N2-Succinylornithine in ornithine catabolism of Pseudomonas aeruginosa

Most Pseudomonas aeruginosa PAO mutants which were unable to utilize l-arginine as the sole carbon and nitrogen source (aru mutants) under aerobic conditions were also affected in l-ornithine utilization. These aru mutants were impaired in one or several enzymes involved in the conversion of N²-succinylornithine to glutamate and succinate, indicating that the latter steps of the arginine succinyltransferase pathway can be used for ornithine catabolism. Addition of aminooxyacetate, an inhibitor of the N²-succinylornithine 5-aminotransferase, to resting cells of P. aeruginosa in ornithine medium led to the accumulation of N²-succinylornithine. In crude extracts of P. aeruginosa an ornithine succinyltransferase (l-ornithine:succinyl-CoA N²-succinyltransferase) activity could be detected. An aru mutant having reduced arginine succinyltransferase activity also had correspondingly low levels of ornithine succinyltransferase. Thus, in P. aeruginosa, these two activities might be due to the same enzyme, which initiates aerobic arginine and ornithine catabolism.Abbreviations OAT ornithine 5-aminotransferase - SOAT N²-succinylornithine 5-aminotransferase - Oru ornithine utilization - Aru arginine utilization     

Identification of a suppressor gene for the arginine-auxotrophic <Emphasis Type="Italic">argJ</Emphasis> mutation in <Emphasis Type="Italic">Corynebacterium glutamicum</Emphasis>

We recently proposed a metabolic engineering strategy for l-ornithine production based on the hypothesis that an increased intracellular supply of N-acetylglutamate may further enhance l-ornithine production in a well-defined recombinant strain of Corynebacterium glutamicum. In this work, an argJ-deficient arginine auxotrophic mutant of C. glutamicum is suppressed by a different locus of C. glutamicum ATCC13032. Overexpression of the NCgl1469 open reading frame (ORF), exhibiting N-acetylglutamate synthase (NAGS) activity, was able to complement the C. glutamicum arginine-auxotrophic argJ strain and showed increased NAGS activity from 0.03 to 0.17 units mg⁻¹ protein. Additionally, overexpression of the NCgl1469 ORF resulted in a 39% increase in excreted l-ornithine. These results indicate that the intracellular supply of N-acetylglutamate is a rate-limiting step during l-ornithine production in C. glutamicum.     

Inhibitors of N α-acetyl-l-ornithine deacetylase: synthesis, characterization and analysis of their inhibitory potency

A series of N ^α-acyl (alkyl)- and N ^α-alkoxycarbonyl-derivatives of l- and d-ornithine were prepared, characterized, and analyzed for their potency toward the bacterial enzyme N ^α-acetyl-l-ornithine deacetylase (ArgE). ArgE catalyzes the conversion of N ^α-acetyl-l-ornithine to l-ornithine in the fifth step of the biosynthetic pathway for arginine, a necessary step for bacterial growth. Most of the compounds tested provided IC₅₀ values in the μM range toward ArgE, indicating that they are moderately strong inhibitors. N ^α-chloroacetyl-l-ornithine (1g) was the best inhibitor tested toward ArgE providing an IC₅₀ value of 85 μM while N ^α-trifluoroacetyl-l-ornithine (1f), N ^α-ethoxycarbonyl-l-ornithine (2b), and N ^α-acetyl-d-ornithine (1a) weakly inhibited ArgE activity providing IC₅₀ values between 200 and 410 μM. Weak inhibitory potency toward Bacillus subtilis-168 for N ^α-acetyl-d-ornithine (1a) and N ^α-fluoro- (1f), N ^α-chloro- (1g), N ^α-dichloro- (1h), and N ^α-trichloroacetyl-ornithine (1i) was also observed. These data correlate well with the IC₅₀ values determined for ArgE, suggesting that these compounds might be capable of getting across the cell membrane and that ArgE is likely the bacterial enzymatic target.     

