Repression of Escherichia coli carbamoylphosphate synthase: relationships with enzyme synthesis in the arginine and pyrimidine pathways.

Cumulative repression of Escherichia coli carbamoylphosphate synthase (CPSase; EC 2.7.2.9) by arginine and pyrimidine was analyzed in relation to control enzyme synthesis in the arginine and pyrimidine pathways. The expression of carA and carB, the adjacent genes that specify the two subunits of the enzyme, was estimated by means of an in vitro complementation assay. The synthesis of each gene product was found to be under repression control. Coordinate expression of the two genes was observed under most conditions investigated. They might thus form an operon. The preparation of strains blocked in the degradation of cytidine and harboring leaky mutations affecting several steps of pyrimidine nucleotide synthesis made it possible to distinguish between the effects of cytidine and uridine compounds in the repression of the pyrimidine pathway enzymes. The data obtained suggest that derivatives of both cytidine and uridine participate in the repression of CPSase. In addition, repression of CPSase by arginine did not appear to occur unless pyrimidines were present at a significant intracellular concentration. This observation, together with our previous report that argR mutations impair the cumulative repression of CPSase, suggests that this control is mediated through the concerted effects of regulatory elements specific for the arginine and pyrimidine pathways.     

Cloning and characterization of argR, a gene that participates in regulation of arginine biosynthesis and catabolism in Pseudomonas aeruginosa PAO1.

Gel retardation experiments indicated the presence in Pseudomonas aeruginosa cell extracts of an arginine-inducible DNA-binding protein that interacts with the control regions for the car and argF operons, encoding carbamoylphosphate synthetase and anabolic ornithine carbamoyltransferase, respectively. Both enzymes are required for arginine biosynthesis. The use of a combination of transposon mutagenesis and arginine hydroxamate selection led to the isolation of a regulatory mutant that was impaired in the formation of the DNA-binding protein and in which the expression of an argF::lacZ fusion was not controlled by arginine. Experiments with various subclones led to the conclusion that the insertion affected the expression of an arginine regulatory gene, argR, that encodes a polypeptide with significant homology to the AraC/XylS family of regulatory proteins. Determination of the nucleotide sequence of the flanking regions showed that argR is the sixth and terminal gene of an operon for transport of arginine. The argR gene was inactivated by gene replacement, using a gentamicin cassette. Inactivation of argR abolished arginine control of the biosynthetic enzymes encoded by the car and argF operons. Furthermore, argR inactivation abolished the induction of several enzymes of the arginine succinyltransferase pathway, which is considered the major route for arginine catabolism under aerobic conditions. Consistent with this finding and unlike the parent strain, the argR::Gm derivative was unable to utilize arginine or ornithine as the sole carbon source. The combined data indicate a major role for ArgR in the control of arginine biosynthesis and aerobic catabolism.     

Arginine-sensitive phenotype of mutations in pyrA of Salmonella typhimurium: role of ornithine carbamyltransferase in the assembly of mutant carbamylphosphate synthetase.

The phenotype of certain mutations in pyrA, the gene encoding carbamylphosphate synthetase (CPSase), is expressed only in the presence od exogenous arginine. In unsupplemented media, synthesis of carbamylphosphate and growth was almost normal; in arginine-containing media, synthesis of carbamylphosphate stopped, as did growth, as a consequence of starvation for pyrimidine. Genetic and biochemical evidence suggests that arginine exerts this inhibition by repressing the synthesis of ornithine carbamyltransferase (OTCase), the intracellular presence of which is required for assembly of the unequal subunits and proper functioning of the mutant CPSase. After the addition of arginine to a culture of the mutant, CPSase activity (glutamine dependent) characteristic of the intact holoenzyme progressively decreased, whereas activity (ammonia dependent) characteristic of the free large (alpha) subunit increased. Extracts of mutant cells contain free small (beta) subunits, as demonstrated directly by in vitro complementation using purified alpha subunits from wild type. The mutant enzyme from cultures grown in the presence of arginine had a markedly decreased affinity for adenosine 5'-triphosphate. Mutations in argR that cause depressed synthesis of OTCase suppressed the phenotype, and a certain mutation in argI, the gene encoding OTCase, enhanced it. In vitro experiments using purified enzyme confirm the stimulatory effect of OTCase on the activity of mutant CPSase.     

