NADH produced during the catabolism of glucose via glycolysis and the TCA cycle needs to be oxidized to NAD+ to provide oxidizing power to the cellular machinery. NADH dehydrogenase is the first enzyme of the respiratory chain, catalyzing the reoxidation of NADH and transferring electrons to ubiquinone, but in B. subtilis, the physiological role of NADH dehydrogenase has not been well understood. Our studies provide evidence that the ndh gene plays an essential role not only in the respiration chain but also in its own expression via a regulatory loop between redox sensing of the NADH/NAD+ ratio by Rex and oxidation of NADH by the NADH dehydrogenase Ndh.
Type I (NDH-1) and type II (NDH-2) NADH dehydrogenases are widely distributed in organisms, both eukaryotic and prokaryotic (16
). E. coli
has both of the two enzymes that can oxidize NADH. During fumarate respiration, NDH-1, encoded by the nuo
genes, is the preferred NADH dehydrogenase, whereas NDH-2, encoded by ndh
, is used during aerobic and nitrate respiration (24
). Interestingly, genes resembling nuoA
encoding NDH-1 of E. coli
were found to be missing in the genome of B. subtilis
). Instead, three genes, i.e., yjlD
, and yumB
, have been predicted to encode NDH-2 in B. subtilis
. In this study we focused on yjlD
) and obtained evidence which proved that Ndh is the major NADH dehydrogenase in B. subtilis
, since an ndh
mutant but not yutJ
mutants exhibited slower growth in LB (Fig. ). We also showed that ndh
is expressed at a much higher level in the exponential phase than in the stationary phase (Fig. and ). The mechanism of repression of ndh
expression during stationary phase is unknown, but it is thought to be independent of Rex (Fig. ). Ndh has been reported to be an anaerobically repressed protein (14
), and the expression of ndh
is elevated during exponential growth under aerobic conditions in a resDE
(oxygen sensor/regulator) mutant (28
). Some additional regulation may result from changes in the expression level of ndh
in the stationary phase and/or anaerobic condition. We have not determined the characterization of the other two gene products, but they may become activated and take over the function of Ndh during stationary phase. However, Ndh most probably plays a central role in the respiratory chain and also maintains the cell NADH/NAD+
balance in the vegetative phase (Fig. ).
Brekasis and Pagget (2
) reported that the S. coelicolor
Rex protein, a novel sensor of the NADH/NAD redox poise, binds to the cis
elements of the cyd
operons in S. coelicolor
. Schau et al. (22
) determined the precise B. subtilis
Rex (YdiH)-binding region in the cyd
operon. However, so far, no study has demonstrated the effect of NADH and NAD+
on the DNA-binding activity of Rex in B. subtilis
. We determined the DNA-binding activity of Rex with NADH or NAD+
. The results showed that NAD+
boosted the binding activity of Rex but that NADH seemed to have a negligible effect or a partial negative effect on DNA-binding activity. These data suggest that DNA-binding determinants of B. subtilis
Rex are distinct from those of S. coelicolor
Rex, since in S. coelicolor
plays a small or negligible role in DNA-binding activity while NADH completely inhibits DNA-binding activity. Interestingly, B. subtilis
Rex and S. coelicolor
Rex regulate respiratory metabolism gene expression via redox sensing of the NADH/NAD+
ratio, although their DNA-binding determinants are distinct.
Microarray analysis performed by Larsson et al. (11
) revealed that during the transition from aerobic to microaerophilic and finally to anaerobic growth, the coordination of certain respiratory genes (e.g., cyd
, encoding cytochorome terminal oxidase; ldh
, encoding lactate dehydrogenase; lctP
, encoding lactate permease; and ywcJ
, encoding a predicted formate nitrate transporter) is negatively regulated by Rex (11
). However, although they emphasized the regulation of the above genes in their microarray analysis, they did not show the regulation of certain important aerobic respiratory genes, such as ndh
, by Rex. They described a regulatory model in which Rex and LDH act coordinately to prevent a large fluctuation in the NADH/NAD+
ratio under fermentative conditions. In this work, however, we have shown an alternative model, which functions under aerobic conditions, in which Rex and Ndh together form a regulatory loop to maintain a constant NADH/NAD+
ratio. More recently, Reents et al. (21
) have also shown a regulatory model for redox regulators ResD, Fnr, and Rex during the transition to anaerobic growth conditions.
We then performed DNA microarray analysis under aerobic conditions and found that Rex, in addition to regulating the above-mentioned genes, including ndh, is also a negative regulator of genes involved in glycerol metabolism and the TCA cycle (data not shown). It would be no surprise if Rex regulated such genes, because they play an important role in the production of NADH. The physiological reason for a global gene regulation system under the control of Rex will be the subject of future studies.
These results support the following model for the maintenance of a constant NADH/NAD+ ratio in the cytoplasm under aerobic conditions. When the NAD+ concentration is high in the cytoplasm, Rex in the complex with NAD+ binds to the upstream region of yjlC-ndh to repress the transcription, leading to a decrease in the oxidation of NADH. When the concentration of NADH becomes higher than that of NAD+, Rex forms a complex with NADH. Since Rex in the complex with NADH has a weaker affinity to DNA, ndh transcription is induced, which results in NADH oxidation in the cytoplasm. Thus, we propose that Ndh and Rex form a “regulatory loop” that maintains the NADH/NAD+ ratio in B. subtilis cells.
The mechanism discovered in this work will provide clues for unraveling the complexity behind the maintenance of a constant NADH/NAD+ ratio in the cytoplasm, but we have not ruled out the existence of other mechanisms that may also be involved in maintaining a constant NADH/NAD+ ratio in the cytoplasm. Whether the Ndh-Rex pair forms a similar regulatory loop in other bacteria or whether this mechanism is exclusive to B. subtilis remains to be clarified. Further work on Ndh and Rex should lead to a better understanding of redox regulation under various conditions in B. subtilis.