabolites based on mass spectrometry imaging (MSI) and microanatomy-based micro-computed X-ray tomography (micro-CT) on the same animal. Both high-resolution MSI and micro-CT allowed us to correlate the distribution of metabolites to the same animal's three-dimensional (3D) histology down to submicrometer resolutions. Our protocol is compatible with tissue-specific DNA sequencing and fluorescence in situ hybridization for the taxonomic identification and localization of the associated micro(organisms). Building CHEMHIST upon in situ imaging, we sampled an earthworm from its natural habitat and created an interactive 3D model of its physical and chemical interactions with bacteria and parasitic nematodes in its tissues. Combining MSI and micro-CT, we present a methodological groundwork for connecting metabolic and anatomic phenotypes of small symbiotic animals that often represent keystone species for ecosystem functioning., Competing Interests: The authors declare no competing interest., (Copyright © 2021 the Author(s). Published by PNAS.)

Spatial metabolomics using mass spectrometry imaging (MSI) is a powerful tool to map hundreds to thousands of metabolites in biological systems. One major challenge in MSI is the annotation of m / z values, which is substantially complicated by background ions introduced throughout the chemicals and equipment used during experimental procedures. Among many factors, the formation of adducts with sodium or potassium ions, or in case of matrix-assisted laser desorption ionization (MALDI)-MSI, the presence of abundant matrix clusters strongly increases total m / z peak counts. Currently, there is a limitation to identify the chemistry of the many unknown peaks to interpret their biological function. We took advantage of the co-localization of adducts with their parent ions and the accuracy of high mass resolution to estimate adduct abundance in 20 datasets from different vendors of mass spectrometers. Metabolites ranging from lipids to amines and amino acids form matrix adducts with the commonly used 2,5-dihydroxybenzoic acid (DHB) matrix like [M + (DHB-H 2 O) + H] + and [M + DHB + Na] + . Current data analyses neglect those matrix adducts and overestimate total metabolite numbers, thereby expanding the number of unidentified peaks. Our study demonstrates that MALDI-MSI data are strongly influenced by adduct formation across different sample types and vendor platforms and reveals a major influence of so far unrecognized metabolite-matrix adducts on total peak counts (up to one third). We developed a software package, mass 2 adduct , for the community for an automated putative assignment and quantification of metabolite-matrix adducts enabling users to ultimately focus on the biologically relevant portion of the MSI data.

Vacuolar myelinopathy is a fatal neurological disease that was initially discovered during a mysterious mass mortality of bald eagles in Arkansas in the United States. The cause of this wildlife disease has eluded scientists for decades while its occurrence has continued to spread throughout freshwater reservoirs in the southeastern United States. Recent studies have demonstrated that vacuolar myelinopathy is induced by consumption of the epiphytic cyanobacterial species Aetokthonos hydrillicola growing on aquatic vegetation, primarily the invasive Hydrilla verticillata Here, we describe the identification, biosynthetic gene cluster, and biological activity of aetokthonotoxin, a pentabrominated biindole alkaloid that is produced by the cyanobacterium A. hydrillicola We identify this cyanobacterial neurotoxin as the causal agent of vacuolar myelinopathy and discuss environmental factors-especially bromide availability-that promote toxin production., (Copyright © 2021 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

Associations between animals and microbes affect not only the immediate tissues where they occur, but also the entire host. Metabolomics, the study of small biomolecules generated during metabolic processes, provides a window into how mutualistic interactions shape host biochemistry. The Hawaiian bobtail squid, Euprymna scolopes , is amenable to metabolomic studies of symbiosis because the host can be reared with or without its species-specific symbiont, Vibrio fischeri In addition, unlike many invertebrates, the host squid has a closed circulatory system. This feature allows a direct sampling of the refined collection of metabolites circulating through the body, a focused approach that has been highly successful with mammals. Here, we show that rearing E. scolopes without its natural symbiont significantly affected one-quarter of the more than 100 hemolymph metabolites defined by gas chromatography mass spectrometry analysis. Furthermore, as in mammals, which harbor complex consortia of bacterial symbionts, the metabolite signature oscillated on symbiont-driven daily rhythms and was dependent on the sex of the host. Thus, our results provide evidence that the population of even a single symbiont species can influence host hemolymph biochemistry as a function of symbiotic state, host sex and circadian rhythm., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2020. Published by The Company of Biologists Ltd.)

