Biology subjects

Liebeke, M.

Publications and source records attributed to Liebeke, M..

At least 19 recordsLinked to original sources

bioRxiv2026

Spatial lipidomics suggests fatty acid scavenging by an intranuclear parasite

Bacteria of the family Endozoicomonadaceae are widely associated with marine animals, yet their in situ metabolism remains mostly unknown. Genomic studies suggest that members of this family engage in extensive metabolic exchange with their hosts, ranging from nutrient provision in mutualistic sponge and coral symbioses to strong dependence on host metabolites like lipids in parasitic associations. Here, we study the lipid phenotype of the parasitic bacterium Candidatus Endonucleibacter childressi in its native tissue environment. The bacterium infects epithelial cells in the gill of the deep-sea mussel Gigantidas childressi and co-occurs with the mussel chemosynthetic symbionts providing a system to compare lipid profiles of symbiotic and parasitic gammaproteobacteria. We linked bacterial identity to metabolic phenotypes by combining mass spectrometry imaging with 16S rRNA fluorescence in situ hybridization. We identified phospholipids characteristic of regions infected with Ca. Endonucleibacter childressi. A subset of these lipids was also detected in symbiont-colonized regions, revealing a partial overlap in the phospholipid profiles of the two gammaproteobacteria. Fatty acids from PEs assigned to Ca. Endonucleibacter childressi contained polyunsaturated fatty acids such as FA 16:3, FA 18:3 or FA 18:4 which have not been reported from cultured close relatives. We hypothesize that the observed fatty acid profile may reflect adaptations to a host-associated lifestyle which involves the acquisition and remodeling of host-derived fatty acids. Our findings demonstrate the value of characterizing bacterial metabolic phenotypes directly in their native tissue environment and highlight the potential of spatial lipidomics to reveal metabolic dependencies in host-microbe associations.

microbiology↗

bioRxiv2026

Predatory bacteria impact C. elegans life-history traits by modulating microbiota community dynamics and thereby vitamin B12 availability

Predatory bacteria such as Bdellovibrio are emerging as ecological modulators in microbial communities by restructuring community composition, yet their roles in host-associated microbiomes remain poorly understood. Using Caenorhabditis elegans as model host and its defined microbiota, we investigated how two Bdellovibrio strains with distinct prey ranges (B. tiberii MYbb2 and B. krueschi MYbb4) affect microbial community composition and host life-history traits. Both strains consistently altered microbiome composition, with MYbb4 causing more pronounced alpha-diversity shifts and MYbb2 selectively enriching strains of the genus Ochrobactrum which coincided with higher host median lifespan. Genome-based predictions indicate that de novo vitamin B12 synthesis by Ochrobactrum underlies the observed host phenotype, which was confirmed through quantitative measurements of the vitamin in mono-cell cultures. Employing the acdh-1p::GFP transcriptional reporter strain, we confirmed that a diet of B12-producing bacteria suppresses the B12-independent propionate detoxification pathway in the host, demonstrating that bacterially produced B12 is bioavailable to C. elegans. Exogenous B12 supplementation assays further confirmed the lifespan-extending effect. Together, these results suggest that predation-driven enrichment of B12-producing bacteria maintains B12 levels sufficient to detoxify propionyl-CoA via the B12-dependent pathway, preventing the accumulation of toxic metabolic byproducts that would otherwise arise under B12-limiting conditions and reduce host lifespan. Our findings demonstrate that predatory bacteria are important drivers of microbiome structure with direct consequences for host physiology, representing an underappreciated ecological mechanism for microbiome modulation.

