Proton Flux Dependence on Glucose Concentration in E. coli Hyd-1 and Hyd-4 Mutants During Fermentation

Abstract

This study examines the effects of E. coli hydrogenase-1 (Hyd-1) and hydrogenase-4 (Hyd-4) on proton flux during fermentation of glucose with different concentrations (2 g L⁻¹ and 8 g L⁻¹). During research, proton flux was (J_H+) measured in the E. coli BW25113 wild-type strain and mutants hyaA-F and hyfA-R using 0.2 mM N,N-dicyclohexylcarbodiimide (DCCD) to evaluate F₀F₁ ATP synthase activity. Cells were grown separately in the absence of both glucose concentrations and during the experiments, either 2 g L⁻¹or 8 g L⁻¹ glucose was supplemented. Our findings indicate that in the hyaA-F mutant, where Hyd-1 is absent, proton flux and F₀F₁-ATPase activity are differently affected depending on glucose concentrations (2 g L⁻¹ and 8 g L⁻¹). Under low glucose conditions (grown in 2 g L⁻¹ and added 2 g L⁻¹), total J_H+ decreased by 55%, both DCCD sensitive and remaining fluxes were decreased as well by 60%. The contribution of F₀F₁-ATPase remained similar, as in wild type. High glucose addition (grown in 2 g L⁻¹ and added 8 g L⁻¹) leads to a 30% decrease in total J_H+ and F₀F₁-ATPase contribution also decreases by 25%. When cells were grown in the presence of 8 g L⁻¹ glucose total J_H+ decreased by 40% and 50% when 2 g L⁻¹ and 8 g L⁻¹ were supplemented, respectively. Meanwhile DCCD-sensitive J_H+ decreased 50% and 80%, respectively. The absence of Hyd-1 significantly reduces total and DCCD-sensitive proton fluxes ATP synthesis. In the hyfA-R mutant, where Hyd-4 is absent and F₀F₁-ATPase activity was significantly dependent on glucose concentration (2 g L⁻¹ and 8 g L⁻¹). Under low glucose conditions (2 g L⁻¹ growth and 2 g L⁻¹ addition), total J_H+ decreased by 60%, but the contribution F₀F₁-ATPase was not affected. High glucose addition reduces total J_H+ by 50%, but F₀F₁-ATPase contribution increases by 40%. In conditions grown with 8 g L⁻¹ glucose total J_H+ decreased by 10% and 40% when 2 g L⁻¹ and 8 g L⁻¹ were supplemented, respectively, while F₀F₁-ATPase contribution increased by 30% and 80%. The absence of Hyd-4 has a more significant impact on activity of F₀F₁-ATPase under high glucose conditions, indicating Hyd-4’s critical role in regulating E. coli’s energy metabolism.