Grigoryan L, Babayan A, Vassilian A, Poladyan A, Sawers G, Trchounian K
Department of Biochemistry, Microbiology and Biotechnology, Faculty of Biology, Yerevan State University, Yerevan, Armenia; Research Institute of Biology, Yerevan State University, Yerevan, Armenia; Microbial Biotechnologies and Biofuel Innovation Center, Yerevan State University, Yerevan, Armenia.
Research Institute of Biology, Yerevan State University, Yerevan, Armenia; Department of Ecology and Nature Protection, Faculty of Biology, Yerevan State University, Yerevan, Armenia.
Biophys Rep (N Y). 2025 Aug 8;5(3):100225. doi: 10.1016/j.bpr.2025.100225.
Escherichia coli translocates formate/formic acid bidirectionally across the cytoplasmic membrane by the FocA/FocB formate channels during fermentation. Depending on the pH and whether formate is supplied exogenously or generated internally, the mechanisms of translocation differ. This study elucidates the role of these channels in dependence on FF ATPase activity in stationary phase cells after cultivation by mixed-carbon fermentation at pH 7.5. In cells cultivated with glucose plus glycerol, exogenously added formate increased the N,N'-dicyclohexylcarbodiimide (DCCD)-sensitive (FF ATPase-dependent) proton flux in single or double foc mutants. Moreover, exogenously supplied formate also increased the DCCD-sensitive potassium flux, but only in mutants where focB was absent. In the cells grown on glucose, glycerol, and formate, addition of formate in the whole-cell assays increased FF ATPase activity by ∼60% compared with cells grown on a mixture of only glucose and glycerol. In a focA mutant cultivated to the stationary phase on glucose, glycerol, and formate, FF ATPase activity was double that compared with cells grown on only glucose and glycerol, while in a focA-focB double-null mutant FF ATPase activity decreased by ∼50% in formate assays. These data suggest that the cell regulates the mechanism of formate translocation depending on whether formate is generated internally or added exogenously. Thus, FF-ATPase activity and the FocA/FocB channels together with formate hydrogenlyase activity combine to balance pH and ion gradients during fermentation in stationary phase cells in response to whether formate is generated metabolically or supplied in high concentration from the environment.
在发酵过程中，大肠杆菌通过FocA/FocB甲酸通道将甲酸/甲酸双向转运穿过细胞质膜。根据pH值以及甲酸是外源供应还是内部产生，转运机制有所不同。本研究阐明了这些通道在pH 7.5条件下通过混合碳发酵培养后，在静止期细胞中依赖FF ATP酶活性的作用。在用葡萄糖加甘油培养的细胞中，外源添加的甲酸增加了单foc或双foc突变体中对N,N'-二环己基碳二亚胺（DCCD）敏感（依赖FF ATP酶）的质子通量。此外，外源供应的甲酸也增加了对DCCD敏感的钾通量，但仅在缺失focB的突变体中。在以葡萄糖、甘油和甲酸生长的细胞中，与仅以葡萄糖和甘油混合物生长的细胞相比，在全细胞测定中添加甲酸使FF ATP酶活性增加了约60%。在以葡萄糖、甘油和甲酸培养至静止期的focA突变体中，FF ATP酶活性是仅以葡萄糖和甘油生长的细胞的两倍，而在focA-focB双缺失突变体中，在甲酸测定中FF ATP酶活性降低了约50%。这些数据表明，细胞根据甲酸是内部产生还是外源添加来调节甲酸转运机制。因此，FF-ATP酶活性、FocA/FocB通道以及甲酸氢酶活性共同作用，以平衡静止期细胞发酵过程中的pH值和离子梯度，这取决于甲酸是代谢产生还是从环境中高浓度供应。