Brown algae are important players in the global carbon cycle by fixing carbon dioxide into 1 Gt of biomass annually, yet the fate of fucoidan-their major cell wall polysaccharide-remains poorly understood. Microbial degradation of fucoidans is slower than that of other polysaccharides, suggesting that fucoidans are more recalcitrant and may sequester carbon in the ocean. This may be due to the complex, branched and highly sulfated structure of fucoidans, which also varies among species of brown algae. Here, we show that 'Lentimonas' sp. CC4, belonging to the Verrucomicrobia, acquired a remarkably complex machinery for the degradation of six different fucoidans. The strain accumulated 284 putative fucoidanases, including glycoside hydrolases, sulfatases and carbohydrate esterases, which are primarily located on a 0.89-megabase pair plasmid. Proteomics reveals that these enzymes assemble into substrate-specific pathways requiring about 100 enzymes per fucoidan from different species of brown algae. These enzymes depolymerize fucoidan into fucose, which is metabolized in a proteome-costly bacterial microcompartment that spatially constrains the metabolism of the toxic intermediate lactaldehyde. Marine metagenomes and microbial genomes show that Verrucomicrobia including 'Lentimonas' are abundant and highly specialized degraders of fucoidans and other complex polysaccharides. Overall, the complexity of the pathways underscores why fucoidans are probably recalcitrant and more slowly degraded, since only highly specialized organisms can effectively degrade them in the ocean.

The connection between monosaccharides influences the structure, solubility, and biological function of carbohydrates. Although tandem mass spectrometry (MS/MS) often enables the compositional identification of carbohydrates, traditional MS/MS fragmentation methods fail to generate abundant cross-ring fragments of intrachain monosaccharides that could reveal carbohydrate connectivity. We examined the potential of helium-charge transfer dissociation (He-CTD) as a method of MS/MS to decipher the connectivity of β-1,4- and β-1,3-linked oligosaccharides. In contrast to collision-induced dissociation (CID), He-CTD of isolated oligosaccharide precursors produced both glycosidic and cross-ring cleavages of each monosaccharide. The radical-driven dissociation in He-CTD induced single cleavage events, without consecutive fragmentations, which facilitated structural interpretation. He-CTD of various standards up to a degree of polymerization of 7 showed that β-1,4- and β-1,3-linked carbohydrates can be distinguished based on diagnostic 3,5 A fragment ions that are characteristic for β-1,4-linkages. Overall, fragment ion spectra from He-CTD contained sufficient information to infer the connectivity specifically for each glycosidic bond. When testing He-CTD to resolve the order of β-1,4- and β-1,3-linkages in mixed-linked oligosaccharide standards, He-CTD spectra sometimes provided less confident assignment of connectivity. Ion mobility spectrometry-mass spectrometry (IMS-MS) of the standards indicated that ambiguity in the He-CTD spectra was caused by isobaric impurities in the mixed-linked oligosaccharides. Radical-driven dissociation induced by He-CTD can thus expand MS/MS to carbohydrate linkage analysis, as demonstrated by the comprehensive fragment ion spectra on native oligosaccharides. The determination of connectivity in true unknowns would benefit from the separation of isobaric precursors, through UPLC or IMS, before linkage determination via He-CTD.

Cold seeps and hydrothermal vents deliver large amounts of methane and other gaseous alkanes into marine surface sediments. Consortia of archaea and partner bacteria thrive on the oxidation of these alkanes and its coupling to sulfate reduction. The inherently slow growth of the involved organisms and the lack of pure cultures have impeded the understanding of the molecular mechanisms of archaeal alkane degradation. Here, using hydrothermal sediments of the Guaymas Basin (Gulf of California) and ethane as the substrate, we cultured microbial consortia of a novel anaerobic ethane oxidizer, " Candidatus Ethanoperedens thermophilum" (GoM-Arc1 clade), and its partner bacterium " Candidatus Desulfofervidus auxilii," previously known from methane-oxidizing consortia. The sulfate reduction activity of the culture doubled within one week, indicating a much faster growth than in any other alkane-oxidizing archaea described before. The dominance of a single archaeal phylotype in this culture allowed retrieval of a closed genome of " Ca. Ethanoperedens," a sister genus of the recently reported ethane oxidizer " Candidatus Argoarchaeum." The metagenome-assembled genome of " Ca. Ethanoperedens" encoded a complete methanogenesis pathway including a methyl-coenzyme M reductase (MCR) that is highly divergent from those of methanogens and methanotrophs. Combined substrate and metabolite analysis showed ethane as the sole growth substrate and production of ethyl-coenzyme M as the activation product. Stable isotope probing demonstrated that the enzymatic mechanism of ethane oxidation in " Ca. Ethanoperedens" is fully reversible; thus, its enzymatic machinery has potential for the biotechnological development of microbial ethane production from carbon dioxide. IMPORTANCE In the seabed, gaseous alkanes are oxidized by syntrophic microbial consortia that thereby reduce fluxes of these compounds into the water column. Because of the immense quantities of seabed alkane fluxes, these consortia are key catalysts of the global carbon cycle. Due to their obligate syntrophic lifestyle, the physiology of alkane-degrading archaea remains poorly understood. We have now cultivated a thermophilic, relatively fast-growing ethane oxidizer in partnership with a sulfate-reducing bacterium known to aid in methane oxidation and have retrieved the first complete genome of a short-chain alkane-degrading archaeon. This will greatly enhance the understanding of nonmethane alkane activation by noncanonical methyl-coenzyme M reductase enzymes and provide insights into additional metabolic steps and the mechanisms underlying syntrophic partnerships. Ultimately, this knowledge could lead to the biotechnological development of alkanogenic microorganisms to support the carbon neutrality of industrial processes., (Copyright © 2020 Hahn et al.)