N5-(l-1-Carboxyethyl)-l-ornithine: NADP+ oxidoreductase inStreptococcus lactis: Distribution,constitutivity, and regulation

N⁵-(l-1-Carboxyethyl)-l-ornithine: NADP⁺ oxidoreductase [N⁵-(CE)ornithine synthase] catalyzes the NADPH-dependent reductive condensation between pyruvic acid and the terminal amino group ofl-ornithine andl-lysine to yield N⁵-(l-1-carboxyethyl)-l-ornithine and N⁶-(l-1-carboxyethyl)-l-lysine respectively. Polyclonal antibodies against N⁵-(CE)ornithine synthase purified fromStreptococcus lactis K1 have been used for the immunochemical (Western blot) detection and sizing of this enzyme in various lactic acid bacteria. The enzyme was confined to about one-half of the strains ofS. lactis examined. N⁵-(CE)ornithine synthase is constitutive, and in strains K1, 6F3, and (plasmid-free)H₁-4125 the native enzyme is a tetramer composed of identical subunits of M_r=38,000. However, in other strains, including 133 (ATCC 11454), C₁₀, and ML₈, the molecular weight of the native enzyme is approximately 130,000 and the corresponding subunit M_r=35,000. Analyses of the amino acid pool components maintained byS. lactis K1 during growth in medium containing [¹⁴C] labeled and unlabeled arginine have revealed that (i) exogenous arginine is the precursor of intracellular ornithine, citrulline, and N⁵-(CE)ornithine, and (ii) the rates of turnover of ornithine and citrulline were considerably faster than that of N⁵-(CE)ornithine. These data account for the biosynthesis and accumulation of N⁵-(CE)ornithine byS. lactis.     

Effect of polyamines and L-ornithine on the development of proembryogenic masses of Cryptomeria japonica

We examined the effects of polyamines, namely, putrescine, spermidine and spermine, and of amino acids, such as l-arginine and l-ornithine, as part of our efforts to identify factors that stimulate the development of proembryogenic masses (PEMs) of Cryptomeria japonica. We maintained two distinct types of PEM designated PEMs A, which consisted of normal embryogenic cells as single embryos with elongated suspensor cells, and PEMs B, which consisted of abnormal embryogenic cells with coalesced embryos on modified Campbell and Durzan medium (mCD) supplemented with individual polyamines at 0–100 μM or amino acids at 0–16.4 mM. All additives had a stimulatory/suppressive effect. Microscopy and image-processing techniques revealed that the regions of authentic embryos of PEMs that were treated with l-ornithine were remarkably enlarged and that the suspensor cells had elongated in the same direction. When all PEMs A were transferred to maturation medium (mCD that contained abscisic acid and maltose at various concentrations), only PEMs that had been treated with l-ornithine matured into somatic embryos and were able to germinate on hormone-free mCD. Our results indicate that l-ornithine is an important stimulator of the development of PEMs to the pre-filamentous stage in C. japonica.     

Direct production of l-ornithine from casein by commercial digestive enzymes and in situ activated arginase

There is a great demand for l-ornithine, which is used as a dietary supplement, and in the pharmaceutical industry. In the present study, when milk casein was hydrolyzed at 37 °C by using commercial digestive enzymes, namely, Pancreatin F and Protease A, a significant accumulation of l-ornithine in the hydrolysate and the simultaneous disappearance of l-arginine was noted. In a radiometric assay, transient but distinct arginase activity, which was sufficiently high for l-ornithine production, was detected in the hydrolysate for a certain period during casein hydrolysis. On the basis of the results of the enzymatic analyses, arginase was thought to be proteolytically generated from an inactive precursor, which may generally be contained in Pancreatin F, and ultimately degraded by further proteolysis. This conversion process using the above-mentioned digestive enzymes is useful for the production of l-ornithine directly from protein sources that are abundant in nature.     

Catabolism of arginine byCarnobacterium spp. isolated from vacuum-packed sugar-salted fish

The ability ofCarnobacterium spp. originally isolated from vacuum-packed, sugar-salted fish to catabolize arginine was examined. All strains were able to produce citrulline, ornithine, and NH₃ from arginine, presumably by the arginine deiminase pathway. The metabolism of arginine was concurrent with acid production from glucose for one strain ofCarnobacterium sp. but delayed for one strain ofCarnobacterium piscicola. The arginine catabolism was not inhibited in the presence of 2% glucose for three strains of carnobacteria during growth in test broth and/or shrimp extract. Growth as well as arginine catabolism was delayed for two strains of carnobacteria by lowering the temperature from 9°C to 4°C. A similar result was obtained by incubating one strain ofC. piscicola in CO₂. None of the compoundsl-citrulline,l-ornithine hydrochloride, and (NH₄)₂SO₄ had any effect on growth or arginine catabolism of this strain. Neither did pH of the medium affect the time for initiation of arginine catabolism.     

