RESUMO
Arginine anaerobic catabolism occurs in Bacillus licheniformis through the arginine deiminase pathway, encoded by the gene cluster arcABDC. We report here the involvement of a new protein, ArcR, in the regulation of the pathway. ArcR is a protein of the Crp/Fnr family encoded by a gene located 109 bp downstream from arcC. It binds to a palindromic sequence, very similar to an Escherichia coli Crp binding site, located upstream from arcA. Residues in the C-terminal domain of Crp that form the DNA binding motif, in particular residues Arg-180 and Glu-181 that make specific bonds with DNA, are conserved in ArcR, suggesting that the complexes formed with DNA by Crp and ArcR are similar. Moreover, the pattern of DNase I hypersensitivity sites induced by the binding of ArcR suggests that ArcR bends the DNA in the same way as Crp. From the absence of anaerobic induction following inactivation of arcR and from the existence of a binding site upstream of the arcA transcription start point, it can be inferred that ArcR is an activator of the arginine deiminase pathway.
Assuntos
Arginina/metabolismo , Bacillus/genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Proteínas de Escherichia coli , Regulação Bacteriana da Expressão Gênica , Sequência de Aminoácidos , Anaerobiose , Bacillus/metabolismo , Proteínas de Bactérias/química , Sequência de Bases , Sítios de Ligação , Proteínas de Transporte , Proteína Receptora de AMP Cíclico/genética , Proteína Receptora de AMP Cíclico/metabolismo , DNA Bacteriano/química , DNA Bacteriano/metabolismo , Proteínas de Ligação a DNA/química , Genes Bacterianos , Proteínas Ferro-Enxofre/genética , Proteínas Ferro-Enxofre/metabolismo , Dados de Sequência Molecular , Família Multigênica , Mutação , Análise de Sequência de DNARESUMO
Pseudomonas cepacia NCTC 10743 utilizes arginine as the sole source of carbon and nitrogen for growth. Arginine is degraded to glutamate via succinyl derivatives. The catabolic sequence in this pathway is L-arginine----N2-succinylarginine----N2-succinylornithine--- -N2-succinylglutamate semialdehyde----N2-succinylglutamate----glutamate + succinate. The formation of the enzymes responsible for arginine degradation is regulated not only by induction but also by both carbon and nitrogen catabolite repression.
Assuntos
Arginina/metabolismo , Pseudomonas/metabolismo , Aciltransferases/metabolismo , Arginina/análogos & derivados , Glutamatos/metabolismo , Ácido Glutâmico , Ornitina/análogos & derivados , Ornitina/metabolismo , Ácido Oxâmico/farmacologia , Pseudomonas/enzimologia , Pseudomonas/crescimento & desenvolvimento , Succinatos/metabolismoRESUMO
The distribution of the arginine succinyltransferase pathway was examined in representative strains of Pseudomonas and related bacteria able to use arginine as the sole carbon and nitrogen source for growth. The arginine succinyltransferase pathway was induced in arginine-grown cells. The accumulation of succinylornithine following in vivo inhibition of succinylornithine transaminase activity by aminooxyacetic acid showed that this pathway is responsible for the dissimilation of the carbon skeleton of arginine. Catabolism of citrulline as a carbon source was restricted to relatively few of the organisms tested. In P. putida, P. cepacia and P. indigofera, ornithine was the main product of citrulline degradation. In most strains which possessed the arginine succinyltransferase pathway, the first step of ornithine utilization as a carbon source was the conversion of ornithine into succinylornithine through an ornithine succinyltransferase. However P. cepacia and P. putida used ornithine by a pathway which proceeded via proline as an intermediate and involved an ornithine cyclase activity.
Assuntos
Arginina/metabolismo , Citrulina/metabolismo , Ornitina/metabolismo , Pseudomonas/metabolismo , Aciltransferases/metabolismo , Aeromonas/enzimologia , Amônia-Liases/metabolismo , Arginina/análogos & derivados , Klebsiella pneumoniae/enzimologia , Pseudomonas/enzimologia , Pseudomonas aeruginosa/metabolismo , Pseudomonas fluorescens/metabolismo , Succinatos/metabolismoRESUMO
The arginine and ornithine succinyltransferase from Pseudomonas aeruginosa, a bifunctional enzyme involved in the aerobic utilization of arginine and ornithine, has been purified to homogeneity. The apparent molecular mass of the native enzyme was 150 kDa by gel filtration and 140 kDa by polyacrylamide gel electrophoresis under non-denaturing conditions. After SDS/PAGE two subunits of 35 kDa and 37 kDa were evident, indicating that the enzyme is a heterotetramer. Microsequence analysis of the electroblotted protein bands gave two different but well-conserved N-terminal amino acid sequences. The L-arginine saturation curve followed Henri-Michaelis kinetics with an apparent Km value of 0.5 mM. The sigmoidal saturation curve for L-ornithine indicated allosteric behaviour. D-Arginine, a competitive inhibitor with respect to L-arginine, reduced L-ornithine cooperativity. In the presence of spermidine, the L-ornithine saturation curve became increasingly sigmoidal, the Hill coefficient shifting from 2.5 in the absence of the inhibitor, to 3.5 in the presence of 20 mM spermidine. The L-arginine analog, L-homoarginine, was also a substrate of the succinyltransferase, and the saturation of the enzyme by this substrate was also cooperative. All these data confirmed the allosteric nature of the enzyme. Moreover, a mutant growing faster on L-ornithine than the parent strain had a modified succinyltransferase with a reduced L-ornithine cooperativity. The fate of L-homoarginine was different depending on whether the succinyltransferase was induced or not; excreted succinylhomoarginine was found in cultures induced for the transferase activity whereas guanidinovalerate was excreted in non-induced cultures. The 'waste' of succinyl CoA, which could not be regenerated from the excreted succinylhomoarginine, explained the inhibition exerted by L-homoarginine on growth when ornithine or arginine was used as the growth medium.
