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Exo-metabolome of Pseudovibrio sp. FO-BEG1 analyzed by ultra-high resolution mass spectrometry and the effect of phosphate limitation.

Romano S, Dittmar T, Bondarev V, Weber RJ, Viant MR, Schulz-Vogt HN - PLoS ONE (2014)

Bottom Line: Oceanic dissolved organic matter (DOM) is an assemblage of reduced carbon compounds, which results from biotic and abiotic processes.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.

View Article: PubMed Central - PubMed

Affiliation: Max Planck Institute for Marine Microbiology, Bremen, Germany.

ABSTRACT
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 Pseudovibrio sp. FO-BEG1 via ultra-high resolution mass spectrometry, comparing phosphate limited with phosphate surplus growth conditions. Bacteria belonging to the Pseudovibrio genus have been isolated worldwide, mainly from marine invertebrates and were described as metabolically versatile Alphaproteobacteria. 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.

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Similarity among the FT-ICR-MS samples analyzed in ESI-negative mode during bacterial growth under +Pi and −Pi conditions.Non metrical multidimensional scaling (NMDS) was performed by employing the Bray-Curtis similarity index and using the data of the unfiltered (A) and filtered (B) datasets. All biological triplicates of +Pi (filled circles) and −Pi (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.
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pone-0096038-g002: Similarity among the FT-ICR-MS samples analyzed in ESI-negative mode during bacterial growth under +Pi and −Pi conditions.Non metrical multidimensional scaling (NMDS) was performed by employing the Bray-Curtis similarity index and using the data of the unfiltered (A) and filtered (B) datasets. All biological triplicates of +Pi (filled circles) and −Pi (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.

Mentions: The raw data obtained from the ESI-negative FT-ICR-MS analysis consisted of 23,892 masses ranging from 154 m/z to 1,930 m/z. After normalization of the ion intensities, we performed a non-metrical multidimensional scaling (NMDS) in order to evaluate the similarities among the samples (Fig. 2A). As the stress value of the NMDS plot was 0.06, it could be considered a good representation of the calculated distance matrix and thus of the similarity among the samples. The samples collected at T1 for each biological triplicate under both −Pi and +Pi conditions clustered together and were clearly separated from the samples collected during the rest of the growth period (Fig. 2A). All biological triplicates of the −Pi conditions collected at the end of the logarithmic phase and in the stationary phase (T2 and T3) were completely divergent from the samples collected under +Pi stationary phase (T2). Moreover, the samples T2 and T3 for the −Pi conditions also clustered separately in the plot (Fig. 2A). The bootstrap analysis of the dendrogram constructed for the Bray-Curtis similarity matrix revealed that the divergence among the samples described above was statistically highly significant, since during the 1000 reiterations always the same clustering occurred (Fig. S1A). In ESI-positive mode 17,859 masses were detected, ranging from 153 m/z to 1,999 m/z. The NMDS plot obtained for this dataset was characterized by a stress value of 0.07, therefore, it could be considered a good representation of the distance matrix as well (Fig. S2A). All samples had a similar clustering as the one observed in the ESI-negative NMDS plot. One of the main difference was the higher divergence between one −Pi replicate (−Pi III T1) and the other replicates collected at the same time point. However, the minimum spanning tree showed that this sample shared the highest degree of similarity with the other samples collected under the same growth stage. Additionally, the samples collected at T2 and T3 under −Pi conditions showed a higher degree of similarity (Fig. S2A). The bootstrap analysis performed on the respective dendrogram revealed that in > 75% of the cases the samples clustered consistently with the NMDS groups, indicating that the divergences described above were statistically significant (Fig. S3A)


Exo-metabolome of Pseudovibrio sp. FO-BEG1 analyzed by ultra-high resolution mass spectrometry and the effect of phosphate limitation.

Romano S, Dittmar T, Bondarev V, Weber RJ, Viant MR, Schulz-Vogt HN - PLoS ONE (2014)

Similarity among the FT-ICR-MS samples analyzed in ESI-negative mode during bacterial growth under +Pi and −Pi conditions.Non metrical multidimensional scaling (NMDS) was performed by employing the Bray-Curtis similarity index and using the data of the unfiltered (A) and filtered (B) datasets. All biological triplicates of +Pi (filled circles) and −Pi (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.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4008564&req=5

pone-0096038-g002: Similarity among the FT-ICR-MS samples analyzed in ESI-negative mode during bacterial growth under +Pi and −Pi conditions.Non metrical multidimensional scaling (NMDS) was performed by employing the Bray-Curtis similarity index and using the data of the unfiltered (A) and filtered (B) datasets. All biological triplicates of +Pi (filled circles) and −Pi (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.
Mentions: The raw data obtained from the ESI-negative FT-ICR-MS analysis consisted of 23,892 masses ranging from 154 m/z to 1,930 m/z. After normalization of the ion intensities, we performed a non-metrical multidimensional scaling (NMDS) in order to evaluate the similarities among the samples (Fig. 2A). As the stress value of the NMDS plot was 0.06, it could be considered a good representation of the calculated distance matrix and thus of the similarity among the samples. The samples collected at T1 for each biological triplicate under both −Pi and +Pi conditions clustered together and were clearly separated from the samples collected during the rest of the growth period (Fig. 2A). All biological triplicates of the −Pi conditions collected at the end of the logarithmic phase and in the stationary phase (T2 and T3) were completely divergent from the samples collected under +Pi stationary phase (T2). Moreover, the samples T2 and T3 for the −Pi conditions also clustered separately in the plot (Fig. 2A). The bootstrap analysis of the dendrogram constructed for the Bray-Curtis similarity matrix revealed that the divergence among the samples described above was statistically highly significant, since during the 1000 reiterations always the same clustering occurred (Fig. S1A). In ESI-positive mode 17,859 masses were detected, ranging from 153 m/z to 1,999 m/z. The NMDS plot obtained for this dataset was characterized by a stress value of 0.07, therefore, it could be considered a good representation of the distance matrix as well (Fig. S2A). All samples had a similar clustering as the one observed in the ESI-negative NMDS plot. One of the main difference was the higher divergence between one −Pi replicate (−Pi III T1) and the other replicates collected at the same time point. However, the minimum spanning tree showed that this sample shared the highest degree of similarity with the other samples collected under the same growth stage. Additionally, the samples collected at T2 and T3 under −Pi conditions showed a higher degree of similarity (Fig. S2A). The bootstrap analysis performed on the respective dendrogram revealed that in > 75% of the cases the samples clustered consistently with the NMDS groups, indicating that the divergences described above were statistically significant (Fig. S3A)

Bottom Line: Oceanic dissolved organic matter (DOM) is an assemblage of reduced carbon compounds, which results from biotic and abiotic processes.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.

View Article: PubMed Central - PubMed

Affiliation: Max Planck Institute for Marine Microbiology, Bremen, Germany.

ABSTRACT
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 Pseudovibrio sp. FO-BEG1 via ultra-high resolution mass spectrometry, comparing phosphate limited with phosphate surplus growth conditions. Bacteria belonging to the Pseudovibrio genus have been isolated worldwide, mainly from marine invertebrates and were described as metabolically versatile Alphaproteobacteria. 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.

Show MeSH
Related in: MedlinePlus