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Most Sym plasmid-localized nodulation genes of Rhizobium leguminosarum bv. viciae are only expressed upon activation of the NodD protein by plant flavonoids, e.g., naringenin (S. A. J. Zaat, C. A. Wijffelman, H. P. Spaink, A. A. N. van Brussel, and B. J. J. Lugtenberg, J. Bacteriol, 169:198-204, 1987). As part of a study on the mechanism of NodD protein activation, the mechanism of uptake and the intracellular fate of [3H]naringenin were studied. Naringenin was accumulated by Rhizobium cells without apparent metabolic conversion to an 80-fold-higher concentration in a process which did not require any of the other Sym plasmid-localized nod genes. Naringenin accumulation was nonsaturable, highly reversible, and not inhibited by the presence of other flavonoids or the metabolic inhibitors potassium cyanide, sodium azide, 2,4-dinitrophenol, and carbonyl cyanide m-chlorophenylhydrazone. These data indicate an accumulation mechanism without high affinity sites which does not use cellular energy. In vitro, naringenin has high affinity for the cytoplasmic membrane. This binding was pH dependent, very high at pH 5.7 and not present anymore at pH 9.7. A similar pH dependency was found for the affinity of naringenin for the olive oil fraction of a biphasic olive oil-water system. pH-dependent changes in the UV spectrum indicate ionization of naringenin at high pH to a negatively charged form. Since it has recently been shown that the nodD gene product is located in the cytoplasmic membrane (H. R. M. Schlaman, H. P. Spaink, R. J. H. Okker, and B. J. J. Lugtenberg, J. Bacteriol., in press), our data are consistent with a model in which the un-ionized form of naringenin accumulates in the cytoplasmic membrane and activates, in a metabolically unaltered form, the NodD protein.

During the last few years the studies about the physiology and bioenergetics of lactic acid bacteria during growth and starvation have evolved from a descriptive level to an analysis of the molecular events in the regulation of various processes. Considerable progress has been made in the understanding of the modes of metabolic energy generation, the mechanism of homeostasis of the internal pH, and the mechanism and regulatory processes of transport systems for sugars, amino acids, peptides, and ions. Detailed studies of these transport processes have been performed in cytoplasmic membrane vesicles of these organisms in which a foreign proton pump has been introduced to generate a high proton motive force.

The role of the membrane lipid composition on the transport protein of branched-chain amino acids of the homofermentative lactic acid bacterium Streptococcus cremoris has been investigated. The major membrane lipid species identified in S. cremoris were acidic phospholipids (phosphatidylglycerol and cardiolipin), glycolipids, and glycerophosphoglycolipids. Phosphatidylethanolamine (PE) was completely absent. Protonmotive force-driven and counterflow transport of leucine was assayed in fused membranes of S. cremoris membrane vesicles and liposomes composed of different lipids obtained by the freeze/thaw-sonication technique. High transport activities were observed with natural S. cremoris and Escherichia coli lipids, as well as with mixtures of phosphatidylcholine (PC) with PE or phosphatidylserine. High transport activities were also observed with mixtures of PC with monogalactosyl diglyceride, digalactosyl diglyceride, or a neutral glycolipid fraction isolated from S. cremoris. PC or mixtures of PC with phosphatidylglycerol, phosphatidic acid, or cardiolipin showed low activities. In mixtures of PC and methylated derivatives of PE, both counterflow and protonmotive force-driven transport activities decreased with increasing degree of methylation of PE. The decreased transport activity in membranes containing PC could be restored by refusion with PE-containing liposomes. These results demonstrate that both aminophospholipids and glycolipids can be activators of the leucine transport system from S. cremoris. It is proposed that aminophospholipids in Gram-negative bacteria and glycolipids in Gram-positive bacteria have similar functions with respect to solute transport.

