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Evaluation and characterization of bacterial metabolic dynamics with a novel profiling technique, real-time metabolotyping.

Fukuda S, Nakanishi Y, Chikayama E, Ohno H, Hino T, Kikuchi J - PLoS ONE (2009)

Bottom Line: Notably, intestinal bacteria maintain homeostatic balance in mammals via multiple dynamic biochemical reactions to produce several metabolites from undigested food, and those metabolites exert various effects on mammalian cells in a time-dependent manner.We identified trans11, cis13 conjugated linoleic acid as the intermediate of linolenic acid hydrogenation by B. fibrisolvens, based on the results of (13)C-labeling RT-MT experiments.RT-MT is useful for the characterization of beneficial bacterium that shows potential for use as probiotic by producing bioactive compounds.

View Article: PubMed Central - PubMed

Affiliation: RIKEN Research Center for Allergy and Immunology, Yokohama, Japan.

ABSTRACT

Background: Environmental processes in ecosystems are dynamically altered by several metabolic responses in microorganisms, including intracellular sensing and pumping, battle for survival, and supply of or competition for nutrients. Notably, intestinal bacteria maintain homeostatic balance in mammals via multiple dynamic biochemical reactions to produce several metabolites from undigested food, and those metabolites exert various effects on mammalian cells in a time-dependent manner. We have established a method for the analysis of bacterial metabolic dynamics in real time and used it in combination with statistical NMR procedures.

Methodology/principal findings: We developed a novel method called real-time metabolotyping (RT-MT), which performs sequential (1)H-NMR profiling and two-dimensional (2D) (1)H, (13)C-HSQC (heteronuclear single quantum coherence) profiling during bacterial growth in an NMR tube. The profiles were evaluated with such statistical methods as Z-score analysis, principal components analysis, and time series of statistical TOtal Correlation SpectroScopY (TOCSY). In addition, using 2D (1)H, (13)C-HSQC with the stable isotope labeling technique, we observed the metabolic kinetics of specific biochemical reactions based on time-dependent 2D kinetic profiles. Using these methods, we clarified the pathway for linolenic acid hydrogenation by a gastrointestinal bacterium, Butyrivibrio fibrisolvens. We identified trans11, cis13 conjugated linoleic acid as the intermediate of linolenic acid hydrogenation by B. fibrisolvens, based on the results of (13)C-labeling RT-MT experiments. In addition, we showed that the biohydrogenation of polyunsaturated fatty acids serves as a defense mechanism against their toxic effects.

Conclusions: RT-MT is useful for the characterization of beneficial bacterium that shows potential for use as probiotic by producing bioactive compounds.

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Related in: MedlinePlus

LNA metabolic dynamics of B. fibrisolvens MDT-10 analyzed by time-dependent 2D-13C-HSQC RT-MT.The NMR tube was anaerobically inoculated with B. fibrisolvens MDT-10 and U-13C18 LNA (2.5 mM) was added to follow LNA hydrogenation reactions in in vivo 2D-13C-HSQC RT-MT. A: Typical 2D HSQC spectra (sequential growth at 0, 2, 3, and 4 hours) are shown. 2D HSQC spectra were observed every 8 minutes. Arrows in upper panel indicate C-H structures corresponding to the signals. Signal intensities were calculated based on their standard curves. Arrows pointing to lower panel (B) indicate 2D HSQC spectra measured at the indicated time point. B: LNA metabolic dynamics of B. fibrisolvens MDT-10 calculated from time-dependent 2D-13C-HSQC RT-MT. LNA (black circles), CLNA (blue triangles), t11, c15–18:2 (pink diamonds), VA (green squares), and bacterial growth (open red squares) are shown. Mean values of triplicate experiments are shown.
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pone-0004893-g005: LNA metabolic dynamics of B. fibrisolvens MDT-10 analyzed by time-dependent 2D-13C-HSQC RT-MT.The NMR tube was anaerobically inoculated with B. fibrisolvens MDT-10 and U-13C18 LNA (2.5 mM) was added to follow LNA hydrogenation reactions in in vivo 2D-13C-HSQC RT-MT. A: Typical 2D HSQC spectra (sequential growth at 0, 2, 3, and 4 hours) are shown. 2D HSQC spectra were observed every 8 minutes. Arrows in upper panel indicate C-H structures corresponding to the signals. Signal intensities were calculated based on their standard curves. Arrows pointing to lower panel (B) indicate 2D HSQC spectra measured at the indicated time point. B: LNA metabolic dynamics of B. fibrisolvens MDT-10 calculated from time-dependent 2D-13C-HSQC RT-MT. LNA (black circles), CLNA (blue triangles), t11, c15–18:2 (pink diamonds), VA (green squares), and bacterial growth (open red squares) are shown. Mean values of triplicate experiments are shown.

