Hydrogen-oxidizing hydrogenases 1 and 2 ofEscherichia coliregulate the onset of hydrogen evolution and ATPase activity, respectively, during glucose fermentation at alkaline pH

Abstract

Simultaneous measurement of redox potential (Eh ) and determination of H2 evolution kinetics using a pair of titanium silicate and platinum redox electrodes in fermenting cultures of Escherichia coli wild type and different mutants lacking hydrogenases 1 (Hyd-1) or 2 (Hyd-2) revealed that Hyd-1 controls the onset of H2 evolution at slightly alkaline pH (pH 7.5) and under oxidizing Eh . In addition, Hyd-2 influences the N,N'-dicyclohexylcarbodiimide-inhibited ATPase activity in fermenting cells and thus regulates the proton F0 F1 -ATPase at the alkaline pH but under reducing Eh .

Optimizing strategy for Escherichia coli growth and hydrogen production during glycerol fermentation in batch culture: Effects of some heavy metal ions and their mixtures

“…In contrast to Pt, Ti-Si electrode readings were not affected by the presence of H 2 (or oxygen) in the medium; this difference allows the determination of H 2 under anaerobic conditions (in bacterial suspension upon fermentation of glucose or glycerol), as reported previously [7,12,14,29]. This electrochemical method for H 2 determination has given reproducible and correct results for cumulative H 2 yield in liquids [2,9].…”

Impact of membrane-associated hydrogenases on the FOF1-ATPase in Escherichia coli during glycerol and mixed carbon fermentation: ATPase activity and its inhibition by N,N′-dicyclohexylcarbodiimide in the mutants lacking hydrogenases

“…Bacteria were grown under anaerobic conditions at 37°C for 18-20 h in highly buffered peptone medium (20 g L À1 peptone, 15 g L À1 K 2 HPO 4 , 1.08 g L À1 KH 2 PO 4, 10 g L À1 NaCl) with glycerol (10 g L À1 ) and glucose and glycerol together (2 g L À1 and 10 g L À1 ) at different pHs during 20-24 h. The medium was filled in glass vessels; oxygen was bubbled out from medium during autoclaving, and then vessels were closed with plastic press caps, as described elsewhere [23][24][25][26]. To check anaerobic conditions, H 2 production was tested, as before [25][26][27]. 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 KCl or 0.1 N NaOH.…”

“…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 KCl or 0.1 N NaOH. The specific growth rate was determined measuring the change of culture absorbance, as detailed previously [23][24][25][26].…”

“…ATPase activity of membrane vesicles was determined by the amount of inorganic phosphate (P i ) produced in the reaction of membrane vesicles with 5 mM ATP (pH 7.5, 6.5 and 5.5) [24][25][26][31][32][33] in the assay mixture (50 mM Tris-HCl buffer (pH 7.5, 6.5 and 5.5) containing 1 mM MgSO 4 ). ATP can reach ATPase in right-side-out vesicles due to membrane peculiarities of cells grown under the mentioned conditions [24,32] or the presence of in-side-out vesicles in the preparations, as suggested [29,34].…”

“…Moreover, ATPase activity of both types of membrane vesicles was markedly inhibited by DCCD (see Table 2). Note, ATPase activity of right-side-out vesicles from E. coli grown during glucose fermentation was determined previously [24][25][26][31][32][33]: it was $70% of that in in-side-out ones, as shown by Adler and Rosen [34] and confirmed by Bagramyan et al [32]. Moreover, DCCD has been shown to inhibit the F O F 1 -ATPase in E. coli since effect of DCCD is absent in the atp mutant lacked this ATPase [23] and, in addition, E. coli has no bc 1 complex which could be inhibited by DCCD [21,34].…”