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Helicobacter pylori displays a worldwide infection rate of about 50%. The Gram-negative bacterium is the main reason for gastric cancer and other severe diseases. Despite considerable knowledge about the metabolic inventory of H. pylori, carbon fluxes through the citrate cycle (TCA cycle) remained enigmatic. In this study, different 13 C-labeled substrates were supplied as carbon sources to H. pylori during microaerophilic growth in a complex medium. After growth, 13 C-excess and 13 C-distribution were determined in multiple metabolites using GC-MS analysis. [U- 13 C 6 ]Glucose was efficiently converted into glyceraldehyde but only less into TCA cycle-related metabolites. In contrast, [U- 13 C 5 ]glutamate, [U- 13 C 4 ]succinate, and [U- 13 C 4 ]aspartate were incorporated at high levels into intermediates of the TCA cycle. The comparative analysis of the 13 C-distributions indicated an adaptive TCA cycle fully operating in the closed oxidative direction with rapid equilibrium fluxes between oxaloacetate-succinate and α-ketoglutarate-citrate. 13 C-Profiles of the four-carbon intermediates in the TCA cycle, especially of malate, together with the observation of an isocitrate lyase activity by in vitro assays, suggested carbon fluxes via a glyoxylate bypass. In conjunction with the lack of enzymes for anaplerotic CO 2 fixation, the glyoxylate bypass could be relevant to fill up the TCA cycle with carbon atoms derived from acetyl-CoA., (© 2021 The Authors. Molecular Microbiology published by John Wiley & Sons Ltd.)