Influence of Carbon or Nitrogen Starvation on Amino Acid Transport in Pseudomonas aeruginosa

Pseudomonas aeruginosa was shown to utilize the majority of commonly occurring amino acids for growth as either the sole carbon or the sole nitrogen source. During carbon or nitrogen deprivation, the rates of transport of most of the amino acids remained unchanged; however, the transport rates for glutamate, alanine, and glycine increased under these conditions and the transport rates for leucine and valine decreased. Normal transport rates for these amino acids were resumed immediately upon the addition of the required nutrient. In the absence of an external source of carbon or of nitrogen, pool amino acids underwent rapid degradation. (14)C-Amino acid pulse experiments indicated that the constitutive amino acid catabolic enzymes, normally present in the organism during growth with glucose as the carbon source, were responsible for rapid pool losses. Nutrient starvation in the presence of chloramphenicol did not prevent amino acid catabolism. This enzymic activity is interpreted as providing P. aeruginosa with a selective advantage for survival during conditions of carbon or nitrogen starvation.     

Amino Acid Pool Formation in Pseudomonas aeruginosa

The accumulation and behavior of various amino acids in the pool of Pseudomonas aeruginosa (ATCC 9027) were investigated. Patterns of pool formation and maintenance varied with different amino acids tested and were dependent, to a considerable extent, upon the ability of the organism to catabolize the particular amino acid. The establishment of steady-state amino acid pool levels depended upon the activity of the amino acid permease involved and upon the rate of protein synthesis. The presence of a relatively large specific amino acid pool did not affect the formation of a pool of a structurally different amino acid, and a preformed steady-state pool was not displaced by structurally unrelated amino acids. Steady-state amino acid pools decreased rapidly in the presence of inhibitors of energy metabolism and at 0 C. Steady-state internal amino acid pools were found to be in equilibrium with the corresponding external amino acid, present at low levels. A multiplicity of proline pools was demonstrated.     

Transport of Aromatic Amino Acids by Pseudomonas aeruginosa

Kinetic studies of the transport of aromatic amino acids by Pseudomonas aeruginosa revealed the existence of two high-affinity transport systems which recognized the three aromatic amino acids. From competition data and studies on the exchange of preformed aromatic amino acid pools, the first transport system was found to be functional with phenylalanine, tyrosine, and tryptophan (in order of decreasing activity), whereas the second system was active with tryptophan, phenylalanine, and tyrosine. The two systems also transported a number of aromatic amino acid analogues but not other amino acids. Mutants defective in each of the two and in both transport systems were isolated and described. When the amino acids were added at low external concentrations to cells growing logarithmically in glucose minimal medium, the tryptophan pool very quickly became saturated. Under identical conditions, phenylalanine and tyrosine each accumulated in the intracellular pool of P. aeruginosa at a concentration which was 10 times greater than that of tryptophan.     

Isolation of Amino Acid Transport-negative Mutants of Pseudomonas aeruginosa and Cells with Repressed Transport Activity

Methods are described for the isolation of amino acid transport-negative mutants of Pseudomonas aeruginosa and for the preparation of cells with repressed, specific amino acid permeases. P. aeruginosa was resistant to high concentrations of the majority of the 53 amino acid analogues examined and was unaffected by low concentrations of any of them. Cells which had been grown in the presence of sublethal concentrations of the few analogues which were inhibitory were subsequently more resistant to the analogues. These cells were also defective in the transport of the corresponding amino acid, as the analogue caused repression of the synthesis of the specific amino acid permease. The cells with repressed transport activity rapidly regained their normal level of constitutive permease when grown in the absence of the analogue. Higher levels of the permeases were induced when these cells were grown in the presence of the appropriate amino acid. The possible mechanisms for the mode of regulation of amino acid permeases are discussed.     

