Nucleotide sequence of the carA gene and regulation of the carAB operon in Salmonella typhimurium

Abstract

The carAB operon of Salmonella typhimurium encoding carbamoyl-phosphate synthetase (CPSase) has been cloned, and the nucleotide sequence of the first gene of the operon, carA, together with 760 base pairs of the 5'-flanking region was determined. The product of the carA gene is the small subunit of CPSase. It catalyzes the transfer of the amide group from glutamine to an NH,-site on the heavy subunit. Primer extension and S1 nuclease mapping of in vivo carAB transcripts revealed that transcription is similar to that of Escherichia co/i [Piette, J. et al. (1984) Proc. Nut1 Acad. Sci. U S A 81, 4134-41381 in its initiation at two promoters, PI and P2, controlled by pyrimidines and arginine, respectively. The arginine control is mediated through binding to the arginine repressor (argR). The involvement of titratable regulatory elements is indicated by the escape from both arginine and pyrimidine control, when the operon is present in multicopies on a plasmid. Measurements of CPSase levels in mutants which allows independent manipulation of the intracellular uracil and cytosine nucleotide pools show, that both uracil and cytosine nucleotides are required for full repression and that limitation of either nucleotide results in derepression of CPSase synthesis. Deletion analyses indicate that regions upstream of the PI promoter are required for normal expression from this promoter but not from P2.Carbamoyl-phosphate synthetase (CPSase) encoded by the carAB (pyrA) operon in Salmonella typhimurium [3] and Escherichia coli [4], catalyzes the synthesis of carbamoyl-phosphate from ATP, bicarbonate and glutamine. It plays a dual role in metabolism by supplying carbamoyl phosphate for both the pyrimidine and the arginine biosynthetic pathways. In accordance with its physiological function, CPSase is allosterically regulated by intermediates of both pathways. The pyrimidine nucleotide UMP inhibits CPSase activity while ornithine, an intermediate in the arginine biosynthetic pathway stimulates it [5].The enzyme from S. typhimurium consists of two nonidentical subunits. The light subunit (Mr = 45000), encoded by the first gene in the operon (carA), converts the amide nitrogen of glutamine to ammonia. The heavy subunit ( M , = IlOOOO), encoded by the second gene of the operon (carB), catalyzes the formation of carbamoyl-phosphate from ammonia, carbon dioxide and ATP, it also contains the binding sites for ornithine. The ammonia-dependent activity of the isolated heavy subunit is only slightly inhibited by high concentrations of UMP and the reconstituted holoenzyme regains sensitivity to this feedback inhibitor [3]. [I]. ,In S. typhimuriurn the homologous locus is designated pyrA [2]. In order to be consistent we propose that the E. coli nomenclature is adopted for the genes encoding S. typhimurium carbamoyl-phosphate synthetase.Expression of carAB is subject to cumulative repression by pyrimidines and arginine. In E. coli [4, 6, 71 transcription of the operon was shown to be initiated at tandem promoters. Transcription from th...

Effect of growth temperature on folding of carbamoylphosphate synthetases of Salmonella typhimurium and a cold-sensitive derivative

“…All available mutants including cold-sensitive mutants map in the same carAB locus (1,37). This operon has been cloned and fully characterized (19,23). Further, there is no evidence for chemical modification of CPSases from enteric bacteria.…”

“…S. typhimurium AA222 (carAB81 pyrB: :TnlO) carrying the carAB operon on a multicopy plasmid (pMK1 [19]) was grown in glucose minimal medium (33) supplemented with uracil (50 ,ug/ml) and used as a source for wild-type CPSase. S. typhimurium 5090 HAN ET AL.…”

“…The large subunit alone can catalyze the synthesis of carbamoylphosphate from ammonia, bicarbonate, and MgATP (2). In accordance with its physiological function, the expression of the carAB operon is subject to control by arginine and pyrimidines (19,23). Further, CPSase activity is subject to allosteric control by intermediates of pyrimidine and arginine biosynthesis: UMP inhibits the reaction, whereas ornithine stimulates it (3).…”

The Salmonella typhimurium uracil-sensitive mutation use is in argU and encodes a minor arginine tRNA

“…Previous studies from this laboratory confirmed the effect of the use-i mutation on carAB expression and showed that this effect is exerted at P1, one of the tandem promoters for the carAB operon (11 (4). A lambda phage clone (Xcya14), generously provided by S. Artz (University of California, Davis), which has an 18-kb chromosomal insert with cya on one end, was examined to determine whether it carries the use gene (Fig.…”

“…The only exception is pyrF, in which this codon is present in 4 of 15 arginine codons. Since the uracil-sensitive phenotype has been shown to be the result of carbamoylphosphate deficiency (3,4,11), the phenotype does not appear to be related to the frequency of usage for the codon among pyr genes.…”