ecology↗

bioRxiv2026

Alkane-oxidizing consortia produce substantial amounts of disaccharides

1Consortia of archaea and partner bacteria couple the anaerobic oxidation of alkanes to sulfate reduction. While catabolic pathways in anaerobic alkane-oxidizing archaea (ANKA) are increasingly understood, their anabolic capacities remain poorly characterized. Here, we examined nine enrichment cultures dominated by ANKA and their partner bacteria for small-molecular compounds using solvent extraction and gas chromatographic analysis of derivatized extracts. All hydrocarbon-degrading cultures contained substantial amounts of disaccharides in their metabolite pools. Cold-adapted methane-oxidizing cultures dominated by ANME-2c and Seep-SRB2 contained up to 1.5 mg of trehalose per mg soluble protein. Trehalose was also abundant in ethane-oxidizing cultures of Candidatus Ethanoperedens and its distinct partner SRBs, accounting for up to 75 % of the extracted metabolites. In contrast, thermophilic ANKA cultures dominated by ANME-1 or Ca. Syntropharchaeum and Ca. Desulfofervidus contained an abundant as-yet-unidentified glucose-containing disaccharide. Metagenomic analysis revealed widespread trehalose metabolism genes among partner Desulfobacterota and in ANME-2c and Ca. Ethanoperedens, but a lower potential in ANME-1 and Syntropharchaeum, consistent with metabolite profiles. If exogenous trehalose was added to the Ethane50 culture, we observed rapid metabolization by heterotrophic microorganisms, but poor assimilation by the Ca. Ethanoperedens/ Ca. Desulfofervidus core community, indicating that ANKA/SRB consortia do not consume externally supplied trehalose. Instead, Ca. Ethanoperedens/ Ca. Desulfofervidus, as well as other ANKA/SRB consortia, may use the disaccharides as energy-storage molecules, osmolytes, or components of the extracellular matrix. Notably, the disaccharides produced by the consortia also sustain ancillary heterotrophs, thereby linking alkane oxidation to broader sedimentary carbon cycling.

microbiology↗

bioRxiv2024

Genomic and proteomic characterization of sulfate-reducing symbionts of gutless marine annelids

Sulfate-reducing bacteria (SRB) are widespread in marine and terrestrial environments, where they often form syntrophic associations with bacteria, archaea, and eukaryotes. Among the most intimate of these are multipartite symbioses in gutless marine oligochaete worms, which host SRB and sulfur-oxidizing endosymbionts that engage in a syntrophic exchange of sulfur compounds. Despite decades of research on free-living SRB, the metabolic traits that enable SRB to persist in symbiosis, and how these differ across hosts and environments, remain poorly understood. Here, we show that a globally distributed clade of symbiotic SRB has a conserved core metabolism that diverges markedly from free-living relatives. Using comparative genomics and metaproteomics, we reveal that these endosymbionts retain key traits of SRB such as sulfate reduction, complete oxidation of acetate to CO2, amino acid degradation for nitrogen acquisition, and transport of essential nutrients. However, they exhibit a more oxygen-tolerant metabolism and lack typical nutrient-scavenging mechanisms of free-living SRB. One symbiont-specific trait, the glyoxylate bypass, was consistently expressed in situ and may serve both in reactive oxygen species defence and in biomass generation. The enrichment and expression of oxygen-tolerant pathways, coupled with the loss of nutrient-scavenging functions, indicate specialization to a host-associated, redox-fluctuating environment distinct from that of free-living SRB. Consistent with this shift, symbiont genomes are larger than those of free-living relatives, contrasting with genome reduction in many endosymbionts and reinforcing the importance of metabolic versatility. Our findings provide a framework for understanding how metabolic flexibility enables SRB to persist in long-term multipartite symbioses across diverse marine ecosystems.

microbiology↗

bioRxiv2024

Selective preservation of fucose-rich oligosaccharides in the North Atlantic Ocean