Spatial metabolomics describes the location and chemistry of small molecules involved in metabolic phenotypes, defence molecules and chemical interactions in natural communities. Most current techniques are unable to spatially link the genotype and metabolic phenotype of microorganisms in situ at a scale relevant to microbial interactions. Here, we present a spatial metabolomics pipeline (metaFISH) that combines fluorescence in situ hybridization (FISH) microscopy and high-resolution atmospheric-pressure matrix-assisted laser desorption/ionization mass spectrometry to image host-microbe symbioses and their metabolic interactions. The metaFISH pipeline aligns and integrates metabolite and fluorescent images at the micrometre scale to provide a spatial assignment of host and symbiont metabolites on the same tissue section. To illustrate the advantages of metaFISH, we mapped the spatial metabolome of a deep-sea mussel and its intracellular symbiotic bacteria at the scale of individual epithelial host cells. Our analytical pipeline revealed metabolic adaptations of the epithelial cells to the intracellular symbionts and variation in metabolic phenotypes within a single symbiont 16S rRNA phylotype, and enabled the discovery of specialized metabolites from the host-microbe interface. metaFISH provides a culture-independent approach to link metabolic phenotypes to community members in situ and is a powerful tool for microbiologists across fields.

Microbial communities exchange molecules with their environment, which plays a major role in regulating global biogeochemical cycles and climate. While extracellular metabolites are commonly measured in terrestrial and limnic ecosystems, the presence of salt in marine habitats limits the nontargeted analyses of the ocean exometabolome using mass spectrometry (MS). Current methods require salt removal prior to sample measurements, which can alter the molecular composition of the metabolome and limit the types of compounds detected by MS. To overcome these limitations, we developed a gas chromatography MS (GC-MS) method that avoids sample altering during salt removal and that detects metabolites down to nanomolar concentrations from less than 1 ml of seawater. We applied our method (SeaMet) to explore marine metabolomes in vitro and in vivo First, we measured the production and consumption of metabolites during the culture of a heterotrophic bacterium, Marinobacter adhaerens Our approach revealed successional uptake of amino acids, while sugars were not consumed. These results show that exocellular metabolomics provides insights into nutrient uptake and energy conservation in marine microorganisms. We also applied SeaMet to explore the in situ metabolome of coral reef and mangrove sediment porewaters. Despite the fact that these ecosystems occur in nutrient-poor waters, we uncovered high concentrations of sugars and fatty acids, compounds predicted to play a key role for the abundant and diverse microbial communities in coral reef and mangrove sediments. Our data demonstrate that SeaMet advances marine metabolomics by enabling a nontargeted and quantitative analysis of marine metabolites, thus providing new insights into nutrient cycles in the oceans. IMPORTANCE Nontargeted approaches using metabolomics to analyze metabolites that occur in the oceans is less developed than those for terrestrial and limnic ecosystems. One of the challenges in marine metabolomics is that salt limits metabolite analysis in seawater to methods requiring salt removal. Building on previous sample preparation methods for metabolomics, we developed SeaMet, which overcomes the limitations of salt on metabolite detection. Considering that the oceans contain the largest dissolved organic matter pool on Earth, describing the marine metabolome using nontargeted approaches is critical for understanding the drivers behind element cycles, biotic interactions, ecosystem function, and atmospheric CO 2 storage. Our method complements both targeted marine metabolomic investigations as well as other "omics" (e.g., genomics, transcriptomics, and proteomics) approaches by providing an avenue for studying the chemical interaction between marine microbes and their habitats., (Copyright © 2019 Sogin et al.)

