A Mutant of Escherichia coli Defective in the Coupling of Metabolic Energy to Active Transport

Abstract

The isolation of a temperature-sensitive mutant of E. coli K12 whose active transport of amino acids and sugars is not coupled to metabolic energy at 420 is described. This The ecfta mutation has been mapped to be in the 54.5-to 60-min region of the E. coli chromosome map. Possible interactions between the metC mutation and the mutated energy coupling factor protein are discussed.The molecular mechanisms by which metabolic energy is coupled to active transport in bacteria have been a subject of considerable interest (1-5). In the Escherichia coli membrane vesicle system the oxidation of D-lactate to pyruvate facilitates the transport of a wide variety of amino acids and sugars against a concentration gradient (1, 6). In whole cells the main energy source utilized for aerobic transport comes from respiration and the generation or utilization of ATP does not seem to be involved (7,8). On the other hand, cells apparently utilize the energy derived from the hydrolysis of ATP for anaerobic transport, as demonstrated in studies on mutants defective in the CaMg-ATPase (7, 9). The form of the metabolic energy, whether it be a high-energy intermediate of oxidative phosphorylation, an energized membrane state, or some other form, has not yet been determined (2, 10-12).In this paper we describe a mutant that is temperaturesensitive in a pleiotropic manner for the active transport of amino acids and sugars and is altered in a component energy coupling factor (ECF) that is necessary for the coupling of metabolic energy to transport. We also present evidence that a derivative of methionine may be interacting with the altered protein. MATERIALS AND METHODSBacterial Strains. All strains used were of E. coli K12 and are listed in Table 1

Characterization of an Escherichia coli Mutant Pleiotropically Altered in Membrane‐Bound Oxidoreductase Activities

“…Their active transport is driven by a respiratory or H±-translocating ATPase generated membrane potential (a component of PMF) across the cytoplasmic membrane (28). Agents (e.g., valinomycin) which collapse this membrane potential also cause a loss of aminoglycoside transport (28), as do mutations affecting either ATPase activity or respiratory chain components (e.g., biosynthesis of heme or ubiquinone) (10,13,24).…”

“…Respiratory deficient mutants of Escherichia coli have proven to be pleiotropically deficient for growth on oxidizable substrates (Suc-phenotype) as a result of mutations that block the biosynthesis of either heme (13,35) or ubiquinone (13,32), or mutations which uncouple electron transfer from membrane associated biological reactions (5,13,15,24). These mutants have been valuable in elucidating the structure, function and organization of the E. coli respiratory chains (13,16,32).…”

A new gene responsible for an energy-transducing system in Escherichia coli.

“…However, ecf mutant strains did not exhibit temperature-sensitive oxygen uptake and they were concluded to carry mutations in other component than respiratory chain (Lieberman and Hong, 1974). Thus the ttr gene seems to specify a factor which is different from proteins encoded by the ecf genes.…”

“…The ecfA (65 min) and ecfB (88 min) genes, which are located in different regions from the ttr gene (82-83 min), are also responsible for an energy-coupling system related to proline-transport (Bachmann, 1983;Lieberman and Hong, 1974). However, ecf mutant strains did not exhibit temperature-sensitive oxygen uptake and they were concluded to carry mutations in other component than respiratory chain (Lieberman and Hong, 1974).…”

“…One approach to this problem is to isolate mutant strains defective in some components involved in the energy transducing system. So far unc mutant strains (Gunsalus et al, 1982) and ecf mutant strains (Lieberman and Hong, 1974) of E. coli have been isolated by genetical approaches.…”