Evaluation and Characterization of Bacterial Metabolic Dynamics with a Novel Profiling Technique, Real-Time Metabolotyping

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

Truffle-Associated Bacteria: Extrapolation from Diversity to Function

A third partner in the symbiosis between the fungus and plant root is represented by natural bacterial communities, which seem to play pivotal role in the complex biological processes of exchange involving nutrients and signaling from the soil hyphae, ectomycorrhizas, ascomata, and stromata. This review summarizes the recent evidence reported in literature showing that ascoma provides a habitat to complex microbial communities that are clearly differentiated from those of the soil and the ectomycorrhizosphere. Although the traditional plate isolation and the culture of microbes are indispensable for vegetative compatibility and/or functional assays, these techniques do not let a real in vivo picture the truffle ecosystem. Advent of the next-generation sequencing methods and recent advances in microarray technologies have increased culture-independent studies. Indeed, most microbiota remains uncultivable in laboratory conditions, and these novel technologies have greatly improved the understanding of microbial diversity and its functioning. This is particularly important for the ectomycorrhizal fungi of Tuber genus, since specific uncultivable-associated prokaryotes may play important roles in the biological system and ontogenetic cycle of these fungi. Success of truffle cultivation may be achieved with co-inoculum of specific bacteria with Tuber spp. upon establishment of commercial plantations; this is particularly relevant to the Tuber species with a high agronomic value