Ubiquitous occurrence of a dimethylsulfoniopropionate ABC transporter in abundant marine bacteria.

Dimethylsulfoniopropionate (DMSP) is a ubiquitous organosulfur compound in marine environments with important functions in both microorganisms and global biogeochemical carbon and sulfur cycling. The SAR11 clade and marine Roseobacter group (MRG) represent two major groups of heterotrophic bacteria in Earth's surface oceans, which can accumulate DMSP to high millimolar intracellular concentrations. However, few studies have investigated how SAR11 and MRG bacteria import DMSP. Here, through comparative genomics analyses, genetic manipulations, and biochemical analyses, we identified an ABC (ATP-binding cassette)-type DMSP-specific transporter, DmpXWV, in Ruegeria pomeroyi DSS-3, a model strain of the MRG. Mutagenesis suggested that DmpXWV is a key transporter responsible for DMSP uptake in strain DSS-3. DmpX, the substrate binding protein of DmpXWV, had high specificity and binding affinity towards DMSP. Furthermore, the DmpX DMSP-binding mechanism was elucidated from structural analysis. DmpX proteins are prevalent in the numerous cosmopolitan marine bacteria outside the SAR11 clade and the MRG, and dmpX transcription was consistently high across Earth's entire global ocean. Therefore, DmpXWV likely enables pelagic marine bacteria to efficiently import DMSP from seawater. This study offers a new understanding of DMSP transport into marine bacteria and provides novel insights into the environmental adaption of marine bacteria.

Aminolipids elicit functional trade-offs between competitiveness and bacteriophage attachment in Ruegeria pomeroyi.

Lipids play a crucial role in maintaining cell integrity and homeostasis with the surrounding environment. Cosmopolitan marine roseobacter clade (MRC) and SAR11 clade bacteria are unique in that, in addition to glycerophospholipids, they also produce an array of amino acid-containing lipids that are conjugated with beta-hydroxy fatty acids through an amide bond. Two of these aminolipids, the ornithine aminolipid (OL) and the glutamine aminolipid (QL), are synthesized using the O-acetyltransferase OlsA. Here, we demonstrate that OL and QL are present in both the inner and outer membranes of the Gram-negative MRC bacterium Ruegeria pomeroyi DSS-3. In an olsA mutant, loss of these aminolipids is compensated by a concurrent increase in glycerophospholipids. The inability to produce aminolipids caused significant changes in the membrane proteome, with the membrane being less permeable and key nutrient transporters being downregulated while proteins involved in the membrane stress response were upregulated. Indeed, the import of 14C-labelled choline and dimethylsulfoniopropionate, as a proxy for the transport of key marine nutrients across membranes, was significantly impaired in the olsA mutant. Moreover, the olsA mutant was significantly less competitive than the wild type (WT) being unable to compete with the WT strain in co-culture. However, the olsA mutant unable to synthesize these aminolipids is less susceptible to phage attachment. Together, these data reveal a critical role for aminolipids in the ecophysiology of this important clade of marine bacteria and a trade-off between growth and avoidance of bacteriophage attachment.

Differential regulation of degradation and immune pathways underlies adaptation of the ectosymbiotic 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 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.

The Proteobacterial Methanotroph Methylosinus trichosporium OB3b Remodels Membrane Lipids in Response to Phosphate Limitation.

