11 research outputs found

    Exo-Metabolome of <i>Pseudovibrio</i> sp. FO-BEG1 Analyzed by Ultra-High Resolution Mass Spectrometry and the Effect of Phosphate Limitation

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    <div><p>Oceanic dissolved organic matter (DOM) is an assemblage of reduced carbon compounds, which results from biotic and abiotic processes. The biotic processes consist in either release or uptake of specific molecules by marine organisms. Heterotrophic bacteria have been mostly considered to influence the DOM composition by preferential uptake of certain compounds. However, they also secrete a variety of molecules depending on physiological state, environmental and growth conditions, but so far the full set of compounds secreted by these bacteria has never been investigated. In this study, we analyzed the exo-metabolome, metabolites secreted into the environment, of the heterotrophic marine bacterium <i>Pseudovibrio</i> sp. FO-BEG1 via ultra-high resolution mass spectrometry, comparing phosphate limited with phosphate surplus growth conditions. Bacteria belonging to the <i>Pseudovibrio</i> genus have been isolated worldwide, mainly from marine invertebrates and were described as metabolically versatile <i>Alphaproteobacteria</i>. We show that the exo-metabolome is unexpectedly large and diverse, consisting of hundreds of compounds that differ by their molecular formulae. It is characterized by a dynamic recycling of molecules, and it is drastically affected by the physiological state of the strain. Moreover, we show that phosphate limitation greatly influences both the amount and the composition of the secreted molecules. By assigning the detected masses to general chemical categories, we observed that under phosphate surplus conditions the secreted molecules were mainly peptides and highly unsaturated compounds. In contrast, under phosphate limitation the composition of the exo-metabolome changed during bacterial growth, showing an increase in highly unsaturated, phenolic, and polyphenolic compounds. Finally, we annotated the detected masses using multiple metabolite databases. These analyses suggested the presence of several masses analogue to masses of known bioactive compounds. However, the annotation was successful only for a minor part of the detected molecules, underlining the current gap in knowledge concerning the biosynthetic ability of marine heterotrophic bacteria.</p></div

    Similarity among the FT-ICR-MS samples analyzed in ESI-negative mode during bacterial growth under +P<sub>i</sub> and −P<sub>i</sub> conditions.

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    <p>Non metrical multidimensional scaling (NMDS) was performed by employing the Bray-Curtis similarity index and using the data of the unfiltered (<b>A</b>) and filtered (<b>B</b>) datasets. All biological triplicates of +P<sub>i</sub> (filled circles) and −P<sub>i</sub> (empty circles) conditions are shown. Nearest neighbor samples (i.e. most similar) are connected to visualize pairwise sample similarities. The stress value for both plots is 0.06.</p

    Bacterial growth (A) and concentrations of solid phase extractable dissolved organic carbon (SPE-DOC) (B).

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    <p>The bars (<b>B</b>) represent the average concentrations of SPE-DOC measured in the solid phase extracts of the biological triplicates collected during growth under both +P<sub>i</sub> (black) and −P<sub>i</sub> conditions (white). The inner panel (<b>A</b>) shows the cell growth, measured as cell density over time, for the two tested conditions. Filled circles represent the cultures growing under +P<sub>i</sub> conditions and empty circles represent the cultures growing under −P<sub>i</sub> conditions. Error bars indicate the standard deviation of biological triplicates</p

    Overview of the data obtained from the ESI-negative FT-ICR-MS analysis.

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    <p>The number of masses detected in all biological triplicates of each time point is shown. The data refer to the dataset obtained after applying the filtration criteria described in the Materials and Methods section. Values in brackets represent the percentages of masses to which a unique molecular formula could be assigned and the percentages of unique molecular formula containing heteroatoms. Isotopologues of assigned molecular formulae are not counted as assigned. Overall, a unique molecular formula could be assigned to 4,122 masses, corresponding to 49% of the obtained <i>m</i>/<i>z</i>.</p

    Substrate Use of <i>Pseudovibrio</i> sp. Growing in Ultra-Oligotrophic Seawater

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    <div><p>Marine planktonic bacteria often live in habitats with extremely low concentrations of dissolved organic matter (DOM). To study the use of trace amounts of DOM by the facultatively oligotrophic <i>Pseudovibrio</i> sp. FO-BEG1, we investigated the composition of artificial and natural seawater before and after growth. We determined the concentrations of dissolved organic carbon (DOC), total dissolved nitrogen (TDN), free and hydrolysable amino acids, and the molecular composition of DOM by electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry (ESI FT-ICR-MS). The DOC concentration of the artificial seawater we used for cultivation was 4.4 μmol C L<sup>-1</sup>, which was eight times lower compared to the natural oligotrophic seawater we used for parallel experiments (36 μmol C L <sup>-1</sup>). During the three-week duration of the experiment, cell numbers increased from 40 cells mL<sup>-1</sup> to 2x10<sup>4</sup> cells mL <sup>-1</sup> in artificial and to 3x10<sup>5</sup> cells mL <sup>-1</sup> in natural seawater. No nitrogen fixation and minor CO<sub>2</sub> fixation (< 1% of cellular carbon) was observed. Our data show that in both media, amino acids were not the main substrate for growth. Instead, FT-ICR-MS analysis revealed usage of a variety of different dissolved organic molecules, belonging to a wide range of chemical compound groups, also containing nitrogen. The present study shows that marine heterotrophic bacteria are able to proliferate with even lower DOC concentrations than available in natural ultra-oligotrophic seawater, using unexpected organic compounds to fuel their energy, carbon and nitrogen requirements.</p></div

    Summary of FT-ICR-MS results: Numbers of assigned formulas, significantly (p < 0.05) changing and decreasing peaks (N-containing compounds were subset of the overall decreasing compounds) in artificial and natural seawater.

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    <p>Summary of FT-ICR-MS results: Numbers of assigned formulas, significantly (p < 0.05) changing and decreasing peaks (N-containing compounds were subset of the overall decreasing compounds) in artificial and natural seawater.</p

    FT-ICR mass spectra (negative electrospray ionization) from the time points t<sub>0</sub> of (A) artificial seawater and (B) natural seawater.

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    <p>Asterisks indicate inorganic trace impurities. Inserts show a zoom into the region of mass 325.0 to 325.2 Da. In the negative mode inorganic contaminations suppressed the ionization of organic substances present in low amounts in the artificial seawater.</p