Institute for Biology/Microbiology, Martin Luther University Halle-Wittenberg, Halle (Saale), Germany.
IMD Laboratory Oderland GmbH, Frankfurt (Oder), Germany.
Microb Physiol. 2022;32(3-4):95-107. doi: 10.1159/000524454. Epub 2022 Apr 7.
The formate channel A (FocA) belongs to the formate-nitrite transporter (FNT) family, members of which permeate small monovalent anions. FocA from Escherichia coli translocates formate/formic acid bi-directionally across the cytoplasmic membrane during fermentative growth. Two residues are particularly well-conserved within the translocation pores of FNTs: threonine-91 and histidine-209, based on E. coli FocA numbering. These residues are located at the tips of two broken transmembrane helices and control anion passage. H209 is the only charged residue within the pore and interacts with T91. Here, we addressed the role of the T91-H209 interaction network in the permeation of formate in vivo through FocA by performing an extensive amino acid-exchange study. Monitoring changes in intracellular formate using a formate-responsive fdhFP::lacZ reporter system revealed that T91 is essential for the ability of FocA to translocate formate bi-directionally. Only exchange for serine was partially tolerated, indicating that the hydroxyl group of T91 is mechanistically important. Substitution of H209 with N or Q was previously shown to convert FocA into a formate efflux channel. We show here that residue exchanges A, I, and T at this position resulted in a similar phenotype. Moreover, efflux function was confirmed for these FocA variants by measuring excreted formate in the culture medium. Substitution of bulky or charged residues for H209 prevented bi-directional formate passage. Studies using hypophosphite, a toxic analogue of formate taken up by FocA, and which causes impaired growth, confirmed that T91 and H209 substitutions essentially abolished, or drastically reduced, FocA's translocation activity, as shown by effects on growth rate. The exceptions were T91S- and T91Y-exchange variants that retained partial ability to take up inhibitory hypophosphite. Together, our findings indicate that T91 is essential for formate permeation in both directions; however, it is particularly important to allow anion efflux. Moreover, H209 is essential for formate uptake by FocA, strongly suggesting that protonation-deprotonation of this residue plays a role in formate uptake. Finally, our results substantiate the premise that efflux and influx of formate by FocA are mechanistically distinct processes that are controlled by the interplay between T91 and H209.
甲酸通道 A（FocA）属于甲酸-亚硝酸盐转运体（FNT）家族，该家族成员可渗透小分子单价阴离子。大肠杆菌中的 FocA 在发酵生长过程中双向转运甲酸/甲酸。在 FNT 的转运孔中，有两个残基特别保守：基于大肠杆菌 FocA 编号，为苏氨酸-91 和组氨酸-209。这些残基位于两个断裂的跨膜螺旋的尖端，控制阴离子通过。H209 是孔内唯一带电荷的残基，与 T91 相互作用。在这里，我们通过进行广泛的氨基酸交换研究，研究了 T91-H209 相互作用网络在体内甲酸渗透过程中对 FocA 的作用。通过使用甲酸响应型 fdhFP::lacZ 报告系统监测细胞内甲酸的变化，结果表明 T91 对于 FocA 双向转运甲酸的能力是必不可少的。只有交换为丝氨酸时部分耐受，表明 T91 的羟基在机械上很重要。之前已显示用 N 或 Q 取代 H209 将 FocA 转化为甲酸外排通道。我们在这里表明，该位置的残基 A、I 和 T 的交换导致了类似的表型。此外，通过测量培养基中排泄的甲酸，证实了这些 FocA 变体具有外排功能。用 H209 的大体积或带电荷的取代物取代阻止了双向甲酸通过。使用亚磷酸盐的研究，亚磷酸盐是 FocA 摄取的有毒甲酸类似物，会导致生长受损，并证实 T91 和 H209 取代基本上消除或大大降低了 FocA 的转运活性，这表现在对生长速率的影响上。例外是 T91S-和 T91Y-交换变体，它们保留了部分摄取抑制性亚磷酸盐的能力。总之，我们的发现表明 T91 对甲酸的双向渗透是必不可少的；然而，允许阴离子外排尤为重要。此外，H209 对 FocA 摄取甲酸是必不可少的，这强烈表明该残基的质子化-去质子化在甲酸摄取中起作用。最后，我们的结果证实了 FocA 摄取和外排甲酸的前提，即这两个过程在机制上是不同的，受 T91 和 H209 的相互作用控制。