Biotechnological production of polyamines by bacteria: recent achievements and future perspectives

In Bacteria, the pathways of polyamine biosynthesis start with the amino acids l-lysine, l-ornithine, l-arginine, or l-aspartic acid. Some of these polyamines are of special interest due to their use in the production of engineering plastics (e.g., polyamides) or as curing agents in polymer applications. At present, the polyamines for industrial use are mainly synthesized on chemical routes. However, since a commercial market for polyamines as well as an industry for the fermentative production of amino acid exist, and since bacterial strains overproducing the polyamine precursors l-lysine, l-ornithine, and l-arginine are known, it was envisioned to engineer these amino acid-producing strains for polyamine production. Only recently, researchers have investigated the potential of amino acid-producing strains of Corynebacterium glutamicum and Escherichia coli for polyamine production. This mini-review illustrates the current knowledge of polyamine metabolism in Bacteria, including anabolism, catabolism, uptake, and excretion. The recent advances in engineering the industrial model bacteria C. glutamicum and E. coli for efficient production of the most promising polyamines, putrescine (1,4-diaminobutane), and cadaverine (1,5-diaminopentane), are discussed in more detail.     

The characteristics of arginine transport by rat cerebellar and cortical synaptosomes

The uptake ofl-[³H]arginine into synaptosomes prepared from rat cerebellum and cortex occurred by a high-affinity carrier-mediated process. The uptake of arginine appeared to be potentiated by removal of extracellular Na⁺, inhibited by high levels of extracellular K⁺, but not by depolarization with veratridine or 4-amino pyridine. The effect of Na⁺ removal or K⁺ elevation did not seem to be due to changes in intracellular Ca²⁺ or pH. In both brain regions, uptake was significantly inhibited byl-arginine,l-lysine,l-ornithine, andl-homoarginine, but not byd-arginine norl-citrulline. Uptake was also inhibited by N^G-monomethyl-l-arginine acetate, but not by N^G-nitro-l-arginine methyl ester nor N^G-nitro-l-arginine except in the cortex at a concentration of 1 mM. The results indicate that the carrier system operating in synaptosomes showed many of the characteristics of the ubiquitous y⁺ system seen in many other tissues, although its apparent sensitivity to variations in extracellular Na⁺ was unusual.     

<Emphasis Type="SmallCaps">l</Emphasis>-Proline nutrition and catabolism in <Emphasis Type="Italic">Staphylococcus saprophyticus</Emphasis>

Staphylococcus saprophyticus strains ATCC 15305, ATCC 35552, and ATCC 49907 were found to require l-proline but not l-arginine for growth in a defined culture medium. All three strains could utilize l-ornithine as a proline source and contained l-ornithine aminotransferase and Δ¹-pyrroline-5-carboxylate reductase activities; strains ATCC 35552 and ATCC 49907 could use l-arginine as a proline source and had l-arginase activity. The proline requirement also could be met by l-prolinamide, l-proline methyl ester, and the dipeptides l-alanyl-l-proline and l-leucyl-l-proline. The bacteria exhibited l-proline degradative activity as measured by the formation of Δ¹-pyrroline-5-carboxylate. The specific activity of proline degradation was not affected by addition of l-proline or NaCl but was highest in strain ATCC 49907 after growth in Mueller–Hinton broth. A membrane fraction from this strain had l-proline dehydrogenase activity as detected both by reaction of Δ¹-pyrroline-5-carboxylate with 2-aminobenzaldehyde (0.79 nmol min⁻¹ mg⁻¹) and by the proline-dependent reduction of p-iodonitrotetrazolium (20.1 nmol min⁻¹ mg⁻¹). A soluble fraction from this strain had Δ¹-pyrroline-5-carboxylate dehydrogenase activity (88.8 nmol min⁻¹ mg⁻¹) as determined by the NAD⁺-dependent oxidation of dl-Δ¹-pyrroline-5-carboxylate. Addition of l-proline to several culture media did not increase the growth rate or final yield of bacteria but did stimulate growth during osmotic stress. When grown with l-ornithine as the proline source, S. saprophyticus was most susceptible to the proline analogues L-azetidine-2-carboylate, 3,4-dehydro-dl-proline, dl-thiazolidine-2-carboxylate, and l-thiazolidine-4-carboxylate. These results indicate that proline uptake and metabolism may be a potential target of antimicrobial therapy for this organism.     

