The Transcription Factor AlsR Binds and Regulates the Promoter of the alsSD Operon Responsible for Acetoin Formation in Bacillus subtilis

Abstract

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.

Distinct Effects of Sorbic Acid and Acetic Acid on the Electrophysiology and Metabolism of Bacillus subtilis

“…When confronted with a lack of an electron acceptor, B. subtilis employs a mixed acid fermentation and has been shown to produce lactate, acetate, acetoin, ethanol, 2,3-butanediol, and succinate as fermentation products when grown on glucose (30), but it also produces a lot of acetate as a result of overflow metabolism (31). In our experiments, only acetate and 2,3-butanediol were found.…”

An Efficient Method Using Gluconacetobacter europaeus To Reduce an Unfavorable Flavor Compound, Acetoin, in Rice Vinegar Production

“…5B and 6D). As described in the introduction, many microorganisms, including G. europaeus, have two types of enzymes that catalyze the condensation of two molecules of pyruvate to yield ␣-acetolactate: one is Als (20), and the other is AHAS (21). AHAS is used during biosynthesis of BCAAs.…”

“…The acetoin pathway shares intermediary metabolites (pyruvate and ␣-acetolactate) with the branchedchain amino acid (BCAA)-biosynthetic pathway, and Als catalyzes the same reaction as acetohydroxyacid synthase (AHAS), which is the rate-limiting enzyme of BCAA biosynthesis (21). The roles of the acetoin pathway are to repress the intracellular and environmental acidification caused by excess carbohydrates and unbalanced carbon fluxes in the central pathway (20,22,23) and to store carbon in the form of acetoin, which can be used in case of nutrient starvation (19). In AAB, it has long been known that the addition of lactic acid to the growth medium results in the formation of acetoin via pyruvate and ␣-acetolactate (24).…”

“…Many microorganisms, including several lactic acid bacteria (LAB) and Bacillus subtilis, produce acetoin as a product of fermentative or overflow metabolism (18)(19)(20). In these microorganisms, acetoin is produced from pyruvate via ␣-acetolactate, and the enzymes responsible for acetoin formation are ␣-acetolactate synthase (Als) and ␣-acetolactate decarboxylase (AldC), as shown in Fig.…”

Selective and Efficient Elimination of Vibrio cholerae with a Chemical Modulator that Targets Glucose Metabolism

“…Of additional note, V. cholerae N16961 produces a LysR-type transcriptional regulator (referred to as AlsR). Expression of alsR gene is induced by environmental factors such as acetate, anaerobiosis, or low environmental pH, and it activates the expression of the acetoin biosynthesis genes (Renna et al, 1993; Mayer et al, 1995; Frädrich et al, 2012). Because acetoin is a neutral fermentation end product and this biosynthetic reaction consumes intracellular protons, bacterial growth can occur on a glucose carbon source without pH decrease (Thomas et al, 2014).…”