Recent advances in mapping environmental microbial metabolisms through¹³C isotopic fingerprints

Abstract

After feeding microbes with a defined 13 C substrate, unique isotopic patterns (isotopic fingerprints) can be formed in their metabolic products. Such labelling information not only can provide novel insights into functional pathways but also can determine absolute carbon fluxes through the metabolic network via metabolic modelling approaches. This technique has been used for finding pathways that may have been mis-annotated in the past, elucidating new enzyme functions, and investigating cell metabolisms in microbial communities. In this review paper, we summarize the applications of 13 C approaches to analyse novel cell metabolisms for the past 3 years. The isotopic fingerprints (defined as unique isotopomers useful for pathway identifications) have revealed the operations of the Entner-Doudoroff pathway, the reverse tricarboxylic acid cycle, new enzymes for biosynthesis of central metabolites, diverse respiration routes in phototrophic metabolism, co-metabolism of carbon nutrients and novel CO 2 fixation pathways. This review also discusses new isotopic methods to map carbon fluxes in global metabolisms, as well as potential factors influencing the metabolic flux quantification (e.g. metabolite channelling, the isotopic purity of 13 C substrates and the isotopic effect). Although 13 C labelling is not applicable to all biological systems (e.g. microbial communities), recent studies have shown that this method has a significant value in functional characterization of poorly understood micro-organisms, including species relevant for biotechnology and human health.

Two Pathways for Glutamate Biosynthesis in the Syntrophic Bacterium Syntrophus aciditrophicus

“…Isotopomer labeling analysis has been used to show the existence of a Re-citrate synthase for glutamate biosynthesis in several microorganisms (9)(10)(11)(12)(13)(14). Detection of a labeled glutamate at the C-1 position when Dehalococcoides ethenogenes was grown in a medium with [1-13 C]acetate (11) was consistent with the operation of a Re-citrate synthase.…”

“…1). Isotopomer labeling patterns of alanine and aspartate were analyzed to infer the labeling patterns in pyruvate and oxaloacetate, which are the 2-oxoacid skeletons of alanine and aspartate, respectively ( fragments of aspartate had a single 13 C atom. These observed labeling percentages up to 22% indicate the use of [1-…”

“…The 1D spectra were acquired with 65,536 data points, whereas 2D spectra were collected using 2,048 points in the F 2 dimension and 512 increments in the F 1 dimension. 13 C spectra were recorded with 32,768 transients. For 2D spectra, 32 to 64 transients were used.…”

“…In addition, the genomic analysis of S. aciditrophicus suggested that acetyl-CoA and bicarbonate (carbon dioxide) were major precursors for the biosynthesis of cellular components. In this study, S. aciditrophicus was grown on 13 C-and 14 C-labeled substrates and the isotopomer labeling patterns of alanine, aspartate, and glutamate were determined by gas chromatography-mass spectrometry (GC-MS), nuclear magnetic resonance (NMR) spectroscopy, and chemical degradation. The obtained patterns were used to delineate the mechanisms for glutamate biosynthesis as well as global utilization of acetate and bicarbonate as carbon sources for S. aciditrophicus.…”

Large-Scale 13 C Flux Profiling Reveals Conservation of the Entner-Doudoroff Pathway as a Glycolytic Strategy among Marine Bacteria That Use Glucose

“…Such studies have provided valuable insight into the intracellular pathways of microorganisms. For example, industrially relevant microbes have been extensively studied (19), and more recently, fluxomic studies have examined pathogenic (16,20) and ecologically important (21) microorganisms. In contrast, there is still limited knowledge on the intracellular pathways of marine bacteria, and only a few isolates have been investigated so far (22)(23)(24).…”