De novo synthesis of thymidylate via deoxycytidine in dcd (dCTP deaminase) mutants of Escherichia coli

Abstract

dcd (dCTP deaminase) mutants of Escherichia coli were reported not to require thymidine for growth even though most of the thymidylate that is synthesized de novo arises from cytosine nucleotides through a pathway involving dCTP deaminase. We found, however, that the fresh introduction of dcd mutations into many strains of E. coli produced a requirement for thymidine for optimum aerobic growth, but the mutants readily reverted to prototrophy via mutations in other genes. One such mutation was in deoA, the gene for deoxyuridine phosphorylase. However, a dcd deo mutant became thymidine dependent once again if a cdd mutation (affecting deoxycytidine deaminase) were introduced. The results indicate that dcd mutants utilize an alternative pathway of TMP synthesis in which deoxycytidine and deoxyuridine are intermediates. A cdd mutation blocks the pathway by preventing the conversion of deoxycytidine to deoxyuridine, whereas a deoA mutation enhances it by sparing deoxyuridine from catabolism. The deoxycytidine must arise from dCTP or dCDP via unknown steps. It is not known to what extent this pathway is utilized in wild-type cells, which, unlike the ded mutants, do not accumulate dCTP.In Escherichia coli, dUTP is an obligatory precursor for the biosynthesis of thymidylate ( Fig. 1) (14,24). Previous studies in E. coli and Salmonella typhimurium suggested that under aerobic conditions, only about 25% of the dUTP is synthesized in a manner analogous to that of the other deoxynucleoside triphosphates, i.e., by the phosphorylation of dUDP that is generated by the reduction of UDP. The remaining 75% of dUTP is produced by dCTP deaminase (15), the product of the dcd gene (27). Accordingly, dcd mutants were noted to have a reduced TTP pool (16) and an increased sensitivity to 5-bromodeoxyuridine (BrdUrd) (7). This sensitivity is consistent with a defect in the synthesis de novo of thymidylate, which would compete with the analog for incorporation into DNA. Nevertheless, dcd mutants have been described as prototrophs (5,14,16,17); they do not require thymidine for growth. Although some dcd mutants with extensive deletions grew slowly and manifested some filamentation, these effects were thymidine independent (16). These results might lead us to the unexpected conclusion that the pathway by which most TMP is synthesized is dispensable. In this study, we examine alternative pathways that may be used by dcd mutants. MATERUILS AND METHODSBacterial strains. The strains used in this study are listed in Table 1. Additional dcd and deoA derivatives were constructed from these strains in the following way. dcd-12::Tnl0dkan was introduced by P1-mediated transduction from BW1040 with selection for kanamycin resistance. deoA22 was introduced by cotransduction with thr-34::TnlO from BW933 with selection for tetracycline resistance. deoA22 transductants were detected by their inability to use thymidine as the sole carbon source. (26) containing thiamine (1 ,ug/ml); glucose (at 0.4%) was used routinely as the carbon source. Amino acids wer...

In Vivo-Expressed Genes ofPasteurella multocida

“…A similar arrangement occurs in E. coli and H. influenzae Rd (7,14). The E. coli and Salmonella enterica serovar Typhimurium dcd genes are involved in the formation of dUTP, a precursor for the de novo synthesis of thymidylate (41,54). Although the udk and dcd genes on MHS17 were separated by only 20 bp, suggesting that they are part of an operon and thus transcribed from a promoter preceding udk, the possibility exists that se-quences upstream of and within the udk gene could allow transcription of dcd in vivo.…”

Gene 1.7 of bacteriophage T7 confers sensitivity of phage growth to dideoxythymidine

“…Our data, however, do not support the role of gp1.7 as an inhibitor of a host metabolic enzyme because T7⌬1.7 is insensitive to ddT in E. coli lacking the deoA, deoB, deoC, deoD, yjjG, dcd, or thyA genes (data not shown). Mutations in any of these genes are believed to increase the ability of E. coli to use exogenous thymidine (23,(27)(28)(29). Elucidation of the exact function of gp1.7 must await its biochemical characterization.…”

Quantitative determination of uracil residues in Escherichia coli DNA: Contribution of ung, dug, and dut genes to uracil avoidance

“…The intracellular concentration of dUTP in E. coli is governed by deoxycytidine triphosphate deaminase (dcd), which converts dCTP directly to dUTP [10], and by dUTP pyrophosphatase (dut) activity, which hydrolyzes dUTP to dUMP and pyrophosphate [11,12]. Together, these reactions generate about 75% of the dUTP pool [13]. The balance of the dUTP pool is produced by the reduction of UDP to dUDP by ribonucleotide reductase, followed by conversion to dUTP by nucleoside diphosphate kinase [13].…”

“…Together, these reactions generate about 75% of the dUTP pool [13]. The balance of the dUTP pool is produced by the reduction of UDP to dUDP by ribonucleotide reductase, followed by conversion to dUTP by nucleoside diphosphate kinase [13].…”