Control of enzyme synthesis in the oxalurate catabolic pathway of Streptococcus faecalis ATCC 11700: evidence for the existence of a third carbamate kinase

Streptococcus faecalis ATCC 11700 uses oxalurate as a sole energy source for growth. An oxamate carbamoyltransferase and a carbamate kinase, both induced by oxalurate, are involved in this process.The oxalurate-induced kinase is specific for the pathway. Its properties are different from those of the previously characterized agmatine and arginine-induced kinases.Glucose, but not arginine, nor agmatine, two other energy sources, represses the oxalurate pathway. In contrast, oxalurate was found to be at least as effective as glucose in repressing the arginine deiminase pathway in arginine grown cells or the agmatine deiminase pathway during growth on agmatine.     

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.     

Control of enzyme synthesis in the arginine deiminase pathway of Streptococcus faecalis

The formation of the arginine deiminase pathway enzymes in Streptococcus faecalis ATCC 11700 was investigated. The addition of arginine to growing cells resulted in the coinduction of arginine diminase (EC 3.5.3.6), ornithine carbamoyltransferase (EC 2.1.3.3), and carbamate kinase (EC 2.7.2.3). Growth on glucose-arginine or on glucose-fumarate-arginine produced a decrease in the specific activity of the arginine fermentation system. Aeration had a weak repressing effect on the arginine deiminase pathway enzymes in cells growing on arginine as the only added substrate. By contrast, depending on the growth phase, a marked repression of the pathway by oxygen was observed in cells growing on glucose-arginine. We hypothesize that, in S. faecalis, the ATP pool is an important signal in the regulation of the arginine deiminase pathway. Mutants unable to utilize arginine as an energy source, isolated from the wild type, exhibited four distinct phenotypes. In group I the three enzymes of the arginine deiminase pathway were present and probably affected in the arginine uptake system. Group II mutants had no detectable arginine deiminase, whereas group III mutants had low levels of ornithine carbamoyltransferase. Group IV mutants were defective for all three enzymes of the pathway.     

The gene cluster for agmatine catabolism of Enterococcus faecalis: study of recombinant putrescine transcarbamylase and agmatine deiminase and a snapshot of agmatine deiminase catalyzing its reaction

Enterococcus faecalis makes ATP from agmatine in three steps catalyzed by agmatine deiminase (AgDI), putrescine transcarbamylase (PTC), and carbamate kinase (CK). An antiporter exchanges putrescine for agmatine. We have cloned the E. faecalis ef0732 and ef0734 genes of the reported gene cluster for agmatine catabolism, overexpressed them in Escherichia coli, purified the products, characterized them functionally as PTC and AgDI, and crystallized and X-ray diffracted them. The 1.65-Angstroms-resolution structure of AgDI forming a covalent adduct with an agmatine-derived amidine reactional intermediate is described. We provide definitive identification of the gene cluster for agmatine catabolism and confirm that ornithine is a genuine but poor PTC substrate, suggesting that PTC (found here to be trimeric) evolved from ornithine transcarbamylase. N-(Phosphonoacetyl)-putrescine was prepared and shown to strongly (K(i) = 10 nM) and selectively inhibit PTC and to improve PTC crystallization. We find that E. faecalis AgDI, which is committed to ATP generation, closely resembles the AgDIs involved in making polyamines, suggesting the recruitment of a polyamine-synthesizing AgDI into the AgDI pathway. The arginine deiminase (ADI) pathway of arginine catabolism probably supplied the genes for PTC and CK but not those for the agmatine/putrescine antiporter, and thus the AgDI and ADI pathways are not related by a single "en bloc" duplication event. The AgDI crystal structure reveals a tetramer with a five-blade propeller subunit fold, proves that AgDI closely resembles ADI despite a lack of sequence identity, and explains substrate affinity, selectivity, and Cys357-mediated-covalent catalysis. A three-tongued agmatine-triggered gating opens or blocks access to the active center.     

Regulation of enzyme synthesis in the arginine deiminase pathway of Pseudomonas aeruginosa.

