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.     

Effect of chloramphenicol and ethidium bromide on the level of ornithine carbamoyltransferase in Neurospora crassa.

The specific activity of the nuclear-gene-encoded, mitochondrial arginine biosynthetic enzyme ornithine carbamoyltransferase (EC 2.1.3.3) in Neurospora crassa was elevated in mycelia treated with chloramphenicol or ethidium bromide. The increase in specific activity was caused by an increase in the number of mature enzyme molecules rather than by the activation of a preexisting enzyme. Chloramphenicol and ethidium bromide appeared to act indirectly via arginine-mediated derepression. However, derepression did not appear to result from a drug-mediated decrease in the arginine pool.     

Repression and derepression of the enzymes of the pyrimidine biosynthetic pathway in Salmonella typhimurium

Activities of five enzymes of the pyrimidine biosynthetic pathway and one enzyme involved in arginine synthesis were measured during batch culture of Salmonella typhimurium. Aspartate carbamoyltransferase, dihydroorotase, and the arginine pathway enzyme, ornithine carbamoyltransferase, remained constant during the growth cycle but showed a sharp decrease in activity after entering the stationary phase. Dihydroorotate dehydrogenase, orotate phosphoribosyltransferase and orotidine-5'-monophosphate (OMP) decarboxylase showed peaks of activity corresponding to the mid-point of the exponential phase of growth while remaining comparatively stable in the stationary phase. Derepression studies carried out by starving individual pyrimidine (Pyr-) deletion mutants for uracil showed that the extent of derepression obtained for aspartate carbamoyltransferase, dihydroorotase and dihydroorotate dehydrogenase depended on the location of the pyr gene mutation. Orotate phosphoribosyltransferase and OMP decarboxylase derepression levels were independent of the location of the pyr mutation. Aspartate carbamoyltransferase showed the greatest degree of derepression of the six enzymes studied, with pyrA strains (blocked in the first step of the pathway) showing about twice as much derepression as pyrF strains (blocked in the sixth step of the pathway). A study of the kinetics of repression on derepressed levels of the pyrimidine enzymes produced data that were compatible with dilution of specific activity by cell division when repressive amounts of uracil were added to the derepression medium.     

Implication of a repression system,homologous to those of other bacteria,in the control of arginine biosynthesis genes inStreptomyces coelicolor

As with most amino acid biosynthetic pathways in streptomycetes, enzymes of arginine biosynthesis inStreptomyces coelicolor show only slight derepression in minimal medium without, as opposed to with, exogenous arginine. However, when an arginine auxotroph was cultured in limiting arginine, ornithine carbamoyltransferase (OCT) activities rose by as much as 100-fold. The response was not due to a general starvation effect. To elucidate the repression-derepression mechanism, a DNA fragment containing the upstream region of the previously isolatedS. coelicolor argCJB cluster was cloned into a multicopy vector and transformed into wild-typeS. coelicolor; a slight transient derepression of OCT was observed in minimal medium without, though not with, added arginine, consistent with titration by the insert of a negatively acting macromolecule such as a repressor. A sub-fragment carrying the 5′ end ofargC and the region immediately upstream showed specific binding, in mobility shift assays, to purified AhrC, the repressor/activator of genes of arginine metabolism inBacillus subtilis. It is therefore likely that inS. coelicolor, expression of arginine biosynthesis genes is controlled by a protein homologous to the well-characterisedB. subtilis andEscherichia coli repressors.     

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.     

Implication of a repression system, homologous to those of other bacteria, in the control of arginine biosynthesis genes inStreptomyces coelicolor

As with most amino acid biosynthetic pathways in streptomycetes, enzymes of arginine biosynthesis inStreptomyces coelicolor show only slight derepression in minimal medium without, as opposed to with, exogenous arginine. However, when an arginine auxotroph was cultured in limiting arginine, ornithine carbamoyltransferase (OCT) activities rose by as much as 100-fold. The response was not due to a general starvation effect. To elucidate the repression-derepression mechanism, a DNA fragment containing the upstream region of the previously isolatedS. coelicolor argCJB cluster was cloned into a multicopy vector and transformed into wild-typeS. coelicolor; a slight transient derepression of OCT was observed in minimal medium without, though not with, added arginine, consistent with titration by the insert of a negatively acting macromolecule such as a repressor. A sub-fragment carrying the 5 end ofargC and the region immediately upstream showed specific binding, in mobility shift assays, to purified AhrC, the repressor/activator of genes of arginine metabolism inBacillus subtilis. It is therefore likely that inS. coelicolor, expression of arginine biosynthesis genes is controlled by a protein homologous to the well-characterisedB. subtilis andEscherichia coli repressors.     