The ocean has a substantial capacity to store carbon dioxide fixed via photosynthesis in dissolved organic molecules. An estimated 20% of the 660 Gt dissolved organic carbon in the ocean pool consists of structurally uncharacterized oligosaccharides, which appear to resist microbial degradation (Aluwihare et al., 1997). Current technologies lack the sensitivity and molecular resolution to identify these oligosaccharides. Here, we adapted graphitized carbon chromatography to extract and separate marine oligosaccharides for liquid chromatography high resolution mass spectrometry analysis. Using a newly-developed de novo annotation tool, we found 110 oligosaccharide structures in surface and deep ocean seawater at two distant locations in the North Atlantic Ocean. One group of the detected oligosaccharides was found only in surface seawater and consisted of larger and more abundant molecules detected by our analysis. A second group of smaller, less abundant oligosaccharides was detected in both the surface and deep ocean seawater of both sampled locations. The composition of oligosaccharides differed between the surface and deep ocean, with deep ocean samples relatively enriched in hard-to-metabolize deoxy-sugars, and xylose, amino sugars and uronic acids compared to simple hexoses. Notably the deoxy-sugar fucose constituted 35-40% of the monomers in deep-sea oligosaccharides, twice the percentage in surface ocean oligosaccharides. The ubiquity of deep ocean oligosaccharides indicates that they represent a preserved fraction of the carbohydrate pool. Their enrichment in specific monosaccharides suggests selective preservation of fucose-rich oligosaccharides in the deep ocean.

ecology↗

bioRxiv2024

Large-Scale Evaluation of Spatial Metabolomics Protocols and Technologies

Spatial metabolomics using imaging mass spectrometry (MS) enables untargeted and label-free metabolite mapping in biological samples. Despite the range of available imaging MS protocols and technologies, our understanding of metabolite detection under specific conditions is limited due to sparse empirical data and predictive theories. Consequently, challenges persist in designing new experiments, and accurately annotating and interpreting data. In this study, we systematically measured the detectability of 172 biologically-relevant metabolites across common imaging MS protocols using custom reference samples. We evaluated 24 MALDI-imaging MS protocols for untargeted metabolomics, and demonstrated the applicability of our findings to complex biological samples through comparison with animal tissue data. We showcased the potential for extending our results to further analytes by predicting metabolite detectability based on molecular properties. Additionally, our interlaboratory comparison of 10 imaging MS technologies, including MALDI, DESI, and IR-MALDESI, showed extensive metabolite coverage and comparable results, underscoring the broad applicability of our findings within the imaging MS community. We share our results and data through a new interactive web application integrated with METASPACE. This resource offers an extensive catalogue of detectable metabolite ions, facilitating protocol selection, supporting data annotation, and benefiting future untargeted spatial metabolomics studies.

systems biology↗

bioRxiv2023

Mechanism of high energy efficiency of carbon fixation by sulfur-oxidizing symbionts revealed by single-cell analyses and metabolic modeling

In chemosynthetic symbioses between marine invertebrates and autotrophic sulfur-oxidizing bacteria the symbionts feed their host by producing organic compounds from CO2 using reduced sulfur compounds as an energy source. One such symbiosis, the gutless marine worm Olavius algarvensis harbors at least five bacterial symbionts of which four have the genetic potential for an autotrophic metabolism. In this study we combined single-cell analyses of CO2 fixation, CO2 release and bulk uptake, with measurements of O2 respiration, sulfur content, and polyhydroxyalkanoate content, as well as mathematical modelling to investigate how energy derived from sulfur oxidation drives carbon fluxes within the symbiosis and between the holobiont and its habitat. We found that under aerobic conditions without external energy sources only the primary symbiont, Ca. Thiosymbion algarvensis, fixed carbon. This symbiont relied on internal sulfur storage for energy production. Our model showed that the apparent efficiency of carbon fixation driven by sulfur oxidation in the symbiosis was higher than thermodynamically feasible if only stored sulfur was considered as source of energy and reducing equivalents. The model and additional calculations showed that reducing equivalents must be derived from a different source than energy. We identified the large amounts of polyhdroxyalkanoate stored by the symbiont as the likely source of reducing equivalents for carbon fixation in the symbiont which boosts the yield of sulfur-driven carbon fixation. The model also showed that heterotrophic carbon fixation by host tissue is not negligible and has to be considered when assessing transfer of carbon from the symbionts to the host.

