Structure and functional properties of the Bacillus subtilis transcriptional repressor Rex

Abstract

SummaryThe transcription factor Rex has been implicated in regulation of the expression of genes important for fermentative growth and for growth under conditions of low oxygen tension in several Gram-positive bacteria. Rex senses the redox poise of the cell through changes in the NADH/NAD + ratio. The crystal structures of two essentially identical Rex proteins, from Thermus aquaticus and T. thermophilus, have previously been determined in complex with NADH. Here we present the crystal structure of the Rex protein from Bacillus subtilis, as well as extensive studies of its affinity for nucleotides and DNA, using surface plasmon resonance, isothermal titration calorimetry and electrophoretic mobility shift assays. We show that Rex has a very high affinity for NADH but that its affinity for NAD + is 20 000 times lower. However, the NAD + affinity is increased by a factor of 30 upon DNA binding, suggesting that there is a positive allosteric coupling between DNA binding and NAD + binding. The crystal structures of two pseudoapo forms (from crystals soaked with NADH and cocrystallized with ATP) show a very different conformation from the previously determined Rex:NADH complexes, in which the N-terminal domains are splayed away from the dimer core. A mechanism is proposed whereby conformational changes in a C-terminal domain-swapped helix mediate the transition from a flexible DNA binding form to a locked NADH-bound form incapable of binding DNA.

Respiration control of multicellularity in Bacillus subtilis by a complex of the cytochrome chain with a membrane-embedded histidine kinase

“…In agreement with the results of Wang et al (2008), we found that removal of PASA and PASC, but not of PASB, impaired sporulation (data not shown). Moreover and importantly, removal of PASA and PASC (but, not so severely, removal of PASB) reduced the amount of colony wrinkling and sinI gene expression (Fig.…”

“…Rex is an NADH-sensitive repressor that inhibits the expression of genes under its control when conditions are aerobic (Wang et al 2008). When the NADH level rises upon oxygen limitation, Rex becomes inactive, leading to derepression of target genes.…”

Suitable extracellular oxidoreduction potential inhibit

“…Rex is a sensor of NADH/NAD + in many Grampositive bacteria, including S. coelicolor [11], S. erythraea [15], and B. subtilits [20]. By sensing cellular NADH/ NAD + , rex regulates the transcription of many genes involved in central carbon metabolism, NADH reoxidation, such as cytochrome bd oxidase (cytAB) and NADH dehydrogenases to maintain cellular redox balance [11].…”

The relationship between redox enzyme activity and electrochemical potential—cellular and mechanistic implications from protein film electrochemistry

“…40,44,45 Testing our hypothesis requires that enzyme activities and electrochemical potentials are correlated in the cellular context. In the first instance, the activities of periplasmically facing enzymes such as NapAB will be most readily measured in chemostat cultures where the rates of substrate consumption and/or product formation can be determined by analysis of the extracellular medium.…”