Partial characterization of ornithine carbamoyltransferase in three microalgae : anabolic role only

Although the existence of isozymes of ornithine carbamoyltransferase (carbamoylphosphate:l-ornithine carbamoyltransferase, EC 2.1.3.3) in higher plants has been reported, and the possibility exists that one or more of these operates catabolically to produce ornithine and carbamoylphosphate from citrulline and inorganic phosphate, no proof has been forthcoming. In view of the fact that many unicellular algae degrade arginine via arginine deiminase to citrulline and ammonium, and that the pathway of utilization of citrulline is unknown, we decided to investigate the possibility of the presence of a catabolic form of ornithine carbamoyltransferase in three microalgae known to have arginine deiminase activity. These were Chlorella autotrophica, Chlorella saccharophila, and Dunaliella tertiolecta. Our results show that the properties of OCT from these three algae are similar to OCTs from many higher plants with respect to general kinetics (K_m values for ornithine and carbamoylphosphate), substrate inhibition by ornithine at high pHs, apparent sequential ordered kinetic mechanisms and paucity of apparent regulatory properties. Our data indicate an exclusively anabolic role of ornithine carbamoyltransferase in these algae.     

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.     

Induction and repression of arginase and ornithine transaminase in baker's yeast

The arginase and the ornithine transaminase of baker's yeast are induced byl-arginine. Both enzymes have been shown to be repressed by nitrogen compounds. This is evidenced primarily by the decrease in specific enzyme activities caused by the addition of readily assimilable nitrogen compounds to a yeast culture with arginine, secondly by the derepression of both enzymes during nitrogen starvation of the yeast grown in various arginine-free media. This derepression equals both in rate and in amount the enzyme synthesis during the adaptation of the yeast to a medium withl-arginine as the sole nitrogen source. It is inhibited by various assimilable and non-assimilable amino acids. The derepression is the result of the nitrogen deficiency itself, since during the starvation of the yeast for sulphate, phosphate or magnesium, neither of the two enzymes is derepressed, and since it is independent of the nature of the carbon source in the nitrogen starvation medium, provided the latter is immediately assimilable.The enzymes are not subject to catabolite repression by glucose metabolites.It is concluded that the synthesis of arginase and ornithine transaminase in yeast is regulated by induction and repression. Arginine induces the enzymes; they are repressed by nitrogen compounds, probably in cooperation with one or more vitamins.Thanks are due to Professor E. G. Mulder for his frequent encouragement, to the Heineken's Brouwerij, Rotterdam and to the Landbouwhogeschoolfonds for research grants, and to Miss H. P. M. Klinkers, to Mr. P. J. Buysman and to Mr. G. J. K. Pesch for their skilful technical assistance.     

Evidence that specific and "general" control of ornithine carbamoyltransferase production occurs at the level of transcription in Saccharomyces cerevisiae.

Ornithine carbamoyltransferase synthesis is subject to two major regulatory systems in Saccharomyces cerevisiae. One system is specific for the arginine biosynthetic enzymes, whereas the other appears to be general, acting on a variety of other amino acid pathways as well. We observed that the synthetic capacity for continued ornithine carbamoyltransferase synthesis had the same short half-life (ca. 5 to 7 min) whether repression of enzyme production was brought about by action of the specific or general control system. We present evidence suggesting that both control systems regulate accumulation or ornithine carbamoyltransferase-specific synthetic capacity, rather than modulating its expression.     

Control of a futile urea cycle by arginine feedback inhibition of ornithine carbamoyltransferase in Agrobacterium tumefaciens and Rhizobia

In Agrobacterium tumefaciens and Rhizobia arginine can be used as the sole nitrogenous nutrient via degradation by an inducible arginase. These microorganisms were found to exhibit arginine inhibition of ornithine carbamoyltransferase activity. This inhibition is competitive with respect to ornithine (Km for ornithine = 0.8 mM; Ki for arginine = 0.05 mM). This type of urea cycle regulation has not been observed among other microorganisms which degrade arginine via an arginase. The competitive pattern of this inhibition leads to its being inoperative in ornithine-grown cells, where the intracellular concentration of ornithine is high. In arginine-grown cells, however, the intracellular arginine and ornithine concentrations are compatible with inhibition and ornithine recycling appears to be effectively blocked in vivo.     

Arginine hydroxamate-resistant mutants of Bacillus subtilis with altered control of arginine metabolism.

Arginine hydroxamate inhibits the growth of Bacillus subtilis. From a large number of mutants isolated as resistant to this arginine analogue, 29 were chosen for further investigation. Most of these shared diminished ability to utilize arginine, citrulline and/or ornithine as sole nitrogen source. All 29 had reduced levels of the catabolic enzymes arginase and ornithine aminotransferase under various conditions in which these enzymes are induced in the parent. In some circumstances, five of the mutants also showed elevated levels of the biosynthetic enzyme ornithine carbamoyltransferase. On the basis of these data, the 29 mutants were divided into six phenotypic classes; in four of these, control of ornithine carbamoyltransferase was the same as in the wild type, while in the other two it was altered. It is suggested that the isolates carry regulatory mutations, and that certain of these may affect simultaneously the formation of arginine catabolic and biosynthetic enzymes. The implication of the latter is that in B. subtilis, as in yeast, controls of the catabolic and biosynthetic pathways are connected. Single representatives of five of the phenotypic classes carry mutations conferring arginine hydroxamate resistance linked to cysA by transduction with phage PBSI; this did not appear to be true for a representative of the sixth class.     