Placozoa is an enigmatic phylum of simple, microscopic, marine metazoans 1,2 . Although intracellular bacteria have been found in all members of this phylum, almost nothing is known about their identity, location and interactions with their host 3-6 . We used metagenomic and metatranscriptomic sequencing of single host individuals, plus metaproteomic and imaging analyses, to show that the placozoan Trichoplax sp. H2 lives in symbiosis with two intracellular bacteria. One symbiont forms an undescribed genus in the Midichloriaceae (Rickettsiales) 7,8 and has a genomic repertoire similar to that of rickettsial parasites 9,10 , but does not seem to express key genes for energy parasitism. Correlative image analyses and three-dimensional electron tomography revealed that this symbiont resides in the rough endoplasmic reticulum of its host's internal fibre cells. The second symbiont belongs to the Margulisbacteria, a phylum without cultured representatives and not known to form intracellular associations 11-13 . This symbiont lives in the ventral epithelial cells of Trichoplax, probably metabolizes algal lipids digested by its host and has the capacity to supplement the placozoan's nutrition. Our study shows that one of the simplest animals has evolved highly specific and intimate associations with symbiotic, intracellular bacteria and highlights that symbioses can provide access to otherwise elusive microbial dark matter.

Since the discovery of symbioses between sulfur-oxidizing (thiotrophic) bacteria and invertebrates at hydrothermal vents over 40 years ago, it has been assumed that autotrophic fixation of CO 2 by the symbionts drives these nutritional associations. In this study, we investigated " Candidatus Kentron," the clade of symbionts hosted by Kentrophoros , a diverse genus of ciliates which are found in marine coastal sediments around the world. Despite being the main food source for their hosts, Kentron bacteria lack the key canonical genes for any of the known pathways for autotrophic carbon fixation and have a carbon stable isotope fingerprint that is unlike other thiotrophic symbionts from similar habitats. Our genomic and transcriptomic analyses instead found metabolic features consistent with growth on organic carbon, especially organic and amino acids, for which they have abundant uptake transporters. All known thiotrophic symbionts have converged on using reduced sulfur to gain energy lithotrophically, but they are diverse in their carbon sources. Some clades are obligate autotrophs, while many are mixotrophs that can supplement autotrophic carbon fixation with heterotrophic capabilities similar to those in Kentron. Here we show that Kentron bacteria are the only thiotrophic symbionts that appear to be entirely heterotrophic, unlike all other thiotrophic symbionts studied to date, which possess either the Calvin-Benson-Bassham or the reverse tricarboxylic acid cycle for autotrophy. IMPORTANCE Many animals and protists depend on symbiotic sulfur-oxidizing bacteria as their main food source. These bacteria use energy from oxidizing inorganic sulfur compounds to make biomass autotrophically from CO 2 , serving as primary producers for their hosts. Here we describe a clade of nonautotrophic sulfur-oxidizing symbionts, " Candidatus Kentron," associated with marine ciliates. They lack genes for known autotrophic pathways and have a carbon stable isotope fingerprint heavier than other symbionts from similar habitats. Instead, they have the potential to oxidize sulfur to fuel the uptake of organic compounds for heterotrophic growth, a metabolic mode called chemolithoheterotrophy that is not found in other symbioses. Although several symbionts have heterotrophic features to supplement primary production, in Kentron they appear to supplant it entirely., (Copyright © 2019 Seah et al.)

Anoxygenic phototrophic sulfide oxidation by green and purple sulfur bacteria (PSB) plays a key role in sulfide removal from anoxic shallow sediments and stratified waters. Although some PSB can also oxidize sulfide with nitrate and oxygen, little is known about the prevalence of this chemolithotrophic lifestyle in the environment. In this study, we investigated the role of these phototrophs in light-independent sulfide removal in the chemocline of Lake Cadagno. Our temporally resolved, high-resolution chemical profiles indicated that dark sulfide oxidation was coupled to high oxygen consumption rates of ~9 μM O 2 ·h -1 . Single-cell analyses of lake water incubated with 13 CO 2 in the dark revealed that Chromatium okenii was to a large extent responsible for aerobic sulfide oxidation and it accounted for up to 40% of total dark carbon fixation. The genome of Chr. okenii reconstructed from the Lake Cadagno metagenome confirms its capacity for microaerophilic growth and provides further insights into its metabolic capabilities. Moreover, our genomic and single-cell data indicated that other PSB grow microaerobically in these apparently anoxic waters. Altogether, our observations suggest that aerobic respiration may not only play an underappreciated role in anoxic environments but also that organisms typically considered strict anaerobes may be involved., (© 2019 Society for Applied Microbiology and John Wiley & Sons Ltd.)

