Molecular mechanisms of microbiome modulation by the eukaryotic secondary metabolite azelaic acid.

Photosynthetic eukaryotes, such as microalgae and plants, foster fundamentally important relationships with their microbiome based on the reciprocal exchange of chemical currencies. Among these, the dicarboxylate metabolite azelaic acid (Aze) appears to play an important, but heterogeneous, role in modulating these microbiomes, as it is used as a carbon source for some heterotrophs but is toxic to others. However, the ability of Aze to promote or inhibit growth, as well as its uptake and assimilation mechanisms into bacterial cells are mostly unknown. Here, we use transcriptomics, transcriptional factor coexpression networks, uptake experiments, and metabolomics to unravel the uptake, catabolism, and toxicity of Aze on two microalgal-associated bacteria, Phycobacter and Alteromonas, whose growth is promoted or inhibited by Aze, respectively. We identify the first putative Aze transporter in bacteria, a 'C4-TRAP transporter', and show that Aze is assimilated through fatty acid degradation, with further catabolism occurring through the glyoxylate and butanoate metabolism pathways when used as a carbon source. Phycobacter took up Aze at an initial uptake rate of 3.8×10-9 nmol/cell/hr and utilized it as a carbon source in concentrations ranging from 10 µM to 1 mM, suggesting a broad range of acclimation to Aze availability. For growth-impeded bacteria, we infer that Aze inhibits the ribosome and/or protein synthesis and that a suite of efflux pumps is utilized to shuttle Aze outside the cytoplasm. We demonstrate that seawater amended with Aze becomes enriched in bacterial families that can catabolize Aze, which appears to be a different mechanism from that in soil, where modulation by the host plant is required. This study enhances our understanding of carbon cycling in the oceans and how microscale chemical interactions can structure marine microbial populations. In addition, our findings unravel the role of a key chemical currency in the modulation of eukaryote-microbiome interactions across diverse ecosystems.

The coral microbiome: towards an understanding of the molecular mechanisms of coral-microbiota interactions.

Corals live in a complex, multipartite symbiosis with diverse microbes across kingdoms, some of which are implicated in vital functions, such as those related to resilience against climate change. However, knowledge gaps and technical challenges limit our understanding of the nature and functional significance of complex symbiotic relationships within corals. Here, we provide an overview of the complexity of the coral microbiome focusing on taxonomic diversity and functions of well-studied and cryptic microbes. Mining the coral literature indicate that while corals collectively harbour a third of all marine bacterial phyla, known bacterial symbionts and antagonists of corals represent a minute fraction of this diversity and that these taxa cluster into select genera, suggesting selective evolutionary mechanisms enabled these bacteria to gain a niche within the holobiont. Recent advances in coral microbiome research aimed at leveraging microbiome manipulation to increase coral's fitness to help mitigate heat stress-related mortality are discussed. Then, insights into the potential mechanisms through which microbiota can communicate with and modify host responses are examined by describing known recognition patterns, potential microbially derived coral epigenome effector proteins and coral gene regulation. Finally, the power of omics tools used to study corals are highlighted with emphasis on an integrated host-microbiota multiomics framework to understand the underlying mechanisms during symbiosis and climate change-driven dysbiosis.

In vitro α-glucosidase inhibitory activity of Tamarix nilotica shoot extracts and fractions.

α-glucosidase inhibitors represent an important class of type 2 antidiabetic drugs and they act by lowering postprandial hyperglycemia. Today, only three synthetic inhibitors exist on the market, and there is a need for novel, natural and more efficient molecules exhibiting this activity. In this study, we investigated the ability of Tamarix nilotica ethanolic and aqueous shoot extracts, as well as methanolic fractions prepared from aqueous crude extracts to inhibit α-glucosidase. Both, 50% ethanol and aqueous extracts inhibited α-glucosidase in a concentration-dependent manner, with IC50 values of 12.5 µg/mL and 24.8 µg/mL, respectively. Importantly, α-glucosidase inhibitory activity observed in the T. nilotica crude extracts was considerably higher than pure acarbose (IC50 = 151.1 µg/mL), the most highly prescribed α-glucosidase inhibitor on the market. When T. nilotica crude extracts were fractionated using methanol, enhanced α-glucosidase inhibitory activity was observed in general, with the highest observed α-glucosidase inhibitory activity in the 30% methanol fraction (IC50 = 5.21 µg/mL). Kinetic studies further revealed a competitive reversible mechanism of inhibition by the plant extract. The phytochemical profiles of 50% ethanol extracts, aqueous extracts, and the methanolic fractions were investigated and compared using a metabolomics approach. Statistical analysis revealed significant differences in the contents of the crude extracts and fractions and potentially identified the molecules that were most responsible for these observed variations. Higher α-glucosidase inhibitory activity was associated with an enrichment of terpenoids, fatty acids, and flavonoids. Among the identified molecules, active compounds with known α-glucosidase inhibitory activity were detected, including unsaturated fatty acids, triterpenoids, and flavonoid glycosides. These results put forward T. nilotica as a therapeutic plant for type 2 diabetes and a source of α-glucosidase inhibitors.

