FIG 4.

Biochemical and transcriptional validation that higher-level resistance (MIC = 8 μg/ml) occurs when mRNA for nifJ, iscR, or xdh is depleted by antisense sequence (AS) in the ΔfeoB1 mutant. (A) Metronidazole (MTZ)-induced DNA damage was reduced in the ΔfeoB1 mutant by expression of antisense sequence compared to the level in the ΔfeoB1 mutant and in the WT carrying the empty vector. As a positive control for DNA fragmentation, the WT was exposed to hydrogen peroxide (0.5%, vol/vol). Captured are lanes from gel photos shown in Fig. S7 in the supplemental material. Resistance was also confirmed by less depletion of low-molecular-weight thiols that form adducts with MTZ (B) and by reduced expression of recA (DNA recombination/repair protein RecA, CD630_13280), hcp (hybrid cluster protein, CD630_21680), and trxA (thioredoxin A, CD630_30330) that respond to activated metronidazole (C). Statistical significance was determined by unpaired t tests in GraphPad Prism, version 8.0. ** and *** indicate P < 0.005 and P < 0.0005, respectively.