Hosts of chemoautotrophic bacteria typically have much higher biomass than their symbionts and consume symbiont cells for nutrition. In contrast to this, chemoautotrophic Candidatus Riegeria symbionts in mouthless Paracatenula flatworms comprise up to half of the biomass of the consortium. Each species of Paracatenula harbors a specific Ca Riegeria, and the endosymbionts have been vertically transmitted for at least 500 million years. Such prolonged strict vertical transmission leads to streamlining of symbiont genomes, and the retained physiological capacities reveal the functions the symbionts provide to their hosts. Here, we studied a species of Paracatenula from Sant'Andrea, Elba, Italy, using genomics, gene expression, imaging analyses, as well as targeted and untargeted MS. We show that its symbiont, Ca R. santandreae has a drastically smaller genome (1.34 Mb) than the symbiont´s free-living relatives (4.29-4.97 Mb) but retains a versatile and energy-efficient metabolism. It encodes and expresses a complete intermediary carbon metabolism and enhanced carbon fixation through anaplerosis and accumulates massive intracellular inclusions such as sulfur, polyhydroxyalkanoates, and carbohydrates. Compared with symbiotic and free-living chemoautotrophs, Ca R. santandreae's versatility in energy storage is unparalleled in chemoautotrophs with such compact genomes. Transmission EM as well as host and symbiont expression data suggest that Ca R. santandreae largely provisions its host via outer-membrane vesicle secretion. With its high share of biomass in the symbiosis and large standing stocks of carbon and energy reserves, it has a unique role for bacterial symbionts-serving as the primary energy storage for its animal host., Competing Interests: The authors declare no conflict of interest., (Copyright © 2019 the Author(s). Published by PNAS.)

We report here that a straightforward change of the standard derivatization procedure for GC⁻MS metabolomics is leading to a strong increase in metabolite signal intensity. Drying samples between methoxymation and trimethylsilylation significantly increased signals by two- to tenfold in extracts of yeast cells, plant and animal tissue, and human urine. This easy step reduces the cost of sample material and the need for expensive new hardware., Competing Interests: Data availability: Datasets are available under metabolights [21] https://www.ebi.ac.uk/metabolights/ MTBLS807.

The enzymatic functionalization of hydrocarbons is a central step in the global carbon cycle initiating the mineralization of methane, isoprenes, and monoterpenes, the most abundant biologically produced hydrocarbons. Also, terpene-modifying enzymes have found many applications in the energy-economic biotechnological production of fine chemicals. Here, we describe a limonene dehydrogenase that was purified from the facultatively anaerobic betaproteobacterium Castellaniella defragrans 65Phen grown on monoterpenes under denitrifying conditions in the absence of molecular oxygen. The purified limonene:ferrocenium oxidoreductase activity hydroxylated the methyl group of limonene (1-methyl-4-(1-methylethenyl)-cyclohex-1-ene) yielding perillyl alcohol ([4-(prop-1-en-2-yl)cyclohex-1-en-1-yl]methanol). The enzyme had a DTT:perillyl alcohol oxidoreductase activity yielding limonene. Mass spectrometry and molecular size determinations revealed a heterodimeric enzyme comprising CtmA and CtmB. Recently, the two proteins had been identified by transposon mutagenesis and proteomics as part of the cyclic terpene metabolism ( ctm ) in C. defragrans and are annotated as FAD-dependent oxidoreductases of the protein domain family phytoene dehydrogenases and related proteins (COG1233). CtmAB is the first heterodimeric enzyme in this protein superfamily. Flavins in the purified CtmAB are oxidized by ferrocenium and are reduced by limonene. Heterologous expression of CtmA, CtmB, and CtmAB in Escherichia coli demonstrated that limonene dehydrogenase activity required both subunits, each carrying a flavin cofactor. Native CtmAB oxidized a wide range of monocyclic monoterpenes containing the allylic methyl group motif (1-methyl-cyclohex-1-ene). In conclusion, we have identified CtmAB as a hydroxylating limonene dehydrogenase and the first heteromer in a family of FAD-dependent dehydrogenases acting on allylic methylene or methyl CH-bonds. We suggest placing in Enzyme Nomenclature as new entry EC 1.17.99.8., (© 2018 by The American Society for Biochemistry and Molecular Biology, Inc.)

The microbial degradation pathways of bicyclic monoterpenes contain unknown enzymes for carbon-carbon cleavages. Such enzymes may also be present in the betaproteobacterium Castellaniella defragrans , a model organism to study the anaerobic monoterpene degradation. In this study, a deletion mutant strain missing the first enzyme of the monocyclic monoterpene pathway transformed cometabolically the bicyclics sabinene, 3-carene and α-pinene into several monocyclic monoterpenes and traces of cyclic monoterpene alcohols. Proteomes of cells grown on bicyclic monoterpenes resembled the proteomes of cells grown on monocyclic monoterpenes. Many transposon mutants unable to grow on bicyclic monoterpenes contained inactivated genes of the monocyclic monoterpene pathway. These observations suggest that the monocyclic degradation pathway is used to metabolize bicyclic monoterpenes. The initial step in the degradation is a decyclization (ring-opening) reaction yielding monocyclic monoterpenes, which can be considered as a reverse reaction of the olefin cyclization of polyenes., Competing Interests: The authors declare no conflict of interest.