The three enzymes of the arginine deiminase pathway in Pseudomonas aeruginosa strain PAO were induced strongly (50- to 100-fold) by a shift from aerobic growth conditions to very low oxygen tension. Arginine in the culture medium was not essential for induction, but increased the maximum enzyme levels twofold. The induction of the three enzymes arginine deiminase (EC 3.5.3.6), catabolic ornithine carbamoyltransferase (EC 2.1.3.3), and carbamate kinase (EC 2.7.2.3) appeared to be coordinate. Catabolic ornithine carbamoyltransferase was studied in most detail. Nitrate and nitrite, which can replace oxygen as terminal electron acceptors in P. aeruginosa, partially prevented enzyme induction by low oxygen tension in the wild-type strain, but not in nar (nitrate reductase-negative) mutants. Glucose was found to exert catabolite repression of the deiminase pathway. Generally, conditions of stress, such as depletion of the carbon and energy source or the phosphate source, resulted in induced synthesis of catabolic ornithine carbamoyltransferase. The induction of the deiminase pathway is thought to mobilize intra- and extracellular reserves of arginine, which is used as a source of adenosine 5'-triphosphate in the absence of respiration.     

Biogenic amine production by Lactobacillus

AIMS: The aim of this work was to demonstrate that strains of Lactobacillus may be able to produce putrescine and agmatine from one of the major amino acids present in fruit juices and wine, arginine, and from amino acid-derived ornithine. METHODS AND RESULTS: Biogenic amines were determined by HPLC. Their production in the culture medium was similar under both microaerophilic and anaerobic conditions. The presence of Mn2+ had a minimal influence on the results, whereas the addition of pyridoxal phosphate increased amine production 10-fold. Lactobacillus hilgardii X1B, isolated from wine, was able to degrade arginine by two pathways: arginine deiminase and arginine decarboxylase. The isolate was able to produce putrescine from ornithine and from agmatine. Lactobacillus plantarum strains N4 and N8, isolated from orange, utilized arginine via the arginine deiminase system. Only the N4 strain was able to produce putrescine from ornithine. CONCLUSION: It has been demonstrated that Lact. hilgardii X1B is able to produce the most important biogenic amine found in wine, putrescine, and also agmatine from arginine and ornithine, and that Lactobacillus plantarum, considered to be an innocuous spoilage micro-organism in fruit juices, is able to produce amines. SIGNIFICANCE AND IMPACT OF THE STUDY: The results have significance in relation to food poisoning caused by beverages that have been contaminated with biogenic amines.     

Pseudomonas aeruginosa mutants affected in anaerobic growth on arginine: evidence for a four-gene cluster encoding the arginine deiminase pathway.

Pseudomonas aeruginosa PAO was able to grow in the absence of exogenous terminal electron acceptors, provided that the medium contained 30 to 40 mM L-arginine and 0.4% yeast extract. Under strictly anaerobic conditions (O2 at less than 1 ppm), growth could be measured as an increase in protein and proceeded in a non-exponential way; arginine was largely converted to ornithine but not entirely consumed at the end of growth. In the GasPak anaerobic jar (Becton Dickinson and Co.), the wild-type strain PAO1 grew on arginine-yeast extract medium in 3 to 5 days; mutants could be isolated that were unable to grow under these conditions. All mutants (except one) were defective in at least one of the three enzymes of the arginine deiminase pathway (arcA, arcB, and arcC mutants) or in a novel function that might be involved in anaerobic arginine uptake (arcD mutants). The mutations arcA (arginine deiminase), arcB (catabolic ornithine carbamoyltransferase), arcC (carbamate kinase), and arcD were highly cotransducible and mapped in the 17-min chromosome region. Some mutations in the arc cluster led to low, noninducible levels of all three arginine deiminase pathway enzymes and thus may affect control elements required for induction of the postulated arc operon. Two fluorescent pseudomonads (P. putida and P. fluorescens) and P. mendocina, as well as one PAO mutant, possessed an inducible arginine deiminase pathway and yet were unable to grow fermentatively on arginine. The ability to use arginine-derived ATP for growth may provide P. aeruginosa with a selective advantage when oxygen and nitrate are scarce.     

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.     

