A universal stress protein acts as a metabolic rheostat controlling carbon flux in mycobacteria

Universal stress proteins (USPs) are ubiquitous amongst prokaryotes and have empirically determined roles in adaptation to stress, while their specific biochemical function in cellular processes is opaque. The most frequently encountered USP is a small ~15kDa protein comprised of a single domain (PF00582). In Mycobacterium tuberculosis Rv1636 is the sole single domain protein amongst its 10 USPs. We were unable to delete rv1636 from M. tuberculosis , supporting previous data that it is essential for growth. We deleted its orthologue, MS3811, from M. smegmatis , and in exponential phase competition experiments we observed that the advantage switched between wildtype and the mutant depending on the growth environment. In rich medium the wildtype had the competitive advantage, but in a defined medium with a single carbon source the competitive advantage switched, with the mutant rapidly taking over the culture. We hypothesised the USP is regulating metabolic flux, with its deletion leading to increased flux and more rapid turn-over of central-carbon metabolites. We tested this by performing 13C-stable isotope tracing, using [U-13C6] glucose as sole carbon source for growth of the parental and deletion strains. The findings were unequivocal; deletion of the USP led to an increase in label incorporation in central carbon metabolites and a profound change in the isotopologue distribution. Furthermore, in vivo protein-crosslinking provided evidence that Rv1636 interacts directly with key central metabolic enzymes, including the glycolytic enzymes pyruvate kinase, pyruvate dehydrogenase, pyruvate synthase. We propose the mycobacterial single domain USP acts as a metabolic rheostat to regulate central carbon metabolism. Importance There are more than 62,000 universal stress proteins (USPs) found mostly in prokaryotes, but very few examples where the role of a USP is unequivocally linked to a specific biological or biochemical function. For the first time we were able to assign a role in metabolic regulation to a bacterial single domain universal stress protein. We demonstrate a mycobacterial USP regulates flux through central catabolic pathways, and our evidence, based on 13C-stable isotope tracing, indicates that the USP acts as a brake on carbon flux, and we assign to it a role as a metabolic rheostat.