Mentions: Our past work has demonstrated that the bacterial growth inhibitory activity of unsaturated FAs increases with increasing degree of unsaturation [47]. Therefore, we next analyzed the metabolic dynamics of LNA, which is more unsaturated than LA, using MDT-10. Similar to the case of LA (SI Fig. S4), MDT-10 growth was suppressed at the initial growth stage when LNA concentration was high, but was gradually improved as LNA was hydrogenated to VA (Fig. 5). The intermediates of LNA hydrogenation were identified as CLNA, trans11, cis15–18:2 (t11, c15–18:2), and VA. When similar analysis was performed with MDT-5 and A38, the final product of LNA hydrogenation was CLNA and t11, c15–18:2, respectively (Fig. 6-A). Previously, we reported that strain TH1 produced CLNA and t11, c15–18:2 as the intermediates of LNA hydrogenation [46], and we also observed that strains ATCC19171 and ATCC51255 produced CLNA and t11, c15–18:2 as intermediates (data not shown). Thus, the LNA hydrogenation pathway may be similar among B. fibrisolvens strains except A38 and MDT-5.


Evaluation and characterization of bacterial metabolic dynamics with a novel profiling technique, real-time metabolotyping.

Fukuda S, Nakanishi Y, Chikayama E, Ohno H, Hino T, Kikuchi J - PLoS ONE (2009)

LNA metabolic dynamics of B. fibrisolvens MDT-10 analyzed by time-dependent 2D-13C-HSQC RT-MT.The NMR tube was anaerobically inoculated with B. fibrisolvens MDT-10 and U-13C18 LNA (2.5 mM) was added to follow LNA hydrogenation reactions in in vivo 2D-13C-HSQC RT-MT. A: Typical 2D HSQC spectra (sequential growth at 0, 2, 3, and 4 hours) are shown. 2D HSQC spectra were observed every 8 minutes. Arrows in upper panel indicate C-H structures corresponding to the signals. Signal intensities were calculated based on their standard curves. Arrows pointing to lower panel (B) indicate 2D HSQC spectra measured at the indicated time point. B: LNA metabolic dynamics of B. fibrisolvens MDT-10 calculated from time-dependent 2D-13C-HSQC RT-MT. LNA (black circles), CLNA (blue triangles), t11, c15–18:2 (pink diamonds), VA (green squares), and bacterial growth (open red squares) are shown. Mean values of triplicate experiments are shown.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2654759&req=5