Occurrence of agmatine pathway for putrescine synthesis in Selenomonas ruminatium

Selenomonas ruminantium synthesizes cadaverine and putrescine from L-lysine and L-ornithine as the essential constituents of its peptidoglycan by a constitutive lysine/ornithine decarboxylase (LDC/ODC). S. ruminantium grew normally in the presence of the specific inhibitor for LDC/ODC, DL-alpha-difluoromethylornithine, when arginine was supplied in the medium. In this study, we discovered the presence of arginine decarboxylase (ADC), the key enzyme in agmatine pathway for putrescine synthesis, in S. ruminantium. We purified and characterized ADC and cloned its gene (adc) from S. ruminantium chromosomal DNA. ADC showed more than 60% identity with those of LDC/ODC/ADCs from Gram-positive bacteria, but no similarity to that from Gram-negative bacteria. In this study, we also cloned the aguA and aguB genes, encoding agmatine deiminase (AguA) and N-carbamoyl-putrescine amidohydrolase (AguB), both of which are involved in conversion from agmatine into putrescine. AguA and AguB were expressed in S. ruminantium. Hence, we concluded that S. ruminantium has both ornithine and agmatine pathways for the synthesis of putrescine.     

Isolation and Characterization of a Mutant of Escherichia coli Blocked in the Synthesis of Putrescine

A mutant of Escherichia coli is described which is defective in the conversion of arginine to putrescine. The activity of the enzyme agmatine ureohydrolase is greatly reduced, whereas the activity of the other two enzymes of the pathway, the constitutive arginine decarboxylase and the inducible arginine decarboxylase, are within the normal range. The growth behavior of the mutant reflects the enzymatic block. It grows well in the absence of arginine, but only poorly in the presence of arginine. Under the former conditions, putrescine can be formed from ornithine as well as arginine, whereas under the latter conditions, because of feedback control, it can be formed only from arginine.     

Oxygen and nitrate in utilization by Bacillus licheniformis of the arginase and arginine deiminase routes of arginine catabolism and other factors affecting their syntheses.

Bacillus licheniformis has two pathways of arginine catabolism. In well-aerated cultures, the arginase route is present, and levels of catabolic ornithine carbamoyltransferase were low. An arginase pathway-deficient mutant, BL196, failed to grow on arginine as a nitrogen source under these conditions. In anaerobiosis, the wild type contained very low levels of arginase and ornithine transaminase. BL196 grew normally on glucose plus arginine in anaerobiosis and, like the wild type, had appreciable levels of catabolic transferase. Nitrate, like oxygen, repressed ornithine carbamoyltransferase and stimulated arginase synthesis. In aerobic cultures, arginase was repressed by glutamine in the presence of glucose, but not when the carbon-energy source was poor. In anaerobic cultures, ammonia repressed catabolic ornithine carbamoyltransferase, but glutamate and glutamine stimulated its synthesis. A second mutant, derived from BL196, retained the low arginase and ornithine transaminase levels of BL196 but produced high levels of deiminase pathway enzymes in the presence of oxygen.     

Factors affecting the production of putrescine from agmatine by Lactobacillus hilgardii XB isolated from wine

Aims:  To elucidate and characterize the metabolic putrescine synthesis pathway from agmatine by Lactobacillus hilgardii X₁B.
Methods and Results:  The putrescine formation from agmatine by resting cells (the normal physiological state in wine) of lactic acid bacteria isolated from wine has been determined for the first time. Agmatine deiminase and N -carbamoylputrescine hydrolase enzymes, determined by HPLC and LC-Ion Trap Mass Spectrometry, carried out the putrescine synthesis from agmatine. The influence of pH, temperature, organic acids, amino acids, sugars and ethanol on the putrescine formation in wine was determined.
Conclusions:  Resting cells of Lact. hilgardii X₁B produce putrescine in wine. The putrescine production was carried out from agmatine through the agmatine deiminase system.
Significance and Impact of the Study:  These results have significance from two points of view, wine quality and toxicological and microbiological aspects, taking account that putrescine, which origin is still controversial, is quantitatively the main biogenic amine found in wine.     

Isolation of Conditionally Putrescine-Deficient Mutants of Escherichia coli

Mutants defective in the conversion of arginine to putrescine were found by screening clones from mutagenized cultures for inability to produce urea during growth in arginine-supplemented media. Two partially blocked mutants were isolated; one was deficient in arginine decarboxylase and the other was deficient in agmatine ureohydrolase. As predicted from the pattern of putrescine synthesis in Escherichia coli, these mutants were conditionally putrescine-deficient. When grown in either minimal or ornithine-supplemented media, conditions which lead to preferential utilization of the ornithine to putrescine pathway, the mutants had normal intracellular polyamine levels. However, when the mutants were placed in arginine-supplemented media, the level of intracellular putrescine was lowered markedly. Under conditions where intracellular putrescine was 1% of normal, the doubling time of the mutants was increased approximately 10%. The putrescine-deficient mutants had wild-type morphology, normal levels of protein and ribonucleic acid (RNA), and stringent amino acid control of RNA synthesis.     

