Redox-Responsive Repressor Rex Modulates Alcohol Production and Oxidative Stress Tolerance in Clostridium acetobutylicum

Abstract

c 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, the tricarboxylic acid cycle, NAD biosynthesis, nitrate and sulfite reduction, and CO 2 /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 the 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 H 2 O 2 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.

Rex in Clostridium kluyveri is a global redox-sensing transcriptional regulator

“…acetobutylicum (Wang et al, 2013;Wietzke and Bahl, 2012;Zhang et al, 2014), nfnAB resides downstream of crt1-bcd-etfAB1-hbd1; however, nfnAB is missing from the genome of C. acetobutylicum. The absence of NfnAB might be accounted for by the activity of NAD + -specific 3-hydroxybutyryl-CoA dehydrogenase (Hbd) of C.…”

“…The 18-bp palindromic Rex-binding consensus sequence, 5′-TTGTTWWWTTWTTAACAA-3′ ("W" represents adenine or thymine), resides 33-bp upstream of the crt start codon. The bcs operon has been proved to be controlled by Rex (Wang et al, 2013;Wietzke and Bahl, 2012;Zhang et al, 2014).…”

“…In these bacteria, Rex was discovered to modulate central carbon metabolism, respiration, solvent and organic acid production, hydrogen formation, tolerance to oxidative stress, biofilm formation, and sulfate and nitrate reduction. Rex regulons were also systematically reconstructed in various bacteria, which identified Rex as a global repressor in response to the cellular NADH/NAD + ratio (Ravcheev et al, 2012;Wang et al, 2013;Zhang et al, 2014). The crystal structures of Rex in complex with NADH, NAD + , and/or the DNA operator indicate that Rex comprises an N-terminal winged helix-turn-helix-fold domain interacting with DNA and a C-terminal Rossmann-fold domain binding NADH and mediating subunit dimerization (McLaughlin et al, 2010;Nakamura et al, 2007;Sickmier et al, 2005;Wang et al, 2008;Zheng et al, 2014).…”

“…Enterococcus faecalis (Vesic and Kristich, 2013) as well as the strict anaerobes Thermoanaerobacter ethanolicus (Pei et al, 2011), C. acetobutylicum (Wang et al, 2013;Wietzke and Bahl, 2012;Zhang et al, 2014), Thermotoga maritima (Ravcheev et al, 2012) and Desulfovibrio vulgaris (Christensen et al, 2015). In these bacteria, Rex was discovered to modulate central carbon metabolism, respiration, solvent and organic acid production, hydrogen formation, tolerance to oxidative stress, biofilm formation, and sulfate and nitrate reduction.…”

Modulation of the Acetone/Butanol Ratio during Fermentation of Corn Stover-Derived Hydrolysate by Clostridium beijerinckii Strain NCIMB 8052

“…Exogenous electron carriers can alter metabolism, the redox balance, and electron flow in clostridia (18,19,31). In the present study, the A/B molar ratio in the end products was approximately 0.5 in a C. beijerinckii batch fermentation with glucose as the carbon source and was 0.95 with DTH as the carbon source ( Table 2).…”