Transport of Glycerol by Pseudomonas aeruginosa

In Pseudomonas aeruginosa, the transport of glycerol was shown to be genetically controlled and to be dependent on induction by glycerol. Accumulation of (14)C-glycerol was almost completely absent in uninduced cells and in a transport-negative mutant. Kinetic studies with induced cells suggested that glycerol may be transported by two systems with different affinities for glycerol. Osmotically shocked cells did not transport glycerol, and the supernatant fluid from shocked cells contained glycerol-binding activity demonstrable by equilibrium dialysis. The binding protein was not glycerol kinase. Binding activity was absent in shock fluids from the transport-negative mutant and from uninduced cells. The glycerol-binding protein was partially purified by precipitation with ammonium sulfate. Mild heat treatment completely eliminated the binding activity of shock fluid and of the partially purified protein. Sodium azide and N-ethylmaleimide inhibited both transport by whole cells and binding of glycerol by shock fluid. It is concluded that transport of glycerol by P. aeruginosa involves a binding protein responsible for recognition of glycerol and may occur by facilitated diffusion or active transport. A requirement for energy has not been demonstrated.     

Amino acid- -naphthylamide hydrolysis by Pseudomonas aeruginosa arylamidase

The intracellular and constitutive arylamidase from Pseudomonas aeruginosa was purified 528-fold by salt fractionation, ion-exchange chromatography, gel filtration, and adsorption chromatography. This enzyme hydrolyzed basic and neutral N-terminal amino acid residues from amino-beta-naphthylamides, dipeptide-beta-naphthylamides, and a variety of polypeptides. Only those substrates having an l-amino acid with an unsubstituted alpha-amino group as the N-terminal residue were susceptible to enzymatic hydrolysis. The molecular weight was estimated to be 71,000 daltons. The lowest K(m) values were associated with substrates having neutral or basic amino acid residues with large side chains with no substitution or branching on the beta carbon atom.     

鲍曼不动杆菌和铜绿假单胞菌在低碱性氨基酸培养基中对抗菌药物的敏感性

为了探讨鲍曼不动杆菌和铜绿假单胞菌在低碱性氨基酸培养基中对抗菌药物的敏感性,本研究利用常规培养基和低碱性氨基酸培养基,采用琼脂稀释法检测140株鲍曼不动杆菌和60株铜绿假单胞菌对亚胺培南、帕尼培南和罗美培南的最低抑菌浓度,利用K-B纸片扩散法进行药敏实验并计算敏感率。结果显示,在低碱性培养基中,铜绿假单胞菌和鲍曼不动杆菌对三种药物的最低抑菌浓度(MIC)值显著降低,铜绿假单胞菌在低碱性氨基酸培养基中对帕尼培南的敏感率显著上升,而鲍曼不动杆菌对三种药物的敏感性均显著上升。研究表明,在低碱性氨基酸培养基中,鲍曼不动杆菌和铜绿假单胞菌对帕尼培南、亚胺培南和美罗培南的敏感性有所增强,在临床检验中需考虑由培养基导致的敏感性差异。     

Amino Acid Racemization in Pseudomonas putida KT2440

d-Amino acids have been shown to play an increasingly diverse role in bacterial physiology, yet much remains to be learned about their synthesis and catabolism. Here we used the model soil- and rhizosphere-dwelling organism Pseudomonas putida KT2440 to elaborate on the genomics and enzymology of d-amino acid metabolism. P. putida KT2440 catabolized the d-stereoisomers of lysine, phenylalanine, arginine, alanine, and hydroxyproline as the sole carbon and nitrogen sources. With the exception of phenylalanine, each of these amino acids was racemized by P. putida KT2440 enzymes. Three amino acid racemases were identified from a genomic screen, and the enzymes were further characterized in vitro. The putative biosynthetic alanine racemase Alr showed broad substrate specificity, exhibiting measurable racemase activity with 9 of the 19 chiral amino acids. Among these amino acids, activity was the highest with lysine, and the k_cat/K_m values with l- and d-lysine were 3 orders of magnitude greater than the k_cat/K_m values with l- and d-alanine. Conversely, the putative catabolic alanine racemase DadX showed narrow substrate specificity, clearly preferring only the alanine stereoisomers as the substrates. However, DadX did show 6- and 9-fold higher k_cat/K_m values than Alr with l- and d-alanine, respectively. The annotated proline racemase ProR of P. putida KT2440 showed negligible activity with either stereoisomer of the 19 chiral amino acids but exhibited strong epimerization activity with hydroxyproline as the substrate. Comparative genomic analysis revealed differences among pseudomonads with respect to alanine racemase genes that may point to different roles for these genes among closely related species.     

