Independent regulation of transport and biosynthesis of arginine in Escherichia coli K-12.

From an arginine auxotrophic strain, a mutant was isolated which is able to utilize d-arginine as a source of l-arginine and shows a high sensitivity to inhibition of growth by canavanine. Transport studies revealed a four- to five-fold increased uptake of arginine and ornithine in cells from the mutant strain. The kinetics of entry of arginine and ornithine evidenced elevated maximal influx values for the arginine- and ornithine-specific transport systems. A close parallel between arginine transport activity and arginine binding activity with one arginine-specific binding periplasmic protein in the mutant strongly suggests that such binding protein is a component of the arginine-specific permease. The affinity between arginine and the binder, isolated from the mutant cells, as well as the electrophoretic mobility of the protein, remain unchanged. The enhanced transport activity of arginine and ornithine with mutant cells is insensitive to repression by arginine or ornithine, whereas the biosynthesis of arginine-forming enzymes is normally repressible. When transport activity was examined in strains with mutations leading to derepression of arginine biosynthesis, the regulation of arginine transport was found to be normal. These studies support the conclusion that arginine transport and arginine biosynthesis, in Escherichia coli K-12, are not regulated in a concerted manner, although both systems may have components in common.     

Mutant of Escherichia coli K-12 Defective in the Transport of Basic Amino Acids

Escherichia coli K-12 possesses two active transport systems for arginine, two for ornithine, and two for lysine. In each case there is a low- and a high-affinity transport system. They have been characterized kinetically and by response to competitive inhibition by arginine, lysine, ornithine and other structurally related amino acids. Competitors inhibit the high-affinity systems of the three amino acids, whereas the low-affinity systems are not inhibited. On the basis of kinetic evidence and competition studies, it is concluded that there is a common high-affinity transport system for arginine, ornithine, and lysine, and three low-affinity specific ones. Repression studies have shown that arginine and ornithine repress each other's specific transport systems in addition to the repression of their own specific systems, whereas lysine represses its own specific transport system. The common transport system was found to be repressible only by lysine. A mutant was studied in which the uptake of arginine, ornithine, and lysine is reduced. The mutation was found to affect both the common and the specific transport systems.     

arcD, the first gene of the arc operon for anaerobic arginine catabolism in Pseudomonas aeruginosa, encodes an arginine-ornithine exchanger.

In the absence of oxygen and nitrate, Pseudomonas aeruginosa metabolizes arginine via the arginine deiminase pathway, which allows slow growth on rich media. The conversion of arginine to ornithine, CO2, and NH3 is coupled to the production of ATP from ADP. The enzymes of the arginine deiminase pathway are organized in the arcDABC operon. The arcD gene encodes a hydrophobic polytopic membrane protein. Translocation of arginine and ornithine in membrane vesicles derived from an Escherichia coli strain harboring a recombinant plasmid carrying the arcD gene was studied. Arginine and ornithine uptake was coupled to the proton motive force with a bias toward the transmembrane electrical potential. Accumulated ornithine was readily exchangeable for external arginine or lysine. The exchange was several orders of magnitude faster than proton motive force-driven transport. The ArcD protein was reconstituted in proteoliposomes after detergent solubilization of membrane vesicles. These proteoliposomes mediate a stoichiometric exchange between arginine and ornithine. It is concluded that the ArcD protein is a transport system that catalyzes an electroneutral exchange between arginine and ornithine to allow high-efficiency energy conversion in the arginine deiminase pathway.     

Control of the ornithine cycle in Neurospora crassa by the mitochondrial membrane.

In Neurospora crassa, the mitochondrial membrane separates ornithine used in arginine biosynthesis from ornithine used in the arginine degradative pathway in the cytosol. Ornithine easily exchanges across the mitochondrial membrane under conditions appropriate for synthesis of the immediate biosynthetic product, citrulline. Neither of the two mitochondrial enzymes required for the ornithine-to-citrulline conversion is feedback inhibitable in vitro. Nevertheless, when arginine is added to cells and cytosolic ornithine increases as arginine degradation begins, the rate of citrulline synthesis drops immediately to about 20% of normal (B. J. Bowman and R. H. Davis, Bacteriol. 130:285-291, 1977). We have studied this phenomenon in citrulline-accumulating strains carrying the arg-1 mutation. Citrulline accumulation is blocked when arginine is added to an arg-1 strain but not to an arg-1 strain carrying a mutation conferring insensitivity of intramitochondrial ornithine synthesis to arginine. Thus, ornithine is evidently unable to enter mitochondria in normal (feedback-sensitive) cells. Other experiments show that cytosolic ornithine enters mitochondria readily except when arginine or other basic amino acids are present at high levels in the cells. We conclude that in N. crassa, the mitochondrial membrane has evolved as a secondary site of feedback inhibition in arginine synthesis and that this prevents a wasteful cycling of catabolic ornithine back through the anabolic pathway. This is compared to the quite different mechanism by which the yeast Saccharomyces cerevisiae prevents a futile ornithine cycle.     

