An alignment of upstream regions of anaerobically induced genes in Staphylococcus aureus revealed the presence of an inverted repeat, corresponding to Rex binding sites in Streptomyces coelicolor. Gel shift experiments of selected upstream regions demonstrated that the redox-sensing regulator Rex of S. aureus binds to this inverted repeat. The binding sequence – TTGTGAAW4TTCACAA – is highly conserved in S. aureus. Rex binding to this sequence leads to the repression of genes located downstream. The binding activity of Rex is enhanced by NAD+ while NADH, which competes with NAD+ for Rex binding, decreases the activity of Rex. The impact of Rex on global protein synthesis and on the activity of fermentation pathways under aerobic and anaerobic conditions was analysed by using a rex-deficient strain. A direct regulatory effect of Rex on the expression of pathways that lead to anaerobic NAD+ regeneration, such as lactate, formate and ethanol formation, nitrate respiration, and ATP synthesis, is verified. Rex can be considered a central regulator of anaerobic metabolism in S. aureus. Since the activity of lactate dehydrogenase enables S. aureus to resist NO stress and thus the innate immune response, our data suggest that deactivation of Rex is a prerequisite for this phenomenon.

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.

We have used DNA microarrays to survey rates of mRNA decay on a genomic scale in early stationary-phase cultures of Bacillus subtilis. The decay rates for mRNAs corresponding to about 1500 genes could be estimated. About 80% of these mRNAs had a half-life of less than 7 min. More than 30 mRNAs, including both mono- and polycistronic transcripts, were found to be extremely stable, i.e. to have a half-life of > or =15 min. Only two such transcripts were known previously in B. subtilis. The results provide the first overview of mRNA decay rates in a gram-positive bacterium and help to identify polycistronic operons. We could find no obvious correlation between the stability of an mRNA and the function of the encoded protein. We have also not found any general features in the 5' regions of mRNAs that distinguish stable from unstable transcripts. The identified set of extremely stable mRNAs may be useful in the construction of stable recombinant genes for the overproduction of biomolecules in Bacillus species.

SummaryIn the soil bacterium Bacillus subtilis Spx is a key regulator that controls expression, positively or negatively, of several genes in response to certain oxidative stresses that lead to the formation of unwanted disulphide bonds. Here we characterized the yjbH gene and show that it encodes a novel effector of Spx. The yjbH gene is part of the yjbIH operon that encodes a truncated haemoglobin (YjbI) and a predicted 34 kDa cytosolic protein of unknown function (YjbH). Deletion of yjbIH or yjbH has pleiotropic effects and affects growth, sporulation and competence development. Cells lacking yjbIH display a reduced sensitivity to the thiol oxidant diamide and show an apparent down-or upregulation of several transcripts that belong to the Spx regulon. Twentytwo suppressor mutations that bypass the defects conferred by yjbH were isolated. These mutations were identified as six deletions, three nonsense and 11 missense substitutions in the spx gene. Real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR) analysis showed that mutations in yjbIH or yjbH do not affect the level of spx transcription. The combined data from the present work show that strains lacking yjbIH or yjbH overproduce Spx under unperturbed growth. The elevated Spx concentration cannot be attributed to an increased spx expression but is likely to result from control at the post-transcriptional level. YjbH is proposed to affect the cellular concentration of Spx by modulating proteolysis via the ClpXP protease.

A copper-containing domain of the caa,-type oxidase from Bacillus subtilis has been expressed as a water-soluble protein in the cytoplasm of Escherichia coli. Electron paramagnetic resonance (EPR) spectra of this purple domain show well-resolved lines in the g, resonance, both at X-band and S-band frequencies. Interpretation of EPR spectra and analytical data indicate a binuclear copper site consisting of one Cu*' and one Cu". This copper site closely resembles Cu, in subunit II of cytochrome c oxidase and is shown here to be a mixed-valence [Cu'+-Cu'+] binuclear centre.