Fig. 3: Altered redox status and susceptibility to oxidizing agent in cydA and icl1 knockdown strains.

A,B CellRox DeepRed staining of (A) cydA and (B) icl1 CRISPRi knockdown strains showing significantly higher fluorescence upon exposure to 10 X MIC rifampicin. Note that the cydA knockdown showed elevated oxidative stress even before the rifampicin exposure in contrast to the icl1 knockdown where oxidative stress was increased only upon by rifampicin exposure. The vector control (C) did not show any difference between the induced and uninduced samples before and after rifampicin exposure. D–F Protein carbonylation assay to measure the oxidative stress of (D) cydA and (E) icl1 CRISPRi knockdown strains before and after rifampicin exposure. The cydA knockdown showed elevated carbonylation before and after rifampicin exposure while the icl1 mutant showed significantly higher levels of carbonylated proteins upon rifampicin exposure. No significant difference was observed in the induced and uninduced samples of vector control (F). G–I Susceptibility of cydA (G) and icl1 (H) knockdown mutants to hydrogen peroxide. Both cydA and icl1 knockdowns were hypersusceptible to H₂O₂ compared to the vector control (I). Exposure to 0.1% H₂O₂ led to sterilization of all cultures in 24 h. Data from triplicate cultures are shown. Error bar indicates standard deviation. *P < 0.05; **P < 0.01; ***P < 0.001, ****P < 0.0001, statistical significance estimated using a two-tailed unpaired t-test, GraphPad Prism 9.1). KD= knockdown.