Assuntos
Aciltransferases/isolamento & purificação , Arginina/metabolismo , Ornitina/metabolismo , Pseudomonas aeruginosa/enzimologia , Aciltransferases/metabolismo , Sequência de Aminoácidos , Cromatografia em Gel , Eletroforese em Gel de Poliacrilamida , Homoarginina/metabolismo , Concentração de Íons de Hidrogênio , Dados de Sequência Molecular , Mutação , Pseudomonas aeruginosa/crescimento & desenvolvimento , Especificidade por SubstratoRESUMO
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.
Assuntos
Ornitina/análogos & derivados , Ornitina/metabolismo , Pseudomonas aeruginosa/metabolismo , Succinatos/metabolismo , Aciltransferases/biossíntese , Aciltransferases/metabolismo , Arginina/metabolismo , Indução Enzimática , Mutação , Pseudomonas aeruginosa/enzimologia , Pseudomonas aeruginosa/genéticaRESUMO
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.
Assuntos
Arginina/metabolismo , Genes Bacterianos , Genes , Hidrolases/genética , Mutação , Pseudomonas aeruginosa/genética , Anaerobiose , Genótipo , Cinética , Pseudomonas aeruginosa/crescimento & desenvolvimento , Especificidade da EspécieRESUMO
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.
Assuntos
Hidrolases/biossíntese , Ornitina Carbamoiltransferase/biossíntese , Fosfotransferases (Aceptor do Grupo Carboxila) , Pseudomonas aeruginosa/enzimologia , Arginina/farmacologia , Carbamatos/biossíntese , Indução Enzimática/efeitos dos fármacos , Nitratos/farmacologia , Nitritos/farmacologia , Oxigênio/fisiologia , Fosfotransferases/biossínteseRESUMO
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.
Assuntos
Aminoácidos/metabolismo , Carboxil e Carbamoil Transferases , Enterococcus faecalis/enzimologia , Ácido Oxâmico/metabolismo , Fosfotransferases (Aceptor do Grupo Carboxila) , Fosfotransferases/metabolismo , Transferases/metabolismo , Agmatina/metabolismo , Agmatina/farmacologia , Arginina/metabolismo , Arginina/farmacologia , Indução Enzimática , Glucose/metabolismo , Glucose/farmacologia , Cinética , Ornitina Carbamoiltransferase/metabolismo , Ácido Oxâmico/análogos & derivadosRESUMO
The arginine deiminase pathway enables Bacillus licheniformis to grow anaerobically on arginine. Both the presence of arginine and anaerobiosis are needed to trigger induction of the pathway. In this study we have cloned and sequenced the arc genes encoding the pathway. They appear clustered in an operon-like structure in the order arcA (arginine deiminase), arcB (ornithine carbamoyltransferase), arcD (putative arginine-ornithine antiporter), arcC (carbamate kinase). It was found that B. licheniformis has an arginine repressor, ArgR, homologous to the B. subtilis arginine repressor AhrC. Mutants affected in argR were isolated. These mutants have lost both repression by arginine of the anabolic ornithine carbamoyltransferase and induction of the arginine deiminase pathway. Electrophoretic band shift experiments and DNase I footprinting revealed that in the presence of arginine, ArgR binds to a site upstream from the arc promoter. The binding site is centered 108 nucleotides upstream from the transcription start point and contains a single Arg box.
Assuntos
Sistemas de Transporte de Aminoácidos , Antiporters/genética , Bacillus/enzimologia , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Hidrolases/genética , Família Multigênica , Ornitina Carbamoiltransferase/genética , Fosfotransferases (Aceptor do Grupo Carboxila)/genética , Proteínas Repressoras/metabolismo , Sequência de Aminoácidos , Bacillus/genética , Proteínas da Membrana Bacteriana Externa/genética , Sequência de Bases , Clonagem Molecular , DNA Bacteriano , Indução Enzimática , Proteínas de Escherichia coli , Regulação Bacteriana da Expressão Gênica , Regulação Enzimológica da Expressão Gênica , Hidrolases/metabolismo , Dados de Sequência Molecular , Mutagênese , Ornitina Carbamoiltransferase/metabolismo , Fosfotransferases (Aceptor do Grupo Carboxila)/metabolismo , Regiões Promotoras Genéticas , Proteínas Repressoras/genéticaRESUMO
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.