Abstract
During fermentation Escherichia coli excrete succinate mainly via Dcu family carriers. Current work reveals the total and N,N’-dicyclohexylcarbodiimide (DCCD) inhibited ATPase activity at pH 7.5 and 5.5 in E. coli wild type and dcu mutants upon glycerol fermentation. The overall ATPase activity was highest at pH 7.5 in dcuABCD mutant. In wild type cells 50% of the activity came from the FOF1-ATPase but in dcuD mutant it reached ~80%. K+ (100 mM) stimulate total but not DCCD inhibited ATPase activity 40% and 20% in wild type and dcuD mutant, respectively. 90% of overall ATPase activity was inhibited by DCCD at pH 5.5 only in dcuABC mutant. At pH 7.5 the H+ fluxes in E. coli wild type, dcuD and dcuABCD mutants was similar but in dcuABC triple mutant the H+ flux decreased 1.4 fold reaching 1.15 mM/min when glycerol was supplemented. In succinate assays the H+ flux was higher in the strains where DcuD is absent. No significant differences were determined in wild type and mutants specific growth rate except dcuD strain. Taken together it is suggested that during glycerol fermentation DcuD has impact on H+ fluxes, FOF1-ATPase activity and depends on potassium ions.
Highlights
20% in wild type and dcuD mutant, respectively. 90% of overall ATPase activity was inhibited by DCCD at pH 5.5 only in dcuABC mutant
Substitution of glucose by glycerol did not affect dcuC expression, it can be assumed that dcuC is not subject to catabolite repression and DcuC is needed for succinate efflux during glucose fermentation[1,3]
To understand what is the role of C4-dicarboxylate carriers (Dcu) during glycerol fermentation the activity of proton FOF1-ATPase has been investigated
Summary
ATPase activity and H+ fluxes of E. coli wild type and dcu mutants at pH 7.5 and pH 5.5. To understand what is the role of C4-dicarboxylate carriers (Dcu) during glycerol fermentation the activity of proton FOF1-ATPase has been investigated For this objective, total and DCCD inhibited ATPase activity at pH 7.5 and pH 5.5 has been defined. It was shown that at pH 7.5 in wild type and dcuABC mutant membrane vesicles DCCD the ATPase activity was inhibited 2 and 1.9 fold, respectively, suggesting that FOF1 contributed to total ATPase activity by 50% under the conditions mentioned. The data suggest that there might be some relationship or interaction between DcuD protein and the FOF1-ATPase at pH 7.5 It had been reviewed[1] that during respiration with the transport of succinate2-3H+ are symported but under fermentative conditions the amount of H+ that are symported during succinate efflux is not known yet. During anaerobic conditions there might be limited possibility of compensatory uptake functions of C4 uptake, an issue that must be further analyzed
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