microbiology↗

bioRxiv2022

Multiplexed neuropeptide mapping in ant brains integrating microtomography and 3D mass spectrometry imaging

Neuropeptides are important regulators of animal physiology and behavior. Hitherto large-scale localization of neuropeptides mainly relied on immunohistochemical methods requiring the availability of antibody panels, while another limiting factor has been the brains opacity for subsequent light or fluorescence microscopy. To address these limitations, we integrated 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 3D distributions of eight conserved neuropeptides throughout the brain micro-anatomy. 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 TK1 and TK4, were widely distributed in many brain areas of both ant species, whereas others, for instance myosuppressin was 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. Our approach provides the basis for investigating fundamental neurobiological processes by visualizing the unbiased 3D neurochemistry in its complex anatomic environment. Significance statementUntil recently, the inability to detect entire molecules such as neuropeptides within their spatial biological context and simultaneously link their occurrence to anatomically and physiologically relevant areas has limited our understanding of complex neurochemical processes. This situation has now changed dramatically with the optimization of a new multiplexed imaging method based on mass spectrometry, which enables us to study previously invisible processes at the microscopic scale. With the marriage of mass spectrometry imaging and microtomography, we show that it has become possible to build high-resolution maps of neuropeptides in complex anatomical structures as small as ant brains. These maps, embedded in the 3D neuroanatomy, expand the understanding of the spatial organization of brain chemistry and provide a baseline for neurobiological and neurochemical studies.

biophysics↗

bioRxiv2022

De novo phytosterol synthesis in animals

Sterols are lipids that regulate multiple processes in eukaryotic cells, and are essential components of cellular membranes. Sterols are currently assumed to be kingdom specific, with phytosterol synthesis restricted to plants while animals are only able to synthesize cholesterol. Here, we challenge this assumption by demonstrating that the marine annelids Olavius and Inanidrilus synthesize the phytosterol sitosterol de novo. Using multi-omics, high-resolution metabolite imaging, heterologous gene expression and enzyme assays, we show that sitosterol is the most abundant (60%) sterol in these animals and characterize its biosynthetic pathway. We show that phytosterol synthesis partially overlaps with cholesterol synthesis and involves a non-canonical C-24 sterol methyltransferase (C24-SMT). C24-SMT is an essential enzyme for sitosterol synthesis in plants, but not known from animals with bilateral symmetry (bilaterians). Our comparative phylogenetic analyses of C24-SMT homologs revealed that these are widely distributed across annelids and other animal phyla, including sponges and rotifers. Our findings show that phytosterol synthesis and use is not restricted to the plant kingdom, and indicate that the evolution of sterols in animals is more complex than previously assumed.

biochemistry↗

bioRxiv2021

Differential regulation of degradation and immune pathways underlies adaptation of the symbiotic nematode Laxus oneistus to oxic-anoxic interfaces

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 upregulated 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 worms Toll- like innate immunity pathway and several immune effectors (e.g., Bacterial Permeability Increasing proteins, fungicides). In conclusion, we hypothesize that, in anoxic sulfidic sand, L. oneistus upregulates degradation processes, rewires oxidative phosphorylation and by 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.

genomics↗

bioRxiv2020

CHEMHIST, connecting structure and function from organisms to molecules in small animal symbioses through chemo-histo-tomography

