title: Uniport of anionic citrate and proton consumption in citrate metabolism generates a proton motive force in Leuconostoc oenos.
abstract: The mechanism and energetics of citrate transport in Leuconostoc oenos were investigated. Resting cells of L. oenos generate both a membrane potential (delta psi) and a pH gradient (delta pH) upon addition of citrate. After a lag time, the internal alkalinization is followed by a continuous alkalinization of the external medium, demonstrating the involvement of proton-consuming reactions in the metabolic breakdown of citrate. Membrane vesicles of L. oenos were prepared and fused to liposomes containing cytochrome c oxidase to study the mechanism of citrate transport. Citrate uptake in the hybrid membranes is inhibited by a membrane potential of physiological polarity, inside negative, and driven by an inverted membrane potential, inside positive. A pH gradient, inside alkaline, leads to the accumulation of citrate inside the membrane vesicles. Kinetic analysis of delta pH-driven citrate uptake over a range of external pHs suggests that the monovalent anionic species (H2cit-) is the transported particle. Together, the data show that the transport of citrate is an electrogenic process in which H2cit- is translocated across the membrane via a uniport mechanism. Homologous exchange (citrate/citrate) was observed, but no evidence for a heterologous antiport mechanism involving products of citrate metabolism (e.g., acetate and pyruvate) was found. It is concluded that the generation of metabolic energy by citrate utilization in L. oenos is a direct consequence of the uptake of the negatively charged citrate anion, yielding a membrane potential, and from H(+)-consuming reactions involved in subsequent citrate metabolism, yielding a pH gradient. The uptake of citrate is driven by its own concentration gradient, which is maintained by efficient metabolic breakdown (metabolic pull).
authors.affiliation: Department of Microbiology, University of Groningen, Haren, The Netherlands.
authors.forename: A
authors.initials: A
authors.lastname: Ramos
authors.forename: B
authors.initials: B
authors.lastname: Poolman
authors.forename: H
authors.initials: H
authors.lastname: Santos
authors.forename: J S
authors.initials: JS
authors.lastname: Lolkema
authors.forename: W N
authors.initials: WN
authors.lastname: Konings
mesh_terms: D000838:Anions; D001693:Biological Transport, Active; D002951:Citrates / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D007934:Leuconostoc / Q000378:metabolism*; D008564:Membrane Potentials
chemical_list: D000838:Anions; D002951:Citrates