pone-0004893-g005: LNA metabolic dynamics of B. fibrisolvens MDT-10 analyzed by time-dependent 2D-13C-HSQC RT-MT.The NMR tube was anaerobically inoculated with B. fibrisolvens MDT-10 and U-13C18 LNA (2.5 mM) was added to follow LNA hydrogenation reactions in in vivo 2D-13C-HSQC RT-MT. A: Typical 2D HSQC spectra (sequential growth at 0, 2, 3, and 4 hours) are shown. 2D HSQC spectra were observed every 8 minutes. Arrows in upper panel indicate C-H structures corresponding to the signals. Signal intensities were calculated based on their standard curves. Arrows pointing to lower panel (B) indicate 2D HSQC spectra measured at the indicated time point. B: LNA metabolic dynamics of B. fibrisolvens MDT-10 calculated from time-dependent 2D-13C-HSQC RT-MT. LNA (black circles), CLNA (blue triangles), t11, c15–18:2 (pink diamonds), VA (green squares), and bacterial growth (open red squares) are shown. Mean values of triplicate experiments are shown.
Mentions: Our past work has demonstrated that the bacterial growth inhibitory activity of unsaturated FAs increases with increasing degree of unsaturation [47]. Therefore, we next analyzed the metabolic dynamics of LNA, which is more unsaturated than LA, using MDT-10. Similar to the case of LA (SI Fig. S4), MDT-10 growth was suppressed at the initial growth stage when LNA concentration was high, but was gradually improved as LNA was hydrogenated to VA (Fig. 5). The intermediates of LNA hydrogenation were identified as CLNA, trans11, cis15–18:2 (t11, c15–18:2), and VA. When similar analysis was performed with MDT-5 and A38, the final product of LNA hydrogenation was CLNA and t11, c15–18:2, respectively (Fig. 6-A). Previously, we reported that strain TH1 produced CLNA and t11, c15–18:2 as the intermediates of LNA hydrogenation [46], and we also observed that strains ATCC19171 and ATCC51255 produced CLNA and t11, c15–18:2 as intermediates (data not shown). Thus, the LNA hydrogenation pathway may be similar among B. fibrisolvens strains except A38 and MDT-5.

Bottom Line: Notably, intestinal bacteria maintain homeostatic balance in mammals via multiple dynamic biochemical reactions to produce several metabolites from undigested food, and those metabolites exert various effects on mammalian cells in a time-dependent manner.We identified trans11, cis13 conjugated linoleic acid as the intermediate of linolenic acid hydrogenation by B. fibrisolvens, based on the results of (13)C-labeling RT-MT experiments.RT-MT is useful for the characterization of beneficial bacterium that shows potential for use as probiotic by producing bioactive compounds.

View Article: PubMed Central - PubMed

Affiliation: RIKEN Research Center for Allergy and Immunology, Yokohama, Japan.

ABSTRACT

Background: Environmental processes in ecosystems are dynamically altered by several metabolic responses in microorganisms, including intracellular sensing and pumping, battle for survival, and supply of or competition for nutrients. Notably, intestinal bacteria maintain homeostatic balance in mammals via multiple dynamic biochemical reactions to produce several metabolites from undigested food, and those metabolites exert various effects on mammalian cells in a time-dependent manner. We have established a method for the analysis of bacterial metabolic dynamics in real time and used it in combination with statistical NMR procedures.

Methodology/principal findings: We developed a novel method called real-time metabolotyping (RT-MT), which performs sequential (1)H-NMR profiling and two-dimensional (2D) (1)H, (13)C-HSQC (heteronuclear single quantum coherence) profiling during bacterial growth in an NMR tube. The profiles were evaluated with such statistical methods as Z-score analysis, principal components analysis, and time series of statistical TOtal Correlation SpectroScopY (TOCSY). In addition, using 2D (1)H, (13)C-HSQC with the stable isotope labeling technique, we observed the metabolic kinetics of specific biochemical reactions based on time-dependent 2D kinetic profiles. Using these methods, we clarified the pathway for linolenic acid hydrogenation by a gastrointestinal bacterium, Butyrivibrio fibrisolvens. We identified trans11, cis13 conjugated linoleic acid as the intermediate of linolenic acid hydrogenation by B. fibrisolvens, based on the results of (13)C-labeling RT-MT experiments. In addition, we showed that the biohydrogenation of polyunsaturated fatty acids serves as a defense mechanism against their toxic effects.

Conclusions: RT-MT is useful for the characterization of beneficial bacterium that shows potential for use as probiotic by producing bioactive compounds.

Show MeSH
Related in: MedlinePlus