Bacillus subtilis 168 mutants resistant to arginine hydroxamate in the presence of ornithine or citrulline

Mutations in Bacillus subtilis 168 have been isolated that confer resistance to arginine hydroxamate in the presence, but not absence, of ornithine. Seven such Ahor mutants have been studied in detail. In common with certain classes of Ahr mutant (resistant to arginine hydroxamate in the absence of arginine precursors) described previously, these Ahor mutants showed little or no inducibility of enzymes of arginine catabolism. Mutants that showed no inducibility were unable to utilize arginine or ornithine as sole nitrogen source. The only biosynthetic enzyme to show any consistent differences in activity from the parent was ornithine carbamoyltransferase, whose level was slightly elevated in cells grown in the presence of ornithine or citrulline. PBS1 transduction crosses showed that two of the ahor mutations map at the ahrA locus, while a third (unique in its resistance to arginine hydroxamate in the presence of citrulline) mapped at a hitherto undescribed locus closely linked to metC, designated ahrD.     

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.     

Cross-Pathway Regulation: Histidine-Mediated Control of Histidine, Tryptophan, and Arginine Biosynthetic Enzymes in Neurospora crassa

In Neurospora crassa, histidine starvation of histidine mutants resulted in derepression of histidine, tryptophan, and arginine biosynthetic enzymes. The same tripartite derepression occurred in wild-type strain 74A when it was grown in medium supplemented with 3-amino-1,2,4-triazole, an inhibitor of histidine biosynthesis. Histidine-mediated derepression of tryptophan and arginine biosynthetic enzymes was not due to a lowered intracellular concentration of tryptophan or arginine, respectively. A discussion of possible mechanisms and of similar studies in prokaryotic and eukaryotic organisms is presented.     

The nature of arginine auxotrophy in cutaneous populations of staphylococci.

L-Arginine was required for growth by a high percentage of strains of Staphylococcus species that were niche-specific and/or host-specific, but was usually not required for growth by species showing a wide host range. Growth stimulation patterns with arginine intermediates indicated that most of the auxotrophic strains had blocks in an early step(s) in arginine biosynthesis. These strains were designated phenotypically as Arg(CHG) according to the Salmonella typhimurium classification scheme. Staphylococcus simulans strains appeared to be either ArgA or Arg I. The ArgI strains of S. simulans and S. capitis had moderate to high ornithine carbamoyltransferase (EC 2.1.3.3) activities and therefore could not be designated as argI mutants. ArgI strains in other species had no or very low ornithine carbamoyltransferase activities. All of the natural Staphylococcus auxotrophs tested grew in the presence of L-citrulline and had moderate to high argininosuccinase (EC 4.3.2.1) activities. Arginine auxotrophs of species with a wide host range were often capable of reverting to arginine-independent or complete prototrophic growth, whereas auxotrophs of species that tended to be niche-specific and/or host-specific were incapable of reversion to arginine-independence, even in the presence of various mutagens. A relationship between the nature of arginine auxotrophy and habitat is suggested.     

Cross-Pathway Regulation: Tryptophan-Mediated Control of Histidine and Arginine Biosynthetic Enzymes in Neurospora crassa

In Neurospora crassa, the starvation of tryptophan mutants for tryptophan resulted in the derepression of tryptophan, histidine, and arginine biosynthetic enzymes. This tryptophan-mediated derepression of histidine and arginine biosynthetic enzymes occurred despite the fact that the tryptophan-starved cells had a higher intracellular concentration of histidine and arginine than did nonstarved cells.     

Depression of enzyme synthesis in response to arginine limitation in Neurospora crassa

Ornithine carbamoyltransferase and argininosuccinase, two enzymes involved in arginine synthesis, are regulated by cross-pathway amino acid control in Neurospora and show derepression in response to limitation of any one of a number of amino acids. The effects of varying the severity of arginine limitation upon the synthesis of these enzymes, in mycelial cultures of an arginine auxotrophic strain, are reported here. Depression occurred at arginine concentrations sufficient to allow normal rates of protein accumulation, leading to increases of not more than fourfold in the absolute rate of enzyme synthesis. On the other hand, differential rates of enzyme synthesis increased progressively up to 20-fold or more under extreme conditions of arginine limitation that also limit net protein synthesis. The major part of the derepression response thus occurred at arginine concentrations that allowed low net rates of protein synthesis. The physiological significance of this is not yet understood. Our evidence suggests that these responses were mediated entirely through the cross-pathway control system, and may not be untypical (allowing for variations in magnitude) of depression resulting through this mechanism in Neurospora.