A cryptic oxidoreductase safeguards oxidative protein folding in Corynebacterium diphtheriae

In many gram-positive Actinobacteria, including Actinomyces oris and Corynebacterium matruchotii, the conserved thiol-disulfide oxidoreductase MdbA that catalyzes oxidative folding of exported proteins is essential for bacterial viability by an unidentified mech-anism. Intriguingly, in Corynebacterium diphtheriae, the deletion of mdbA blocks cell growth only at 37 & DEG;C but not at 30 & DEG;C, suggesting the presence of alternative oxidoreduc-tase enzyme(s). By isolating spontaneous thermotolerant revertants of the mdbA mutant at 37 & DEG;C, we obtained genetic suppressors, all mapped to a single T-to-G mutation within the promoter region of tsdA, causing its elevated expression. Strikingly, increased expression of tsdA-via suppressor mutations or a constitutive promoter-rescues the pilus assembly and toxin production defects of this mutant, hence compensating for the loss of mdbA. Structural, genetic, and biochemical analyses demonstrated TsdA is a membrane-tethered thiol-disulfide oxidoreductase with a conserved CxxC motif that can substitute for MdbA in mediating oxidative folding of pilin and toxin substrates. Together with our observation that tsdA expression is upregulated at nonpermissive temperature (40 & DEG;C) in wild-type cells, we posit that TsdA has evolved as a compensatory thiol-disulfide oxidoreductase that safeguards oxidative protein folding in C. diphtheriae against thermal stress.

Automated summary

In many gram-positive Actinobacteria, MdbA plays a crucial role in bacterial viability by catalyzing oxidative folding of exported proteins. However, in Corynebacterium diphtheriae, the deletion of mdbA only affects cell growth at 37°C, suggesting the existence of alternative oxidoreductase enzymes. Through genetic suppressor mutations, we discovered that elevated expression of tsdA can compensate for the loss of mdbA by mediating oxidative folding of pilin and toxin substrates.