Fermentation of Agmatine in Streptococcus faecalis: Occurrence of Putrescine Transcarbamoylase

Putrescine transcarbamoylase, EC 2.1.3.x (carbamoylphosphate:putrescine transcarbamoylase), has been purified from Streptococcus faecalis 10C1 grown on agmatine as primary energy source. The formation of N-carbamoylputrescine from putrescine and carbamoylphosphate serves as a convenient and sensitive assay for this enzymatic activity. The enzyme catalyzes both the phosphorolysis arsenolysis of N-carbamoylputrescine. Arginine does not induce the synthesis of putrescine transcarbamoylase in S. faecalis. Furthermore, the putrescine transcarbamoylase activity is easily separated from ornithine transcarbamoylase activity by gel filtration on Sephadex G-100 indicating that the two activities are associated with different proteins. The significance of this new enzyme in the fermentation of agmatine and its relation to the other known transcarbamoylases are discussed.     

Mapping of the arginine deiminase gene in Pseudomonas aeruginosa

A mutant of Pseudomonas aeruginosa PAO lacking arginine deiminase activity (arcA) was isolated by screening for a derivative of an arcB mutant (deficient in catabolic ornithine carbamoyltransferase) that did not excrete citrulline under conditions of limited aeration. The arcA mutation was highly cotransducible with arcB.     

Arginine metabolism in the deep sea tube worm Riftia pachyptila and its bacterial endosymbiont

The present study describes the distribution and properties of enzymes involved in arginine metabolism in Riftia pachyptila, a tubeworm living around deep sea hydrothermal vents and known to be engaged in a highly specific symbiotic association with a bacterium. The results obtained show that the arginine biosynthetic enzymes, carbamyl phosphate synthetase, ornithine transcarbamylase, and argininosuccinate synthetase are present in all of the tissues of the worm and in the bacteria. Thus, Riftia and its bacterial endosymbiont can assimilate nitrogen and carbon via this arginine biosynthetic pathway. The kinetic properties of ornithine transcarbamylase strongly suggest that neither Riftia nor the bacteria possess the catabolic form of this enzyme belonging to the arginine deiminase pathway, the absence of this pathway being confirmed by the lack of arginine deiminase activity. Arginine decarboxylase and ornithine decarboxylase are involved in the biosynthesis of polyamines such as putrescine and agmatine. These activities are present in the trophosome, the symbiont-harboring tissue, and are higher in the isolated bacteria than in the trophosome, indicating that these enzymes are of bacterial origin. This finding indicates that Riftia is dependent on its bacterial endosymbiont for the biosynthesis of polyamines that are important for its metabolism and physiology. These results emphasize a particular organization of the arginine metabolism and the exchanges of metabolites between the two partners of this symbiosis.     

Structural Characterization of the Enzymes Composing the Arginine Deiminase Pathway in Mycoplasma penetrans

The metabolism of arginine towards ATP synthesis has been considered a major source of energy for microorganisms such as Mycoplasma penetrans in anaerobic conditions. Additionally, this pathway has also been implicated in pathogenic and virulence mechanism of certain microorganisms, i.e. protection from acidic stress during infection. In this work we present the crystal structures of the three enzymes composing the gene cluster of the arginine deiminase pathway from M. penetrans: arginine deiminase (ADI), ornithine carbamoyltransferase (OTC) and carbamate kinase (CK). The arginine deiminase (ADI) structure has been refined to 2.3 Å resolution in its apo-form, displaying an “open” conformation of the active site of the enzyme in comparison to previous complex structures with substrate intermediates. The active site pocket of ADI is empty, with some of the catalytic and binding residues far from their active positions, suggesting major conformational changes upon substrate binding. Ornithine carbamoyltransferase (OTC) has been refined in two crystal forms at 2.5 Å and 2.6 Å resolution, respectively, both displaying an identical dodecameric structure with a 23-point symmetry. The dodecameric structure of OTC represents the highest level of organization in this protein family and in M.penetrans it is constituted by a novel interface between the four catalytic homotrimers. Carbamate kinase (CK) has been refined to 2.5 Å resolution and its structure is characterized by the presence of two ion sulfates in the active site, one in the carbamoyl phosphate binding site and the other in the β-phosphate ADP binding pocket of the enzyme. The CK structure also shows variations in some of the elements that regulate the catalytic activity of the enzyme. The relatively low number of metabolic pathways and the relevance in human pathogenesis of Mycoplasma penetrans places the arginine deiminase pathway enzymes as potential targets to design specific inhibitors against this human parasite.     

