Tanaka Y, Kimata K, Inada T, Tagami H, Aiba H
Division of Biological Science, Graduate School of Science, Nagoya University, Chikusa, Nagoya 464-8601, Japan.
Genes Cells. 1999 Jul;4(7):391-9. doi: 10.1046/j.1365-2443.1999.00268.x.
The pts operon of Escherichia coli consists of three genes ptsH, ptsI and crr, each encoding for central components of the phosphoenolpyruvate: carbohydrate phosphotransferase system, HPr, enzyme I and IIAGlc, respectively. Transcription of the pts operon is stimulated when glucose is present in the culture medium. One of the two major promoters, P0, is responsible for this glucose induction. However, no regulatory protein responsible for the glucose induction of the pts operon has been identified yet and molecular mechanism by which glucose stimulates the pts transcription is not known.
We found by Northern blotting that the pts mRNA levels in cells lacking Mlc, a new global repressor of carbohydrate metabolism, were increased without external glucose and that the addition of glucose had no effect on the pts mRNA levels in the mutant cells. Western blotting revealed that the enzyme I level in the mlc- cells was also elevated without glucose and no further increase in the enzyme I level was observed in the presence of glucose. S1 analysis revealed that transcription of the glucose-sensitive promoter, P0, occurs constitutively in the mlc- cells independently from the external glucose. In vitro transcription studies indicated that Mlc strongly inhibited P0 transcription. DNase I footprinting experiment revealed that Mlc bound to P0 promoter region to prevent RNA polymerase binding at P0.
We conclude that Mlc is a repressor for the pts transcription acting as a major regulatory protein involved in the glucose induction of pts operon. We propose that glucose induces the pts transcription by modulating the Mlc activity. The mechanism by which glucose modulates the Mlc action remains to be studied.
大肠杆菌的pts操纵子由三个基因ptsH、ptsI和crr组成，分别编码磷酸烯醇丙酮酸：糖磷酸转移酶系统的核心组分，即HPr、酶I和IIAGlc。当培养基中存在葡萄糖时，pts操纵子的转录会受到刺激。两个主要启动子之一P0负责这种葡萄糖诱导作用。然而，尚未鉴定出负责pts操纵子葡萄糖诱导作用的调控蛋白，葡萄糖刺激pts转录的分子机制也尚不清楚。
我们通过Northern印迹法发现，在缺乏Mlc（一种新的碳水化合物代谢全局阻遏物）的细胞中，pts mRNA水平在无外源葡萄糖的情况下升高，并且添加葡萄糖对突变细胞中的pts mRNA水平没有影响。Western印迹显示，mlc -细胞中的酶I水平在无葡萄糖时也升高，并且在有葡萄糖存在的情况下未观察到酶I水平进一步增加。S1分析表明，葡萄糖敏感启动子P0的转录在mlc -细胞中组成性发生，与外源葡萄糖无关。体外转录研究表明，Mlc强烈抑制P0转录。DNase I足迹实验表明，Mlc与P0启动子区域结合，以阻止RNA聚合酶在P0处结合。
我们得出结论，Mlc是pts转录的阻遏物，作为参与pts操纵子葡萄糖诱导的主要调控蛋白。我们提出葡萄糖通过调节Mlc活性来诱导pts转录。葡萄糖调节Mlc作用的机制仍有待研究。
