Redox-responsive repressor Rex modulates alcohol production and oxidative stress tolerance in Clostridium acetobutylicum

Rex, a transcriptional repressor that modulates its DNA binding activity in response to
NADH/NAD+ ratio, has recently been found to play a role in the solventogenic shift of
Clostridium acetobutylicum. Here we combined a comparative genomic reconstruction of
Rex regulons in 11 diverse clostridial species with detailed experimental characterization of
Rex-mediated regulation in C. acetobutylicum. The reconstructed Rex regulons in clostridia
included the genes involved in fermentation, hydrogen production, tricarboxylic acid cycle,
NAD biosynthesis, nitrate and sulphite reduction, and CO2/CO fixation. The predicted Rex
binding sites in the genomes of Clostridium spp. were verified by in vitro binding assays with
purified Rex protein. Novel members of C. acetobutylicum Rex regulon were identified and
experimentally validated by comparing the transcript levels between the wild-type and rex
inactivated mutant strains. Furthermore, the effects of exposure to methyl viologen or H2O2
on intracellular NADH and NAD+ concentrations, expression of Rex regulon genes, and
physiology of the wild-type and rex-inactivated mutant were comparatively analyzed. Our
results indicate that Rex responds to NADH/NAD+ ratio in vivo to regulate gene expression
and modulates fermentation product formation and oxidative stress tolerance in C.
acetobutylicum. It is suggested that Rex plays an important role in maintaining NADH/NAD+
homeostasis in clostridia.