Discovery of an operon that participates in agmatine metabolism and regulates biofilm formation in Pseudomonas aeruginosa

Agmatine is the decarboxylation product of arginine and a number of bacteria have devoted enzymatic pathways for its metabolism. Pseudomonas aeruginosa harbours the aguBA operon that metabolizes agmatine to putrescine, which can be subsequently converted into other polyamines or shunted into the TCA cycle for energy production. We discovered an alternate agmatine operon in the P. aeruginosa strain PA14 named agu2ABCA′ that contains two genes for agmatine deiminases (agu2A and agu2A′). This operon was found to be present in 25% of clinical P. aeruginosa isolates. Agu2A′ contains a twin‐arginine translocation signal at its N‐terminus and site‐directed mutagenesis and cell fractionation experiments confirmed this protein is secreted to the periplasm. Analysis of the agu2ABCA′ promoter demonstrates that agmatine induces expression of the operon during the stationary phase of growth and during biofilm growth and agu2ABCA′ provides only weak complementation of aguBA, which is induced during log phase. Biofilm assays of mutants of all three agmatine deiminase genes in PA14 revealed that deletion of agu2ABCA′, specifically its secreted product Agu2A′, reduces biofilm production of PA14 following addition of exogenous agmatine. Together, these findings reveal a novel role for the agu2ABCA′ operon in the biofilm development of P. aeruginosa.     

L-arginine utilization by Pseudomonas species

The utilization of arginine was studied in several different Pseudomonas species. The arginine decarboxylase and agmatine deiminase pathways were found to be characteristic of Pseudomonas species of group I as defined by Palleroni et al. (1974). Pseudomonas putida strains had three distinct arginine catabolic pathways initiated by arginine decarboxylase, arginine deiminase and arginine oxidase, respectively. The two former routes were also present in P. fluorescens and P. mendocina and in P. aeruginosa which also used arginine by a further unknown pathway. None of these pathways occurred in P. cepacia strains; agmatine catabolism seemed to follow an unusual route involving guanidinobutyrate as intermediate.     

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.     

The Arginine Decarboxylase Pathways of Host and Pathogen Interact to Impact Inflammatory Pathways in the Lung

The arginine decarboxylase pathway, which converts arginine to agmatine, is present in both humans and most bacterial pathogens. In humans agmatine is a neurotransmitter with affinities towards α2-adrenoreceptors, serotonin receptors, and may inhibit nitric oxide synthase. In bacteria agmatine serves as a precursor to polyamine synthesis and was recently shown to enhance biofilm development in some strains of the respiratory pathogen Pseudomonas aeruginosa. We determined agmatine is at the center of a competing metabolism in the human lung during airways infections and is influenced by the metabolic phenotypes of the infecting pathogens. Ultra performance liquid chromatography with mass spectrometry detection was used to measure agmatine in human sputum samples from patients with cystic fibrosis, spent supernatant from clinical sputum isolates, and from bronchoalvelolar lavage fluid from mice infected with P. aeruginosa agmatine mutants. Agmatine in human sputum peaks during illness, decreased with treatment and is positively correlated with inflammatory cytokines. Analysis of the agmatine metabolic phenotype in clinical sputum isolates revealed most deplete agmatine when grown in its presence; however a minority appeared to generate large amounts of agmatine presumably driving sputum agmatine to high levels. Agmatine exposure to inflammatory cells and in mice demonstrated its role as a direct immune activator with effects on TNF-α production, likely through NF-κB activation. P. aeruginosa mutants for agmatine detection and metabolism were constructed and show the real-time evolution of host-derived agmatine in the airways during acute lung infection. These experiments also demonstrated pathogen agmatine production can upregulate the inflammatory response. As some clinical isolates have adapted to hypersecrete agmatine, these combined data would suggest agmatine is a novel target for immune modulation in the host-pathogen dynamic.     