Basic Amino Acid Transport in Escherichia coli: Properties of Canavanine-Resistant Mutants

A mutant of Escherichia coli strain CanR 22 has been isolated which is resistant to growth inhibition by canavanine, an analogue of arginine. The properties of this strain and of another canavanine-resistant mutant, JC182-5 (isolated by Celis et al. [5]), were studied. The mutation is pleiotropic in that it results in a reduction in the activity of two distinct permeases, the arginine-specific and lysine-arginine-ornithine transport systems. The lesion maps at min 56 of the E. coli linkage map, at or near the argP locus. Although strain CanR 22 excretes arginine, this excretion appears to result from reduced ability to concentrate arginine, rather than the loss of transport ability being the result of excretion. This conclusion is based on findings with a canavanine-resistant strain auxotrophic for arginine, which exhibits transport properties similar to those of the prototrophic strains. Additionally, growth in the presence of arginine or ornithine results in a repression of the activity of the two basic amino acid transport systems. Neither the arginine-specific nor the lysine-arginine-ornithine binding proteins of the mutant cells show significant alterations in terms of amount, physical properties, or kinetic parameters. These observations lead to the proposal of a model for the two basic amino acid transport systems in which two carrier proteins with different specificities interact with a common energy coupling mechanism. A lesion in the gene (or one of the genes) for this coupling mechanism can confer canavanine resistance.     

Amino acid transport and metabolism in mycobacteria: cloning, interruption, and characterization of an L-Arginine/gamma-aminobutyric acid permease in Mycobacterium bovis BCG

Genes encoding L-arginine biosynthetic and transport proteins have been shown in a number of pathogenic organisms to be important for metabolism within the host. In this study we describe the cloning of a gene (Rv0522) encoding an amino acid transporter from Mycobacterium bovis BCG and the effects of its deletion on L-arginine transport and metabolism. The Rv0522 gene of BCG was cloned from a cosmid library by using primers homologous to the rocE gene of Bacillus subtilis, a putative arginine transporter. A deletion mutant strain was constructed by homologous recombination with the Rv0522 gene interrupted by a selectable marker. The mutant strain was complemented with the wild-type gene in single copy. Transport analysis of these strains was conducted using (14)C-labeled substrates. Greatly reduced uptake of L-arginine and gamma-aminobutyric acid (GABA) but not of lysine, ornithine, proline, or alanine was observed in the mutant strain compared to the wild type, grown in Middlebrook 7H9 medium. However, when the strains were starved for 24 h or incubated in a minimal salts medium containing 20 mM arginine (in which even the parent strain does not grow), L-[(14)C]arginine uptake by the mutant but not the wild-type strain increased strongly. Exogenous L-arginine but not GABA, lysine, ornithine, or alanine was shown to be toxic at concentrations of 20 mM and above to wild-type cells growing in optimal carbon and nitrogen sources such as glycerol and ammonium. L-Arginine supplied in the form of dipeptides showed no toxicity at concentrations as high as 30 mM. Finally, the permease mutant strain showed no defect in survival in unactivated cultured murine macrophages compared with wild-type BCG.     

Escherichia coli regulatory mutation affecting lysine transport and lysine decarboxylase

A spontaneous thiosine-resistant mutant of Escherichia coli was shown to have the following characteristics: lowered initial rate of lysine uptake and lowered plateau level of accumulation of exogenous lysine by both the lysine-specific and the general basic amino acid transport systems; altered repressibility of these two lysine transport systems; a derepressed level of lysine decarboxylase; normal growth rate; parental levels of lysyl-transfer ribonucleic acid synthetase and the inducible and constitutive arginine and ornithine decarboxylases. Both the mutant (lysP) and its parent (lysP+) feed a lysine auxotroph when they are plated in proximity on solid medium. However, the feeding response was observable after 1 day less of incubation when the mutant was the feeding strain. Despite the derepressed level of lysine decarboxylase in exponential cultures of the mutant extracts of these cultures had no detectable cadaverine pool. Conjugation experiments established the following gene order: gyrA (formerly nalA) lysP metG his. All thiosine-resistant recombinants assayed showed reduced lysine transport. In many of these recombinants the derepression of lysine decarboxylase was not expressed.     

