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Copyright of Proceedings of the YSU B: Chemical & Biological Sciences / Gitakan Teghekagir. K'imia, Kensabanut'yun is the property of Publishing House of Yerevan State University and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

This study examines how F O F 1 -ATPase, hydrogenases (Hyd-1 and Hyd-2), and potassium transport systems (TrkA) interact to maintain the proton motive force (pmf) in E. coli during fermentation of different glucose concentrations (2 g L -1 and 8 g L -1 ). Our findings indicate that mutants lacking the hyaA-hyaC genes exhibited a 30 % increase in total proton flux compared to the wild type when grown with 2 g L -1 glucose. This has been observed during assays where similar glucose levels were supplemented. Disruptions in proton pumping, particularly in hyaB and hyaC single mutants, led to increased potassium uptake. The hyaB mutant showed a threefold increase in the contribution of F O F 1 -ATPase to proton flux, suggesting a significant role for Hyd-1 in proton translocation. In the hybC mutant grown in 2 g L -1 glucose conditions, DCCD-sensitive fluxes decreased by 70 %, indicating critical role of Hyd-2 in proton transport and F O F 1 function. When cells were grown with 8 g L -1 glucose, the 2H + /1K + ratio was significantly disturbed in both wild type and mutants. Despite these perturbances, mutants with disruptions in Hyd-1 and Hyd-2 maintained constant F O F 1 function, suggesting that this enzyme remains stable in glucose-rich environments. These results provide valuable insights into how Hyd-1 and Hyd-2 contribute to the regulation of ion transport, particularly proton translocation, in response to glucose concentration. Our study uncovered potential complementary mechanisms between Hyd-1 and Hyd-2 subunits, suggesting a complex interplay between these enzymes via metabolic cross talk with F O F 1 in response to glucose concentrations to maintain pmf., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Pentameric FocA permeates either formate or formic acid bidirectionally across the cytoplasmic membrane of anaerobically growing Escherichia coli. Each protomer of FocA has its own hydrophobic pore, but it is unclear whether formate or neutral formic acid is translocated in vivo. Here, we measured total and dicyclohexylcarbodiimide (DCCD)-inhibited proton flux out of resting, fermentatively grown, stationary-phase E. coli cells in dependence on FocA. Using a wild-type strain synthesizing native FocA, it was shown that using glucose as a source of formate, DCCD-independent proton efflux was ∼2.5 mmol min -1 , while a mutant lacking FocA showed only DCCD-inhibited, F O F 1 -ATPase-dependent proton-efflux. A strain synthesizing a chromosomally-encoded FocA H209N variant that functions exclusively to translocate formic acid out of the cell, showed a further 20 % increase in FocA-dependent proton efflux relative to the parental strain. Cells synthesizing a FocA T91A variant, which is unable to translocate formic acid out of the cell, showed only DCCD-inhibited proton efflux. When exogenous formate was added, formic acid uptake was shown to be both FocA- and proton motive force-dependent. By measuring rates of H 2 production, potassium ion flux and ATPase activity, these data support a role for coupling between formate, proton and K + ion translocation in maintaining pH and ion gradient homeostasis during fermentation. FocA thus plays a key role in maintaining this homeostatic balance in fermenting cells by bidirectionally translocating formic acid., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Escherichia coli anaerobically ferment glucose and perform proton/potassium exchange at pH 7.5. The role of hyf (hydrogenase 4) subunits (HyfBDF) in sensing different concentrations of glucose (2 g L -1 or 8 g L -1 ) via regulating H + /K + exchange was studied. HyfB, HyfD and HyfF part of a protein family of NADH-ubiquinone oxidoreductase ND2, ND4 and ND5 subunits is predicted to operate as proton pump. Specific growth rate was optimal in wild type and mutants grown on 2 g L -1 glucose reaching ~ 0.8 h -1 . It was shown that in wild type cells proton but not potassium fluxes were stimulated ~ 1.7 fold reaching up to 1.95 mmol/min when cells were grown in the presence of 8 g L -1 glucose. Interestingly, cells grown on peptone only had similar proton/potassium fluxes as grown on 2 g L -1 glucose. H + /K + fluxes of the cells grown on 2 g L -1 but not 8 g L -1 glucose depend on externally added glucose concentration in the assays. DCCD-sensitive H + fluxes were tripled and K + fluxes doubled in wild type cells grown on 8 g L -1 glucose compared to 2 g L -1 when in the assays 2 g L -1 glucose was added. Interestingly, in hyfF mutant when cells were grown on 2 g L -1 glucose and in 2 g L -1 assays DCCD-sensitive fluxes were not determined compared to wild type while in hyfD mutant it was doubled reaching up to 0.657 mmol/min. In hyf mutants DCCD-sensitive K + fluxes were stimulated in hyfD and hyfF mutants compared to wild type but depend on external glucose concentration. DCCD-sensitive H + /K + ratio was equal to ~ 2 except hyfF mutant grown and assayed on 2 g L -1 glucose while in 8 g L -1 conditions role of hyfB and hyfD is considered. Taken together it can be concluded that Hyd-4 subunits (HyfBDF) play key role in sensing glucose concentration for regulation of DCCD-sensitive H + /K + fluxes for maintaining proton motive force generation., (© 2022. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

After brewing roasted coffee, spent coffee grounds (SCGs) are generated being one of the daily wastes emerging in dominant countries with high rate and big quantity. Escherichia coli BW25113 wild-type strain, mutants with defects in hydrogen (H2)-producing/oxidizing four hydrogenases (Hyd) (ΔhyaB ΔhybC, ΔhycE, ΔhyfG) and septuple mutant (ΔhyaB ΔhybC ΔhycA ΔfdoG ΔldhA ΔfrdC ΔaceE) were investigated by measuring change of external pH, bacterial growth and H2 production during the utilization of SCG hydrolysate. In wild type, H2 was produced with rate of 1.28 mL H2 (g sugar)-1 h-1 yielding 30.7 mL H2 (g sugar)-1 or 2.75 L (kg SCG)-1 during 24 h. In septuple mutant, H2 production yield was 72 mL H2 (g sugar)-1 with rate of 3 mL H2 (g sugar)-1 h-1. H2 generation was absent in hycE single mutant showing the main role of Hyd-3 in H2 production. During utilization of SCG wild type, specific growth rate was 0.72 ± 0.01 h-1 with biomass yield of 0.3 g L-1. Genetic modifications and control of external parameters during growth could lead to prolonged and enhanced microbiological H2 production by organic wastes, which will aid more efficiently global sustainable energy needs resulting in diversification of mobile and fixed energy sources., (© FEMS 2020.)