Salt sensitivity and arginine toxicity in Aspergillus nidulans

Biochimica et Biophysica Acta (BBA) - General Subjects, 1967

I. The catabolism of exogenous and endogenously biosynthesized L-arginine has been studied in Neurospora erassa by growing the strains wild-type 74 A and the mutant auxotroph for L-proline, arg-g, in L-arginine or L-citrulline, with or withou~ NH~NO a. 2. A culture of N. crassa 74 A or arg-8 growing in the presence of L-citrulline (as nitrogen source, and nitrogen/L-proline source, respectively) develops an ammoniurn deficiency, as judged by a lower pool of almost all the amino acids, an increase in the ratio of dry weight to protein, and a low rate of growth. 3-The ammonium starvation occurs in the presence of a highly elevated pool of L-citrulline and L-arginine, and of a ful!y induced arginase, ornithine transaminase and urease. 4. NH4NOa or L-arginine when present in the medium reverse the ammonium deficiency. The citrulline and arginine pool, and the arginase, ornithine transaminase and urease activities, are then restored to basal levels and the catabolism of endogenous arginine is accelerated. 5. The basis for the preferential utilization of exogenous arginme versus endogenous arginine, that occurs in the absence of NH4NO a, is discussed in relation to channeling and cell organization.

European Journal of Biochemistry, 1970

The pools of arginine, ornithine and citrulline have been measured under different conditions of growth in wild type and argR mutants (non-repressible for ornithine transcarbamylase and four other enzymes of the arginine biosynthetic pathway). Comparing these pools with the level of ornithine transcarbamylase and the rate of growth one may conclude:1The arginine pool of a wild type strain growing on ammonium as nitrogen source is 18 mM; it increases to 72 mM by addition of arginine to the medium and causes 95% repression of ornithine transcarbamylase synthesis. In decreasing the pool to 3.7 mM by a bradytrophic mutation the differential rate of ornithine transcarbamylase synthesis increases 6 times.2When the pool of ornithine is increased by external addition of this amino acid or by using ornithine as the only nitrogen source a repression occurs which reaches 50 to 70% simulataneously with an intense reduction of the arginine pool (≤ 1.2 mM). This low pool may reach a growth limiting value. This shows the existence of a double control by arginine and ornithine. Lysine mimics ornithine.3The primary effect of argR mutation is not at the level of the arginine pool.4A double regulatory mutant, argR and cpaO (which confers non-respressibility to carbamoyl-phosphate synthetase) shows that the double control of arginine and ornithine also reaches this enzyme.5The strain bearing simultaneously the argR and cpaO mutations shows an intense distrurbance of metabolism leading to cessation of macromolecules biosyntheses and a loss of viability when basic amino acids such as lysine, ornithine, α-γ-diamino butyric acid or arginine are added to a culture growing on glutamate as nitrogen source.6Citrulline is shown to be used as nitrogen source via arginine, under these conditions the arginine pool increases to 190 mM, but does not produce the expected high repression. Citrulline or a derivative attenine repression.The arginine pool of a wild type strain growing on ammonium as nitrogen source is 18 mM; it increases to 72 mM by addition of arginine to the medium and causes 95% repression of ornithine transcarbamylase synthesis. In decreasing the pool to 3.7 mM by a bradytrophic mutation the differential rate of ornithine transcarbamylase synthesis increases 6 times.When the pool of ornithine is increased by external addition of this amino acid or by using ornithine as the only nitrogen source a repression occurs which reaches 50 to 70% simulataneously with an intense reduction of the arginine pool (≤ 1.2 mM). This low pool may reach a growth limiting value. This shows the existence of a double control by arginine and ornithine. Lysine mimics ornithine.The primary effect of argR mutation is not at the level of the arginine pool.A double regulatory mutant, argR and cpaO (which confers non-respressibility to carbamoyl-phosphate synthetase) shows that the double control of arginine and ornithine also reaches this enzyme.The strain bearing simultaneously the argR and cpaO mutations shows an intense distrurbance of metabolism leading to cessation of macromolecules biosyntheses and a loss of viability when basic amino acids such as lysine, ornithine, α-γ-diamino butyric acid or arginine are added to a culture growing on glutamate as nitrogen source.Citrulline is shown to be used as nitrogen source via arginine, under these conditions the arginine pool increases to 190 mM, but does not produce the expected high repression. Citrulline or a derivative attenine repression.

