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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

Development of real-time metabolotyping (RT-MT) to analyze bacterial metabolic dynamics.In vivo1H-NMR (right) and two-dimensional 1H, 13C-HSQC with 13C labeled substrate technique (2D-13C-HSQC) (left) were continuously conducted during bacterial growth in an NMR tube at 37°C, and the profiling data were processed and analyzed by several statistical methods, such as Z-score analysis, principal components analysis (PCA), and time series of statistical total correlation spectroscopy (STOCSY). From the results of statistical analyses of In vivo1H-NMR profiling data, we extracted meaningful data related to time-varying information. Furthermore, from the results of in vivo 2D-13C-HSQC profiling data, we revealed the metabolic kinetics of specific metabolic reactions.
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pone-0004893-g001: Development of real-time metabolotyping (RT-MT) to analyze bacterial metabolic dynamics.In vivo1H-NMR (right) and two-dimensional 1H, 13C-HSQC with 13C labeled substrate technique (2D-13C-HSQC) (left) were continuously conducted during bacterial growth in an NMR tube at 37°C, and the profiling data were processed and analyzed by several statistical methods, such as Z-score analysis, principal components analysis (PCA), and time series of statistical total correlation spectroscopy (STOCSY). From the results of statistical analyses of In vivo1H-NMR profiling data, we extracted meaningful data related to time-varying information. Furthermore, from the results of in vivo 2D-13C-HSQC profiling data, we revealed the metabolic kinetics of specific metabolic reactions.

Mentions: We have developed RT-MT to understand and evaluate the metabolic dynamics of several bacterial strains (Fig. 1). The most important point of this method is that the time-dependent metabolic profiles from 1H-NMR and 1H, 13C-HSQC sequential observations during bacterial growth in an NMR tube are calculated and evaluated with several statistical methods, such as Z-score analysis, principal components analysis (PCA), and statistical total correlated spectroscopy (STOCSY). It is considered that the statistical analyses of time-dependent metabolic profiles would show meaningful biological data related to metabolic dynamics. In addition, time-dependent two-dimensional 1H, 13C-HSQC with 13C labeled substrates (2D-13C-HSQC) profiles could be used to generate three-dimensional metabolic kinetic profiles. Time-dependent 2D-13C-HSQC can provide very detailed information of specific metabolic dynamics. As anaerobic conditions can be easily created in NMR tubes, RT-MT can be applied to the analysis of anaerobic environments, such as soil, industrial plant, and animal gut. RT-MT is expected to improve our understanding of metabolic dynamics that would be necessary for the extraction of particular characteristics of metabolic changes.


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)

Development of real-time metabolotyping (RT-MT) to analyze bacterial metabolic dynamics.In vivo1H-NMR (right) and two-dimensional 1H, 13C-HSQC with 13C labeled substrate technique (2D-13C-HSQC) (left) were continuously conducted during bacterial growth in an NMR tube at 37°C, and the profiling data were processed and analyzed by several statistical methods, such as Z-score analysis, principal components analysis (PCA), and time series of statistical total correlation spectroscopy (STOCSY). From the results of statistical analyses of In vivo1H-NMR profiling data, we extracted meaningful data related to time-varying information. Furthermore, from the results of in vivo 2D-13C-HSQC profiling data, we revealed the metabolic kinetics of specific metabolic reactions.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0004893-g001: Development of real-time metabolotyping (RT-MT) to analyze bacterial metabolic dynamics.In vivo1H-NMR (right) and two-dimensional 1H, 13C-HSQC with 13C labeled substrate technique (2D-13C-HSQC) (left) were continuously conducted during bacterial growth in an NMR tube at 37°C, and the profiling data were processed and analyzed by several statistical methods, such as Z-score analysis, principal components analysis (PCA), and time series of statistical total correlation spectroscopy (STOCSY). From the results of statistical analyses of In vivo1H-NMR profiling data, we extracted meaningful data related to time-varying information. Furthermore, from the results of in vivo 2D-13C-HSQC profiling data, we revealed the metabolic kinetics of specific metabolic reactions.
Mentions: We have developed RT-MT to understand and evaluate the metabolic dynamics of several bacterial strains (Fig. 1). The most important point of this method is that the time-dependent metabolic profiles from 1H-NMR and 1H, 13C-HSQC sequential observations during bacterial growth in an NMR tube are calculated and evaluated with several statistical methods, such as Z-score analysis, principal components analysis (PCA), and statistical total correlated spectroscopy (STOCSY). It is considered that the statistical analyses of time-dependent metabolic profiles would show meaningful biological data related to metabolic dynamics. In addition, time-dependent two-dimensional 1H, 13C-HSQC with 13C labeled substrates (2D-13C-HSQC) profiles could be used to generate three-dimensional metabolic kinetic profiles. Time-dependent 2D-13C-HSQC can provide very detailed information of specific metabolic dynamics. As anaerobic conditions can be easily created in NMR tubes, RT-MT can be applied to the analysis of anaerobic environments, such as soil, industrial plant, and animal gut. RT-MT is expected to improve our understanding of metabolic dynamics that would be necessary for the extraction of particular characteristics of metabolic changes.

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