Mutations affecting regulation of the anabolic argF and the catabolic aru genes in Pseudomonas aeruginosa PAO

Summary The nucleotide sequence required for a fully functional promoter and operator of the Pseudomonas aeruginosa argF gene (argF _po), the arginine-repressible gene for anabolic ornithine carbamoyltransferase, was defined within a 160 by region. The streptomycin (Sm) resistance genes strAB of plasmid RSF1010 were fused to argF _po. This construct in P. aeruginosa strain PAO conferred resistance to Sm. Mutants of strain PAO were selected which were resistant to Sm in the presence of arginine due to constitutive expression of argF _po —strAB. These mutants were designated argR. They were unable to grow or grew poorly on arginine or ornithine as the sole carbon and nitrogen source. This growth defect (Aru^–/Oru^– phenotype) was correlated with a reduced level of N-succinylornithine aminotransferase, an enzyme participating in the major aerobic pathway for arginine and ornithine catabolism in this organism. The argR mutants were classified into four groups by transduction analysis and three argR mutations were mapped on the PAO chromosome. argR9901 and argR9902 were co-transducible with car-9 (at 1 min) and thus close to the oru-310 locus; argR9906 was localized in the oruI (=aru) gene cluster (67 min). Some aru mutants, which have been isolated previously and which produce very low amounts of all enzymes in the arginine succinyltransferase pathway, were unable to repress the argF gene in an arginine medium. Thus, P. aeruginosa PAO appears to have multiple genes that are involved in the regulation of both the anabolic argF and the catabolic aru genes.Abbreviations Arg^– arginine auxotrophy - Aru arginine utilization - Oru ornithine utilization     

Enzymes of agmatine degradation and the control of their synthesis in Streptococcus faecalis.

Streptococcus faecalis ATCC 11700 uses agmatine as its sole energy source for growth. Agmatine deiminase and putrescine carbamoyltransferase are coinduced by growth on agmatine. Glucose and arginine were found to exert catabolite repression on the agmatine deiminase pathway. Four mutants unable to utilize agmatine as an energy source, isolated from the wild-type strain, exhibited three distinct phenotypes. Two of these strains showed essentially no agmatine deiminase, one mutant showed negligible activity of putrescine carbamoyltransferase, and one mutant was defective in both activities. Two carbamate kinases are present in S. faecalis, one belonging to the arginine deiminase pathway, the other being induced by growth on agmatine. These two enzymes have the same molecular weight, 82,000, and seem quite different in size from the kinases isolated from other streptococci.     

Arginine metabolism in Chlamydomonas reinhardi. Conditional expression of arginine-requiring mutants

Summary In Chlamydomonas reinhardi, the arginine-requiring mutants directly derived from the wild-type strain are not able to grow on media containing the routinely used concentration of ammonia. The biochemical basis of this phenomenon has been investigated.The results show that the uptake of arginine is severely depressed by NH ₊ ⁴ and that arginine which succeeds in entering the cells under these conditions is immediately broken down by the first enzyme of the catabolic pathway, arginine deiminase.Accordingly, growth is inhibited as the result of non-availability of exogenous arginine for protein synthesis.It is suggested that similar interactions could be responsible for the lack of other types of amino acid-requiring mutants in Chlamydomonas.Chargé de Recherches du Fonds National Belge de la Recherche Scientifique.     

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.     

Enzymes of arginine utilization and their formation in Aeromonas formicans NCIB 9232

In Aeromonas formicans two inducible catabolic pathways of L-arginine have been characterized. The arginine decarboxylase is induced by arginine which also induces the three enzymes of the arginine deiminase pathway but only in stress conditions such as a shift from aerobic growth conditions to very low oxygen tension. Addition of glucose to medium containing arginine leads to repression of the enzymes involved in the arginine deiminase pathway while exogenous cAMP prevents that repression of enzyme synthesis by glucose. This suggests that the induction of arginine deiminase pathway is regulated by carbon catabolite repression and the energetic state of the cell.     