Agmatine deiminase from cucumber seedlings is a mono-specific enzyme: purification and characteristics

Agmatine deiminase was purified to homogeneity from cucumber seedlings. The purification procedures included treatment with DE52, ammonium sulfate precipitation, DE52 column chromatography, Superdex 200 column chromatography, and agmatine-(CNBr)-diaminohexane-CNBr-activated-Sepharose 4B column chromatography. The purified agmatine deiminase exhibited a specific activity of 242nkat/mg protein at 30 degrees C, pH 7.0, with a yield of 33%. The molecular mass of the native enzyme was 67kDa, as estimated by Superdex 200 column chromatography. On the other hand, SDS-PAGE showed that the molecular masses of the subunits with 1% SDS and 5% of 2-mercaptoethanol treatment and with additional N-glycosidase F treatment were 47 and 36kDa, respectively. These results suggest that agmatine deiminase from cucumber is a glycoprotein. The Km of the enzyme for agmatine was 16microM and arcaine was a potent competitive inhibitor of the enzyme, with a Ki of 7.1microM. The enzyme was stable for 2 months at 4 degrees C. The enzyme does not have putrescine synthase activity or the activities of its components ornithine and putrescine transcarbamylase. The characteristics of the enzyme purified from cucumber were like those of the enzyme from maize. These results indicate that agmatine deiminase is distinctly different from putrescine synthase in higher plants.     

Expression of the arginine deiminase pathway genes in Lactobacillus sakei is strain dependent and is affected by the environmental pH

The adaptation of Lactobacillus sakei to a meat environment is reflected in its metabolic potential. For instance, the ability to utilize arginine through the arginine deiminase (ADI) pathway, resulting in additional ATP, represents a competitive benefit. In L. sakei CTC 494, the arc operon (arcABCTDR) shows the same gene order and organization as that in L. sakei 23K, the genome sequence of which is known. However, differences in relative gene expression were found, and these seemed to be optimal in different growth phases, namely, the highest relative gene expression level was in the end exponential growth phase in the case of L. sakei CTC 494 and in the mid-exponential growth phase of L. sakei 23K. Also, the environmental pH influenced the relative expression level of the arc operon, as shown for L. sakei CTC 494, with the highest relative expression level occurring at the optimal pH for growth (pH 6.0). Deviations from this optimal pH (pH 5.0 and pH 7.0) resulted in an overall decline of the relative expression level of all genes of the arc operon. Furthermore, a differential relative expression of the individual genes of the arc operon was found, with the highest relative gene expression occurring for the first two genes of the arc operon (arcA and arcB). Finally, it was shown that some L. sakei strains were able to convert agmatine into putrescine, suggesting an operational agmatine deiminase pathway in these strains, a metabolic trait that is undesirable in meat fermentations. This study shows that this metabolic trait is most probably encoded by a previously erroneously annotated second putative arc operon.     

Arginine degradation in Pseudomonas aeruginosa mutants blocked in two arginine catabolic pathways

Summary Pseudomonas aeruginosa mutants defective in agmatine utilization (agu) were isolated. The genes encoding agmatine deiminase (aguA) and N-carbamoylputrescine amidinohydrolase (aguB) were 98% cotransducible and mapped between gpu and ser-3 in the 30 min region of the chromosome. Constructed agu arc double mutants (blocked in the arginine decarboxylase and arginine deiminase pathways) used arginine efficiently as the sole carbon and nitrogen source. This suggests the existence of a further arginine catabolic pathway in P. aeruginosa. The mapping data of this study confirm that in P. aeruginosa the chromosomal genes with catabolic functions do not show supraoperonic clustering as found in P. putida.