Substrate-dependent CO2-fixation in heterotrophic bacteria revealed by stable isotope labelling

M. Spona-Friedl, A. Braun, C. Huber, W. Eisenreich, C. Griebler, A. Kappler, M. Elsner

FEMS Microbiology Ecology https://doi.org/10.1093/femsec/fiaa080

Abstract

Virtually all heterotrophs incorporate carbon dioxide by anaplerotic fixation. Little explored, however, is the interdependency of pathways and rates of CO₂-fixation on the concurrent usage of organic substrate(s). Potentially, this could reveal which substrates out of a pool of dissolved organic carbon (DOC) are utilised by environmental microorganisms. To explore this possibility, Bacillus subtilis W23 was grown in a minimal medium with normalised amounts of either glucose, lactate or malate as only organic substrates, each together with 1 g/ L NaH¹³CO₃. Incorporation of H¹³CO₃- was traced by elemental analysis-isotope ratio mass spectrometry (EA-IRMS) of biomass and gas chromatography-mass spectrometry (GC-MS) of protein-derived amino acids. Until the late logarithmic phase, ¹³C-incorporation into the tricarboxylic acid cycle increased with time and occurred via [4-¹³C]oxaloacetate formed by carboxylation of pyruvate. The levels of ¹³C-incorporation were highest for growth on glucose and lowest on malate. ¹³C-Incorporation into gluconeogenesis products was mainly detected in the lactate and malate experiment, whereas glucose down-regulated this path. A proof-of-principle study with a natural groundwater community confirmed the ability to determine incorporation from H¹³CO₃- by natural communities leading to specific labelling patterns. This underlines the potential of the labelling approach to characterise carbon sources of heterotrophic microorganisms in their natural environments.