Relationship between phospholipid compositions and transport activities of amino acids in Escherichia coli membrane vesicles.

Cells of Escherichia coli were incubated in broth medium in the presence of 5 mM of hydroxylamine which completely inhibited growth but did not affect viabilities. Hydroxylamine is known to inhibit phosphatidylserine decarboxylase. A large amount of phosphatidylserine (up to 20% of total phospholipids), which did not occur in normal cells, accumulated accompanied with a decrease in phosphatidylethanolamine. Higher uptake activities of serine and glutamate were observed with the hydroxylamine-treated cells than control cells. When membrane vesicles from hydroxylamine-treated cells were prepared, they also displayed higher uptake activities of serine, proline, glutamate, and threonine than those of normal membranes. When hydroxylamine-treated cells were incubated with chloramphenicol, at concentrations which almost completely inhibited protein synthesis, the composition of phosphatidylserine decreased with a concomitant increase in that of phosphatidylethanolamine. The phospholipid composition of these cells incubated for 5 h with chloramphenicol became almost normal. Membranes vesicles prepared from such cells displayed reduced uptake activities, which were close to those of normal vesicles. These results were interpreted as indicating the altered transport activities due to the altered phospholipid composition.     

Transport of branched-chain amino acids in membrane vesicles of Streptococcus cremoris.

The kinetics, specificity, and mechanism of branched-chain amino acid transport in Streptococcus cremoris were studied in a membrane system of S. cremoris in which beef heart mitochondrial cytochrome c oxidase was incorporated as a proton motive force (delta p)-generating system. Influx of L-leucine, L-isoleucine, and L-valine can occur via a common transport system which is highly selective for the L-isomers of branched chain amino acids and analogs. The pH dependency of the kinetic constants of delta p-driven L-leucine transport and exchange (counterflow) was determined. The maximal rate of delta p-driven transport of L-leucine (Vmax) increased with increasing internal pH, whereas the affinity constant increased with increasing external pH. The affinity constant for exchange (counterflow) varied in a similar fashion with pH, whereas Vmax was pH independent. Further analysis of the pH dependency of various modes of facilitated diffusion, i.e., efflux, exchange, influx, and counterflow, suggests that H+ and L-leucine binding and release to and from the carrier proceed by an ordered mechanism. A kinetic scheme of the translocation cycle of H+-L-leucine cotransport is suggested.     

NADH oxidation in phospholipid-enriched cytoplasmic membrane vesicles from Escherichia coli

NADH oxidation in Escherichia coli cytoplasmic membrane vesicles enriched in anionic phospholipids by de novo synthesis of lipid in the vesicles from acyl-CoA esters and sn-glycerol 3-phosphate has been studied. NADH-oxidase but not NADH-dehydrogenase activity was found to decrease during synthesis and accumulation of phospholipid in the vesicles. Density gradient fractionation showed that NADH-oxidase activity was reduced to approximately 30% in vesicles with a 3-6 fold increase in anionic phospholipid, whereas vesicles with a greater than 10-fold increase in phospholipid had virtually no NADH oxidase activity.     

Transport of basic amino acids by membrane vesicles of Lactococcus lactis.

The uptake of the basic amino acids arginine, ornithine, and lysine was studied in membrane vesicles derived from cells of Lactococcus lactis which were fused with liposomes in which beef heart mitochondrial cytochrome c oxidase was incorporated as a proton motive force (PMF)-generating system. In the presence of ascorbate N,N,N'N'-tetramethylphenylenediamine-cytochrome c as the electron donor, these fused membranes accumulated lysine but not ornithine or arginine under aerobic conditions. The mechanism of energy coupling to lysine transport was examined in membrane vesicles of L. lactis subsp. cremoris upon imposition of an artificial electrical potential (delta psi) or pH gradient or both and in fused membranes of these vesicles with cytochrome c oxidase liposomes in which the delta psi and delta pH were manipulated with ionophores. Lysine uptake was shown to be coupled to the PMF and especially to the delta psi, suggesting a proton symport mechanism. The lysine carrier appeared to be specific for L and D isomers of amino acids with a guanidine or NH2 group at the C6 position of the side chain. Uptake of lysine was blocked by p-chloromercuribenzene sulfonic acid but not by maleimides. Counterflow of lysine could not be detected in L. lactis subsp. cremoris, but in the arginine-ornithine antiporter-containing L. lactis subsp. lactis, rapid counterflow occurred. Homologous exchange of lysine and heterologous exchange of arginine and lysine were mediated by this antiporter. PMF-driven lysine transport in these membranes was noncompetitively inhibited by arginine, whereas the uptake of arginine was enhanced by lysine. These observations are compatible with a model in which circulation of lysine via the lysine carrier and the arginine-ornithine antiporter leads to accumulation of arginine.     