arg—13可能参与鸟氨酸在粗糙脉孢霉线粒体的过膜转运

arg-13 is a leaky mutation involved in arginine metabolism. A tight selection is developed using similar amount of lysine and ornithine replacing other nitrogen source in minimal medium. This selection strongly inhibits the growth of arg-13 under stringent sorbose/glucose condition but allows arg-13 to grow under spot test conditions. As ornithine is build up through mitochondrial ornithine biosynthesis and transport from cytoplasm to mitochondria, arg-13 is combined in genetic crosses with arg-4 which blocks mitochondrial ornithine synthesis. Under spot test conditions, double mutant arg-4, arg-13 is able to use ornithine as sole nitrogen source and arginine biosynthesis precursor, but subject to strong lysine and canavanine inhibition. While the usage of ornithine in arg-4 single mutant with intact ornithine transport function is only slightly inhibited by lysine. All available data suggest arg-13 plays a major role in mitochondrial ornithine transport. The strain carrying the mutation at the arg-13 locus allows inefficient mitochondrial ornithine trafficking, possibly mediated by another distinct basic amino acid carrier.     

Arginine Auxotrophic Phenotype of Mutations in pyrA of Salmonella typhimurium: Role of N-Acetylornithine in the Maturation of Mutant Carbamylphosphate Synthetase

Mutations in pyrA that abolish catalytic activity of carbamylphosphate synthetase cause auxotrophy for both arginine and a pyrimidine. Eight pyrA mutants auxotrophic only for arginine (AUX) were isolated by the mutagenized phage technique; three of these required arginine only at low temperature (20 degrees C). Explanations of the AUX phenotype based on bradytrophy were eliminated by the discovery that blocking the utilization of carbamylphosphate for pyrimidine biosynthesis by insertion of an additional mutation in pyrB (encoding aspartic transcarbamylase) did not reduce the requirement for arginine. In contrast, mutational blocks in the arginine biosynthetic pathway before N-acetylornithine (argB, argC, argG, or argH) did suppress the mutation in pyrA. This suggests that exogenous arginine permits growth of the AUX mutants by inhibiting the first step in the arginine pathway, thereby preventing accumulation of an intermediate that antagonizes mutant pyrA function. A mutation in argA (N-acetylornithinase) failed to suppress AUX, indicating that N-acetylornithine was the inhibitory intermediate. This intermediate had no effect on the catalytic or regulatory properties of carbamylphosphate synthetase from mutant cells grown under permissive conditions (37 degrees C). However, the regulatory properties of carbamylphosphate synthetase synthesized under restrictive conditions (20 degrees C) were demonstrably defective (insensitive to activation by ornithine); the enzyme synthesized under permissive conditions was activated by ornithine. A strain carrying an additional mutation (argC), which prevents the accumulation of N-acetylornithine, produced an ornithine-activatable enzyme at both growth temperatures. These results suggest that N-acetylornithine antagonizes the proper preconditioning or maturation of the mutant carbamylphosphate synthetase.     

Phosphorylation of the Periplasmic Binding Protein in Two Transport Systems for Arginine Incorporation in Escherichia coli K-12 Is Unrelated to the Function of the Transport System