A metabolomics approach to evaluate the effect of lyophilization versus oven drying on the chemical composition of plant extracts.

Lyophilization is the "gold standard" for drying plant extracts, which is important in preserving their quality and extending their shelf-life. Compared to other methods of drying plant extracts, lyophilization is costlier due to equipment, material and operational expenses. An alternative method is post-extraction oven-drying, but the effects of this process on extract quality are unknown. In this study, crude extracts from Arthrocnemum macrostachyum shoots were compared using three post-extraction drying methods (lyophilization and oven drying at 40 and 60 °C) and two extraction solvents (water and aqueous 50% ethanol). Untargeted metabolomics coupled with chemometrics analysis revealed that post extraction oven-drying resulted in the loss of up to 27% of molecular features when compared to lyophilization in water extracts only. In contrast, only 3% of molecular features were lost in aqueous 50% ethanol extracts when subjected to oven drying. That is to say, ethanol used as a solvent has a stabilizing effect on metabolites and enhances their resistance to thermal transformation in the oven. Collectively, oven-drying of extracts was as effective as lyophilization in preserving metabolites in extracts only when 50% ethanol was used as a solvent. The results presented in this paper demonstrate the value of selecting solvent-appropriate post-extraction drying methods.

Diatom modulation of select bacteria through use of two unique secondary metabolites.

Unicellular eukaryotic phytoplankton, such as diatoms, rely on microbial communities for survival despite lacking specialized compartments to house microbiomes (e.g., animal gut). Microbial communities have been widely shown to benefit from diatom excretions that accumulate within the microenvironment surrounding phytoplankton cells, known as the phycosphere. However, mechanisms that enable diatoms and other unicellular eukaryotes to nurture specific microbiomes by fostering beneficial bacteria and repelling harmful ones are mostly unknown. We hypothesized that diatom exudates may tune microbial communities and employed an integrated multiomics approach using the ubiquitous diatom Asterionellopsis glacialis to reveal how it modulates its naturally associated bacteria. We show that A. glacialis reprograms its transcriptional and metabolic profiles in response to bacteria to secrete a suite of central metabolites and two unusual secondary metabolites, rosmarinic acid and azelaic acid. While central metabolites are utilized by potential bacterial symbionts and opportunists alike, rosmarinic acid promotes attachment of beneficial bacteria to the diatom and simultaneously suppresses the attachment of opportunists. Similarly, azelaic acid enhances growth of beneficial bacteria while simultaneously inhibiting growth of opportunistic ones. We further show that the bacterial response to azelaic acid is numerically rare but globally distributed in the world's oceans and taxonomically restricted to a handful of bacterial genera. Our results demonstrate the innate ability of an important unicellular eukaryotic group to modulate select bacteria in their microbial consortia, similar to higher eukaryotes, using unique secondary metabolites that regulate bacterial growth and behavior inversely across different bacterial populations.

MH-ICP-MS Analysis of the Freshwater and Saltwater Environmental Resources of Upolu Island, Samoa.

The elemental composition of freshwater and saltwater samples around the South Pacific island of Upolu, Samoa has been investigated together with other indicators of water quality. Up to 69 elements from Li (3) to U (92) are measured in each sample, analyzed by Mattauch-Herzog-inductively coupled plasma-mass spectrometry (MH-ICP-MS). One hundred and seventy-six samples were collected from surface freshwater sources (24 rivers, two volcanic lakes, one dam) and from seawater sources from the surface to 30 m depth (45 inner reef, reef, and outer reef locations) around Upolu Island, including river mouths and estuaries. Principal component and hierarchical clustering correlation analyses were performed on quantile normalized log transformed elemental composition data to identify groups of samples with similar characteristics and to improve the visualization of the full spectrum of elements. Human activities, such as the use of herbicides and pesticides, may relate to observed elevated concentrations of some elements contained in chemicals known to have deleterious obesogenic effects on humans that may also cause coral reef decline. Furthermore, the salinity of some saltwater samples tested were very high, possibly due to climate variability, which may additionally harm the health and biodiversity of coral reefs.

Quorum sensing regulates 'swim-or-stick' lifestyle in the phycosphere.