Polyamine metabolism in <Emphasis Type="Italic">Leishmania</Emphasis>: from arginine to trypanothione

Polyamines (PAs) are essential metabolites in eukaryotes, participating in a variety of proliferative processes, and in trypanosomatid protozoa play an additional role in the synthesis of the critical thiol trypanothione. The PAs are synthesized by a metabolic process which involves arginase (ARG), which catalyzes the enzymatic hydrolysis of l-arginine (l-Arg) to l-ornithine and urea, and ornithine decarboxylase (ODC), which catalyzes the enzymatic decarboxylation of l-ornithine in putrescine. The S-adenosylmethionine decarboxylase (AdoMetDC) catalyzes the irreversible decarboxylation of S-adenosylmethionine (AdoMet), generating the decarboxylated S-adenosylmethionine (dAdoMet), which is a substrate, together with putrescine, for spermidine synthase (SpdS). Leishmania parasites and all the other members of the trypanosomatid family depend on spermidine for growth and survival. They can synthesize PAs and polyamine precursors, and also scavenge them from the microenvironment, using specific transporters. In addition, Trypanosomatids have a unique thiol-based metabolism, in which trypanothione (N1-N8-bis(glutathionyl)spermidine, T(SH)₂) and trypanothione reductase (TR) replace many of the antioxidant and metabolic functions of the glutathione/glutathione reductase (GR) and thioredoxin/thioredoxin reductase (TrxR) systems present in the host. Trypanothione synthetase (TryS) and TR are necessary for the protozoa survival. Consequently, enzymes involved in spermidine synthesis and its utilization, i.e. ARG, ODC, AdoMetDC, SpdS and, in particular, TryS and TR, are promising targets for drug development.     

Altered control of glutamate dehydrogenases in ornithine utilization mutants of Pseudomonas aeruginosa

Two classes of ornithine-nonutilizing (oru) mutants of Pseudomonas aeruginosa PAO were investigated. Strains carrying the oru-310 mutation were entirely unable to grow on l-ornithine as the only carbon and nitrogen source and were affected in the assimilation of a variety of nitrogen sources (e.g., amino acids, nitrate). The oru-310 mutation caused changes in the regulation of the catabolic NAD-dependent glutamate dehydrogenase; this enzyme was no longer inducible by glutamate but instead could be induced by ammonia. The oru-310 locus was cotransducible with car-9 and tolA in the 10 min region of the chromosome. An oru-314 mutant was severely handicapped in ornithine medium but could grow when a good carbon source was added; the mutant also showed pleiotropic growth effects related to nitrogen metabolism. The oru-314 mutation affected the regulation of the anabolic NADP-dependent glutamate dehydrogenase, which was no longer repressed by glutamate but showed normal derepression in the presence of ammonia. The oru-314 locus was mapped by transduction near met-9011 at 55 min. Both oru mutants could grow on l-glutamate, l-proline, or l-ornithine amended with 2-oxoglutarate, albeit slowly. We speculate that insufficient 2-oxoglutarate concentrations might account, at least in part, for the Oru^- phenotype of the mutants.     

A novel crude oil emulsifier excreted in the culture supernatant of a marine bacterium, Myroides sp. strain SM1

A marine bacterium, Myroides sp. SM1, can grow on weathered crude oil and show emulsification of it. The biosurfactant able to emulsify crude oil was excreted in culture supernatant of Myroides sp. SM1 grown on marine broth, which was extracted with chloroform/methanol (1:1) at pH 7 and purified by normal and reverse phase silica gel column chromatographies. The compound was ninhydrin-positive, and the chemical structure was elucidated by nuclear magnetic resonance (NMR), infrared spectroscopy (IR), fast atom bombardment mass spectrometry, and gas chromatography–mass spectrometry (GC-MS) to be a mixture of l-ornithine lipids, which were composed of l-ornithine and a different couple of iso-3-hydroxyfatty acid (C₁₅–C₁₇) and iso-fatty acid (C₁₅ or C₁₆) in a ratio of 1:1:1. The critical micelle concentration for a mixture of ornithine lipids was measured to be approximately 40 mg/l. A mixture of ornithine lipids exhibited emulsifying activity for crude oil in a broad range of pH, temperature, and salinity and showed higher surface activity for oil displacement test than other several artificial surfactants and a biosurfactant, surfactin.     