Amino Acid and Sugar Transport in Rabbit Ileum

L-Alanine and 3-O-methyl-D-glucose accumulation by mucosal strips from rabbit ileum has been investigated with particular emphasis on the interaction between Na and these transport processes. L-Alanine is rapidly accumulated by mucosal tissue and intracellular concentrations of approximately 50 mM are reached within 30 min when extracellular L-alanine concentration is 5 mM. Evidence is presented that intracellular alanine exists in an unbound, osmotically active form and that accumulation is an active transport process. In the absence of extracellular Na, the final ratio of intracellular to extracellular L-alanine does not differ significantly from unity and the rate of net uptake is markedly inhibited. Amino acid accumulation is also inhibited by 5 x 10^-5 M ouabain. 3-O-methyl-D-glucose accumulation by this preparation is similarly affected by ouabain and by incubation in a Na-free medium. The effects of amino acid accumulation, of ouabain, and of incubation in a Na-free medium on cell water content and intracellular Na and K concentrations have also been investigated. These results are discussed with reference to the two hypotheses which have been suggested to explain the interaction between Na and intestinal nonelectrolyte transport.     

Transport systems for branched-chain amino acids in Pseudomonas aeruginosa.

The cells of Pseudomonas aeruginosa showed high activity for leucine transport in the absence of Na+, giving a Km value of 0.34 microM. In the presence of Na+, however, two Km values, 0.37 microM (LIV-I system) and 7.6 microM (LIV-II system), were obtained. The former system seemed to serve not only for the entry of leucine, isoleucine, and valine, but also for that of alanine and threonine, although less effectively. However, the LIV-II system served for the entry of branched-chain amino acids only. The LIV-II system alone was operative in membrane vesicles, for the transport of branched-chain amino acids in membrane vesicles required Na+ and gave single Km values for the respective amino acids. When cells were osmotically shocked, the activity of the LIV-I system decreased, whereas the LIV-II system remained unaffected. The shock fluid from P. aeruginosa cells showed leucine-binding activity with a dissociation constant of 0.25 microM. The specificity of the activity was very similar to that of the LIV-I system. These results suggest that a leucine-binding protein(s) in the periplasmic space may be required for the transport process via the LIV-I system of P. aeruginosa.     

Small Intestinal Sugar and Amino Acid Transport in Semistarvation

The effect of semistarvation on small intestinal transport of D-glucose, L-valine, and NaCl was studied in an in vitro system of isolated rat brush border membrane vesicles. Whereas semistarvation enhanced the transport rate for L-valine by 19-29%, there was no change in D-glucose transport. When energy in the form of a NaSCN gradient was supplied to the membrane vesicles prepared from semistarved animals, L-valine was concentrated to a greater extent than those from well-fed animals. Strain differences were observed in the manner semistarvation affected NaCl transport across the brush border membrane. Semistarvation increased the NaCl transport rate by a factor of 3.5 in one rat strain and not at all in another. These results provide a partial explanation for the cellular basis of elevated neutral amino acid absorption by the small intestine in semistarvation.     

Specificity of Amino Acid Transport in Trypanosoma equiperdum*

Uptake of [¹⁴C] alanine, arginine, glutamic acid and phenylalanine by Trypanosoma equiperdum occurred by both a mediated mechanism and diffusion. Twenty amino acids were studied as inhibitors of absorption of the above amino acids. Results suggested that at least 4 distinct transport loci are involved in amino acid transport. These 4 loci have overlapping affinities for amino acids and seem to be involved, respectively, in the absorption of (a) arginine and phenylalanine; (b) arginine; (c) alanine, phenylalanine, and glutamic acid; (d) glutamic acid. The data also showed that multiple sites for substrate binding occur on each of 2 transport systems.     