Regulation of arginine and pyrimidine biosynthesis in Pseudomonas putida.

Repression of biosynthetic enzyme synthesis in Pseudomonas putida is incomplete even when the bacteria are growing in a nutritionally complex environment. The synthesis of four of the enzymes of the arginine biosynthetic pathway (N-acetyl-alpha-glutamokinase/N-acetylglutamate-gamma-semialdehyde dehydrogenase, ornithine carbamoyltransferase and acetylornithine-delta-transaminase) could be repressed and derepressed, but the maximum difference observed between repressed and derepressed levels for any enzyme of the pathway was only 5-fold (for ornithine carbamoyltransferase). No repression of five enzymes of the pyrimidine biosynthetic pathway (aspartate carbamoyltransferase, dihydro-orotase, dihydro-orotate dehydrogenase, orotidine-5'-phosphate pyrophosphorylase and orotidine-5'-phosphate decarboxylase) could be detected on addition of pyrimidines to minimal asparagine cultures of P. putida A90, but a 1-5- to 2-fold degree of derepression was found following pyrimidine starvation of pyrimidine auxotrophic mutants of P. putida A90. Aspartate carbamoyltransferase in crude extracts of P. putida A90 was inhibited in vitro by (in order of efficiency) pyrophosphate, CTP, UTP and ATP, at limiting but not at saturating concentrations of carbamoyl phosphate.     

钝齿棒杆菌argR基因克隆、表达及其重组菌发酵产精氨酸研究

钝齿棒杆菌(Corynebacterium crenatum)AS.M7是筛选获得的一株高产精氨酸生产菌株。ArgR是精氨酸合成过程中的一种调控蛋白。为进一步验证其在钝齿棒杆菌中对精氨酸合成量的影响,利用特异性引物,分别扩增标准菌C. creantum AS 1.542和诱变菌C. creantum AS.M7的argR全长基因,测序后比较二者的差异;结果表明标准菌argR基因ORF全长516 bp,编码一个含172个氨基酸残基的蛋白;而诱变菌argR基因的109位碱基由C替换为T,导致ArgR蛋白在钝齿诱变菌中表达被提前终止。同时,将来源于标准菌的argR基因连接到穿梭表达载体pXMJ19中,电击转化至诱变菌C. crenatum AS.M7 得到重组菌株,用摇瓶发酵的方法观测重组菌产精氨酸量的变化。SDS-PAGE和Western blot分析证明标准菌的argR基因在诱变菌中得到了表达。对重组诱变菌产精氨酸量进行了测定,结果显示:产精氨酸能力由原来7.8 mg/ml下降至2.5 mg/ml,下降了约67.9%。     

Construction of an L-arginine-producing mutant in Serratia marcescens. Use of the wide substrate specificity of acetylornithinase.

L-Arginine biosynthesis in Serratia marcescens Sr41 was found to be controlled by (a) feedback inhibition of N-acetylglutamate synthetase and (b) repression of some L-arginine biosynthetic enzymes, and an L-arginine-degrading system was found to exist. Accordingly, an L-arginine-producing mutant (aru argR argA) of S. marcescens Sr41 was constructed as follows. A mutant incapable of L-arginine utilization (aru) was obtained from the wild strain. Subsequently, from the lysine auxotroph (lysA) of aru mutant, a mutant having derepressed L-arginine biosynthetic enzymes (argR) was isolated by screening for colonies that could utilize Nalpha-acetyl-L-lysine in the presence of L-arginine. This selection was based on the finding that acetylornithinase of S. marcescens hydrolyzed Nalpha-acetyl-L-lysine. On the other hand, to obtain a mutant with feedback-resistant N-acetylglutamate synthetase (argA), the proAB argD argR triple mutant was isolated from the indirectly suppressed revertant (proAB argD) of the proline auxotroph (proAB). Next, the argA mutant was isolated from the triple mutant by selection for resistance to 3,4-dehydro-DL-proline in the presence of L-arginine. The argA mutation was introduced into the aru lysA argR strain by PS20-mediated cotransduction with lysA+. The aru argR argA lysA+ transductant produced 25 mg/ml of L-arginine in the medium.     