The reversible ring-opening addition and fragmentation reaction of p-cresol-based N-phenylbenzoxazine with aliphatic and aromatic thiols was investigated in solvent-mediated and solvent-free reactions. Independently of the used thiol, N-phenylbenzoxazine and the thiols reacted to equilibrium with comparable amounts of reactants and products in aprotic solvent, whereas in protic solvent almost full conversions were reached. In contrast, thiol reactivity was a crucial factor in solvent-free reactions yielding fast and complete conversions for a more acidic thiol and balanced equilibrium concentrations in case of thiols with high pK a values. The strong influence of thiols with low pK a values emphasizes the relevance of the protonation step in the ring-opening reactions of 1,3-benzoxazines with thiols in absence of solvents where acidity predominates nucleophilicity. The reverse reactions, namely adduct dissociation and benzoxazine recovery, were successfully conducted at elevated temperatures and reduced pressure facilitated by the removal of the formed thiols yielding up to 95% recovered 1,3-benzoxazine. These results provide deeper understanding of the reversible ring-opening reaction mechanism of 1,3-benzoxazine with thiols.

NMR spectroscopy and mass spectrometry are the two major analytical platforms for metabolomics, and both generate substantial data with hundreds to thousands of observed peaks for a single sample. Many of these are unknown, and peak assignment is generally complex and time-consuming. Statistical correlations between data types have proven useful in expediting this process, for example, in prioritizing candidate assignments. However, this approach has not been formally assessed for the comparison of direct-infusion mass spectrometry (DIMS) and NMR data. Here, we present a systematic analysis of a sample set (tissue extracts), and the utility of a simple correlation threshold to aid metabolite identification. The correlations were surprisingly successful in linking structurally related signals, with 15 of 26 NMR-detectable metabolites having their highest correlation to a cognate MS ion. However, we found that the distribution of the correlations was highly dependent on the nature of the MS ion, such as the adduct type. This approach should help to alleviate this important bottleneck where both 1D NMR and DIMS data sets have been collected.

Fluorescence in situ hybridization (FISH) has become a vital tool for environmental and medical microbiology and is commonly used for the identification, localization, and isolation of defined microbial taxa. However, fluorescence signal strength is often a limiting factor for targeting all members in a microbial community. Here, we present the application of a multilabeled FISH approach (MiL-FISH) that (i) enables the simultaneous targeting of up to seven microbial groups using combinatorial labeling of a single oligonucleotide probe, (ii) is applicable for the isolation of unfixed environmental microorganisms via fluorescence-activated cell sorting (FACS), and (iii) improves signal and imaging quality of tissue sections in acrylic resin for precise localization of individual microbial cells. We show the ability of MiL-FISH to distinguish between seven microbial groups using a mock community of marine organisms and its applicability for the localization of bacteria associated with animal tissue and their isolation from host tissues using FACS. To further increase the number of potential target organisms, a streamlined combinatorial labeling and spectral imaging-FISH (CLASI-FISH) concept with MiL-FISH probes is presented here. Through the combination of increased probe signal, the possibility of targeting hard-to-detect taxa and isolating these from an environmental sample, the identification and precise localization of microbiota in host tissues, and the simultaneous multilabeling of up to seven microbial groups, we show here that MiL-FISH is a multifaceted alternative to standard monolabeled FISH that can be used for a wide range of biological and medical applications., (Copyright © 2015 Schimak et al.)

All higher plants produce polyphenols, for defence against above-ground herbivory. These polyphenols also influence the soil micro- and macro-fauna that break down plant leaf litter. Polyphenols therefore indirectly affect the fluxes of soil nutrients and, ultimately, carbon turnover and ecosystem functioning in soils. It is unknown how earthworms, the major component of animal biomass in many soils, cope with high-polyphenol diets. Here, we show that earthworms possess a class of unique surface-active metabolites in their gut, which we term 'drilodefensins'. These compounds counteract the inhibitory effects of polyphenols on earthworm gut enzymes, and high-polyphenol diets increase drilodefensin concentrations in both laboratory and field populations. This shows that drilodefensins protect earthworms from the harmful effects of ingested polyphenols. We have identified the key mechanism for adaptation to a dietary challenge in an animal group that has a major role in organic matter recycling in soils worldwide.