In Escherichia coli K-12, the accumulation of arginine is mediated by two distinct periplasmic binding protein-dependent transport systems, one common to arginine and ornithine (AO system) and one for lysine, arginine, and ornithine (LAO system). Each of these systems includes a specific periplasmic binding protein, the AO-binding protein for the AO system and the LAO-binding protein for the LAO system. The two systems include a common inner membrane transport protein which is able to hydrolyze ATP and also phosphorylate the two periplasmic binding proteins. Previously, a mutant resistant to the toxic effects of canavanine, with low levels of transport activities and reduced levels of phosphorylation of the two periplasmic binding proteins, was isolated and characterized (R. T. F. Celis, J. Biol. Chem. 265:1787–1793, 1990). The gene encoding the transport ATPase enzyme (argK) has been cloned and sequenced. The gene possesses an open reading frame with the capacity to encode 268 amino acids (mass of 29.370 Da). The amino acid sequence of the protein includes two short sequence motifs which constitute a well-defined nucleotide-binding fold (Walker sequences A and B) present in the ATP-binding subunits of many transporters. We report here the isolation of canavanine-sensitive derivatives of the previously characterized mutant. We describe the properties of these suppressor mutations in which the transport of arginine, ornithine, and lysine has been restored. In these mutants, the phosphorylation of the AO- and LAO-binding proteins remains at a low level. This information indicates that whereas hydrolysis of ATP by the transport ATPase is an obligatory requirement for the accumulation of these amino acids in E. coli K-12, the phosphorylation of the periplasmic binding protein is not related to the function of the transport system.     

arg-13可能参与鸟氨酸在粗糙脉孢霉线粒体的过膜转运(英文)

arg-13为精氨酸代谢途径里的一个渗露型突变。经研究发展了该突变的严格选择方法。该法省略了基本培养基的氮源而加上相似浓度的鸟氨酸与赖氨酸。此法在严紧山梨糖/葡萄糖条件下能强烈抑制arg-13突变株生长,但在斑点试验条件下允许arg-13突变株生长。由于鸟氨酸是通过线粒体合成和由细胞质至线粒体的过膜转运而积累,我们构建了arg-4,arg-13双突变株,其中arg-4阻断了线粒体鸟氨酸合成。在斑点试验条件下,arg-4,arg-13双突变株能利用鸟氨酸作为唯一氮源与精氨酸合成前体,但受赖氨酸与刀豆氨酸强烈抑制。具正常鸟氨酸转运功能的arg-4单突变株在鸟氨酸基本培养基的生长只受微弱的赖氨酸抑制。已有报道arg-13为嘧啶合成代谢途径里pyr-3(CPSACT~ )突变的部分抑制基因,序列分析表明arg-13编码一线粒体转运酶。本文数据提示arg-13在线粒体鸟氨酸过膜转运过程中起主要作用。arg-13突变株仍携带一定的线粒体鸟氨酸转运功能并受碱性氨基酸赖氨酸、刀豆氨酸抑制,可能为另一线粒体碱性氨基酸转运酶介导。     

Effects of ornithine or arginine administration on serum amino acid levels

In healthy humans after overnight fasting, an oral administration of ornithine induced a new steady state: an accumulation of serum alanine and proline, a decrease in serum valine concentration, transient reductions in serum urea and urinary urea contents, and then an increased urea excretion. On the other hand, an oral administration of arginine resulted in an anabolic state: decreases in serum leucine and isoleucine concentrations, reductions in serum glucose and free fatty acid contents and a rapid increase in serum insulin level. It was assumed that the effect of ornithine administration may be exerted through an activation of hepatic System A transport and that of arginine is an insulin-mediated action.     

Kinetic mechanism and specificity of the arginine-ornithine antiporter of Lactococcus lactis

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.     

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.     

Role of Pseudomonas putida tol-oprL gene products in uptake of solutes through the cytoplasmic membrane

Proteins of the Tol-Pal (Tol-OprL) system play a key role in the maintenance of outer membrane integrity and cell morphology in gram-negative bacteria. Here we describe an additional role for this system in the transport of various carbon sources across the cytoplasmic membrane. Growth of Pseudomonas putida tol-oprL mutant strains in minimal medium with glycerol, fructose, or arginine was impaired, and the growth rate with succinate, proline, or sucrose as the carbon source was lower than the growth rate of the parental strain. Assays with radiolabeled substrates revealed that the rates of uptake of these compounds by mutant cells were lower than the rates of uptake by the wild-type strain. The pattern and amount of outer membrane protein in the P. putida tol-oprL mutants were not changed, suggesting that the transport defect was not in the outer membrane. Consistently, the uptake of radiolabeled glucose and glycerol in spheroplasts was defective in the P. putida tol-oprL mutant strains, suggesting that there was a defect at the cytoplasmic membrane level. Generation of a proton motive force appeared to be unaffected in these mutants. To rule out the possibility that the uptake defect was due to a lack of specific transporter proteins, the PutP symporter was overproduced, but this overproduction did not enhance proline uptake in the tol-oprL mutants. These results suggest that the Tol-OprL system is necessary for appropriate functioning of certain uptake systems at the level of the cytoplasmic membrane.     