The Action of Ethylenediaminetetra-acetic Acid on Pseudomonas aeruginosa

It has been shown that ethylenediaminetetra-acetic acid (EDTA) has a direct bactericidal action against strains of Pseudomonas aeruginosa and Alcaligenes faecalis . The action against Ps. aeruginosa is considered to take the form of competition for a metal essential to the integrity of the cells: some of the factors influencing this antibacterial action have been discussed. From a study of the release of solutes from cells of Ps. aeruginosa , it was concluded that the action of EDTA takes place at the cell wall of the organism: an effect by EDTA on the isolated walls of sensitive organisms has been demonstrated.     

Changes in Amino Acid Transport During Red Cell Maturation

We studied amino acid transport in sheep red blood cells (RBCs) as a function of cell maturation. Transport of amino acids is decreased strikingly in the mature mammalian RBC compared to the immature reticulocyte. Blood obtained 5-6 days after massive bleeding was fractionated on dextran gradients. In the mature erythrocyte amino acids are taken up only slowly, and in the normal experimental interval (60 min) the concentration in the cell does not reach that of the medium. In contrast, the reticulocyte-rich (top) fraction (50-90% reticulocytes) accumulates certain amino acids, particularly histidine, methionine, and leucine. The underlying process is ATP-independent and Na⁺-insensitive, and has properties consistent with exchange diffusion, i.e., accelerated uptake or efflux when unlabeled solute is present on the trans side. The process is apparent not only in intact cells but also in resealed ghosts. The decrease in activity of amino acid transport is a function of red cell maturation. Thus it can be shown that (a) separation of cells according to their density 1, 2, and 3 weeks after bleeding leads to progressively lower amino acid transport activity with increasing cell density; and (b) during in vitro long-term incubation at 37°C of reticulocyte-rich, unfractionated blood (5–10% reticulocytes), amino acid transport decreases while red cell integrity is maintained, as evidenced by the retention of a normal K⁺ gradient and the absence of hemolysis. The progressive loss is seen with resealed ghosts as well as with intact cells. Not all the amino acids examined participate in this exchange process. The most actively exchanged are histidine, leucine, methionine, and phenylalanine. Glycine, proline, arginine, and a-amino isobutyric acid do not participate in the exchange process.     

Branched Chain Amino Acid Aminotransferase of Pseudomonas sp

Branched chain amino acid aminotransferase was partially purified from Pseudomonas sp. by ammonium sulfate fractionation, aminohexyl-agarose and Bio-Gel A-0.5 m column chromatography.

This enzyme showed different substrate specificity from those of other origins, namely lower reactivity for l-isoleucine and higher reactivity for l-methionine.

Km values at pH 8.0 were calculated to be 0.3 mm for l-leucine, 0.3 mm for α-ketoglutarate, 1.1 mm for α-ketoisocaproate and 3.2 mm for l-glutamate.

This enzyme was activated with β-mercaptoethanol, and this activated enzyme had different kinetic properties from unactivated enzyme, namely, Km values at pH 8.0 were calculated to be 1.2 mm for l-leucine, 0.3 mm for α-ketoglutarate.

Isocaproic acid which is the substrate analog of l-leucine was competitive inhibitor for pyridoxal form of unactivated and activated enzymes, and inhibitor constants were estimated to be 6 mm and 14 mm, respectively.     

Inhibition of Amino Acid Transport in Rabbit Intestine by p-Chloromercuriphenyl Sulfonic Acid

Influx of phenylalanine across the brush border of rabbit intestine is markedly reduced by treatment with 5 mM p-chloromercuriphenyl sulfonate (PCMBS). The effect is rapidly and completely reversed by dithiothreitol. Phenylalanine influx into PCMBS-treated tissue can be competitively inhibited by other neutral amino acids and follows saturation kinetics. PCMBS causes an increase in the apparent Michaelis constant from the value observed in control tissue but does not alter the maximal influx significantly. Treatment of the tissue with PCMBS leads to a significant reduction in the Na-sensitivity of the transport, and a number of results indicate that the major effect of the reagent is to cause a marked reduction in the affinity of the transport system for Na. The transport system can be partially protected against reaction with PCMBS by phenylalanine and tryptophan but not by methionine or norleucine. The results suggest that PCMBS reacts with a sulfhydryl group in the region of the transport site and may alter conformational changes associated with the binding of substrates.     

