Limits...
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.

Show MeSH

Related in: MedlinePlus

Z-score analysis of time-dependent 1H-NMR profiles of three B. fibrisolvens strains incubated with or without LA or LNA.A total of 120 continuously acquired in vivo1H-NMR spectra were used in each Z-score analysis. MDT-10, MDT-5, and A38 were grown in anaerobic culture (control), with 2.5 mM LA, or with 2.5 mM LNA. Asterisks mean low concentration (0.6 mM) of FAs, because MDT-5 did not grow in the presence of 2.5 mM LA or 2.5 mM LNA, and A38 did not grow in the presence of 2.5 mM LNA. Data are representative of three independent experiments. Contour levels (Z-score values) defined by different colors are indicated at the bottom.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2654759&req=5

pone-0004893-g002: Z-score analysis of time-dependent 1H-NMR profiles of three B. fibrisolvens strains incubated with or without LA or LNA.A total of 120 continuously acquired in vivo1H-NMR spectra were used in each Z-score analysis. MDT-10, MDT-5, and A38 were grown in anaerobic culture (control), with 2.5 mM LA, or with 2.5 mM LNA. Asterisks mean low concentration (0.6 mM) of FAs, because MDT-5 did not grow in the presence of 2.5 mM LA or 2.5 mM LNA, and A38 did not grow in the presence of 2.5 mM LNA. Data are representative of three independent experiments. Contour levels (Z-score values) defined by different colors are indicated at the bottom.

Mentions: Z-score analysis of three B. fibrisolvens strains revealed their metabolic dynamics, including time-varying factors (Fig. 2). All samples showed remarkable changes of the chemical shifts in the 1 to 4 ppm region, which were derived from lipids, organic acids, and sugars, and around 8 ppm, which were derived from formic acid. In particular, signals around 2 ppm, which were attributed mainly to acetic acid present in abundance, were slightly shifted downfield with decreasing culture pH. A negative correlation was observed between glucose concentration, which was calculated from the 1H-NMR standard curve (data not shown), and bacterial growth rate (SI Fig. S1). It was possible to calculate the transition to acidic culture pH due to organic acid production by bacteria from the chemical shift changes of bacterial metabolites containing a carboxylic group (SI Fig. S2). Furthermore, Z-score analysis clearly showed changes in the chemical shifts of metabolites present in small amounts, which appeared as unidentified signals in the low field region (8.3–8.6 ppm) during bacterial growth.


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)

Z-score analysis of time-dependent 1H-NMR profiles of three B. fibrisolvens strains incubated with or without LA or LNA.A total of 120 continuously acquired in vivo1H-NMR spectra were used in each Z-score analysis. MDT-10, MDT-5, and A38 were grown in anaerobic culture (control), with 2.5 mM LA, or with 2.5 mM LNA. Asterisks mean low concentration (0.6 mM) of FAs, because MDT-5 did not grow in the presence of 2.5 mM LA or 2.5 mM LNA, and A38 did not grow in the presence of 2.5 mM LNA. Data are representative of three independent experiments. Contour levels (Z-score values) defined by different colors are indicated at the bottom.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0004893-g002: Z-score analysis of time-dependent 1H-NMR profiles of three B. fibrisolvens strains incubated with or without LA or LNA.A total of 120 continuously acquired in vivo1H-NMR spectra were used in each Z-score analysis. MDT-10, MDT-5, and A38 were grown in anaerobic culture (control), with 2.5 mM LA, or with 2.5 mM LNA. Asterisks mean low concentration (0.6 mM) of FAs, because MDT-5 did not grow in the presence of 2.5 mM LA or 2.5 mM LNA, and A38 did not grow in the presence of 2.5 mM LNA. Data are representative of three independent experiments. Contour levels (Z-score values) defined by different colors are indicated at the bottom.
Mentions: Z-score analysis of three B. fibrisolvens strains revealed their metabolic dynamics, including time-varying factors (Fig. 2). All samples showed remarkable changes of the chemical shifts in the 1 to 4 ppm region, which were derived from lipids, organic acids, and sugars, and around 8 ppm, which were derived from formic acid. In particular, signals around 2 ppm, which were attributed mainly to acetic acid present in abundance, were slightly shifted downfield with decreasing culture pH. A negative correlation was observed between glucose concentration, which was calculated from the 1H-NMR standard curve (data not shown), and bacterial growth rate (SI Fig. S1). It was possible to calculate the transition to acidic culture pH due to organic acid production by bacteria from the chemical shift changes of bacterial metabolites containing a carboxylic group (SI Fig. S2). Furthermore, Z-score analysis clearly showed changes in the chemical shifts of metabolites present in small amounts, which appeared as unidentified signals in the low field region (8.3–8.6 ppm) during bacterial growth.

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