Methane is a potent greenhouse gas in the atmosphere, and its concentration has continued to increase in recent decades. Aerobic methanotrophs, bacteria that use methane as the sole carbon source, are an important biological sink for methane, and they are widely distributed in the natural environment. However, relatively little is known on how methanotroph activity is regulated by nutrients, particularly phosphorus (P). P is the principal nutrient constraining plant and microbial productivity in many ecosystems, ranging from agricultural land to the open ocean. Using a model methanotrophic bacterium, Methylosinus trichosporium OB3b, we demonstrate here that this bacterium can produce P-free glycolipids to replace membrane phospholipids in response to P limitation. The formation of the glycolipid monoglucuronic acid diacylglycerol requires plcP-agt genes since the plcP-agt mutant is unable to produce this glycolipid. This plcP-agt-mediated lipid remodeling pathway appears to be important for M. trichosporium OB3b to cope with P stress, and the mutant grew significantly slower under P limitation. Interestingly, comparative genomics analysis shows that the ability to perform lipid remodeling appears to be a conserved trait in proteobacterial methanotrophs; indeed, plcP is found in all proteobacterial methanotroph genomes, and plcP transcripts from methanotrophs are readily detectable in metatranscriptomics data sets. Together, our study provides new insights into the adaptation to P limitation in this ecologically important group of bacteria. IMPORTANCE Methane is a potent greenhouse gas in the atmosphere, and its concentration has continued to increase steadily in recent decades. In the natural environment, bacteria known as methanotrophs help mitigate methane emissions at no cost to human beings. However, relatively little is known regarding how methane oxidation activity in methanotrophs is regulated by soil nutrients, particularly phosphorus. Here, we show that methanotrophs can modify their membrane in response to phosphorus limitation and that the ability to change membrane lipids is important for methanotroph activity. Genome and metatranscriptome analyses suggest that such an adaptation strategy appears to be strictly conserved in all proteobacterial methanotrophs and is used by these bacteria in the natural environment. Together, our study provides a plausible molecular mechanism for better understanding the role of phosphorus on methane oxidation in the natural environment.

Anaerobic Sulfur Oxidation Underlies Adaptation of a Chemosynthetic Symbiont to Oxic-Anoxic Interfaces.

Chemosynthetic symbioses occur worldwide in marine habitats, but comprehensive physiological studies of chemoautotrophic bacteria thriving on animals are scarce. Stilbonematinae are coated by thiotrophic Gammaproteobacteria. As these nematodes migrate through the redox zone, their ectosymbionts experience varying oxygen concentrations. However, nothing is known about how these variations affect their physiology. Here, by applying omics, Raman microspectroscopy, and stable isotope labeling, we investigated the effect of oxygen on "Candidatus Thiosymbion oneisti." Unexpectedly, sulfur oxidation genes were upregulated in anoxic relative to oxic conditions, but carbon fixation genes and incorporation of 13C-labeled bicarbonate were not. Instead, several genes involved in carbon fixation were upregulated under oxic conditions, together with genes involved in organic carbon assimilation, polyhydroxyalkanoate (PHA) biosynthesis, nitrogen fixation, and urea utilization. Furthermore, in the presence of oxygen, stress-related genes were upregulated together with vitamin biosynthesis genes likely necessary to withstand oxidative stress, and the symbiont appeared to proliferate less. Based on its physiological response to oxygen, we propose that "Ca. T. oneisti" may exploit anaerobic sulfur oxidation coupled to denitrification to proliferate in anoxic sand. However, the ectosymbiont would still profit from the oxygen available in superficial sand, as the energy-efficient aerobic respiration would facilitate carbon and nitrogen assimilation. IMPORTANCE Chemoautotrophic endosymbionts are famous for exploiting sulfur oxidization to feed marine organisms with fixed carbon. However, the physiology of thiotrophic bacteria thriving on the surface of animals (ectosymbionts) is less understood. One longstanding hypothesis posits that attachment to animals that migrate between reduced and oxic environments would boost sulfur oxidation, as the ectosymbionts would alternatively access sulfide and oxygen, the most favorable electron acceptor. Here, we investigated the effect of oxygen on the physiology of "Candidatus Thiosymbion oneisti," a gammaproteobacterium which lives attached to marine nematodes inhabiting shallow-water sand. Surprisingly, sulfur oxidation genes were upregulated under anoxic relative to oxic conditions. Furthermore, under anoxia, the ectosymbiont appeared to be less stressed and to proliferate more. We propose that animal-mediated access to oxygen, rather than enhancing sulfur oxidation, would facilitate assimilation of carbon and nitrogen by the ectosymbiont.

A novel class of sulfur-containing aminolipids widespread in marine roseobacters.

