The deoxycytidine pathway for thymidylate synthesis in Escherichia coli

Abstract

When thymidylate production is diminished by a mutation affecting dCTP deaminase, Escherichia coli is known to use an alternate pathway involving deoxycytidine as an intermediate. The pathway requires the gene for any of three nucleoside diphosphate kinases (ndk, pykA, or pykF) and the gene for a 5'-nucleotidase (yfbR).

FIG. 1.

dCTP and dUDP pathways for de novo synthesis of dUMP. The major (dCTP) pathway is shown with bold arrows. The genes and their products are as follows: dcd, dCTP deaminase; dut, dUTPase (dUTP pyrophosphohydrolase); ndk, nucleoside diphosphate kinase; nrdAB, ribonucleoside diphosphate reductase; pykA, pyruvate kinase I; and pykF, pyruvate kinase II. Abbreviations: (d)NDP and (d)NTP, any ribo- or deoxyribonucleoside diphosphate and triphosphate, respectively; PEP, phosphoenolpyruvate.

FIG. 2.

The dCyd pathway, an alternative, salvage pathway for the synthesis of dUMP in a dcd deoA mutant. The broad shaded arrows cover identified steps, including those established in this work. The genes and their corresponding products are as follows: cdd, cytidine (or dCyd) deaminase; cdh, (d)CTP-diglyceride hydrolase; cds, (d)CDP-diglyceride synthetase; cmk, (d)CMP kinase; deoA, Thd (dUrd) phosphorylase; nudG, Nudix (d)CTP pyrophosphohydrolase; tmk, Thd (dUrd) kinase; and yfbR, a cytoplasmic 5′-nucleotidase. Other symbols and abbreviations are as described in the legend to Fig. 1.

FIG. 3.

Thd requirement of strain BW1929 (deoA dcd) and mutant derivatives. The strains were constructed by phage P1-mediated transduction (25) of mutations from the strains listed in Table 1. Saturated cultures were diluted in 10 mM MgSO₄ and plated on a minimal agar containing 0.4% glucose and 1% Norit-treated Difco Casamino Acids (28), both with and without Thd at 125 μg/ml. After 24 h of growth at 37°C, from 25 to 50 uncrowded colonies were measured. Each bar represents the mean colony area ± standard error of the mean. The relative area (+Thd/−Thd) is the ratio of the lengths of the corresponding bars.