In situ diversity of metabolism and carbon use efficiency among soil bacteria

Abstract

The central carbon (C) metabolic network harvests energy to power the cell and feed biosynthesis for growth. In pure cultures, bacteria use some but not all of the network's major pathways, such as glycolysis and pentose phosphate and Entner-Doudoroff pathways. However, how these pathways are used in microorganisms in intact soil communities is unknown. Here, we analyzed the incorporation of ¹³C from glucose isotopomers into phospholipid fatty acids. We showed that groups of Gram-positive and Gram-negative bacteria in an intact agricultural soil used different pathways to metabolize glucose. They also differed in C use efficiency (CUE), the efficiency with which a substrate is used for biosynthesis. Our results provide experimental evidence for diversity among microbes in the organization of their central carbon metabolic network and CUE under in situ conditions. These results have important implications for our understanding of how community composition affects soil C cycling and organic matter formation.

Fig. 1.. Atom percent excess enrichment of ¹³C of fragment and entire molecule of PLFAs (C₁₄ to C₁₈) after a 10-day incubation with position-specific¹³C-labeled glucose.

Fragments include ethanoate (Eth), propionate (Pro), and the molecular ion (M). C1 to C6 refer to glucose-1-¹³C, glucose-2-¹³C, glucose-3-¹³C, glucose-4-¹³C, glucose-5-¹³C, and glucose-6-¹³C. P values above figure indicate differences in ¹³C among fragments and entire molecules with significant ¹³C difference relative to ethanoate being marked with star (*) (repeated-measure ANOVA, df = 15,2, P < 0.01). Box plots indicate interquartile ranges with each line (from down to up) representing minimum, 25%, median, 75%, and maximum value, while dots represent outliers.

Fig. 2.. Schematic representation of carbon incorporation from glucose into acetyl-CoA, the precursor for fatty acid biosynthesis.

Carbon atom numbering for metabolites (pyruvate, PYR; acetyl-CoA, AcCoA) is ordered from left to right, while numbers indicate the C position in the original glucose molecule. ED indicates Entner-Doudoroff pathway, EMP is Embden-Meyerhof-Parnas glycolysis, and PP is pentose phosphate pathway. Gray background shading indicates those C positions released by pyruvate dehydrogenase as CO₂.

Fig. 3.. Analysis of the metabolic flux patterns across PLFAs.

NMDS (A) and cluster analysis of modeled central C metabolic fluxes (B) based on ¹³C incorporation patterns into PLFA. To compare the observations with pure culture studies, the results for B. licheniformis and P. fluorescens from the study by Wu et al. (20) and S. pungens from the study by Scandellari et al. (55) are displayed (see the Supplementary Materials). Cluster analysis was based on the average Euclidean distance of fluxes between PLFAs. The heatmap shows the activity of the different pathways for individual PLFAs with red for pathway activities above and blue below the mean value. v₁ to v₁₈ and br₁ to br₈ are reactions in the model of the central C metabolic network (fig. 1). TCA, tricarboxylic acid.

Fig. 4.. Relative fluxes through the major metabolic pathways and carbon use efficiency of soil bacterial groups.

Fraction of glucose directed to (A) Embden-Meyerhof-Parnas glycolysis and pentose phosphate and Entner-Doudoroff pathways, (B) flux rate of TCA cycle reaction (v₆) relative to the flux v₁ (glucose➔glucose-6-phosphate), and (C) CUE for cluster A, cluster B, and cluster C PLFAs. Means and SEs were calculated from the grouped PLFAs in Fig. 3.

Fig. 5.. Correlation between TCA cycle activity (v₆) and CUE.

Each data point represents an individual PLFA in this study defining not only the correlation but also pure strains data (20, 55) that are displayed in Fig. 3. Clusters A, B, and C are assigned as Gram-positive bacteria A (Gram Pos_A), Gram-positive bacteria B (Gram Pos_B), and Gram-negative bacteria (Gram Neg), respectively.