Ornithine dissimilation byTreponema denticola

Treponema denticola convertedl-ornithine, a product ofl-arginine catabolism, to putrescine via a decarboxylation reaction and to proline via a deamination reaction. Ornithine decarboxylation byT. denticola extracts was stimulated by pyridoxal 5′-phosphate. In the absence of pyridoxal 5′-phosphate, (NH₄)₂SO₄-fractionated extracts converted ornithine to proline and ammonia. This activity was not stimulated by α-keto acids, nicotinamide adenine dinucleotide, reduced nicotinamide adenine dinucleotide or ADP. Neither ornithine δ-transaminase (l-ornithine: 2-oxoacid aminotransferase, EC 2.6.1.13) nor Δ¹ reductase [l-proline: NAD(P) 5-oxidoreductase, EC 1.5.1.2.] activity was detectable in cell extracts. These results indicate that formation of proline from ornithine inT. denticola is catalyzed by an enzyme system analogous to the ornithine cyclase (deaminating) ofClostridium sporogenes. Exogenous ornithine inhibited the growth ofT. denticola. Thus, in addition to generating putrescine and proline, the ornithine dissimilatory pathways may serve to prevent accumulation of inhibitory concentrations of ornithine in the spirochete's environment.     

Characterization of cationic amino acid transporters (hCATs) 1 and 2 in human skin

In the present study, we characterized the distribution of human cationic amino acid transporters 1 (hCAT1) and 2 (hCAT2) in healthy skin and compared it to psoriatic skin lesions by means of immunohistochemistry. Moreover, we tested the hypothesis that l-arginine and l-ornithine influence the expression and synthesis of hCAT1 and hCAT2 in cell culture experiments by means of real-time-PCR and Western blot. Immunohistochemical comparison between healthy and psoriatic skin revealed a decreased amount of hCAT1, especially in the stratum granulosum of psoriatic skin; the distribution pattern of hCAT2 was not significantly affected in psoriatic skin. Cell culture experiments showed that supraphysiological concentrations of 15 mM l-arginine (72 h) lead to a significant increase of the hCAT1-mRNA and protein expression, whereas other concentrations had no significant influence. In contrast, l-arginine concentrations of 2 mM led to a significant increase of the hCAT2B mRNA-expression after 24 h. However, 48 and 72 h revealed no significant changes and high concentrations (15 mM l-arginine) led to a significant downregulation of the hCAT2B transporter over all time points analyzed. l-ornithine had no effect on the hCAT1 expression of mRNA and protein level. On the other hand the expression of hCAT2B was significantly up regulated at a 5-mM concentration of l-ornithine at all analyzed time points. Other concentrations had no effect. For the first time, the findings yield data about hCAT1 and hCAT2 on protein-level and suggest that l-arginine is a worthwhile object of studies, which investigated l-arginine as a possible therapeutic agent to reduce psoriatic symptoms.     

Effect of poly-l-ornithine on isolated tobacco mesophyll protoplasts: Evidence against stimulated pinocytosis

Summary The effects of poly-l-ornithine on the surface membrane of isolated tobacco protoplasts have been examined in the electron microscope using a colloidal metal oxide and a spherical virus as marker substances. No evidence was found to suggest that isolated protoplasts take up either of these markers by a pinocytotic process. Poly-l-ornithine increased the degree of damage observed in fixed preparations, and specifically caused lesions of the plasmalemma which were favoured sites for the binding of both external marker substances. It is suggested that the function of poly-l-ornithine and other treatments used to obtain virus infection of protoplasts is to stress the cell membrane to allow a non-physiological entry of high molecular weight materials. Pinocytosis appears not to occur nor to be necessary for uptake of these materials under conditions of membrane stress.     

