Dual role of LldR in regulation of the lldPRD operon, involved in L-lactate metabolism in Escherichia coli
Dual role of LldR in regulation of the lldPRD operon, involved in L-lactate metabolism in Escherichia coli
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DOI：
10.1128/jb.02013-07
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发表时间：
2008-04-01
影响因子：
3.2
通讯作者：
Baldoma, Laura
中科院分区：
文献类型：
--
作者：
Aguilera, Laura;Campos, Evangelina;Baldoma, Laura
参与 L-乳酸代谢的大肠杆菌的 lldPRD 操纵子是由该化合物的生长诱导的。我们通过实验确定该系统是从单个启动子转录的，其起始位点位于 ATG 起始密码子上游 110 个核苷酸处。根据计算数据，有人提出 LldR 及其同源物 PdhR 作为 lldPRD 操纵子的调节因子。然而，到目前为止，还没有关于这些调节器功能的实验数据的报道。在这里，我们发现 L-乳酸对 lldP-lacZ 融合的诱导在 Delta lldR 突变体中消失，表明 LldR 在这种诱导中的作用。对该构建体在 pdhR 突变体中的表达分析排除了 PdhR 参与 lldPRD 的控制。凝胶位移实验表明，LldR 结合两个操纵位点：O1（位置 -105 至 -89）和 O2（位置 +22 至 +38），其中 O1 以较低浓度的 LldR 填充。 L-乳酸诱导 LldR 构象变化，但不会改变其 DNA 结合活性。 O1 和 O2 的突变增强了基础转录水平。然而，只有 O1 突变才能消除 L-乳酸的诱导作用。螺旋定相在 O1 和 O2 之间发生变化的突变体表现得与 O2 突变体相似。这些结果与 LldR 具有双重作用的假设相一致，即作为 lldPRD 的抑制子或激活子。我们提出，在缺乏L-乳酸的情况下，LldR结合O1和O2，可能导致DNA成环和转录抑制。 L-乳酸与 LldR 的结合会促进构象变化，从而破坏 DNA 环，从而形成转录开放复合物。
The lldPRD operon of Escherichia coli, involved in L-lactate metabolism, is induced by growth in this compound. We experimentally identified that this system is transcribed from a single promoter with an initiation site located 110 nucleotides upstream of the ATG start codon. On the basis of computational data, it had been proposed that LldR and its homologue PdhR act as regulators of the lldPRD operon. Nevertheless, no experimental data on the function of these regulators have been reported so far. Here we show that induction of an lldP-lacZ fusion by L-lactate is lost in an Delta lldR mutant, indicating the role of LldR in this induction. Expression analysis of this construct in a pdhR mutant ruled out the participation of PdhR in the control of lldPRD. Gel shift experiments showed that LldR binds to two operator sites, O1 (positions -105 to -89) and O2 (positions +22 to +38), with O1 being filled at a lower concentration of LldR. L-Lactate induced a conformational change in LldR that did not modify its DNA binding activity. Mutations in O1 and O2 enhanced the basal transcriptional level. However, only mutations in O1 abolished induction by L-lactate. Mutants with a change in helical phasing between O1 and O2 behaved like O2 mutants. These results were consistent with the hypothesis that LldR has a dual role, acting as a repressor or an activator of lldPRD. We propose that in the absence Of L-lactate, LldR binds to both O1 and O2, probably leading to DNA looping and the repression of transcription. Binding Of L-lactate to LldR promotes a conformational change that may disrupt the DNA loop, allowing the formation of the transcription open complex.