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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

Time series of STOCSY analysis of B. fibrisolvens MDT-10 incubated with LNA.During these one-hour experiments, a total of twelve continuously acquired in vivo1H-NMR spectra were used for STOCSY analysis. Spectra appearing on the right side are the sum of the twelve in vivo1H-NMR spectra measured in every one-hour experiment. Contour levels (STOCSY correlations) defined by different colors are indicated at the bottom. Blue represents high positive correlation and red represents low. Optical density (OD) is a measure of bacterial growth. Arrows indicate butyrate (black), lactate (orange), acetate (pink), and ethanol (purple). Signals surrounded by green squares are assumed to be derived from LNA or its metabolites. Data are representative of three independent experiments.
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pone-0004893-g004: Time series of STOCSY analysis of B. fibrisolvens MDT-10 incubated with LNA.During these one-hour experiments, a total of twelve continuously acquired in vivo1H-NMR spectra were used for STOCSY analysis. Spectra appearing on the right side are the sum of the twelve in vivo1H-NMR spectra measured in every one-hour experiment. Contour levels (STOCSY correlations) defined by different colors are indicated at the bottom. Blue represents high positive correlation and red represents low. Optical density (OD) is a measure of bacterial growth. Arrows indicate butyrate (black), lactate (orange), acetate (pink), and ethanol (purple). Signals surrounded by green squares are assumed to be derived from LNA or its metabolites. Data are representative of three independent experiments.

Mentions: Analyzing the metabolic response of MDT-10 to LNA, signals in the 1 to 3 ppm region were greatly changed and some of the compounds were successfully identified. For example, several organic acids produced from glucose were identified, and some signals, such as those at 1.2, 1.5, 1.8, and nearby 2.8 ppm, showed a positive correlation with glucose consumption (Fig. 4). As those signals were assumed to be derived from LNA or its metabolites, we next performed 2D 1H, 13C-HSQC analysis using U-13C18 LA and U-13C18 LNA.


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)

Time series of STOCSY analysis of B. fibrisolvens MDT-10 incubated with LNA.During these one-hour experiments, a total of twelve continuously acquired in vivo1H-NMR spectra were used for STOCSY analysis. Spectra appearing on the right side are the sum of the twelve in vivo1H-NMR spectra measured in every one-hour experiment. Contour levels (STOCSY correlations) defined by different colors are indicated at the bottom. Blue represents high positive correlation and red represents low. Optical density (OD) is a measure of bacterial growth. Arrows indicate butyrate (black), lactate (orange), acetate (pink), and ethanol (purple). Signals surrounded by green squares are assumed to be derived from LNA or its metabolites. Data are representative of three independent experiments.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2654759&req=5

pone-0004893-g004: Time series of STOCSY analysis of B. fibrisolvens MDT-10 incubated with LNA.During these one-hour experiments, a total of twelve continuously acquired in vivo1H-NMR spectra were used for STOCSY analysis. Spectra appearing on the right side are the sum of the twelve in vivo1H-NMR spectra measured in every one-hour experiment. Contour levels (STOCSY correlations) defined by different colors are indicated at the bottom. Blue represents high positive correlation and red represents low. Optical density (OD) is a measure of bacterial growth. Arrows indicate butyrate (black), lactate (orange), acetate (pink), and ethanol (purple). Signals surrounded by green squares are assumed to be derived from LNA or its metabolites. Data are representative of three independent experiments.
Mentions: Analyzing the metabolic response of MDT-10 to LNA, signals in the 1 to 3 ppm region were greatly changed and some of the compounds were successfully identified. For example, several organic acids produced from glucose were identified, and some signals, such as those at 1.2, 1.5, 1.8, and nearby 2.8 ppm, showed a positive correlation with glucose consumption (Fig. 4). As those signals were assumed to be derived from LNA or its metabolites, we next performed 2D 1H, 13C-HSQC analysis using U-13C18 LA and U-13C18 LNA.

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