The Transcription Factor AlsR Binds and Regulates the Promoter of the alsSD Operon Responsible for Acetoin Formation in Bacillus subtilis
The Transcription Factor AlsR Binds and Regulates the Promoter of the alsSD Operon Responsible for Acetoin Formation in Bacillus subtilis
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DOI：
10.1128/jb.06425-11
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发表时间：
2012-03-01
影响因子：
3.2
通讯作者：
Haertig, Elisabeth
中科院分区：
文献类型：
--
作者：
Fraedrich, Claudia;March, Anika;Haertig, Elisabeth
枯草芽孢杆菌在厌氧发酵生长条件下形成乙偶姻，并作为有氧碳溢流代谢的产物。由丙酮酸形成乙偶姻需要α-乙酰乳酸合酶和乙酰乳酸脱羧酶，两者均由alsSD操纵子编码。的alsR基因，编码的LysR型转录调节因子AlsR，被认为是必不可少的在体内表达的alsSD响应厌氧乙酸积累，添加乙酸，低pH值，和有氧静止期。alsSD操纵子和alsR调节基因的表达独立于厌氧调节网络的其他调节剂，包括ResDE，Fnr和ArfM。观察到alsR的负性自身调节。使用纯化的B从alsSD启动子进行体外转录。枯草杆菌RNA聚合酶需要AlsR。用纯化的重组AlsR结合启动子诱变实验的DNA结合研究鉴定了alsSD转录起始位点上游位置-76至-58（调节结合位点[RBS]）处的19-bp高亲和力回文结合位点（TA AT-N-11-ATTA）和位置-41至-27（激活剂结合位点[ABS]）处的低亲和力位点（AT-N-11-AT）。RBS和ABS被发现是体内alsSD转录所必需的。AlsR结合到这两个网站诱导形成的高阶，转录能力的复合物。在潜在的共诱导物结合位点携带S100 A取代的AlsR蛋白仍然与RBS和ABS结合。然而，AlsR（S100 A）未能形成更高级的复合物，并启动在体内和体外转录。推导出AlsR启动子结合和转录激活的模型。
Bacillus subtilis forms acetoin under anaerobic fermentative growth conditions and as a product of the aerobic carbon overflow metabolism. Acetoin formation from pyruvate requires a-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.