Membrane vesicles derived from the strictly fermentative lactic acid bacterium Streptococcus cremoris have been fused with proteoliposomes containing the beef heart mitochondrial cytochrome c oxidase by means of a freeze/thaw-sonication technique. Evidence that fusion has taken place was obtained by freeze-etch electron microscopy, showing a less-dense intramembranous particle distribution in the fused membranes than in the bacterial membranes, and by sucrose gradient centrifugation, indicating a buoyant density of the majority of the membranes after fusion that was between the buoyant densities of the starting membrane preparations. In the fused membranes, 55-60% of the cytochrome c oxidase molecules are oriented with the cytochrome c binding site at the outer surface of the membrane. With the electron-donor system ascorbate/N,N,N',N'-tetramethyl-p-phenylenediamine/cytochrome c, a high proton-motive force (greater than 130 mV), inside negative and alkaline, can be generated in the fused membrane, and this proton-motive force can drive secondary transport of several amino acids. The procedure described can be used for incorporating a proton-motive force-generating system in isolated membrane vesicles from bacterial or eukaryotic origin that lack a suitable primary proton pump.

The energetics of neutral and branched chain amino acid transport by membrane vesicles from Streptococcus cremoris have been studied with a novel model system in which beef heart mitochondrial cytochrome c oxidase functions as a proton-motive force (delta p) generating system. In the presence of reduced cytochrome c, a large delta p was generated with a maximum value at pH 6.0. Apparent H+/amino acid stoichiometries (napp) have been determined at external pH values between 5.5 and 8.0 from the steady state levels of accumulation and the delta p. For L-leucine napp (0.8) was nearly independent of the pH. For L-alanine and L-serine napp decreased from 0.9-1.0 at pH 5.5 to 0-0.2 at pH 8.0. The napp for the different amino acids decreased with increasing external amino acid concentration. At pH 6.0, first order rate constants for amino acid exit (kex) under steady state conditions for L-leucine, L-alanine, and L-serine were 1.1-1.3, 0.084, and 0.053 min-1, respectively. From the pH dependence of kex it is concluded that amino acid exit in steady state is the sum of two processes, pH-dependent carrier-mediated amino acid exit and pH-independent passive diffusion (external leak). The first order rate constant for passive diffusion increased with increasing hydrophobicity of the side chain of the amino acids. As a result of these processes the kinetic steady state attained is less than the amino acid accumulation ratio predicted by thermodynamic equilibrium. The napp determined from the steady state accumulation represents, therefore, a lower limit. It is concluded that the mechanistic stoichiometry (n) for L-leucine, L-alanine, and L-serine transport most likely equals 1.

Lactobacillus casei 393 cells which were energized with glucose (pH 6.0) took up glutamine, asparagine, glutamate, aspartate, leucine, and phenylalanine. Little or no uptake of several essential amino acids (valine, isoleucine, arginine, cysteine, tyrosine, and tryptophan) was observed. Inhibition studies indicated that there were at least five amino acid carriers, for glutamine, asparagine, glutamate/aspartate, phenylalanine, or branched-chain amino acids. Transport activities had pH optima between 5.5 and 6.0, but all amino acid carriers showed significant activity even at pH 4.0. Leucine and phenylalanine transport decreased markedly when the pH was increased to 7.5. Inhibitors which decreased proton motive force (delta p) nearly eliminated leucine and phenylalanine uptake, and studies with de-energized cells and membrane vesicles showed that an artificial electrical potential (delta psi) of at least -100 mV was needed for rapid uptake. An artificial delta p was unable to drive glutamine, asparagine, or glutamate uptake, and transport of these amino acids was sensitive to a decline in intracellular pH. When intracellular pH was greater than 7.7, glutamine, asparagine, or glutamate was transported rapidly even though the proton motive force had been abolished by inhibitors.

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.