Marine roseobacter group bacteria are numerically abundant and ecologically important players in ocean ecosystems. These bacteria are capable of modifying their membrane lipid composition in response to environmental change. Remarkably, a variety of lipids are produced in these bacteria, including phosphorus-containing glycerophospholipids and several amino acid-containing aminolipids such as ornithine lipids and glutamine lipids. Here, we present the identification and characterization of a novel sulfur-containing aminolipid (SAL) in roseobacters. Using high resolution accurate mass spectrometry, a SAL was found in the lipid extract of Ruegeria pomeroyi DSS-3 and Phaeobacter inhibens DSM 17395. Using comparative genomics, transposon mutagenesis and targeted gene knockout, we identified a gene encoding a putative lyso-lipid acyltransferase, designated salA, which is essential for the biosynthesis of this SAL. Multiple sequence analysis and structural modeling suggest that SalA is a novel member of the lysophosphatidic acid acyltransferase (LPAAT) family, the prototype of which is the PlsC acyltransferase responsible for the biosynthesis of the phospholipid phosphatidic acid. SAL appears to play a key role in biofilm formation in roseobacters. salA is widely distributed in Tara Oceans metagenomes and actively expressed in Tara Oceans metatranscriptomes. Our results raise the importance of sulfur-containing membrane aminolipids in marine bacteria.

High CO2 concentration and iron availability determine the metabolic inventory in an Emiliania huxleyi-dominated phytoplankton community.

Ocean acidification (OA), a consequence of anthropogenic carbon dioxide (CO2 ) emissions, strongly impacts marine ecosystems. OA also influences iron (Fe) solubility, affecting biogeochemical and ecological processes. We investigated the interactive effects of CO2 and Fe availability on the metabolome response of a natural phytoplankton community. Using mesocosms we exposed phytoplankton to ambient (390 µatm) or future CO2 levels predicted for the year 2100 (900 µatm), combined with ambient (4.5 nM) or high (12 nM) dissolved iron (dFe). By integrating over the whole phytoplankton community, we assigned functional changes based on altered metabolite concentrations. Our study revealed the complexity of phytoplankton metabolism. Metabolic profiles showed three stages in response to treatments and phytoplankton dynamics. Metabolome changes were related to the plankton group contributing respective metabolites, explaining bloom decline and community succession. CO2 and Fe affected metabolic profiles. Most saccharides, fatty acids, amino acids and many sterols significantly correlated with the high dFe treatment at ambient pCO2 . High CO2 lowered the abundance of many metabolites irrespective of Fe. However, sugar alcohols accumulated, indicating potential stress. We demonstrate that not only altered species composition but also changes in the metabolic landscape affecting the plankton community may change as a consequence of future high-CO2 oceans.

A new family of uncultivated bacteria involved in methanogenesis from the ubiquitous osmolyte glycine betaine in coastal saltmarsh sediments.

BACKGROUND: Coastal environments are dynamic and rapidly changing. Living organisms in coastal environments are known to synthesise large quantities of organic osmolytes, which they use to cope with osmotic stresses. The organic osmolyte glycine betaine (GBT) is ubiquitously found in marine biota from prokaryotic Bacteria and Archaea to coastal plants, marine protozoa, and mammals. In intertidal coastal sediment, GBT represents an important precursor of natural methane emissions and as much as 90% of total methane production in these ecosystems can be originated from methanogenesis from GBT and its intermediate trimethylamine through microbial metabolism. RESULTS: We set out to uncover the microorganisms responsible for methanogenesis from GBT using stable isotope labelling and metagenomics. This led to the recovery of a near-complete genome (2.3 Mbp) of a novel clostridial bacterium involved in anaerobic GBT degradation. Phylogenetic analyses of 16S rRNA gene, functional marker genes, and comparative genomics analyses all support the establishment of a novel family Candidatus 'Betainaceae' fam. nov. in Clostridiales and its role in GBT metabolism. CONCLUSIONS: Our comparative genomes and metagenomics analyses suggest that this bacterium is widely distributed in coastal salt marshes, marine sediments, and deep subsurface sediments, suggesting a key role of anaerobic GBT metabolism by this clostridial bacterium in these ecosystems.