Molecular genetic methods can distinguish divergent evolutionary lineages in what previously appeared to be single species, but it is not always clear what functional differences exist between such cryptic species. We used a metabolomic approach to profile biochemical phenotype (metabotype) differences between two putative cryptic species of the earthworm Lumbricus rubellus. There were no straightforward metabolite biomarkers of lineage, i.e. no metabolites that were always at higher concentration in one lineage. Multivariate methods, however, identified a small number of metabolites that together helped distinguish the lineages, including uncommon metabolites such as Nε-trimethyllysine, which is not usually found at high concentrations. This approach could be useful for characterizing functional trait differences, especially as it is applicable to essentially any species group, irrespective of its genome sequencing status., (© 2014 The Author(s) Published by the Royal Society. All rights reserved.)

Microorganisms preserve their metabolic function against a wide range of external perturbations including biotic or abiotic factors by utilizing cellular adaptations to maintain cell homeostasis. Functional genomics aims to detect such adaptive alterations on the level of transcriptome, proteome and metabolome to understand system wide changes and to identify interactions between the different levels of biochemical organization. Microbial metabolomics measures metabolites, the direct biochemical response to the environment, and is pivotal to the understanding of the variability and dynamics of bacterial cell metabolism. Metabolomics can measure many different types of compounds including primary metabolites, secondary metabolites, second messengers, quorum sensing compounds and others, which all contribute to the complex bacterial response to an environmental change. Recent data confirmed that many metabolic processes in pathogenic bacteria are linked to virulence and invasive capabilities. Deciphering bacterial metabolism in response to specific environmental conditions and in specific genetic backgrounds will help map the complex network between the metabolome and the other "-omes". Here, we will review a selection of case studies for the pathogenic Gram-positive bacterium Staphylococcus aureus and summarize the current state of metabolomics literature covering staphylococci metabolism under different physiological states., (Copyright © 2013 Elsevier GmbH. All rights reserved.)

Colonization of the human nose by Staphylococcus aureus in one-third of the population represents a major risk factor for invasive infections. The basis for adaptation of S. aureus to this specific habitat and reasons for the human predisposition to become colonized have remained largely unknown. Human nasal secretions were analyzed by metabolomics and found to contain potential nutrients in rather low amounts. No significant differences were found between S. aureus carriers and non-carriers, indicating that carriage is not associated with individual differences in nutrient supply. A synthetic nasal medium (SNM3) was composed based on the metabolomics data that permits consistent growth of S. aureus isolates. Key genes were expressed in SNM3 in a similar way as in the human nose, indicating that SNM3 represents a suitable surrogate environment for in vitro simulation studies. While the majority of S. aureus strains grew well in SNM3, most of the tested coagulase-negative staphylococci (CoNS) had major problems to multiply in SNM3 supporting the notion that CoNS are less well adapted to the nose and colonize preferentially the human skin. Global gene expression analysis revealed that, during growth in SNM3, S. aureus depends heavily on de novo synthesis of methionine. Accordingly, the methionine-biosynthesis enzyme cysteine-γ-synthase (MetI) was indispensable for growth in SNM3, and the MetI inhibitor DL-propargylglycine inhibited S. aureus growth in SNM3 but not in the presence of methionine. Of note, metI was strongly up-regulated by S. aureus in human noses, and metI mutants were strongly abrogated in their capacity to colonize the noses of cotton rats. These findings indicate that the methionine biosynthetic pathway may include promising antimicrobial targets that have previously remained unrecognized. Hence, exploring the environmental conditions facultative pathogens are exposed to during colonization can be useful for understanding niche adaptation and identifying targets for new antimicrobial strategies.

Data processing for 1D NMR spectra is a key bottleneck for metabolomic and other complex-mixture studies, particularly where quantitative data on individual metabolites are required. We present a protocol for automated metabolite deconvolution and quantification from complex NMR spectra by using the Bayesian automated metabolite analyzer for NMR (BATMAN) R package. BATMAN models resonances on the basis of a user-controllable set of templates, each of which specifies the chemical shifts, J-couplings and relative peak intensities for a single metabolite. Peaks are allowed to shift position slightly between spectra, and peak widths are allowed to vary by user-specified amounts. NMR signals not captured by the templates are modeled non-parametrically by using wavelets. The protocol covers setting up user template libraries, optimizing algorithmic input parameters, improving prior information on peak positions, quality control and evaluation of outputs. The outputs include relative concentration estimates for named metabolites together with associated Bayesian uncertainty estimates, as well as the fit of the remainder of the spectrum using wavelets. Graphical diagnostics allow the user to examine the quality of the fit for multiple spectra simultaneously. This approach offers a workflow to analyze large numbers of spectra and is expected to be useful in a wide range of metabolomics studies.