Our understanding of metabolic interactions between small symbiotic animals and bacteria or parasitic eukaryotes that reside within their body 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. To close this gap, we developed chemo-histo-tomography (CHEMHIST), a culture-independent approach to connect anatomic structure and metabolic function in millimeter-sized symbiotic animals. CHEMHIST combines spatial metabolomics based on mass spectrometry imaging (MSI) and microanatomy-based micro-computed X-ray tomography (microCT) on the same animal. Both high-resolution MSI and microCT allowed us to correlate the distribution of metabolites to the same animals three-dimensional (3D) histology down to sub-micrometer resolutions. Our protocol is compatible with tissue specific DNA sequencing and fluorescence in situ hybridization (FISH) 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 microCT, we introduce a workflow to connect metabolic and anatomic phenotypes of small symbiotic animals that often represent keystone species for ecosystem-functioning. SignificanceMetabolites mediate the establishment and persistence of most inter-kingdom symbioses. Still, to pinpoint the metabolites each partner displays upon interaction remains the biggest challenge in studying multi-organismal assemblages. Addressing this challenge, we developed a correlative imaging workflow to connect the in situ production of metabolites with the organ-scale and cellular 3D distributions of mutualistic and pathogenic (micro)organisms in the same host animal. Combining mass spectrometry imaging and micro-computed X-ray tomography provided a culture-independent approach, which is essential to include the full spectrum of naturally occurring interactions. To introduce the potential of combining high-resolution tomography with metabolite imaging, we resolve the metabolic interactions between an invertebrate host, its symbiotic bacteria and tissue parasites at unprecedented detail for model and non-model symbioses.

ecology↗

bioRxiv2020

Candidatus Ethanoperedens, a thermophilic genus of archaea mediating the anaerobic oxidation of ethane

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 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 retrieving 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 for 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 sole growth substrate and production of ethyl-coenzyme M as activation product. Stable isotope probing showed that the enzymatic mechanisms 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. IMPORTANCEIn 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 non-methane alkane activation by non-canonical 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. EtymologyEthanoperedens. ethano, (new Latin): pertaining to ethane; peredens (Latin): consuming, devouring; thermophilum. (Greek): heat-loving. The name implies an organism capable of ethane oxidation at elevated temperatures. LocalityEnriched from hydrothermally heated, hydrocarbon-rich marine sediment of the Guaymas Basin at 2000 m water depth, Gulf of California, Mexico. DiagnosisAnaerobic, ethane-oxidizing archaeon, mostly coccoid, about 0.7 m in diameter, forms large irregular cluster in large dual-species consortia with the sulfate-reducing partner bacterium Candidatus Desulfofervidus auxilii.

microbiology↗

bioRxiv2019

Seagrass excretes sugars to their rhizosphere making them the sweet spots in the sea

Seagrasses are one of the most efficient sinks of carbon dioxide on Earth1: They bury carbon 35 times faster than tropical rainforests on a per unit area basis2. While we know that carbon sequestration in terrestrial plants is intimately linked to the microorganisms living in their soils3-6, the interactions of seagrasses with their rhizospheres are poorly understood. We show that three seagrass species from two oceans excrete simple sugars, mainly sucrose, into their rhizosphere that accumulate to over 200 {micro}M. Such high concentrations are at least 80 times higher than previously observed in the ocean, and surprising, as sugars are quickly consumed by microorganisms. In situ analyses and incubation experiments indicated that phenolic compounds from the seagrass inhibited microbial consumption of sucrose. Metagenomic and metatranscriptomic analyses of the microbial communities in the seagrass rhizosphere revealed that many members had the genes for degrading sucrose, but these were only expressed by a few specialists that also expressed genes for degrading phenolics. Our results explain why sucrose accumulates under seagrass meadows, where it comprises as much as 40% of the dissolved organic carbon. Destruction of extant seagrass canopies would allow sediment microorganisms to consume the tremendous deposits of sucrose buried underneath their meadows, thereby releasing large amounts of CO2 into the oceans and atmosphere.

microbiology↗

bioRxiv2019

The Trichoplax microbiome: the simplest animal lives in an intimate symbiosis with two intracellular bacteria