Microbiology and Ecology of Methylated Amine Metabolism in Marine Ecosystems.

Methylated amines (MAs) are ubiquitous in marine ecosystems, found from surface seawaters to sediment pore waters. These volatile ammonium analogs play important roles in biogeochemical cycles of carbon and nitrogen in the marine water column. They also contribute to the release of climate-active gases, being precursors of the potent greenhouse gas methane through methanogenesis in coastal sediments. Very recently, it also became acknowledged that MAs are important precursors for new particle growth, hence forming cloud-condensation nuclei in the marine atmosphere. Microbial metabolism of MAs has been demonstrated in the marine ecosystems for both Archaea and Bacteria. In this chapter, we summarize the latest developments in analytical methods for quantifying MA concentrations in marine surface water and sediments. We discuss the metabolic pathways leading to the formation and degradation of MAs by marine microbes and the novel biochemistry and structural biology of the enzymes for MA transformation. Lastly, we highlight the need for future research toward a better understanding of the microbiology and ecology of oceanic MA cycles.

Microbial trimethylamine metabolism in marine environments.

Trimethylamine (TMA) is common in marine environments. Although the presence of this compound in the oceans has been known for a long time, unlike the mammalian gastrointestinal tract, where TMA metabolism by microorganisms has been studied intensely, many questions remain unanswered about the microbial metabolism of marine TMA. This minireview summarizes what is currently known about the sources and fate of TMA in marine environments and the different pathways and enzymes involved in TMA metabolism in marine bacteria. This review also raises several questions about microbial TMA metabolism in the marine environments and proposes potential directions for future studies.

Elucidation of glutamine lipid biosynthesis in marine bacteria reveals its importance under phosphorus deplete growth in Rhodobacteraceae.

Marine microorganisms employ multiple strategies to cope with transient and persistent nutrient limitation, one of which, for alleviating phosphorus (P) stress, is to substitute membrane glycerophospholipids with non-P containing surrogate lipids. Such a membrane lipid remodelling strategy enables the most abundant marine phytoplankton and heterotrophic bacteria to adapt successfully to nutrient scarcity in marine surface waters. An important group of non-P lipids, the aminolipids which lack a diacylglycerol backbone, are poorly studied in marine microbes. Here, using a combination of genetic, lipidomics and metagenomics approaches, we reveal for the first time the genes (glsB, olsA) required for the formation of the glutamine-containing aminolipid. Construction of a knockout mutant in either glsB or olsA in the model marine bacterium Ruegeria pomeroyi DSS-3 completely abolished glutamine lipid production. Moreover, both mutants showed a considerable growth cost under P-deplete conditions and the olsA mutant, that is unable to produce the glutamine and ornithine aminolipids, ceased to grow under P-deplete conditions. Analysis of sequenced microbial genomes show that glsB is primarily confined to the Rhodobacteraceae family, which includes the ecologically important marine Roseobacter clade that are key players in the marine sulphur and nitrogen cycles. Analysis of the genes involved in glutamine lipid biosynthesis in the Tara ocean metagenome dataset revealed the global occurrence of glsB in marine surface waters and a positive correlation between glsB abundance and N* (a measure of the deviation from the canonical Redfield ratio), suggesting glutamine lipid plays an important role in the adaptation of marine Rhodobacteraceae to P limitation.

Identification of dimethylamine monooxygenase in marine bacteria reveals a metabolic bottleneck in the methylated amine degradation pathway.

Methylated amines (MAs) are ubiquitous in the marine environment and their subsequent flux into the atmosphere can result in the formation of aerosols and ultimately cloud condensation nuclei. Therefore, these compounds have a potentially important role in climate regulation. Using Ruegeria pomeroyi as a model, we identified the genes encoding dimethylamine (DMA) monooxygenase (dmmABC) and demonstrate that this enzyme degrades DMA to monomethylamine (MMA). Although only dmmABC are required for enzyme activity in recombinant Escherichia coli, we found that an additional gene, dmmD, was required for the growth of R. pomeroyi on MAs. The dmmDABC genes are absent from the genomes of multiple marine bacteria, including all representatives of the cosmopolitan SAR11 clade. Consequently, the abundance of dmmDABC in marine metagenomes was substantially lower than the genes required for other metabolic steps of the MA degradation pathway. Thus, there is a genetic and potential metabolic bottleneck in the marine MA degradation pathway. Our data provide an explanation for the observation that DMA-derived secondary organic aerosols (SOAs) are among the most abundant SOAs detected in fine marine particles over the North and Tropical Atlantic Ocean.