Rightside-out membrane vesicles of Streptococcus cremoris were fused with proteoliposomes containing the light-driven proton pump bacteriorhodopsin by a low-pH fusion procedure reported earlier [Driessen, A.J.M., Hellingwerf, K.J. & Konings, W.N. (1985) Biochim. Biophys. Acta 808, 1-12]. In these fused membranes a proton motive force, interior positive and acid, can be generated in the light and this proton motive force can drive the uptake of Ca2+. Collapsing delta psi with a concomitant increase in delta pH stimulates Ca2+ uptake while dissipation of the delta pH results in a reduced rate of Ca2+ uptake. Also an artificially generated delta pH, interior acid, can drive Ca2+ uptake in S. cremoris membrane vesicles. Ca2+ uptake depends strongly on the presence of external phosphate while Ca2+-efflux-induced proton flux is independent of the presence of external phosphate. Ca2+ accumulation is abolished by the divalent cation ionophore A23187. Calcium extrusion from intact cells is accelerated by lactose. Collapse of the proton motive force by the uncoupler carbonylcyanide p-trifluoromethoxyphenylhydrazone or inhibition of the membrane-bound ATPase by N,N'-dicyclohexylcarbodiimide strongly inhibits Ca2+ release. Further studies on Ca2+ efflux at different external pH values in the presence of either valinomycin or nigericin suggested that Ca2+ exit from intact cells is an electrogenic process. It is concluded that Ca2+ efflux in S. cremoris is mediated by a secondary transport system catalyzing exchange of calcium ions and protons.

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.

The kinetic mechanism and specificity of the arginine-ornithine antiporter was investigated in membrane vesicles derived from Lactococcus lactis. Membrane vesicles loaded with ornithine, and diluted into an arginine-free medium, rapidly released a limited amount of ornithine during the first seconds of incubation. The amount of ornithine released was independent of the amount initially present on the inside and roughly matched the number of ornithine-binding sites in the membrane. Net flow of ornithine was only observed in membrane vesicles derived from induced cells and blocked by p-chloromercuribenzene sulfonic acid. These results suggest that net flow of ornithine is caused by a single turnover of the antiporter. With saturating concentrations of arginine in the external medium, efflux of ornithine was stoichiometrically coupled to uptake of arginine. Arginine-ornithine exchange and net flow of ornithine are electrically silent and not regulated by the electrical potential. The kinetics of the homologous exchange reactions indicate that the Vmax values for arginine and ornithine uptake are comparable, whereas the apparent Kt values differ. No major sidedness of the apparent Kt values are observed for both surfaces of the cytoplasmic membrane. Various basic amino acid analogues, including optical isomers, are transported as well, albeit with different efficiencies (Vmax/Kt). Evidence for a competitive character of arginine and ornithine interactions for binding sites on the antiporter are provided by transport and binding measurements. The Vmax and apparent Kt for arginine uptake increases with increasing internal ornithine, with little effect on the ratio of Vmax to apparent Kt. These results are discussed in terms of a simple carrier model in which the substrate-binding site is presented alternately to the two surfaces of the membrane as in a Ping Pong mechanism for enzyme kinetics.

Streptococcus bovis JB1 cells were able to transport serine, threonine, or alanine, but only when they were incubated in sodium buffers. If glucose-energized cells were washed in potassium phosphate and suspended in potassium phosphate buffer, there was no detectable uptake. Cells deenergized with 2-deoxyglucose and incubated in sodium phosphate buffer were still able to transport serine, and this result indicated that the chemical sodium gradient was capable of driving transport. However, when the deenergized cells were treated with valinomycin and diluted into sodium phosphate to create both an artificial membrane potential and a chemical sodium gradient, rates of serine uptake were fivefold greater than in cells having only a sodium gradient. If deenergized cells were preloaded with sodium (no membrane potential or sodium gradient), there was little serine transport. Nigericin and monensin, ionophores capable of reversing sodium gradients across membranes, strongly inhibited sodium-dependent uptake of the three amino acids. Membrane vesicles loaded with potassium and diluted into either lithium or choline chloride were unable to transport serine, but rapid uptake was evident if sodium chloride was added to the assay mixture. Serine transport had an extremely poor affinity for sodium, and more than 30 mM was needed for half-maximal rates of uptake. Serine transport was inhibited by an excess of threonine, but an excess of alanine had little effect. Results indicated that S. bovis had separate sodium symport systems for serine or threonine and alanine, and either the membrane potential or chemical sodium gradient could drive uptake.