Deregulation of arginine biosynthesis in Synechococcus sp. PCC7942

Summary In order to deregulate arginine biosynthesis in Synechococcus sp. PCC7942, d-arginine-resistant cell lines were selected following ethyl methanesulfonate mutagenesis of wild-type (WT) cells. Three of these arginine-producing mutant (APM) cell lines, APM1, APM31 and APM40, were putative regulatory mutants based upon secretion of l-arginine into their growth medium. HPLC of lyophilized post-harvest supernatants of APM 31 and 40 resolved two predominant amino acids, arginine and citrulline. In-vitro activity of N-acetylglutamate kinase (NAGK), the proposed regulatory enzyme of the arginine pathway, was about 100-fold less sensitive to l-arginine inhibition in extracts from APM 31 and 40 than the enzyme in WT extracts. The enzyme from APM 1 was 20-fold less sensitive to l-arginine inhibition than WT. The most likely site of mutation in each of the APM cell lines is in the gene for NAGK, rendering the enzymes insensitive to l-arginine feedback control. These strains can be utilized for the phototrophic production of arginine. Offprint requests to: S. E. Bingham     

Proline production via the arginine biosynthetic pathway: transfer of regulatory mutations of arginine biosynthesis into a proline-producing strain ofSerratia marcescens

Summary Proline production via a part of the arginine biosynthetic pathway was examined. About 20 mg/ml ofl-proline was produced by using arginine biosynthetic enzymes. Accordingly, three mutations of arginine biosynthesis, namely, derepression of arginine biosynthetic enzymes (assigned byargR2), feedback inhibition-resistant N-acetylglutamate synthase (assigned byargA2) and defectiveness in N-acetylornithine aminotransferase (assigned byargD ^–) were introduced by three transductional crosses into a proline-producing strain which produced about 55 mg/ml ofl-proline. The constructed strain produced 62 mg/ml ofl-proline, although about 10 mg/ml ofl-arginine and 1 mg/ml of N-acetylglutamate--semialdehyde were produced as by-products.     

The mechanism of infection of plant protoplasts by viruses

Summary The process of virus infection of protoplasts isolated from tobacco leaves has been examined by means of electron microscopy. Immediately after inoculation, virus particles appear at two types of site: trapped in complex surface lesions of the plasmalemma, or in peripheral cytoplasmic vesicles. The complex lesions are only visible after treatment of the protoplasts with inocula containing poly-l-ornithine. With infection by tobacco mosaic virus and cowpea chlorotic mottle virus, which require poly-l-ornithine, the majority of virus particles occur at lesion sites. Pea enation mosaic virus, which does not require poly-l-ornithine for infection to become established, is found predominantly and in high numbers in peripheral vesicles. The behaviour of these three viruses is discussed in terms of a probable mechanism for infection of the protoplasts.     

Metabolic interactions between restraint stress and <Emphasis Type="SmallCaps">l</Emphasis>-lysine: The effect on urea cycle components

Summary.  We studied the effects of l-lysine on wrap-restraint stress-induced changes in ureagenesis. An exposure to wrap-restraint stress did not affect the plasma concentration of l-lysine, but did decrease plasma urea and arginine. Oral l-lysine (1 g/kg) blocked the effect of stress on ureagenesis, and enhanced the effect of stress on l-arginine. No influence of l-lysine were found in controls. The results imply a stress-specific, ureagenesis-stimulating effect of l-lysine, and suggest an increased requirement for l-arginine during the above conditions. Received December 9, 2002 Accepted January 21, 2003 Published online April 3, 2003 Acknowledgement Authors thank Dr. M. Miura (Ajinomoto Co.) for his help with amino acid analysis and Dr. T. Kimura (Ajinomoto Co.) for discussions on amino acid metabolism. Authors' address: Dr. M. Smriga, Ajinomoto Co. Inc., Institute of Life Sciences, 1-1 Suzuki-cho, 210-8681 Kawasaki, Japan, Fax +81-44-210-5893, E-mail: miroslav_smriga@ajinomoto.com Abbreviations: Arg, l-arginine; Orn, l-ornithine; Lys, l-lysine; p.o., oral; WRS, wrap-restraint stress