Control of Pyrimidine Biosynthesis in Synchronously Dividing Cells of Helianthus tuberosus

Factors with potential for regulating pyrimidine biosynthesis in plant tissue have been explored in quiescent cells of Helianthus tuberosus induced to divide by auxin addition. Investigations confined to the first highly synchronous cell cycle of the tuber explants revealed that the relative activity of asparate carbamoyltransferase (ACTase) to ornithinecarbamoyltransferase (OCTase) (enzymes competing for carbamoyl phosphate for the pyrimidine and arginine pathways, respectively) changes from 0.5 in quiescent cells to 3.0 by the end of the first cell cycle. This was interpreted as a change in the state of cell function from accumulation of storage arginine to cell division with a concomitant demand for pyrimidine nucleotides for nucleic acid synthesis. The rise in ACTase activity began at the same time as the initiation of DNA synthesis and was dependent on continued DNA synthesis. OCTase activity declined whether or not auxin was added to the medium, whereas ACTase activity was observed to decline only in the absence of DNA synthesis.     

ArgR and AhrC are both required for regulation of arginine metabolism in Lactococcus lactis

The DNA binding proteins ArgR and AhrC are essential for regulation of arginine metabolism in Escherichia coli and Bacillus subtilis, respectively. A unique property of these regulators is that they form hexameric protein complexes, mediating repression of arginine biosynthetic pathways as well as activation of arginine catabolic pathways. The gltS-argE operon of Lactococcus lactis encodes a putative glutamate or arginine transport protein and acetylornithine deacetylase, which catalyzes an important step in the arginine biosynthesis pathway. By random integration knockout screening we found that derepression mutants had ISS1 integrations in, among others, argR and ahrC. Single as well as double regulator deletion mutants were constructed from Lactococcus lactis subsp. cremoris MG1363. The three arginine biosynthetic operons argCJDBF, argGH, and gltS-argE were shown to be repressed by the products of argR and ahrC. Furthermore, the arginine catabolic arcABD1C1C2TD2 operon was activated by the product of ahrC but not by that of argR. Expression from the promoter of the argCJDBF operon reached similar levels in the single mutants and in the double mutant, suggesting that the regulators are interdependent and not able to complement each other. At the same time they also appear to have different functions, as only AhrC is involved in activation of arginine catabolism. This is the first study where two homologous arginine regulators are shown to be involved in arginine regulation in a prokaryote, representing an unusual mechanism of regulation.     

Induction of holomycin production and complex metabolic changes by the argR mutation in Streptomyces clavuligerus NP1

In bacteria, arginine biosynthesis is tightly regulated by a universally conserved regulator, ArgR, which regulates the expression of arginine biosynthetic genes, as well as other important genes. Disruption of argR in Streptomyces clavuligerus NP1 resulted in complex phenotypic changes in growth and antibiotic production levels. To understand the metabolic changes underlying the phenotypes, comparative proteomic studies were carried out between NP1 and its argR disruption mutant (designated CZR). In CZR, enzymes involved in holomycin biosynthesis were overexpressed; this is consistent with its holomycin overproduction phenotype. The effects on clavulanic acid (CA) biosynthesis are more complex. Several proteins from the CA cluster were moderately overexpressed, whereas several proteins from the 5S clavam biosynthetic cluster and from the paralog cluster of CA and 5S clavam biosynthesis were severely downregulated. Obvious changes were also detected in primary metabolism, which are mainly reflected in the altered expression levels of proteins involved in acetyl-coenzyme A (CoA) and cysteine biosynthesis. Since acetyl-CoA and cysteine are precursors for holomycin synthesis, overexpression of these proteins is consistent with the holomycin overproduction phenotype. The complex interplay between primary and secondary metabolism and between secondary metabolic pathways were revealed by these analyses, and the insights will guide further efforts to improve production levels of CA and holomycin in S. clavuligerus.     

