H+-Translocating Membrane-Bound Pyrophosphatase from Rhodospirillum rubrum Fuels Escherichia coli Cells via an Alternative Pathway for Energy Generation

Abstract

Inorganic pyrophosphatases (PPases) catalyze an essential reaction, namely, the hydrolysis of PPi, which is formed in large quantities as a side product of numerous cellular reactions. In the majority of living species, PPi hydrolysis is carried out by soluble cytoplasmic PPase (S-PPases) with the released energy dissipated in the form of heat. In Rhodospirillum rubrum, part of this energy can be conserved by proton-pumping pyrophosphatase (H+-PPaseRru) in the form of a proton electrochemical gradient for further ATP synthesis. Here, the codon-harmonized gene hppaRru encoding H+-PPaseRru was expressed in the Escherichia coli chromosome. We demonstrate, for the first time, that H+-PPaseRru complements the essential native S-PPase in E. coli cells. 13C-MFA confirmed that replacing native PPase to H+-PPaseRru leads to the re-distribution of carbon fluxes; a statistically significant 36% decrease in tricarboxylic acid (TCA) cycle fluxes was found compared with wild-type E. coli MG1655. Such a flux re-distribution can indicate the presence of an additional method for energy generation (e.g., ATP), which can be useful for the microbiological production of a number of compounds, the biosynthesis of which requires the consumption of ATP.