PLoS ONE, 2013

The opportunistic fungal pathogen Aspergillus fumigatus produces siderophores for uptake and storage of iron, which is essential for its virulence. The main precursor of siderophore biosynthesis (SB), ornithine, can be produced from glutamate in the mitochondria or by cytosolic hydrolysis of ornithine-derived arginine. Here, we studied the impact of mitochondrial versus cytosolic ornithine biosynthesis on SB by comparison of the arginine auxotrophic mutants DargEF and DargB, which lack and possess mitochondrial ornithine production, respectively. Deficiency in argEF (encoding acetylglutamate kinase and acetylglutamyl-phosphate-reductase), but not argB (encoding ornithine transcarbamoyl transferase) decreased (i) the cellular ornithine content, (ii) extra-and intracellular SB, (iii) growth under harsh iron starvation, (iv) resistance to the ornithine decarboxylase inhibitor eflornithine, and (v) virulence in the Galleria mellonella larvae model. These lines of evidence indicate that SB is mainly fueled by mitochondrial rather than cytosolic ornithine production and underline the role of SB in virulence. Ornithine content and SB of DargB increased with declining arginine supplementation indicating feedback-inhibition of mitochondrial ornithine biosynthesis by arginine. In contrast to SB, the arginine and polyamine contents were only mildly affected in DargEF, indicating prioritization of the latter two ornithine-consuming pathways over SB. These data highlight the metabolic differences between the two arginine auxotrophic mutants DargEF and DargB and demonstrate that supplementation of an auxotrophic mutant does not restore the wild type metabolism at the molecular level, a fact to be considered when working with auxotrophic mutants. Moreover, cross pathway control-mediating CpcA was found to influence the ornithine pool as well as biosynthesis of siderophores and polyamines.

Journal of Bacteriology, 2004

To help clarify the control of arginine synthesis in Thermotoga maritima, the putative gene (argB) for N-acetyl-L-glutamate kinase (NAGK) from this microorganism was cloned and overexpressed, and the resulting protein was purified and shown to be a highly thermostable and specific NAGK that is potently and selectively inhibited by arginine. Therefore, NAGK is in T. maritima the feedback control point of arginine synthesis, a process that in this organism involves acetyl group recycling and appears not to involve classical acetylglutamate synthase. The inhibition of NAGK by arginine was found to be pH independent and to depend sigmoidally on the concentration of arginine, with a Hill coefficient (N) of ϳ4, and the 50% inhibitory arginine concentration (I 0.5) was shown to increase with temperature, approaching above 65°C the I 0.50 observed at 37°C with the mesophilic NAGK of Pseudomonas aeruginosa (the best-studied arginine-inhibitable NAGK). At 75°C, the inhibition by arginine of T. maritima NAGK was due to a large increase in the K m for acetylglutamate triggered by the inhibitor, but at 37°C arginine also substantially decreased the V max of the enzyme. The NAGKs of T. maritima and P. aeruginosa behaved in gel filtration as hexamers, justifying the sigmoidicity and high Hill coefficient of arginine inhibition, and arginine or the substrates failed to disaggregate these enzymes. In contrast, Escherichia coli NAGK is not inhibited by arginine and is dimeric, and thus the hexameric architecture may be an important determinant of arginine sensitivity. Potential thermostability determinants of T. maritima NAGK are also discussed.