Interactions between phytoplankton and bacteria play major roles in global biogeochemical cycles and oceanic nutrient fluxes. These interactions occur in the microenvironment surrounding phytoplankton cells, known as the phycosphere. Bacteria in the phycosphere use either chemotaxis or attachment to benefit from algal excretions. Both processes are regulated by quorum sensing (QS), a cell-cell signalling mechanism that uses small infochemicals to coordinate bacterial gene expression. However, the role of QS in regulating bacterial attachment in the phycosphere is not clear. Here, we isolated a Sulfitobacter pseudonitzschiae F5 and a Phaeobacter sp. F10 belonging to the marine Roseobacter group and an Alteromonas macleodii F12 belonging to Alteromonadaceae, from the microbial community of the ubiquitous diatom Asterionellopsis glacialis. We show that only the Roseobacter group isolates (diatom symbionts) can attach to diatom transparent exopolymeric particles. Despite all three bacteria possessing genes involved in motility, chemotaxis, and attachment, only S. pseudonitzschiae F5 and Phaeobacter sp. F10 possessed complete QS systems and could synthesize QS signals. Using UHPLC-MS/MS, we identified three QS molecules produced by both bacteria of which only 3-oxo-C16:1 -HSL strongly inhibited bacterial motility and stimulated attachment in the phycosphere. These findings suggest that QS signals enable colonization of the phycosphere by algal symbionts.

Coral metabolite gradients affect microbial community structures and act as a disease cue.

Corals are threatened worldwide due to prevalence of disease and bleaching. Recent studies suggest the ability of corals to resist disease is dependent on maintaining healthy microbiomes that span coral tissues and surfaces, the holobiont. Although our understanding of the role endosymbiotic microbes play in coral health has advanced, the role surface-associated microbes and their chemical signatures play in coral health is limited. Using minimally invasive water sampling, we show that the corals Acropora and Platygyra harbor unique bacteria and metabolites at their surface, distinctly different from surrounding seawater. The surface metabolites released by the holobiont create concentration gradients at 0-5 cm away from the coral surface. These molecules are identified as chemo-attractants, antibacterials, and infochemicals, suggesting they may structure coral surface-associated microbes. Further, we detect surface-associated metabolites characteristic of healthy or white syndrome disease infected corals, a finding which may aid in describing effects of diseases.

Bacterial Communities of Diatoms Display Strong Conservation Across Strains and Time.

Interactions between phytoplankton and bacteria play important roles in shaping the microenvironment surrounding these organisms and in turn influence global biogeochemical cycles. This microenvironment, known as the phycosphere, is presumed to shape the bacterial diversity around phytoplankton and thus stimulate a diverse array of interactions between both groups. Although many studies have attempted to characterize bacterial communities that associate and interact with phytoplankton, bias in bacterial cultivation and consistency and persistence of bacterial communities across phytoplankton isolates likely impede the understanding of these microbial associations. Here, we isolate four strains of the diatom Asterionellopsis glacialis and three strains of the diatom Nitzschia longissima and show through metabarcoding of the bacterial 16S rDNA gene that though each species possesses a unique bacterial community, the bacterial composition across strains from the same species are highly conserved at the genus level. Cultivation of all seven strains in the laboratory for longer than 1 year resulted in only small changes to the bacterial composition, suggesting that despite strong pressures from laboratory culturing conditions associations between these diatoms and their bacterial communities are robust. Specific operational taxonomic units (OTUs) belonging to the Roseobacter-clade appear to be conserved across all strains and time, suggesting their importance to diatoms. In addition, we isolate a range of cultivable bacteria from one of these cultures, A. glacialis strain A3, including several strains of Shimia marina and Nautella sp. that appear closely related to OTUs conserved across all strains and times. Coculturing of A3 with some of its cultivable bacteria as well as other diatom-associated bacteria shows a wide range of responses that include enhancing diatom growth. Cumulatively, these findings suggest that phytoplankton possess unique microbiomes that are consistent across strains and temporal scales.

The role of floridoside in osmoadaptation of coral-associated algal endosymbionts to high-salinity conditions.

The endosymbiosis between Symbiodinium dinoflagellates and stony corals provides the foundation of coral reef ecosystems. The survival of these ecosystems is under threat at a global scale, and better knowledge is needed to conceive strategies for mitigating future reef loss. Environmental disturbance imposing temperature, salinity, and nutrient stress can lead to the loss of the Symbiodinium partner, causing so-called coral bleaching. Some of the most thermotolerant coral-Symbiodinium associations occur in the Persian/Arabian Gulf and the Red Sea, which also represent the most saline coral habitats. We studied whether Symbiodinium alter their metabolite content in response to high-salinity environments. We found that Symbiodinium cells exposed to high salinity produced high levels of the osmolyte 2-O-glycerol-α-d-galactopyranoside (floridoside), both in vitro and in their coral host animals, thereby increasing their capacity and, putatively, the capacity of the holobiont to cope with the effects of osmotic stress in extreme environments. Given that floridoside has been previously shown to also act as an antioxidant, this osmolyte may serve a dual function: first, to serve as a compatible organic osmolyte accumulated by Symbiodinium in response to elevated salinities and, second, to counter reactive oxygen species produced as a consequence of potential salinity and heat stress.

