Rosenberg Jonathan, Müller Peter, Lentes Sabine, Thiele Martin J, Zeigler Daniel R, Tödter Dominik, Paulus Henry, Brantl Sabine, Stülke Jörg, Commichau Fabian M
Department of General Microbiology, Georg August University Göttingen, Grisebachstr. 8, Göttingen, 37077, Germany.
Department of Genetics, Bacterial Genetics, Friedrich Schiller University Jena, Jena, Germany.
Mol Microbiol. 2016 Sep;101(5):879-93. doi: 10.1111/mmi.13429. Epub 2016 Jun 27.
The threonine dehydratase IlvA is part of the isoleucine biosynthesis pathway in the Gram-positive model bacterium Bacillus subtilis. Consequently, deletion of ilvA causes isoleucine auxotrophy. It has been reported that ilvA pseudo-revertants having a derepressed hom-thrCB operon appear in the presence of threonine. Here we have characterized two classes of ilvA pseudo-revertants. In the first class the hom-thrCB operon was derepressed unmasking the threonine dehydratase activity of the threonine synthase ThrC. In the second class of mutants, threonine biosynthesis was more broadly affected. The first class of ilvA pseudo-revertants had a mutation in the Phom promoter (P*hom ), resulting in constitutive expression of the hom-thrCB operon. In the second class of ilvA pseudo-revertants, the thrR gene encoding a putative DNA-binding protein was inactivated, also resulting in constitutive expression of the hom-thrCB operon. Here we demonstrate that ThrR is indeed a DNA-binding transcription factor that regulates the hom-thrCB operon and the thrD aspartokinase gene. DNA binding assays uncovered the DNA-binding site of ThrR and revealed that the repressor competes with the RNA polymerase for DNA binding. This study also revealed that ThrR orthologs are ubiquitous in genomes from the Gram-positive phylum Firmicutes and in some Gram-negative bacteria.
苏氨酸脱水酶IlvA是革兰氏阳性模式细菌枯草芽孢杆菌中异亮氨酸生物合成途径的一部分。因此，ilvA基因的缺失会导致异亮氨酸营养缺陷。据报道，在苏氨酸存在的情况下会出现具有去阻遏的hom-thrCB操纵子的ilvA假回复突变体。在此，我们对两类ilvA假回复突变体进行了表征。在第一类中，hom-thrCB操纵子被去阻遏，从而揭示了苏氨酸合酶ThrC的苏氨酸脱水酶活性。在第二类突变体中，苏氨酸生物合成受到更广泛的影响。第一类ilvA假回复突变体在Phom启动子（P*hom）中发生了突变，导致hom-thrCB操纵子组成型表达。在第二类ilvA假回复突变体中，编码假定DNA结合蛋白的thrR基因失活，同样导致hom-thrCB操纵子组成型表达。在此我们证明，ThrR确实是一种调节hom-thrCB操纵子和thrD天冬氨酸激酶基因的DNA结合转录因子。DNA结合分析揭示了ThrR的DNA结合位点，并表明该阻遏物与RNA聚合酶竞争DNA结合。这项研究还表明，ThrR直系同源物在革兰氏阳性厚壁菌门的基因组以及一些革兰氏阴性细菌中普遍存在。