Fungal Genetics and Biology, 2003

The arginine catabolism gene otaA encoding ornithine transaminase (OTAse) is specifically induced by arginine and is under the control of the broad-domain carbon and nitrogen repression systems. Arginine induction is mediated by a product of arcA gene coding for Zn 2 C 6 activator. We have identified a region responsible for arginine induction in the otaA promoter (AnUAS arg ). Deletions within this region result in non-inducibility of OTAse by arginine, whether in an arcA þ strain or in the presence of the arcA d 47 gain of function allele. AnUAS arg is very similar to the Saccharomyces cerevisiae UAS arg , a sequence bound by the Zn 2 C 6 activator (ArgRIIp), acting in a complex with two MADS-box proteins (McmIp and ArgRIp).We demonstrate here that two CREA in vitro binding sites in the otaA promoter are functional in vivo. CREA is directly involved in carbon repression of the otaA gene and it also reduces its basal level of expression. Although AREA binds to the otaA promoter in vitro, it probably does not participate in nitrogen metabolite repression of the gene in vivo. We show here that another putative negatively acting GATA factor AREB participates directly or indirectly in otaA nitrogen repression. We also demonstrate that the high levels of OTAse activity are an important factor in the suppression of proline auxotrophic mutations. This suppression can be achieved neither by growing of the proline auxotroph under carbon/nitrogen derepressing conditions nor by introducing of a creA d mutation.

Microbiology, 2015

L-Ornithine production in the alfalfa microsymbiont Sinorhizobium meliloti occurs as an intermediate step in arginine biosynthesis. Ornithine is required for effective symbiosis but its synthesis in S. meliloti has been little studied. Unlike most bacteria, S. meliloti 1021 is annotated as encoding two enzymes producing ornithine: N-acetylornithine (NAO) deacetylase (ArgE) hydrolyses NAO to acetate and ornithine, and glutamate N-acetyltransferase (ArgJ) transacetylates L-glutamate with the acetyl group from NAO, forming ornithine and N-acetylglutamate (NAG). NAG is the substrate for the second step of arginine biosynthesis catalysed by NAG kinase (ArgB). Inactivation of argB in strain 1021 resulted in arginine auxotrophy. The activity of purified ArgB was significantly inhibited by arginine but not by ornithine. The purified ArgJ was highly active in NAO deacetylation/glutamate transacetylation and was significantly inhibited by ornithine but not by arginine. The purified ArgE protein (with a 6His-Sumo affinity tag) was also active in deacetylating NAO. argE and argJ single mutants, and an argEJ double mutant, are arginine prototrophs. Extracts of the double mutant contained aminoacylase (Ama) activity that deacetylated NAO to form ornithine. The purified products of three candidate ama genes (smc00682 (hipO1), smc02256 (hipO2) and smb21279) all possessed NAO deacetylase activity. hipO1 and hipO2, but not smb21279, expressed in trans functionally complemented an Escherichia coli DargE : : Km mutant. We conclude that Ama activity accounts for the arginine prototrophy of the argEJ mutant. Transcriptional assays of argB, argE and argJ, fused to a promoterless gusA gene, showed that their expression was not significantly affected by exogenous arginine or ornithine.

Fungal Genetics and …, 2003

Fems Microbiology Letters, 1981

Journal of Bacteriology, 1975

The accumulation or ornithine, citrulline, and possibly acetylornithine by Escherichia coli K-12 arginineless mutants provided with acetylarginine as source of arginine causes severe growth inhibition. This occurs under conditions where comparable derivatives of E. coli W (Bollon and Vogel, 1973) show little or no growth inhibition. The same conditions, which have been reported to cause noncorrelative synthesis of acetylornithinase and argininosuccinase in E. coli W (Bollon and Vogel, 1973), do not alter the correlative pattern of enzyme synthesis observed in E. coli K-12. Moreover, previously reported effects of ornithine and citrulline on repression of the arginine regulon in E. coli W are not observed in the K-12 strains examined. The bearing of these observations on possible differences between the mechanism of enzyme repression operating in the two types of strains cannot yet be fully evaluated; it is, however, clear that considerable care should be exercised before extrapolati...

Journal of General Microbiology, 1976

Arginine and methionine transport by Aspergillus nidulans mycelium was investigated. A single uptake system is responsible for the transport of arginine, lysine and ornithine. Transport is energy-dependent and specific for these basic amino acids.