A Suggested Mechanism for the Selective Procedure for Isolating Thymine-Requiring Mutants of <i>Escherichia coli</i>

In a minimal medium, trimethoprim is merely bacteriostatic on the prototroph Escherichia coli 114. The drug was bactericidal when the amino acids methionine and glycine, plus a purine or purine nucleoside, were also present. This response could be reversed completely when thymine and lysine were added to the culture. Methionine, glycine, and the purine are thought to maintain the integrity of the tetrahydrofolate pool under trimethoprim treatment and prevent the thymidylate synthetase reaction. Thus, the organism behaves phenotypically as a thymineless mutant. The mechanisms by which thymine and lysine reverse the bactericidal effect of trimethoprim in a minimal medium containing methionine, glycine, and adenine is discussed. (27). 169 We have shown (J. Med. Microbiol., in press) that a prototrophic organism treated with Tm in a complex medium lacking any derivative of thymine behaved similarly to a thymineless mutant starved of thymine in a minimal medium, in that a rapid bactericidal effect was observed (2). In both cases, when thymine was added multiplication occurred. However, in this present work, when these experiments were repeated in minimal media the prototroph did not "die" rapidly in the presence of Tm nor was it able to multiply when thymine was added to the culture. We find, as did Angehrn and Then (1), that, provided certain metabolites (a purine, methionine, and glycine) are added, the prototrophic organism does die rapidly when treated with Tm in minimal medium. We additionally show, however, that these metabolites are not sufficient to allow complete recovery of the organism when thymine is added to the minimal medium. The additional requirement for thymine reversal of Tm-induced death in a minimal medium was investigated and found to be lysine. We propose a model based on the metabolism of THF to explain the differences in the requirements for thymineless death and Tm-induced death and their reversal by thymine derivatives in minimal media.MATERIALS AND METHODS Organisms. The bacteria used were Escherichia coli 114 (thy+) and its thymineless (thy-)

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“…whether a minimal or complex medium is used (6,33), just as it had little effect on thymine starvation (Fig. 5).…”

Section: Resultsmentioning

confidence: 92%

Trimethoprim Action and Its Analogy with Thymine Starvation

Amyes

Smith

1974

Antimicrob Agents Chemother

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“…In prokaryotes, loss of TS activity confers transient resistance to antifolates if thymidine is provided (5,62). This stems from a sparing of reduced-folate pools, since TS is the only significant enzyme whose activity oxidizes tetrahydrofolates.…”

Section: Resultsmentioning

confidence: 99%

“…Thus, the "classical" anti-TS selection works in Leishmania spp. as well as it has in bacteria, yeasts, and cultured mammalian cells (3,5,11,62). In Leishmania spp.…”

Section: Resultsmentioning

confidence: 99%

Selection against the Dihydrofolate Reductase-Thymidylate Synthase (DHFR-TS) Locus as a Probe of Genetic Alterations in Leishmania major

Gueiros‐Filho

Beverley

1996

Molecular and Cellular Biology

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The genome of the trypanosomatid protozoan genus Leishmania has been shown to undergo a number of changes relevant to drug resistance and virulence, such as gene amplification, chromosomal rearrangement, and variation in ploidy. Experimental approaches to the study of genomic changes have in some cases been limited by the fact that Leishmania cells are asexual diploids, as are some other trypanosomatids, pathogenic fungi, and cultured mammalian cells. Here we report upon a system which permits the measurement of several types of genomic change occurring at the dihydrofolate reductase-thymidylate synthase (DHFR-TS) locus. First, we show that DHFR-TS can function as a positive/negative marker. We used selection against DHFR-TS on a heterozygous line (؉/HYG) to generate colonies exhibiting both loss of heterozygosity and structural mutations in DHFR-TS, permitting the first measurement of mutation frequencies in this parasite. Loss of heterozygosity occurred at a frequency ranging from 10 ؊4 to 10 ؊6 and was elevated 24-fold by treatment with ␥-irradiation, while the frequency of other events was less than 10 ؊6 and was increased more than 1,000-fold by nitrosoguanidine treatment. The frequency of loss of heterozygosity relative to other processes such as mutation and gene replacement has important implications for genetic variability in natural Leishmania populations and the generation of both targeted and random mutations. We also developed a protocol for null targeting of diploid cells, in which transfection of a DHFR-TS deletion construct into Leishmania cells followed by negative selection yielded parasites lacking DHFR-TS or foreign sequences. The null-targeting method can be applied to any diploid cell, at any locus for which a negative selection exists. Such marker-free auxotrophic Leishmania cells show potential as an attenuated vaccine, and the methods developed here provide a new approach for manipulating and characterizing the plasticity of the Leishmania genome.