Basic and neutral amino acid transport in Aspergillus nidulans.

Arginine and methionine transport by Aspergillus nidulans mycelium was investigated. A single uptake system is responsible for the transport of arginine, lysine and ornithine. Transport is energy-dependent and specific for these basic amino acids. The Km value for arginine is 1 X 10(-5) M, and Vmax is 2-8 nmol/mg dry wt/min; Km for lysine is 8 X 10(-6) M; Kt for lysine as inhibitor of arginine uptake is 12 muM, and Ki for ornithine is mM. On minimal medium, methionine is transported with a Km of 0-I mM and Vmax about I nmol/mg dry wt/min; transport is inhibited by azide. Neutral amnio acids such as serine, phenylalanine and leucine are probably transported by the same system, as indicated by their inhibition of methionine uptake and the existence of a mutant specifically impaired in their transport. The recessive mutant nap3, unable to transport neutral amino acids, was isolated as resistant to selenomethionine and p-fluorophenylanine. This mutant has unchanged transport of methionine by general and specific sulphur-regulated permeases.     

Transport of diamines by Enterococcus faecalis is mediated by an agmatine-putrescine antiporter.

Enterococcus faecalis ATCC 11700 is able to use arginine and the diamine agmatine as a sole energy source. Via the highly homologous deiminase pathways, arginine and agmatine are converted into CO2, NH3, and the end products ornithine and putrescine, respectively. In the arginine deiminase pathway, uptake of arginine and excretion of ornithine are mediated by an arginine-ornithine antiport system. The translocation of agmatine was studied in whole cells grown in the presence of arginine, agmatine, or glucose. Rapid uncoupler-insensitive uptake of agmatine was observed only in agmatine-grown cells. A high intracellular putrescine pool was maintained by these cells, and this pool was rapidly released by external putrescine or agmatine but not by arginine or ornithine. Kinetic analysis revealed competitive inhibition for uptake between putrescine and agmatine. Agmatine uptake by membrane vesicles was observed only when the membrane vesicles were preloaded with putrescine. Uptake of agmatine was driven by the outwardly directed putrescine concentration gradient, which is continuously sustained by the metabolic process. Uptake of agmatine and extrusion of putrescine by agmatine-grown cells of E. faecalis appeared to be catalyzed by an agmatine-putrescine antiporter. This transport system functionally resembled the previously described arginine-ornithine antiport, which was exclusively induced when the cells were grown in the presence of arginine.     

Energy coupling for methionine transport in Escherichia coli.

The source of metabolic energy for the accumulation of methionine in cells of Escherichia coli was shown to differ from that for proline uptake. In contrast to proline uptake, methionine accumulation was sensitive to arsenate, and relatively resistant to azide or dinitrophenol. Adenosine triphosphatase mutant strains also differentiated between the two systems, consistent with the conclusion that, although proline uptake is driven directly by the energized membrane state, methionine uptake is not. Methionine transport is similar to that of other osmotic shock-sensitive systems in its direct utilization of adenosine 5'-triphosphate or a related compound as energy source.     

Genetic and physiological characterization of Pseudomonas aeruginosa mutants affected in the catabolic ornithine carbamoyltransferase.

In Pseudomonas aeruginosa arginine can be degraded by the arginine "dihydrolase" system, consisting of arginine deiminase, catabolic ornithine carbamoyltransferase, and carbamate kinase. Mutants of P. aeruginosa strain PAO affected in the structural gene (arcB) of the catabolic ornithine carbamoyltransferase were isolated. Firt, and argF mutation (i.e., a block in the anabolic ornithine carbamoyltransferase) was suppressed specifically by a mutationally altered catabolic ornithine carbamoyltransferase capable of functioning in the anabolic direction. The suppressor locus arcB (Su) was mapped by transduction between hisII and argA. Second, mutants having lost suppressor activity were obtained. The Su- mutations were very closely linked to arcB (Su) and caused strongly reduced ornithine carbamoyltransferase activities in vitro. Under aerobic conditions, a mutant (PA0630) which had less than 1% of the wild-type catabolic ornithine carbamoyltransferase activity grew on arginine as the only carbon and nitrogen source, at the wild-type growth rate. When oxygen was limiting, strain PA0630 grown on arginine excreted citrulline in the stationary growth phase. These observations suggest that during aerobic growth arginine is not degraded exclusively via the dihydrolase pathway.