Escherichia coli is able to utilize the mixture of carbon sources and produce molecular hydrogen (H 2 ) via formate hydrogen lyase (FHL) complexes. In current work role of transcriptional activator of formate regulon FhlA in generation of fermentation end products and proton motive force, N 0 N 0dicyclohexylcarbodiimide (DCCD)-sensitive ATPase activity at 20 and 72 hr growth during utilization of mixture of glucose, glycerol, and formate were investigated. It was shown that in fhlA mutant specific growth rate was~1.5 fold lower compared to wt, while addition of DCCD abolished the growth in fhlA but not in wt. Formate was not utilized in fhlA mutant but wt cells simultaneously utilized formate with glucose. Glycerol utilization started earlier (from 2 hr) in fhlA than in wt. The DCCD-sensitive ATPase activity in wt cells membrane vesicles increased~2 fold at 72 hr and was decreased 70% in fhlA. Addition of formate in the assays increased proton ATPase activity in wt and mutant strain. FhlA absence mainly affected the ΔpH but not ΔΨ component of Δp in the cells grown at 72 hr but not in 24 hr. The Δp in wt cells decreased from 24 to 72 hr of growth~40 mV while in fhlA mutant it was stable. Taken together, it is suggested that FhlA regulates the concentration of fermentation end products and via influencing F O F 1 -ATPase activity contributes to the proton motive force generation.

Escherichia coli is able to ferment mixed carbon sources and produce various fermentation end products. In this study, the function of FhlA protein in the specific growth rate (µ), metabolism, regulation of ΔpH and proton ATPase activity was investigated. It was shown reduced µ in fhlA mutant by ∼25% suggesting the role of FhlA in the growth process. The utilization rate of glycerol is decreased in fhlA ∼ 2 fold depending on the ORP values. Bacteria regulate Hyd enzymes activity during the growth depending on the external pH, which manifests the lack of H2 generation during the glycerol utilization at pH below 5.9. It is suggested that cells maintain ΔpH during the fermentative growth via formate-lactate-succinate exchange. The decrement of the value of pHin, but not pHex in mutant cells is regulating ΔpH and consequently proton motive force generation.