Transport of lactate and acetate through the energized cytoplasmic membrane of Escherichia coli

Escherichia coli produces lactate and acetate in significant amounts during both aerobic and anaerobic glycolysis. A model describing the mechanism of protein mediated lactate transport has previously bee proposed. A simple theoretical analysis here indicates that the proposed model would be drain cellular energy resources by catalytically dissipating the proton-motive force. An experimental analysis of lactate and acetate transport employ nuclear magnetic resonance (NMR) spectroscopy to measure the relative concentration of these end products on the two sides of the cytoplasmic membrane of anaerobically glycolyzing cells. Comparison of measured concentration rations to those expected at equilibrium for various transport modes indicates that acetate is a classical uncoupling agent, permeating the membrane oat comparable rates in the dissociated and undissociated forms. The lactate concentration ratio changes market markedly after an initial period of sustained glycolysis. This change is most readily explained as resulting from a lactate transport system that responds to an indicator of glycolytic activity. The data further indicates that lactate permeates the membrane in both dissociated and undissociated forms. Both acids, then are capable of catalytically dissipating the proton-motives force. (c) 1995 John Wiley & Sons, Inc.     

Transport of sugars and amino acids in bacteria. XIV. Preferential inhibition of oxidase activities and active transport reactions for amino acids by azidebenzenes.

4-Methylazidebenzene and various azidebenzene derivatives were prepared, and the effects of these compounds on oxidase activities and active transport reactions for amino acids in Escherichia coli cells were studied. Azidebenzenes inhibited succinate oxidation by intact cells preferentially to glycerol oxidation. However, the azidebenzenes could not inhibit succinate oxidation which was not coupled to phosphorylation. The compounds inhibited succinate driven proline uptake much more strongly than isoleucine uptake. Unlike sodium azide and diphenyl phosphorazidate, azidebenzenes did not inhibit membrane-bound, Mg2+-requiring ATPase [EC 3.6.1.3] of E. coli. Reactivities of various azide compounds in the mechanism of inhibition for energy transducing and energy transforming reactions were discussed briefly.     

Cytoplasmic membrane vesicles of Escherichia coli. A simple method for preparing the cytoplasmic and outer membranes.

A simple preparative method is described for isolation of the cytoplasmic and outer membranes from E. coli. The characteristics of both membrane fractions were studied chemically, biologically, and morphologically. Spheroplasts of E. coli K-12 strain W3092, prepared by treating cells with EDTA-lysozyme [EC 3.2.1.17], were disrupted in a French press. The crude membrane fraction was washed with 3 mM EDTA-10% (w/v) sucrose, pH 7.2, and the cytoplasmic membranes and outer membranes were separated by sucrose isopycnic density gradient centrifugation. The crude membrane fraction contained approximately 10% of the protein of the whole cells, 0.3% of the DNA, 0.7% of the RNA, 0.3% of the peptidoglycan, and about 30% of the lipopolysaccharide. The cytoplasmic membrane fraction was rich in phospholipid, while the outer membrane fraction contained much lipopolysaccharide and carbohydrate; the relative contents of lipopolysaccharide and carbohydrate per mg protein in the cytoplasmic membrane fraction were 12 and 40%, respectively, of the contents in the outer membrane fraction. Cytochrome b1, NADH oxidase, D-lactate dehydrogenase [EC 1.1.1.28], succinate dehydrogenase [EC 1.3.99.1], ATPase [EC 3.5.1.3], and activity for concentrative uptake of proline were found to be localized mainly in the cytoplasmic membranes; their specific activities in the outer membrane fraction were 1.5 to 3% of those in the cytoplasmic membrane fraction. In contrast, a phospholipase A appeared to be localized mainly in the outer membranes and its specific activity in the cytoplasmic membrane fraction was only 5% of that in the outer membrane fraction. The cytoplasmic and outer membrane fractions both appeared homogeneous in size and shape and show vesicular structures by electron microscopy. The advantages of this method for large scale preparation of the cytoplasmic and outer membrane fractions are discussed.     

Secondary transport of amino acids by membrane vesicles derived from lactic acid bacteria

Lactococci are fastidious bacteria which require an external source of amino acids and many other nutrients. These compounds have to pass the membrane. However, detailed analysis of transport processes in membrane vesicles has been hampered by the lack of a suitable protonmotive force (pmf)-generating system in these model systems. A membrane-fusion procedure has been developed by which pmf-generating systems can be functionally incorporated into the bacterial membrane. This improved model system has been used to analyze the properties of amino acid transport systems in lactococci. Detailed studies have been made of the specificity and kinetics of amino acid transport and also of the interaction of the transport systems with their lipid environment. The properties of a pmf-independent, arginine-catabolism specific transport system in lactococci will be discussed.Abbreviations pmf protonmotive force - transmembrane electrical potential - pH transmembrane pH gradient - PE phosphatidylethanolamine - PC phosphatidylcholine Paper adapted from a treatise Secondary Transport of Amino Acids by Membrane Vesicles Derived from Lactic Acid Bacteria and awarded the Kluyver Prize 1988 by the Netherlands Society of Microbiology.