Dynamic exometabolome analysis reveals active metabolic pathways in non‐replicating mycobacteria

An organism's metabolic activity leaves an extracellular footprint and dynamic changes in this exometabolome inform about nutrient uptake, waste disposal and signalling activities. Using non‐targeted mass spectrometry, we report exometabolome dynamics of hypoxia‐induced, non‐replicating mycobacteria that are thought to play a role in latent tuberculosis. Despite evidence of active metabolism, little is known about the mechanisms enabling obligate aerobic mycobacteria to cope with hypoxia, resulting in long‐term survival and increased chemotherapeutic tolerance. The dynamics of 379 extracellular compounds of Mycobacterium smegmatis were deconvoluted with a genome‐scale metabolic reaction‐pair network to generate hypotheses about intracellular pathway usage. Time‐resolved ¹³C‐tracing and mutant experiments then demonstrated a crucial, energy‐generating role of asparagine utilization and non‐generic usage of the glyoxylate shunt for hypoxic fitness. Experiments with M. bovis and M. tuberculosis revealed the general relevance of asparagine fermentation and a variable contribution of the glyoxylate shunt to non‐replicative, hypoxic survival between the three species.