Phytochelatins are small cysteine-rich non-ribosomal peptides that chelate soft metal and metalloid ions, such as cadmium and arsenic. They are widely produced by plants and microbes; phytochelatin synthase genes are also present in animal species from several different phyla, but there is still little known about whether these genes are functional in animals, and if so, whether they are metal-responsive. We analysed phytochelatin production by direct chemical analysis in Lumbricus rubellus earthworms exposed to arsenic for a 28 day period, and found that arsenic clearly induced phytochelatin production in a dose-dependent manner. It was necessary to measure the phytochelatin metabolite concentrations directly, as there was no upregulation of phytochelatin synthase gene expression after 28 days: phytochelatin synthesis appears not to be transcriptionally regulated in animals. A further untargetted metabolomic analysis also found changes in metabolites associated with the transsulfuration pathway, which channels sulfur flux from methionine for phytochelatin synthesis. There was no evidence of biological transformation of arsenic (e.g. into methylated species) as a result of laboratory arsenic exposure. Finally, we compared wild populations of earthworms sampled from the field, and found that both arsenic-contaminated and cadmium-contaminated mine site worms had elevated phytochelatin concentrations.

Metabolic footprinting of supernatants has been proposed as a tool for assigning gene function. We used NMR spectroscopy to measure the exometabolome of 86 single-gene transposon insertion mutant strains (mutants from central carbon metabolism and regulatory mutants) of the opportunistic pathogen Pseudomonas aeruginosa, grown on a medium designed to represent the nutritional content of cystic fibrosis sputum. Functionally related genes had similar metabolic profiles. E.g. for two-component system mutants, the cognate response regulator and sensor kinase genes clustered tightly together. Some strains had metabolic phenotypes (metabotypes) that could be related to the known gene function. E.g. pyruvate dehydrogenase mutants accumulated large amounts of pyruvate in the medium. In other cases, the metabolic phenotypes were not easily interpretable. The rpoN mutant, which lacks the alternative σ factor RpoN (σ(54)), accumulated high levels of gluconate in the medium. In addition, endometabolome profiling of intracellular metabolites identified a number of systemic metabolic changes. We linked this to indirect regulation of the catabolite repression protein Crc via the non-coding RNA crcZ and found that a crcZ (but not crc) mutant also shared the high-gluconate phenotype. We profiled an additional set of relevant metabolic enzymes and transporters, including Crc targets, and showed that the Crc-regulated edd mutant (gluconate-6-phosphate dehydratase) had similar gluconate levels as the rpoN mutant. Finally, a set of clinical isolates showed patient- and random amplification of polymorphic DNA (RAPD) type-specific differences in gluconate production, which were associated significantly with resistance across four antibiotics (tobramycin, ciprofloxacin, aztreonam, and imipenem), indicating that this has potential clinical relevance.

One-dimensional (1)H NMR spectra are widely used for metabolic profiling. Such data sets often contain hundreds or thousands of spectra, which typically have variation in their sample chemistry, which leads to chemical shift variation "positional noise". This is a severe problem for metabolite quantification and data analysis, as peak integrals do not necessarily correspond across all spectra in a set. Various alignment algorithms have been developed to address this problem, but different studies have taken different approaches to evaluating the performance of NMR alignment routines and can be subjective or rely on an arbitrary cutoff. Furthermore, most alignment approaches completely fail to deal with peaks that overlap. We adopt the simple and robust method of ordering spectra with respect to an internally varying peak and use this to compare different alignment algorithms. Furthermore, we use the information from this procedure to help improve a Bayesian approach to automated peak deconvolution by restricting the prior probability distribution of the peak position in a model-free manner and compare the performance to manual peak deconvolution and to binning. This combination of spectral ordering and compound deconvolution improved the quality of the data for quantitative metabolomics.

The ability to accumulate osmoprotectant compounds, such as betaines, is an important evolutionary feature in many organisms. This is particularly the case for organisms that live in variable environments, which may have fluctuations in moisture and salinity levels. There is, surprisingly, very little known about betaines in soil invertebrates in general, and there is almost no information about earthworms - a group that are important 'ecosystem engineers' and key indicators of soil health. Here, we describe a fast and reliable (1)H-(13)C heteronuclear single quantum coherence (HSQC) 2D NMR approach for the metabolic profiling of a series of betaines and related metabolites in tissue extracts, and list (1)H and (13)C chemical shifts for the trimethylammonium signal for 23 such compounds. The analysis of ten different species from three different families (Lumbricidae, Megascolecidae and Glossoscolecidae) showed an unexpected diversity of betaines present in earthworms. In total ten betaines were identified, including hydroxyproline-betaine, proline-betaine, taurine-betaine, GABA-betaine and histidine-betaine, and a further eleven as-yet unassigned putative betaine metabolites detected. The findings clearly indicate a hitherto-unappreciated important role for betaine metabolism in earthworms., (Copyright © 2012 Elsevier Inc. All rights reserved.)