Trimethoprim Action and Its Analogy with Thymine Starvation

Abstract

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-)

Role of purine biosynthetic intermediates in response to folate stress in Escherichia coli

“…The lack of thymidylate for DNA synthesis leads to cell death (3,4). The effect of histidine on viability in this medium needs to be interpreted with caution.…”

“…Trimethoprim is bacteriostatic under normal culture conditions. A source of purine bases (e.g., inosine, xanthine, and hypoxanthine), methionine, and glycine are required for continued growth (3,4). Trimethoprim then becomes bactericidal (4).…”

Influence of specific mutations on the thermal stability of the td group I intron in vitro and on its splicing efficiency in vivo: A comparative study

“…We used a plating assay (Belfort et al+, 1987) to determine the phenotype at 37 8C of the mutants we had chosen to investigate+ Splicing efficiency was assayed by the ability of a plasmid-borne td gene to provide thymidylate synthase (TS) to Escherichia coli cells lacking this enzyme because of the disruption of the endogenous thy A gene (Galloway-Salvo et al+, 1990)+ E. coli C600 thyA::Km r cells were transformed with td plasmids containing the mutant intron+ Transformants were replated on minimal medium (MM), MM with thymine (MMT) and MM with trimethoprim and thymine (TTM)+ Cells containing introns that cannot splice fail to produce TS and thus cannot grow on MM, but are able to grow on TTM+ Inefficient splicing results in the synthesis of small amounts of TS and is reflected by growth on both MM and TTM, whereas cells that are splicing efficiently can grow on MM, but are unable to grow on TTM (Amyes & Smith, 1974)+ The location and nature of substitutions we introduced in the td intron, in the td ⌬P6-2 context, are shown in Figure 1, as well as the mutants recovered by Belfort et al+ (1987) in the td ⌬P6-1 context+ Figure 2 shows a representative plating assay for the different td constructs and summarizes the results of plating assays for all the td intron mutants+ As expected, the Thy Ϫ control, harboring a pTZ18U plasmid lacking the td gene, grew on trimethoprim-containing TTM medium, in contrast to the Thy ϩ control containing the unmutagenized td ϩ plasmid that was sensitive to trimethoprim+ On the other hand, the Thy ϩ control grew on medium lacking thymine, whereas the Thy Ϫ control did not+ Two other td mutants with mutations in the P7 helix of the catalytic core, namely C873U (Belfort et al+, 1987) and C870U (Schroeder et al+, 1991), were used as additional splicing-deficient controls+ These mutants also exhibit the expected phenotype+ Although C873U did not grow on thymine-less media, C870U grows slightly in the absence of thymine; both constructs grew on TTM medium+ The G944A mutant, which also harbors a mutation in P7, exhibits a Thy Ϫ phenotype, whereas mutants U912C (in helix P3) and G948A (in an isolated tertiary Watson-Crick pair called P9+0a), which grew on both MM and TTM media, have to be considered as leaky Thy Ϫ mutants, producing enough TS to grow in the absence of thymine, but too little to become sensitive to trimethoprim+ As illustrated by the fact that they can grow on MM but are unable to grow on TTM, all the other mutants have a Thy ϩ phenotype+…”

Rifampin plus trimethoprim: bactericidal activity and suppression of resistance in human urine in vitro

“…showed the expected variability, even in synthetic media with different purine and amino acid supplements (3,4). We therefore decided to study the activity of Ramp+Tmp in human urine, which reportedly contains the required amino acids and purines (17) Bactericidal activity.…”

“…Because urine samples from infected individuals sometimes contain thymine derivatives (18,19,25) which adversely affect Tmp activity (3,4), it was of interest to us to assess the effect of thymine on the antibacterial activity of Ramp+Tmp in urine. Figure 4 compares the activities of Ramp, Tmp, and Ramp+Tmp on E. coli L766 growing in urine with and without the addition of 2 jig of thymine per ml.…”