Phenotypic diversity of diploid and haploid Emiliania huxleyi cells and of cells in different growth phases revealed by comparative metabolomics.

In phytoplankton a high species diversity of microalgae co-exists at a given time. But diversity is not only reflected by the species composition. Within these species different life phases as well as different metabolic states can cause additional diversity. One important example is the coccolithophore Emiliania huxleyi. Diploid cells play an important role in marine ecosystems since they can form massively abundant algal blooms but in addition the less abundant haploid life phase of E. huxleyi occurs in lower quantities. Both life phases may fulfill different functions in the plankton. We hypothesize that in addition to the functional diversity caused by this life phase transition the growth stage of cells can also influence the metabolic composition and thus the ecological impact of E. huxleyi. Here we introduce a metabolomic survey in dependence of life phases as well as different growth phases to reveal such changes. The comparative metabolomic approach is based on the extraction of intracellular metabolites from intact microalgae, derivatization and analysis by gas chromatography coupled to mass spectrometry (GC-MS). Automated data processing and statistical analysis using canonical analysis of principal coordinates (CAP) revealed unique metabolic profiles for each life phase. Concerning the correlations of metabolites to growth phases, complex patterns were observed. As for example the saccharide mannitol showed its highest concentration in the exponential phase, whereas fatty acids were correlated to stationary and sterols to declining phase. These results are indicative for specific ecological roles of these stages of E. huxleyi and are discussed in the context of previous physiological and ecological studies.

Rewiring Host Lipid Metabolism by Large Viruses Determines the Fate of Emiliania huxleyi, a Bloom-Forming Alga in the Ocean.

Marine viruses are major ecological and evolutionary drivers of microbial food webs regulating the fate of carbon in the ocean. We combined transcriptomic and metabolomic analyses to explore the cellular pathways mediating the interaction between the bloom-forming coccolithophore Emiliania huxleyi and its specific coccolithoviruses (E. huxleyi virus [EhV]). We show that EhV induces profound transcriptome remodeling targeted toward fatty acid synthesis to support viral assembly. A metabolic shift toward production of viral-derived sphingolipids was detected during infection and coincided with downregulation of host de novo sphingolipid genes and induction of the viral-encoded homologous pathway. The depletion of host-specific sterols during lytic infection and their detection in purified virions revealed their novel role in viral life cycle. We identify an essential function of the mevalonate-isoprenoid branch of sterol biosynthesis during infection and propose its downregulation as an antiviral mechanism. We demonstrate how viral replication depends on the hijacking of host lipid metabolism during the chemical "arms race" in the ocean.

Diatom derived polyunsaturated aldehydes do not structure the planktonic microbial community in a mesocosm study.

Several marine and freshwater diatoms produce polyunsaturated aldehydes (PUA) in wound-activated processes. These metabolites are also released by intact diatom cells during algal blooms. Due to their activity in laboratory experiments, PUA are considered as potential mediators of diatom-bacteria interactions. Here, we tested the hypothesis that PUA mediate such processes in a close-to-field mesocosm experiment. Natural plankton communities enriched with Skeletonema marinoi strains that differ in their PUA production, a plankton control, and a plankton control supplemented with PUA at natural and elevated concentrations were observed. We monitored bacterial and viral abundance as well as bacterial community composition and did not observe any influence of PUA on these parameters even at elevated concentrations. We rather detected an alternation of the bacterial diversity over time and differences between the two S. marinoi strains, indicating unique dynamic bacterial communities in these algal blooms. These results suggest that factors other than PUA are of significance for interactions between diatoms and bacteria.