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“…The thyA::Tn10dtet mutation was selected by trimethoprim resistance, a procedure that is highly specific for tight thyA mutants (1,5). The mutant required more than 150 M of thymine for optimum growth, thereby classifying it as a highthymine-requiring mutant (7).…”

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confidence: 99%

YjjG, a dUMP Phosphatase, Is Critical for Thymine Utilization by Escherichia coli K-12

Weiss

2007

J Bacteriol

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Exogenous thymine must be converted to thymidine to enable a thyA (thymidylate synthase) mutant to grow. The deoxyribose in the thymidine comes from dUMP, which must first be dephosphorylated. The nucleotidase YjjG is critical for this step. A yjjG thyA mutant cannot use thymine for growth on a glucose minimal medium.Radiolabeled thymine is not efficiently incorporated into the DNA of wild-type Escherichia coli (reviewed in reference 7). Thymidine is better but only in the short term, before it is degraded by phosphorylase activity in the periplasm. However, thymine uptake may be greatly enhanced by supplying the cell with deoxyadenosine or by introducing a thyA (thymidylate synthase) mutation. The explanation is that in order for the thymine to be salvaged, it must have a source of deoxyribose-1-phosphate with which to condense. The resulting thymidine is then converted to TMP by thymidine kinase. Deoxyadenosine helps because it can produce deoxyribose-1-phosphate (plus adenine) through the action of purine nucleoside phosphorylase. In a thyA mutant, however, the source for deoxyribose-1-phosphate is dUMP, which accumulates in the cell because its conversion to TMP is blocked. dUMP must first be hydrolyzed to deoxyuridine, which is degraded by thymidine (deoxyuridine) phosphorylase to uracil and deoxyribose-1-phosphate (Fig. 1). The same enzyme then catalyzes the condensation of exogenous thymine and deoxyribose-1-phosphate to produce thymidine. In support of this mechanism, it was observed that the thymine requirement of thyA mutants was greatly reduced by the acquisition of mutations in deoB and deoC, which block the catabolism of deoxyribose-1-phosphate. However, the first step in this salvage pathway has been largely overlooked. The hydrolysis of dUMP to deoxyuridine is catalyzed by an unidentified enzyme.E. coli has at least five enzymes with 5Ј nucleotidase (nucleoside 5Ј phosphomonoesterase) activity. The product of the ushA gene, which was originally designated a 5Ј nucleotidase, was found to be primarily a UDP-sugar hydrolase. However, it is located in the periplasmic space, where it would be inaccessible to phosphorylated cytoplasmic compounds, and it has a cytoplasmic inhibitor (15). Similarly, alkaline phosphatase (phoA gene) is located mainly in the periplasm, and it is both repressed and inhibited by levels of phosphate above growthlimiting conditions (17). A dUMP phosphatase was first described in 1973 (16). However, its gene was unknown and there were no available mutants, so its putative role in thymine salvage could not be tested. It had almost equal specificities for UMP, dUMP, and TMP. When tested with Mg 2ϩ , it had an extraordinarily high K m (ϳ10 mM) for each of its three substrates. More recently, three other nucleotidases were discovered in a broad screen that used p-nitrophenylphosphate as a substrate (9). One, the YjjG protein, which was characterized in the form of a hexahistidine-tagged protein, had a substrate specificity that was similar to that of the earlier described dUMP phosp...

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A Suggested Mechanism for the Selective Procedure for Isolating Thymine-Requiring Mutants of Escherichia coli

Cited by 48 publications

References 11 publications

Trimethoprim Action and Its Analogy with Thymine Starvation

Trimethoprim Action and Its Analogy with Thymine Starvation

Selection against the Dihydrofolate Reductase-Thymidylate Synthase (DHFR-TS) Locus as a Probe of Genetic Alterations in Leishmania major

YjjG, a dUMP Phosphatase, Is Critical for Thymine Utilization by Escherichia coli K-12

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