Bacillus subtilis forms acetoin under anaerobic fermentative growth conditions and as a product of the aerobic carbon overflow metabolism. Acetoin formation from pyruvate requires ␣-acetolactate synthase and acetolactate decarboxylase, both encoded by the alsSD operon. The alsR gene, encoding the LysR-type transcriptional regulator AlsR, was found to be essential for the in vivo expression of alsSD in response to anaerobic acetate accumulation, the addition of acetate, low pH, and the aerobic stationary phase. The expressions of the alsSD operon and the alsR regulatory gene were independent of other regulators of the anaerobic regulatory network, including ResDE, Fnr, and ArfM. A negative autoregulation of alsR was observed. In vitro transcription from the alsSD promoter using purified B. subtilis RNA polymerase required AlsR. DNA binding studies with purified recombinant AlsR in combination with promoter mutagenesis experiments identified a 19-bp high-affinity palindromic binding site (TA AT-N 11 -ATTA) at positions ؊76 to ؊58 (regulatory binding site [RBS]) and a low-affinity site (AT-N 11 -AT) at positions ؊41 to ؊27 (activator binding site [ABS]) upstream of the transcriptional start site of alsSD. The RBS and ABS were found to be essential for in vivo alsSD transcription. AlsR binding to both sites induced the formation of higher-order, transcription-competent complexes. The AlsR protein carrying the S100A substitution at the potential coinducer binding site still bound to the RBS and ABS. However, AlsR(S100A) failed to form the higher-order complex and to initiate in vivo and in vitro transcription. A model for AlsR promoter binding and transcriptional activation was deduced.

Eukaryotic life is adapted to an oxygen-dependent energy generation. In contrast, many prokaryotes populate their ecological niches by the ability to survive and multiply under conditions of low oxygen tension. Anaerobic growth of these organisms is mediated by a broad spectrum of proton gradient-generating alternative respiration strategies. In addition, substrate level phosphorylation employing highly diverse fermentation processes allows for ATP formation and growth under anaerobic conditions. Moreover, this anaerobic energy metabolism was found to be essential for many pathogenic microorganisms during infection and biofilm formation. Because of the better energy yield, organisms prefer oxygen-dependent growth over anaerobic energy generation strategies. Additionally, several components of the anaerobic respiratory machineries were found to be oxygen-sensitive. Consequently, a strict regulation of the aerobic-anaerobic transition is advised. For oxygen sensing, many different types of sensors have been identified in bacteria and archaea, most of which react directly with oxygen by oxygen-reactive groups, like heme, FeS clusters, cysteine pairs, and FAD (1).A key regulatory protein in bacteria is Fnr, named after the fumarate and nitrate reduction-negative phenotype of an fnr gene-defective Escherichia coli strain (2). The N-terminal part of the E. coli Fnr protein contains four cysteine residues, Cys-20, Cys-23, Cys-29, and Cys-122, which are essential for Fnr function by coordinating a [4Fe-4S] 2ϩ cluster responsible for oxygen sensing. The C-terminal DNA-binding domain recognizes specific binding sites located in Fnr-controlled promoters (2).In contrast to E. coli Fnr, the [4Fe-4S] 2ϩ cluster of the redox regulator from the Gram-positive model bacterium Bacillus subtilis possesses only three cysteine ligands localized at the C terminus of the protein and a fourth unknown non-cysteinyl ligand (3). In most cases, the iron atoms of 2ϩ clusters are coordinated by four cysteine residues as found for E. coli Fnr. However, there are reported precedents of iron-sulfur clusters with only three cysteine ligands. Histidine residues are known to be involved in iron-sulfur cluster coordination in so-called "Rieske clusters" that are usually part of membrane-localized electron-transferring proteins (4). However, alanine substitutions of all conserved histidine residues of B. subtilis Fnr failed to abolish Fnr function and excluded histidines as the fourth ligand (3).

Clostridium difficile infections can induce mild to severe diarrhoea and the often associated characteristic pseudomembranous colitis. Two protein toxins, the large glucosyltransferases TcdA and TcdB, are the main pathogenicity factors that can induce all clinical symptoms in animal models. The classical molecular mode of action of these homologous toxins is the inhibition of Rho GTPases by mono-glucosylation. Rho-inhibition leads to breakdown of the actin cytoskeleton, induces stress-activated and pro-inflammatory signaling and eventually results in apoptosis of the affected cells. An increasing number of reports, however, have documented further qualities of TcdA and TcdB, including the production of reactive oxygen species (ROS) by target cells. This review summarizes observations dealing with the production of ROS induced by TcdA and TcdB, dissects pathways that contribute to this phenomenon and speculates about ROS in mediating pathogenesis. In conclusion, ROS have to be considered as a discrete, glucosyltransferase-independent quality of at least TcdB, triggered by different mechanisms.

The LysR-type transcriptional regulator (LTTR) AlsR from Bacillus subtilis activates the transcription of the alsSD operon encoding enzymes for acetoin formation in response to the presence of acetate. The structural basis for effector binding, oligomerization, DNA binding, higher ordered complex formation, DNA bending and transcriptional control by B. subtilis AlsR was functionally characterized. The binding of two molecules of acetate per molecule AlsR was determined. Acetate-dependent transcription complex formation was observed. A structural model of AlsR was used to identify the amino acid residues V98, S100, H147 of the binding site 1, which were experimentally verified. The second binding site formed by T193, V194, A196, T201 and L202 mediated high acetate responsive induction. Residues L124, E225 Q74, I79 and R111 contributed to dimerization of AlsR. A22, Q29, P30, S33, K37, L39, E46, R50 and R53 of the winged helix-turn-helix motif were important for promoter recognition. The DNA binding domain alone dimerized and effectively bound the promoter. The LTTR promoter elements RBS and ABS had to be localized on the same site of the DNA. Higher ordered complex formation resulted in bending of promoter DNA and transcriptional activation.