Article Figures & Data
Figures
- 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.
Tables
- TABLE 1.
Bacterial strains and plasmids used
Strain or plasmid Relevant genotypea Source or referenceb Strains BW934 KL16 deoA22 8 BW1146 KL16 dcd-12::Tn10d kan deoA22 cdd-50 cir-52::Tn10 8 BW1670 λcI857 Δ(cro-bioA) hsdR2 mdoB202::Tn10 28 BW1670 dcd BW1670 Δdcd::(FRT-Sp-FRT) PCR(pUC18::Sp) × BW1670 BW1670 nudG BW1670 ΔnudG::(FRT-Tp-FRT) PCR(BW1915) × BW1670 BW1679 pykF::Tn10dcat This lab; random insertion of element 105 (14) BW1697 BW1670 ΔyggV::(FRT-kan-FRT) PCR(pKD4) × BW1670 BW1914 BW1670 ΔpykA::(FRT-Gm-FRT) PCR(pKD4::Gm) × BW1670 BW1915 BW1670 ΔyggV::(FRT-Tp-FRT) PCR(EZ-Tn<DHFR-1>Tnpc) × BW1697 BW1929 KL16 Δdcd::(FRT-Sp-FRT) deoA22 P1(BW1670 dcd) × BW934 JW0893_11 Δcmk::(FRT-kan-FRT) Keio collection (1) JW2288_11 ΔyfbR::(FRT-kan-FRT) Keio collection (1) JW2502_11 Δndk::(FRT-kan-FRT) Keio collection (1) JW2714_11 ΔsurE::(FRT-kan-FRT) Keio collection (1) JW3889-1 Δcdh::(FRT-kan-FRT) Keio collection (1) KL16 Hfr KL16 (PO-45) thi-1 relA1 spoT1 2 Plasmids pKD4 oriγ bla FRT-kan-FRT 6 pKD4::Gm oriγ bla FRT-Gm-FRT This labd pUCGm pUC18::Gm bla; Gm cassette in multiple cloning site 24 pUC18::Sp pUC18::(FRT-Sp-FRT) bla This labe ↵ a All strains are derivatives of E. coli K-12 and are F− λ− unless indicated otherwise. cat, kan, Sp, Gm, and Tp indicate resistance to chloramphenicol, kanamycin, spectinomycin, gentamicin, and trimethoprim, respectively. FRT (FLP recombinase target) sequences enable excision of the bounded resistance gene by site-specific recombination (6).
↵ b Transductions with phage P1 dam rev6 (25) are described as follows: P1(donor) × recipient. Deletion/substitution mutations were produced by allelic replacement with PCR-amplified DNA and are described as follows: PCR(donor template) × recipient. Details are available on request.
↵ c Transposon DNA from Epicentre Biotechnologies, Madison, WI.
↵ d An 855-bp SmaI Gm cassette of pUCGm (24) was cloned into pKD4, replacing a 360-bp PvuII segment containing the kan gene.
↵ e A HindIII segment of pKD3 (6) that contains an FRT-cat-FRT cassette was cloned in pUC18 (30), and then the cat gene was excised by digestion with AscI and replaced by a PCR product of the Sp determinant of pKRP13 (22).
- TABLE 2.
Doubling times in liquid culture of some mutant derivatives of BW1929 (dcd deoA) grown with and without Thd
Strain Additional mutation(s) Doubling time (min)a +Thd −Thd BW1929 None 38 44 BW1930 yfbR 42 340 BW1935 ndk, pykA, pykF 60 148 ↵ a The liquid medium was similar to the agar medium used for colony size measurements. Cells at an initial concentration of about 2 × 107/ml were aerated by shaking at 37°C in flasks with sidearm cuvettes, and growth was monitored by determining the optical density at 560 nm by using a Klett colorimeter. Readings were taken periodically at cell densities between 1 × 108 and 4 × 108/ml.
