Fig. 3: The reduced form of TrxA is required for TreX activity.

a Mutations of five independent evolved strains resistant to the chimeric CDI::TreX-TriX system selected in bacterial competition. Genes and corresponding mutations are indicated (f.s., frameshift after the residue indicated). Asterisks indicate an incomplete list of mutations identified (see Supplementary Table 4 for the complete list of mutations). b E. coli MC4100 wild-type (WT), trxA, trxA C36A, and trxB mutant strains expressing, or not, treX from the pBAD33 vector, were serially diluted and spotted on LB plates supplemented with 0.2% L-arabinose. c In vitro ADP-ribosylation of FtsZ by the TreX toxin in various conditions. Purified FtsZ was incubated with the TreX toxin, biotinyl-NAD⁺, wild-type or C36A mutant TrxA, reducing agent DTT or TrxB and NADPH, as indicated, for 15 min at 37 °C. Reactions were then subjected to SDS-PAGE, transfer onto nitrocellulose, and stained with Ponceau Red (top panel). Biotinyl-ADP-ribosylation was detected using streptavidin-alkaline phosphate conjugate (lower panel). Molecular weight markers (in kDa) are indicated on the left.