초록
<▼1><P><B>Significance</B></P><P>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 <I>mdbA</I> 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, <I>Corynebacterium diphtheriae</I> cells lacking <I>mdbA</I> are viable at 30 °C, suggesting the presence of alternative oxidoreductase(s) recompensing the loss of <I>mdbA</I>. Using genetic suppressor, structural, and biochemical analyses, we provide evidence to support that <I>C. diphtheriae</I> 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.</P></▼1><▼2><P>In many gram-positive Actinobacteria, including <I>Actinomyces oris</I> and <I>Corynebacterium matruchotii</I>, the conserved thiol-disulfide oxidoreductase MdbA that catalyzes oxidative folding of exported proteins is essential for bacterial viability by an unidentified mechanism. Intriguingly, in <I>Corynebacterium diphtheriae</I>, the deletion of <I>mdbA</I> blocks cell growth only at 37 °C but not at 30 °C, suggesting the presence of alternative oxidoreductase enzyme(s). By isolating spontaneous thermotolerant revertants of the <I>mdbA</I> mutant at 37 °C, we obtained genetic suppressors, all mapped to a single T-to-G mutation within the promoter region of <I>tsdA</I>, causing its elevated expression. Strikingly, increased expression of <I>tsdA</I>—via suppressor mutations or a constitutive promoter—rescues the pilus assembly and toxin production defects of this mutant, hence compensating for the loss of <I>mdbA</I>. 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 <I>tsdA</I> expression is upregulated at nonpermissive temperature (40 °C) in wild-type cells, we posit that TsdA has evolved as a compensatory thiol-disulfide oxidoreductase that safeguards oxidative protein folding in <I>C. diphtheriae</I> against thermal stress.</P></▼2>