Department of Environmental Protection, Consejo Superior de Investigaciones Científicas, Calle Profesor Albareda 1, Granada, Spain.
J Bacteriol. 2010 Sep;192(17):4357-66. doi: 10.1128/JB.00520-10. Epub 2010 Jun 25.
Metabolic flux analysis revealed that in Pseudomonas putida KT2440 about 50% of glucose taken up by the cells is channeled through the 2-ketogluconate peripheral pathway. This pathway is characterized by being compartmentalized in the cells. In fact, initial metabolism of glucose to 2-ketogluconate takes place in the periplasm through a set of reactions catalyzed by glucose dehydrogenase and gluconate dehydrogenase to yield 2-ketogluconate. This metabolite is subsequently transported to the cytoplasm, where two reactions are carried out, giving rise to 6-phosphogluconate, which enters the Entner-Doudoroff pathway. The genes for the periplasmic and cytoplasmic set of reactions are clustered in the host chromosome and grouped within two independent operons that are under the control of the PtxS regulator, which also modulates its own synthesis. Here, we show that although the two catabolic operons are induced in vivo by glucose, ketogluconate, and 2-ketogluconate, in vitro we found that only 2-ketogluconate binds to the regulator with an apparent K(D) (equilibrium dissociation constant) of 15 muM, as determined using isothermal titration calorimetry assays. PtxS is made of two domains, a helix-turn-helix DNA-binding domain located at the N terminus and a C-terminal domain that binds the effector. Differential scanning calorimetry assays revealed that PtxS unfolds via two events characterized by melting points of 48.1 degrees C and 57.6 degrees C and that, in the presence of 2-ketogluconate, the unfolding of the effector binding domain occurs at a higher temperature, providing further evidence for 2-ketogluconate-PtxS interactions. Purified PtxS is a dimer that binds to the target promoters with affinities in the range of 1 to 3 muM. Footprint analysis revealed that PtxS binds to an almost perfect palindrome that is present within the three promoters and whose consensus sequence is 5'-TGAAACCGGTTTCA-3'. This palindrome overlaps with the RNA polymerase binding site.
代谢通量分析表明，在恶臭假单胞菌 KT2440 中，细胞摄取的葡萄糖约有 50%通过 2-酮葡萄糖酸盐的外围途径进行代谢。该途径的特点是在细胞中分隔开。事实上，葡萄糖最初代谢为 2-酮葡萄糖酸是在周质中通过一组由葡萄糖脱氢酶和葡糖酸脱氢酶催化的反应进行的，生成 2-酮葡萄糖酸。这种代谢物随后被转运到细胞质中，在那里进行两个反应，生成 6-磷酸葡萄糖酸，它进入 Entner-Doudoroff 途径。周质和细胞质反应的基因簇在宿主染色体上，并分为两个独立的操纵子，受 PtxS 调节剂的控制，该调节剂也调节自身的合成。在这里，我们表明，尽管两个分解代谢操纵子在体内被葡萄糖、酮葡萄糖酸和 2-酮葡萄糖酸诱导，但在体外我们发现只有 2-酮葡萄糖酸与调节剂结合，表观 K(D)（平衡解离常数）为 15 μM，如使用等温滴定量热法测定。PtxS 由两个结构域组成，一个位于 N 端的螺旋-转角-螺旋 DNA 结合结构域和一个结合效应物的 C 端结构域。差示扫描量热法测定表明，PtxS 通过两个事件展开，其特征熔点分别为 48.1°C 和 57.6°C，并且在存在 2-酮葡萄糖酸的情况下，效应物结合结构域的展开发生在更高的温度下，为 2-酮葡萄糖酸-PtxS 相互作用提供了进一步的证据。纯化的 PtxS 是一种二聚体，与靶启动子的亲和力在 1 到 3 μM 范围内。足迹分析表明，PtxS 结合到存在于三个启动子中的几乎完美的回文序列上，其保守序列为 5'-TGAAACCGGTTTCA-3'。这个回文序列与 RNA 聚合酶结合位点重叠。
