Understanding the Role of Escherichia coli Hydrogenases and Formate Dehydrogenases in the F_OF₁‐ATPase Activity during the Mixed Acid Fermentation of Mixture of Carbon Sources

Abstract

During fermentation Escherichia coli produces di-hydrogen (H ) via reversible membrane-bound [Ni-Fe]-hydrogenases (Hyd). This study describes the total and N,N'-dicyclohexylcarbodiimide (DCCD) inhibited ATPase activity and H production at various pHs in E. coli wild type and mutants encoding Hyd enzymes and formate dehydrogenases (FDH) on fermentation of glucose, glycerol, and formate. The highest total ATPase activity was detected at pH 7.5 in hyaB hybC selC (lacking large subunits of Hyd-1 and Hyd-2 and FDH, respectively) triple mutant. This ATPase activity was mainly due to the proton-translocating ATPase but in FDH mutant the DCCD inhibition was less compared to wild type. Potassium ions stimulated total ATPase activity at pH 5.5 ~50% and ~35% in wild type and hypF (lacking all Hyd enzymes) mutant, respectively. Moreover, K also stimulated DCCD inhibited ATPase activity ~1.7-fold-2-fold in strains where FDH was absent only at pH 5.5. DCCD inhibited H production only at pH 5.5 in all assays. Taken together it is suggested that at low pH, FDH, and Hyd enzymes are linked with the F F -ATPase for regulating and maintaining the cytoplasmatic pH and thus proton motive force generation. FDH and Hyd enzymes have impact on the F F -ATPase activity depending on external pH and potassium ions. © 2018 IUBMB Life, 70(10):1040-1047, 2018.

Formate and potassium ions affect Escherichia coli proton ATPase activity at low pH during mixed carbon fermentation

“…The growth medium pH was measured by a pH‐meter with a selective pH‐electrode (HJ1131B, Hanna Instruments, Portugal) and adjusted using of 0.1 M HCl or 0.1 N NaOH. Membrane vesicles (MV) were isolated from bacteria, treated with lysozyme and ethylenediaminetetraacetic acid, by the osmotic lysis of spheroplasts, as described previously …”

“…The ATPase activity was expressed in nmol P i (min μg protein) −1 . P i was determined spectrophotometrically (UV–VIS spectrophotometer, Cary 60, Agilent Technologies) by the method, as described . The DCCD‐inhibited ATPase activity was calculated as a difference between activities in the absence and in the presence of the inhibitor.…”

“…Membrane vesicles (MV) were isolated from bacteria, treated with lysozyme and ethylenediaminetetraacetic acid, by the osmotic lysis of spheroplasts, 29 as described previously. 19…”

“…16,17 In addition, recently it was shown that during mixed carbon (glucose and glycerol) sources fermentation DCCDinhibited proton ATPase activity depends also on Hyd enzymes. 18,19 During single-carbon source (glucose or glycerol) fermentative conditions, role of different Hyd enzymes, proton ATPase, formate dehydrogenase activities is generally described. 1,[18][19][20] However, in natural environments, Enterobacterium spp.…”

“…18,19 During single-carbon source (glucose or glycerol) fermentative conditions, role of different Hyd enzymes, proton ATPase, formate dehydrogenase activities is generally described. 1,[18][19][20] However, in natural environments, Enterobacterium spp. such as E. coli always encounters complex mixtures of carbon sources.…”