Long-term salinity tolerance is accompanied by major restructuring of the coral bacterial microbiome.

Scleractinian corals are assumed to be stenohaline osmoconformers, although they are frequently subjected to variations in seawater salinity due to precipitation, freshwater run-off and other processes. Observed responses to altered salinity levels include differences in photosynthetic performance, respiration and increased bleaching and mortality of the coral host and its algal symbiont, but a study looking at bacterial community changes is lacking. Here, we exposed the coral Fungia granulosa to strongly increased salinity levels in short- and long-term experiments to disentangle temporal and compartment effects of the coral holobiont (i.e. coral host, symbiotic algae and associated bacteria). Our results show a significant reduction in calcification and photosynthesis, but a stable microbiome after short-term exposure to high-salinity levels. By comparison, long-term exposure yielded unchanged photosynthesis levels and visually healthy coral colonies indicating long-term acclimation to high-salinity levels that were accompanied by a major coral microbiome restructuring. Importantly, a bacterium in the family Rhodobacteraceae was succeeded by Pseudomonas veronii as the numerically most abundant taxon. Further, taxonomy-based functional profiling indicates a shift in the bacterial community towards increased osmolyte production, sulphur oxidation and nitrogen fixation. Our study highlights that bacterial community composition in corals can change within days to weeks under altered environmental conditions, where shifts in the microbiome may enable adjustment of the coral to a more advantageous holobiont composition.

Changes to lipid droplet configuration in mCMV-infected fibroblasts: live cell imaging with simultaneous CARS and two-photon fluorescence microscopy.

We have performed multimodal imaging of live fibroblast cells infected by murine cytomegalovirus (mCMV). The infection process was monitored by imaging the two-photon fluorescence signal from a GFP-expressing strain of mCMV, whilst changes to lipid droplet configuration were observed by CARS imaging. This allowed us to identify three visually distinct stages of infection. Quantitative analysis of lipid droplet number and size distributions were obtained from live cells, which showed significant perturbations across the different stages of infection. The CARS and two-photon images were acquired simultaneously and the experimental design allowed incorporation of an environmental control chamber to maintain cell viability. Photodamage to the live cell population was also assessed.

Redox potential dependence of peptide structure studied using surface enhanced Raman spectroscopy.

We describe a novel surface enhanced Raman spectroscopy (SERS) sensing approach utilizing modified gold nanoshells and demonstrate its application to analysis of critical redox-potential dependent changes in antigen structure that are implicated in the initiation of a human autoimmune disease. In Goodpasture's disease, an autoimmune reaction is thought to arise from incomplete proteolysis of the autoantigen, α3(IV)NC1(67-85) by proteases including Cathepsin D. We have used SERS to study conformational changes in the antigen that correlate with its oxidation state and to show that the antigen must be in the reduced state in order to undergo proteolysis. Our results demonstrate that a redox potential of ∼-200 mV was sufficient for reduction and subsequent productive processing of the antigenic fragment α3(IV)NC1(67-85). Moreover, we demonstrate that the peptide bonds subsequently cleaved by Cathepsisn D can be identified by comparison with a SERS library of short synthetic peptides.

Probing biomolecular interactions using surface enhanced Raman spectroscopy: label-free protein detection using a G-quadruplex DNA aptamer.

We demonstrate a strategy for label-free protein detection through monitoring the Surface Enhanced Raman Spectrum of an aptamer probe attached to a gold nanoshell. Low limit of detection and minimal non-specific binding show potential for in vitro and in vivo assays.

Nanoshells for surface-enhanced Raman spectroscopy in eukaryotic cells: cellular response and sensor development.

The application of gold nanoshells (NS) as a surface-enhanced Raman (SER) platform for intracellular sensing in NIH-3T3 fibroblast cells was studied by using a near-infrared Raman system. To show the feasibility of using these 151 +/- 5 nm sized solution-stable nanoparticles inside living cells, we investigated the uptake, cellular response, and the health of the cell population. We show that NS are taken up voluntarily and can be found in the cytosol by transmission electron microscopy (TEM), which also provides detailed information about location and immediate surrounding of the NS. The internalization into cells has been found to be independent of active cellular mechanisms, such as endocytosis, and can be suggested to be of passive nature. Uptake of NS into cells can be controlled, and cells show no increase in necrosis or apoptosis as a result; we show that NS-based intracytosolic SER spectra can be measured on biological samples using short acquisition times and low laser powers. We demonstrate its application using 4-mercaptobenzoic acid (4-MBA)-functionalized nanoshells as a pH sensor.