Subcellular Localization of the Enzymes of the Arginine Dihydrolase Pathway in Trichomonas vaginalis and Tritrichomonas foetus

The enzymes of the arginine dihydrolase pathway were demonstrated in Tritrichomonas foetus and their subcellular localization determined for both T. foetus and Trichomonas vaginalis. Ornithine carbamyltransferase (anabolic and catabolic activities), ornithine decarboxylase and carbamate kinase activity were localized predominately (56–80%) in the non sedimentable fraction of both species. A large proportion (35–40%) of the arginine deiminase was, however, recovered in the large granular fraction, and this distribution was unchanged by increasing the ionic strength of the buffer. Upon density gradient centrifugation the particles containing arginine deiminase activity had an isopycnic density of 1.09 g/ml in percoll, and separated from hydrogenosomes (1.18 g/ml) and lysosomes (1.12 g/ml). Arginine deiminase was also the only enzyme of the dihydrolase pathway which demonstrated latency upon treatment of the 1.09 g/ml fraction with non-ionic detergents. The results demonstrate the presence of the arginine dihydrolase pathway in T. foetus and indicate that at least a portion of the arginine deiminase in trichomonads is membrane associated.     

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     

Arginine Deiminase in Staphylococcus epidermidis Functions To Augment Biofilm Maturation through pH Homeostasis

Allelic replacement mutants were constructed within arginine deiminase (arcA1 and arcA2) to assess the function of the arginine deiminase (ADI) pathway in organic acid resistance and biofilm formation of Staphylococcus epidermidis 1457. A growth-dependent acidification assay (pH ∼5.0 to ∼5.2) determined that strain 1457 devoid of arginine deiminase activity (1457 ΔADI) was significantly less viable than the wild type following depletion of glucose and in the presence of arginine. However, no difference in viability was noted for individual 1457 ΔarcA1 (native) or ΔarcA2 (arginine catabolic mobile element [ACME]-derived) mutants, suggesting that the native and ACME-derived ADIs are compensatory in S. epidermidis. Furthermore, flow cytometry and electron paramagnetic resonance spectroscopy results suggested that organic acid stress resulted in oxidative stress that could be partially rescued by the iron chelator dipyridyl. Collectively, these results suggest that formation of hydroxyl radicals is partially responsible for cell death via organic acid stress and that ADI-derived ammonia functions to counteract this acid stress. Finally, static biofilm assays determined that viability, ammonia synthesis, and pH were reduced in strain 1457 ΔADI following 120 h of growth in comparison to strain 1457 and the arcA1 and arcA2 single mutants. It is hypothesized that ammonia synthesis via the ADI pathway is important to reduce pH stress in specific microniches that contain high concentrations of organic acids.     

Catabolism of biogenic amines in Pseudomonas species

Biogenic amines (BAs; 2-phenylethylamine, tyramine, dopamine, epinephrine, norepinephrine, octopamine, histamine, tryptamine, serotonin, agmatine, cadaverine, putrescine, spermidine, spermine and certain aliphatic amines) are widely distributed organic molecules that play basic physiological functions in animals, plants and microorganisms. Pseudomonas species can grow in media containing different BAs as carbon and energy sources, a reason why these bacteria are excellent models for studying such catabolic pathways. In this review, we analyse most of the routes used by different species of Pseudomonas (P. putida, P. aeruginosa, P. entomophila and P. fluorescens) to degrade BAs. Analysis of these pathways has led to the identification of a huge number of genes, catabolic enzymes, transport systems and regulators, as well as to understanding of their hierarchy and functional evolution. Knowledge of these pathways has allowed the design and collection of genetically manipulated microbes useful for eliminating BAs from different sources, highlighting the biotechnological applications of these studies.     

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.     