Summary paragraphPlacozoa is an enigmatic phylum of simple, microscopic, marine metazoans. 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. We used metagenomic and metatranscriptomic sequencing of single host individuals, plus metaproteomic and imaging analyses, to show that the placozoan Trichoplax H2 lives in symbiosis with two intracellular bacteria. One symbiont forms a new genus in the Midichloriaceae (Rickettsiales) and has a genomic repertoire similar to that of rickettsial parasites, but does not appear to express key genes for energy parasitism. Correlative microscopy and 3-D electron tomography revealed that this symbiont resides in an unusual location, the rough endoplasmic reticulum of its hosts internal fiber cells. The second symbiont belongs to the Margulisbacteria, a phylum without cultured representatives and not known to form intracellular associations. This symbiont lives in the ventral epithelial cells of Trichoplax, likely metabolizes algal lipids digested by its host, and has the capacity to supplement the placozoans nutrition. Our study shows that even the simplest animals known have evolved highly specific and intimate associations with symbiotic, intracellular bacteria, and highlights that symbioses with microorganisms are a basal trait of animal life.

microbiology↗

bioRxiv2019

Spatial metabolomics of in situ, host-microbe interactions

Spatial metabolomics describes the location and chemistry of small molecules involved in metabolic phenotypes, defense 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 mass spectrometry imaging (AP-MALDI-MSI) to image host-microbe symbioses and their metabolic interactions. metaFISH aligns and integrates metabolite and fluorescent images at the micrometer-scale for 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, a variation in metabolic phenotypes in one symbiont type, and novel symbiosis metabolites. metaFISH provides a culture-independent approach to link metabolic phenotypes to community members in situ - a powerful tool for microbiologists across fields.

microbiology↗

bioRxiv2019

Drying enhances signal intensity for global GC-MS metabolomics.

We report here that a straightforward change of 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.

biochemistry↗

bioRxiv2019

Sulfur-oxidizing symbionts without canonical genes for autotrophic CO2 fixation

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 CO2 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 lack the key canonical genes for any of the known pathways for autotrophic fixation, and have a carbon stable isotope fingerprint 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 generate 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. We have shown that Kentron 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 reverse tricarboxylic acid cycles for autotrophy. Significance StatementMany 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 CO2, serving as primary producers for their hosts. Here we describe apparently non-autotrophic sulfur symbionts called 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.

microbiology↗

bioRxiv2019

Marine metabolomics: measurement of metabolites in seawater by gas chromatography mass spectrometry

All life exchanges molecules with its environment. While these metabolites are commonly measured in terrestrial and limnic ecosystems, the presence of salt in marine habitats has hampered quantitative analyses of the ocean metabolome. To overcome these limitations, we developed SeaMet, a gas chromatography-mass spectrometry (GC-MS) method that detects hundreds of metabolites down to nano-molar concentrations in less than one milliliter of seawater. Using a set of metabolites dissolved in artificial seawater to benchmark our method, we show metabolite signal detection increased on average across ions by 324 fold in comparison to standard GC-MS methods. Our observed signal improvement occurred across tested metabolite classes and provides reproducible and quantifiable results. To showcase the capabilities of our method, we used SeaMet to explore the production and consumption of metabolites during culture of a heterotrophic bacteria that is widespread in the North Sea. Our approach revealed successional uptake of amino acids, while sugars were not consumed, and highlight the power of metabolomics in providing 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 a remarkable diversity and abundance of sugars and fatty acids, compounds predicted to be rapidly consumed by marine microorganisms. Our method advances marine metabolomics by enabling the unbiased, and quantitative analysis of marine metabolites, and will help provide new insights into carbon cycle dynamics and ocean biogeochemistry. SignificanceMetabolites are the chemical currency of cellular metabolism across all domains of life. However, describing metabolites that occur in the oceans is lagging behind similar studies conducted on land. The central challenge in marine metabolomics is that salt prevents the comprehensive analysis of metabolites in seawater. We developed a method, SeaMet, that overcomes the limitations of salt on metabolite detection. SeaMet provides a time and cost efficient method, using gas chromatography-mass spectrometry, for the reproducible identification and quantification of a broad range of marine compounds. Considering the oceans contain the largest organic carbon pool on Earth, describing the marine metabolome is critical for understanding the drivers behind element cycles, biotic interactions, ecosystem function, and atmospheric CO2 storage.

biochemistry↗