Carbon Catabolite Repression and the Related Genes of ccpA, ptsH and hprK in Thermoanaerobacterium aotearoense.
Carbon Catabolite Repression and the Related Genes of ccpA, ptsH and hprK in Thermoanaerobacterium aotearoense.
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奥特罗热厌氧杆菌中碳分解代谢物抑制及 ccpA、ptsH 和 hprK 相关基因。
DOI：
10.1371/journal.pone.0142121
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发表时间：
2015
期刊：
影响因子：
3.7
通讯作者：
Wang X
中科院分区：
文献类型：
--
作者：
Zhu M;Lu Y;Wang J;Li S;Wang X
严格厌氧的革兰氏阳性细菌，热厌氧细菌aotearoense SCUT27，能够通过直接发酵葡聚糖、木聚糖和各种木质纤维素来源的糖来生产乙醇、氢和乳酸。通过使用非代谢糖和代谢糖作为底物，我们发现2-脱氧葡萄糖（2-DG）的存在强烈抑制了纤维二糖、半乳糖、阿拉伯糖和淀粉的利用。然而，当2-DG浓度为可代谢糖的十分之一时，木糖和甘露糖的消耗量不受显著影响。因此，T. aotearose SCUT27可以在葡萄糖存在的情况下消耗木糖和甘露糖。通过基因克隆和蛋白鉴定，证实了T. aotearoense SCUT27中存在碳分解代谢抑制（CCR）相关基因ccpA、ptsH和hprK。高纯度的含组氨酸蛋白（HPr）无需在反应混合物中添加果糖-1,6-二磷酸（FBP）或葡萄糖-6-磷酸（Glc-6-P），即可被HPr激酶/磷酸酶（HPrK/P）特异性磷酸化丝氨酸46位点。在无甲醛条件下，通过亲和层析证实了分解代谢控制蛋白A （CcpA）与磷酸化HPr的特异性蛋白相互作用。表面等离子体共振（SPR）测定CcpA和HPrSerP的平衡结合常数（K D）为2.22±0.36 nM，表明两者具有较高的亲和力。
The strictly anaerobic, Gram-positive bacterium, Thermoanaerobacterium aotearoense SCUT27, is capable of producing ethanol, hydrogen and lactic acid by directly fermenting glucan, xylan and various lignocellulosically derived sugars. By using non-metabolizable and metabolizable sugars as substrates, we found that cellobiose, galactose, arabinose and starch utilization was strongly inhibited by the existence of 2-deoxyglucose (2-DG). However, the xylose and mannose consumptions were not markedly affected by 2-DG at the concentration of one-tenth of the metabolizable sugar. Accordingly, T. aotearoense SCUT27 could consume xylose and mannose in the presence of glucose. The carbon catabolite repression (CCR) related genes, ccpA, ptsH and hprK were confirmed to exist in T. aotearoense SCUT27 through gene cloning and protein characterization. The highly purified Histidine-containing Protein (HPr) could be specifically phosphorylated at Serine 46 by HPr kinase/phosphatase (HPrK/P) with no need to add fructose-1,6-bisphosphate (FBP) or glucose-6-phosphate (Glc-6-P) in the reaction mixture. The specific protein-interaction of catabolite control protein A (CcpA) and phosphorylated HPr was proved via affinity chromatography in the absence of formaldehyde. The equilibrium binding constant (K D) of CcpA and HPrSerP was determined as 2.22 ± 0.36 nM by surface plasmon resonance (SPR) analysis, indicating the high affinity between these two proteins.