Ornithine cycle in Nostoc PCC 73102. Arginase,OCT and arginine deiminase,and the effects of addition of external arginine,ornithine, or citrulline

Arginase, ornithine carbamoyl transferase (OCT) and arginine deiminase activities were found in cell-free extracts of Nostoc PCC 73102, a free-living cyanobacterium originally isolated from the cycad Macrozamia. Addition of either arginine, ornithine or citrulline to the growth medium induced significant changes in their in vitro activities. Moreover, growth in darkness, compared to in light, induced higher in vitro activities. The in vitro activities of arginase and arginine deiminase, two catabolic enzymes primarily involved in the breakdown of arginine, increased substantially by a combination of growth in darkness and addition of either arginine, or ornithine, to the growth medium. The most significant effects on the in vitro OCT activities where observed in cells grown with the addition of ornithine. Cells grown in darkness exhibited about 6% of the in vivo nitrogenase activity observed in cells grown in light. However, addition of external carbon (glucose and fructose) to cells grown in darkness resulted in in vivo nitrogenase activity levels similar to, or even higher than, cells grown in light. Growth with high in vivo nitrogenase activity or in darkness with the addition of external carbon, resulted in repressed levels of in vitro arginase and arginine deiminase activities. It is suggested that nitrogen starvation induces a mobilization of the stored nitrogen, internal release of the amino compound arginine, and an induction of two catabolic enzymes arginase and arginine deiminase. A similar and even more pronunced induction can be observed by addition of external arginine to the growth medium.     

Arginine deiminases: Therapeutic tools in the etiology and pathogenesis of Alzheimer's disease

There is, at present, no definitive pre-mortem diagnostic tool for Alzheimer's disease, (AD) which relates to a poor understanding of its etiology. Brains of AD patients contain large amounts of the toxic plaque-forming β-amyloid_1–42 fragment in addition to elevated concentrations of the amino acid l-arginine. This work proposes that lowering levels of arginine in the astrocytes surrounding amyloid plaques may serve as a therapeutic tool in this neurodegenerative disorder. Arginine deiminase (ADI), from Pseudomonas aeruginosa, and peptidylarginine deiminase [PAD II], from bovine brain, are inhibited by amyloid peptides that contain arginine (amyloid_1–42) and those that have no arginine (amyloid_{12–28/22–35}). Enhanced activity of PAD II is noted with free l-arginine.     

The arginine deiminase pathway in Rhizobium etli: DNA sequence analysis and functional study of the arcABC genes.

Sequence analysis upstream of the Rhizobium etli fixLJ homologous genes revealed the presence of three open reading frames homologous to the arcABC genes of Pseudomonas aeruginosa. The P. aeruginosa arcABC genes code for the enzymes of the arginine deiminase pathway: arginine deiminase, catabolic ornithine carbamoyltransferase (cOTCase), and carbamate kinase. OTCase activities were measured in free-living R. etli cells and in bacteroids isolated from bean nodules. OTCase activity in free-living cells was observed at a different pH optimum than OTCase activity in bacteroids, suggesting the presence of two enzymes with different characteristics and different expression patterns of the corresponding genes. The characteristics of the OTCase isolated from the bacteroids were studied in further detail and were shown to be similar to the properties of the cOTCase of P. aeruginosa. The enzyme has a pH optimum of 6.8 and a molecular mass of approximately 450 kDa, is characterized by a sigmoidal carbamoyl phosphate saturation curve, and exhibits a cooperativity for carbamoyl phosphate. R. etli arcA mutants, with polar effects on arcB and arcC, were constructed by insertion mutagenesis. Bean nodules induced by arcA mutants were still able to fix nitrogen but showed a significantly lower acetylene reduction activity than nodules induced by the wild type. No significant differences in nodule dry weight, plant dry weight, and number of nodules were found between the wild type and the mutants. Determination of the OTCase activity in extracts from bacteroids revealed a strong decrease in activity of this enzyme in the arcA mutant compared to the wild-type strain. Finally, we observed that expression of an R. etli arcA-gusA fusion was strongly induced under anaerobic conditions.     

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.