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Radioactive labeling techniques are of major importance in the study of DNA synthesis in bacteria. In order to label DNA in Escherichia coli, either radioactive thymine or thymidine is generally used because these compounds are specifically incorporated into DNA. The availability of thyA mutants unable to synthesize thymidylate makes it possible to control the specific activity of the isotopic labeling of newly synthesized DNA by adjusting the specific activity of exogenous thymine or thymidine. For this reason it is very common to obtain thyA mutants for continuous DNA labeling. thyA mutants are high-thymine-requiring strains and require between 20 and 50 µg of thymine/ml for normal growth; however, in most of the thyA mutants, deoB and deoC mutations arise spontaneously, and thus they become low-thymine-requiring strains that can grow in media with 1 to 5 µg of thymine/ml (14).

On the one hand, the use of any thymidine concentration lower than that required slows down replication velocity without changing the growth rate, and as replication initiates once every cell cycle, the consequence is an increasing number of replication forks along the chromosome. Using a variety of techniques, Pritchard et al. (15, 16) demonstrated that the rate of chain elongation can be reduced in Thy strains by decreasing the concentration of thymine in the growth medium and that this reduction in replication velocity does not lead to a detectable change in the growth rate. This also has consequences for the DNA-to-mass ratio, the mass-to-cell ratio, and cell composition in general (15, 21).

On the other hand, the use of a thymidine concentration higher than that required can affect nucleotide metabolism by allosterically inhibiting ribonucleoside diphosphate reductase, decreasing dCTP pools (20). Furthermore, TTP pools are decreased in most of the strains when they grow at high thymidine concentrations, and some mutants requiring low concentrations of thymine (thyA deoC mutants) are very sensitive to thymidine, most likely due to the inhibition of TMP kinase (14). Finally, the use of any thymidine concentration higher than that required decreases the specific activity of the labeling, and therefore a higher radioactive concentration must be used.

Nevertheless, procedures for securing the optimal thymidine concentration have not always been carried out properly or have even been ignored. In studies using thyA deo mutants, the use of thymine concentrations ranging from 2 to 50 µg/ml can be found (3, 10-12).

Finding the optimal thymidine concentration, i.e., the minimal thymidine concentration giving the minimal C period, is therefore an important factor for determining the required growth medium of a Thy mutant, and it is essential for any analysis related to DNA replication and the cell cycle. But in contrast to any other requirements for bacterial growth, optimal thymidine concentration cannot be determined by ascertaining the concentration of thymidine giving the highest growth rate (16). In this work we show how the results from runout experiments can be used to obtain the optimal thymidine concentration for any Thy strain.

Studying the bacterial cell cycle, we have determined the mass doubling time, DNA duplication time, and runout replication of strain CR34 (thr leu thyA deoC lac tonA strA) at 37°C in M9 minimal medium containing different thymidine concentrations (0.8, 1, 2, 5, and 10 µg/ml) and [methyl-³H]thymidine (20 Ci/mmol) at 1 µCi/ml to label DNA. By the time the cultures reached 0.1 OD₄₅₀ (optical density at 450 nm) unit after a 1:200 dilution, a portion of the culture was treated with rifampin (150 µg/ml) in order to inhibit initiation of chromosome replication, and runout synthesis was measured as trichloroacetic acid-precipitable material. From the amount of runout synthesis, G, the number of replication forks per chromosome equivalent, n, was obtained by the algorithm G = [2ⁿ · n · ln2/(2ⁿ  1)]  1 (16, 18) (Table 1). From this we obtained the length of the C period by the equation C = n (Table 1), where  is the time for mass doubling and DNA duplication. Mass doubling and doubling of DNA content took around 60 min for CR34 at all thymidine concentrations. Otherwise, runout synthesis and the length of the C period increased with decreasing thymidine concentrations in the growth medium (Fig. 1a; Table 1), as expected for a Thy phenotype, where the thymidine concentration limits the replication velocity.

View larger version (15K): [in this window] [in a new window]   FIG. 1.   C period as a function of the thymidine concentration for CR34 () (a), MG1655 () (b), and NF859 () (b) growing in M9 minimal medium at 37°C.

From these data we obtained a biphasic curve with two regions (Fig. 1a): the first one within the low thymidine concentrations, where a minimal variation in these concentrations gave rise to a maximal variation in the C period, and a second one where thymidine concentration can be increased up to 5 times without a significant change in the C period. From this kind of plotting the optimal thymidine concentration can easily be obtained, as the minimal thymidine concentration giving the minimal C period. Thus, in the case of CR34, this concentration is 2 µg/ml.

As a control of this protocol to determine the optimal thymidine concentration, we applied the same method (but in medium containing 1.5 mM uridine for DNA labeling [14]) in two Thy⁺ strains, NF859 (metB pro argA) and the "wild-type" MG1655 (F rph) (8). Mass doubling and DNA duplication times were around 40 min for NF859 and 54 min for MG1655 with all tested thymidine concentrations (Table 1). Changing the thymidine concentration in the growth medium of NF859 did not change either the runout synthesis or the length of the C period (Fig. 1b; Table 1), as expected for a Thy⁺ strain, where thymidine concentration does not limit the replication velocity. Higher concentrations of thymidine might affect replication velocity and increase the C period due to inhibition of the TTP pool, but this effect is not observed at the concentrations used in this work.

Surprisingly, the C period of MG1655 was affected by the thymidine concentration and was reduced from 79 to 49 min when the thymidine concentration was increased to 5 µg/ml (Fig. 1b; Table 1). Since the time of mass doubling and duplication of DNA content was the same under all conditions and the replication velocity in MG1655 increased with increasing thymidine concentrations, we conclude that this strain behaves in a manner expected for a Thy strain.

MG1655 has been used as a genetic background for characterizing the phenotypes of several RNA polymerase mutations (9), for studies on the control of ribosome synthesis and the effects of ppGpp (6, 7, 19), as the host for a collection of Tn10 insertions to facilitate genetic mapping (17), for total-genome sequencing (5), and, also as a control strain in many experiments involving DNA replication of E. coli growing without thymidine (1, 2, 4, 13). Data presented in this work show that MG1655 requires 5 µg of thymidine/ml for optimal growth. This thymidine response, therefore, should be taken into consideration.