LAUSR

Significance Oxidative protein folding via disulfide bond formation is an important process in bacteria, although it can be dispensable in various organisms. In many gram-positive Actinobacteria, deletion of mdbA coding for the conserved thiol-disulfide oxidoreductase MdbA that catalyzes oxidative folding of exported proteins is lethal for cell growth by an uncharacterized mechanism. However, Corynebacterium diphtheriae cells lacking mdbA are viable at 30 °C, suggesting the presence of alternative oxidoreductase(s) recompensing the loss of mdbA. Using genetic suppressor, structural, and biochemical analyses, we provide evidence to support that C. diphtheriae encodes TsdA as a compensatory thiol-disulfide oxidoreductase safeguarding oxidative protein folding in this actinobacterium against thermal stress. This study expands our understanding of oxidative protein folding mechanisms in the understudied Actinobacteria.