Translation of Pseudomonas aeruginosa Bacteriophage PP7 RNA by a Cell-Free Amino Acid Incorporating System from Escherichia coli

We have compared the activities of the RNA genomes of Pseudomonas aeruginosa phage PP7 and coliphages Qbeta and f2 in a cell-free amino acid incorporating system derived from Escherichia coli. The rate of incorporation of [(14)C]leucine in the PP7 RNA-directed system is greater than in the systems directed by either Qbeta or f2 RNA. The response to changes in phage RNA concentrations is similar in all the systems, reaching a saturation level at 0.75 to 1.0 mg of RNA per ml of reaction mixture. Analysis of complete reaction mixtures of the PP7 RNA and of the Qbeta RNA systems by sucrose gradient centrifugation shows generally similar patterns for both RNAs. The principal differences are that in the PP7 system a slightly higher percentage of RNA forms ribosome complexes and that the polysomes are somewhat smaller. PP7 RNA is also degraded more extensively during the reaction than is Qbeta RNA. Analysis of the products of the reactions by acrylamide gel electrophoresis shows that PP7 coat protein is the only identifiable product of the PP7 RNA-directed system, suggesting that only the coat protein cistron is translated by E. coli ribosomes.     

Amino Acid Neurotransmitters in the CNS: Properties of Diaminobutyric Acid Transport

Uptake of L-2,4-diaminobutyric acid (DABA), a positively charged analogue of gamma-aminobutyric acid (GABA), by a synaptosomal fraction isolated from rat brain occurred with a Km of 54 +/- 12 microM and a Vmax of 1.3 +/- 0.2 nmol/min/mg protein. The transport of DABA was inhibited competitively by GABA whereas that of GABA was affected in the same manner by addition of DABA. The maximal accumulation of DABA ([DABA]i/[DABA]c) was observed to increase as the second power of the transmembrane electrical potential ([K+]i/[K+]e) and the first power of the sodium ion concentration gradient. These findings indicate that DABA is transported on the GABA carrier with a net charge of +2, where one charge is provided by the cotransported Na+ and the second is contributed by the amino acid itself. Since uptake of GABA, an electroneutral molecule, is accompanied by transfer of two sodium ions, the results obtained with DABA suggest that one of the sodium binding sites on the GABA transporter is in proximity to the amino acid binding site.     

Crystal Structure of Dihydro-Heme d1 Dehydrogenase NirN from Pseudomonas aeruginosa Reveals Amino Acid Residues Essential for Catalysis

Many bacteria can switch from oxygen to nitrogen oxides, such as nitrate or nitrite, as terminal electron acceptors in their respiratory chain. This process is called “denitrification” and enables biofilm formation of the opportunistic human pathogen Pseudomonas aeruginosa, making it more resilient to antibiotics and highly adaptable to different habitats. The reduction of nitrite to nitric oxide is a crucial step during denitrification. It is catalyzed by the homodimeric cytochrome cd₁ nitrite reductase (NirS), which utilizes the unique isobacteriochlorin heme d₁ as its reaction center. Although the reaction mechanism of nitrite reduction is well understood, far less is known about the biosynthesis of heme d₁. The last step of its biosynthesis introduces a double bond in a propionate group of the tetrapyrrole to form an acrylate group. This conversion is catalyzed by the dehydrogenase NirN via a unique reaction mechanism. To get a more detailed insight into this reaction, the crystal structures of NirN with and without bound substrate have been determined. Similar to the homodimeric NirS, the monomeric NirN consists of an eight-bladed heme d₁-binding β-propeller and a cytochrome c domain, but their relative orientation differs with respect to NirS. His147 coordinates heme d₁ at the proximal side, whereas His323, which belongs to a flexible loop, binds at the distal position. Tyr461 and His417 are located next to the hydrogen atoms removed during dehydrogenation, suggesting an important role in catalysis. Activity assays with NirN variants revealed the essentiality of His147, His323 and Tyr461, but not of His417.