Salmonella typhimurium mutants with altered expression of the pyrA gene due to changes in RNA polymerase

Rifampicin-resistant mutants of Salmonella typhimurium were isolated and tested for pleiotropic defects in the regulation of pyr gene expression. Seven per cent of all the Rifr mutants were inhibited in growth by addition of uracil (uracil-sensitive). The uracil-sensitive phenotype ( UraS ) was reversed by arginine or citrulline, but not by ornithine, and it was suppressed by mutations in either argR or pyrH , which causes increased expression of pyrA . It was shown that the basal levels of carbamoylphosphate synthase (the pyrA gene product) was reduced to approximately 60% in the mutants, and that addition of arginine and/or uracil to the growth medium caused hyperrepression of pyrA expression. The expression of other genes of the arginine and pyrimidine biosynthetic pathways was not affected significantly in the mutants. The mutations were located in the rpoB gene coding for the beta-subunit of RNA polymerase, suggesting a regulatory function of RNA polymerase in the control of pyrA expression.     

C. elegans pharyngeal morphogenesis requires both de novo synthesis of pyrimidines and synthesis of heparan sulfate proteoglycans

The C. elegans pharynx undergoes elongation and morphogenesis to its characteristic bi-lobed shape between the 2- and 3-fold stages of embryogenesis. During this period, the pharyngeal muscles and marginal cells forming the isthmus between the anterior and posterior pharyngeal bulbs elongate and narrow. We have identified the spontaneous mutant pyr-1(cu8) exhibiting defective pharyngeal isthmus elongation, cytoskeletal organization defects, and maternal effect lethality. pyr-1 encodes CAD, a trifunctional enzyme required for de novo pyrimidine synthesis, and pyr-1(cu8) mutants are rescued by supplying exogenous pyrimidines. Similar pharyngeal defects and maternal effect lethality were found in sqv-1, sqv-8, rib-1 and rib-2 mutants, which affect enzymes involved in heparan sulfate proteoglycan (HSPG) synthesis. rib-1 mutant lethality was enhanced in a pyr-1 mutant background, indicating that HSPG synthesis is very sensitive to decreased pyrimidine pools, and HS disaccharides are moderately decreased in both rib-1 and pyr-1 mutants. We hypothesize that HSPGs are necessary for pharyngeal isthmus elongation, and pyr-1 functions upstream of proteoglycan synthesizing enzymes by providing precursors of UDP-sugars essential for HSPG synthesis.     

The arginine regulatory protein mediates repression by arginine of the operons encoding glutamate synthase and anabolic glutamate dehydrogenase in Pseudomonas aeruginosa

The arginine regulatory protein of Pseudomonas aeruginosa, ArgR, is essential for induction of operons that encode enzymes of the arginine succinyltransferase (AST) pathway, which is the primary route for arginine utilization by this organism under aerobic conditions. ArgR also induces the operon that encodes a catabolic NAD(+)-dependent glutamate dehydrogenase (GDH), which converts l-glutamate, the product of the AST pathway, in alpha-ketoglutarate. The studies reported here show that ArgR also participates in the regulation of other enzymes of glutamate metabolism. Exogenous arginine repressed the specific activities of glutamate synthase (GltBD) and anabolic NADP-dependent GDH (GdhA) in cell extracts of strain PAO1, and this repression was abolished in an argR mutant. The promoter regions of the gltBD operon, which encodes GltBD, and the gdhA gene, which encodes GdhA, were identified by primer extension experiments. Measurements of beta-galactosidase expression from gltB::lacZ and gdhA::lacZ translational fusions confirmed the role of ArgR in mediating arginine repression. Gel retardation assays demonstrated the binding of homogeneous ArgR to DNA fragments carrying the regulatory regions for the gltBD and gdhA genes. DNase I footprinting experiments showed that ArgR protects DNA sequences in the control regions for these genes that are homologous to the consensus sequence of the ArgR binding site. In silica analysis of genomic information for P. fluorescens, P. putida, and P. stutzeri suggests that the findings reported here regarding ArgR regulation of operons that encode enzymes of glutamate biosynthesis in P. aeruginosa likely apply to other pseudomonads.