Regulation of the Expression of De Novo Pyrimidine Biosynthesis Genes in Corynebacterium glutamicum

Abstract

Expression of pyrimidine de novo biosynthesis is downregulated by an exogenous uracil in many bacteria. In this study, we show that a putative binding motif sequence of PyrR is required for uracil-mediated repression of pyrR-lacZ translational fusion. However, the uracil response was still observed in the strain with the pyrR gene deleted, implying the existence of a uracil response factor other than PyrR which also acts through the PyrR binding loop region. Deletion of rho, encoding the transcription termination factor Rho, resulted in an increase in the expression of pyrR-lacZ. Moreover, the strain with a double deletion of pyrR and rho showed elimination of the uracil-responsive downregulation of the pyrR-lacZ. Therefore, expression of the pyrimidine biosynthetic gene cluster in Corynebacterium glutamicum is controlled by two different mechanisms mediated by PyrR and Rho. IMPORTANCEThe pyr genes of C. glutamicum are downregulated in the presence of uracil in culture medium. The mRNA binding regulator PyrR represses the expression of pyr genes, as reported previously. However, the uracil response was still observed in the pyrR deletion strain. Deletion of rho in addition to pyrR deletion results in the elimination of the uracil response. Therefore, we identified the factors that are involved in the uracil response. Involvement of Rho in the regulation of pyrimidine de novo biosynthesis genes has not been reported. Pyrimidine de novo biosynthesis starts with the synthesis of a pyrimidine ring assembled from aspartate, bicarbonate, and glutamine (Fig. 1C). The pyrimidine ring reacts with phosphoribosyl pyrophosphate (PRPP) to generate orotidine 5=-monophosphate (OMP). Subsequently, decarboxylation of OMP results in the formation of UMP, which is converted to other essential pyrimidine nucleotides. Carbamoyl phosphate is also used for biosynthesis of arginine, connecting the pyrimidine biosynthesis with arginine biosynthesis. The pyrimidine-biosynthetic pathway consists of six enzymatic steps, and the genes coding for these enzymes (carA, carB, pyrB, pyrC, pyrD, pyrE, and pyrF) are widely conserved in bacteria (1).Regulation of the expression of pyrimidine de novo biosynthesis genes has been well studied in Escherichia coli and Bacillus subtilis. Many regulatory mechanisms that are not dependent on DNA-binding transcriptional regulators have been demonstrated. Expression of the pyrBI operons in E. coli is proposed to be controlled by transcriptional attenuation (2-4). The regulation at the transcription initiation step has also been reported. In the presence of high concentrations of UTP, nascent transcript slips from the template DNA at the consecutive T residues, resulting in the reiterative addition of UTP to the transcript (5). In B. subtilis, pyrimidine de novo biosynthesis genes are clustered (pyr operon). Expression of the pyr operon is regulated by the transcriptional termination-antitermination mechanism involving the RNAbinding protein PyrR (6, 7). In the absence of PyrR, formation of the antit...

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“…Moreover, studies have shown that uracil is an allosteric regulator and coenzyme in many important biochemical reactions, and participates in a variety of enzyme reactions. 45 Uracil can be phosphorylated by various kinases to produce UMP, UDP, and UTP. UDP and UTP regulate the activity of animal carbamoyl phosphate synthase II, and uracil is also involved in the biosynthesis of polysaccharides and the transport of aldose‐containing sugars.…”

“…Moreover, studies have shown that uracil is an allosteric regulator and coenzyme in many important biochemical reactions, and participates in a variety of enzyme reactions 45 . Uracil can be phosphorylated by various kinases to produce UMP, UDP, and UTP.…”

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“…In the presence of the UMP coregulator, PyrR binds to the 5 ' UTR of the pyrR mRNA transcript and disrupts the anti-terminator stemloop, resulting in decreased downstream gene expression. In contrast, when the UMP concentration is low, PRPP antagonizes UMP termination by binding to the PyrR protein [10][11][12] . The expression of de novo synthesis of pyrimidine genes is regulated by RNA-binding proteins, but it remains unclear whether it is regulated by DNA-binding proteins.…”

“…The pyrimidine de novo synthesis pathway consists of six enzymatic steps, and the genes encoding these enzymes (carA, carB, pyrB, pyrC, pyrD, pyrE, and pyrF) are widely conserved in bacteria [6][7][8] . CarAB (spd_1132,spd_1131)encodes two subunits of carbamyl phosphate synthetase in Escherichia coli, and this enzyme catalyzes the formation of carbamyl phosphate, an intermediate in the pyrimidine nucleotide and arginine biosynthetic pathways 9 . It has been reported that carA mutation reduces the expression of capsule operon promoter (P cps ) in medium lacking uracil in the exponential growth period 7 , but it remains unclear whether several other genes affect the expression of P cps in S. pneumoniae.…”