Increased levels of dihydrofolate reductase in rifampin-resistant mutants of Bacillus subtilis.

Several independent, spontaneous rifampin-resistant mutants of Bacillus subtilis were isolated and found to have an increased resistance to trimethoprim, an inhibitor of dihydrofolate reductase. This increased resistance in the rif mutants was the result of a specific threefold increase in the activity of dihydrofolate reductase, since six other enzymes examined remained unchanged. This increased level of dihydrofolate reductase and the trimethoprim resistance were cotransformed (100%) with the rif marker. These results suggest that the RNA polymerase is altered in its recognition of the gene that specifies dihydrofolate reductase.     

Trimethoprim resistance in enterococci: microbiological and biochemical aspects

Synergy was found between sulphonamide and trimethoprim in ratios 1:1 and 20:1 against both trimethoprim-sensitive enterococci (17 strains) and trimethoprim-resistant enterococci (23 strains). Many of these strains were resistant to kanamycin, tetracycline, streptomycin and/or erythromycin. Resistance to kanamycin, but not to trimethoprim, was clearly associated with the presence of a plasmid of molecular weight 35-45 Md. Elimination of this plasmid in three out of four highly trimethoprim resistant strains brought about loss of resistance to both kanamycin and trimethoprim. Furthermore, it was possible to transfer trimethoprim resistance from three of five highly resistant strains, but not from three strains with low-grade resistance. It is concluded that resistance to trimethoprim in enterococci can be encoded on a plasmid, and that the gene responsible may be on a transposon. No significant differences were found between the specific activities of dihydrofolate reductase from trimethoprim-sensitive and -resistant strains. The enzyme from resistant strains was several orders of magnitude less susceptible to inhibition by trimethoprim than was the enzyme from sensitive strains.     

A new mechanism of plasmid trimethoprim resistance. Characterization of an inducible dihydrofolate reductase

The dihydrofolate reductase encoded by plasmid pUK1123, which confers only a moderate level of trimethoprim resistance on its host, has been isolated and characterized. This enzyme, designated type IV, differs markedly from all previously described plasmid dihydrofolate reductases. It has a relatively high molecular weight of 46,700 as measured by gel filtration and, unlike previous plasmid dihydrofolate reductases, its synthesis is induced in the presence of increasing concentrations of trimethoprim. It is only slightly resistant to trimethoprim but is competitively inhibited by this drug with an inhibitor binding constant of 63 nM. In addition, the enzyme has a relatively low affinity for the substrate, dihydrofolate (Km = 37 microM). This is the first report of a plasmid trimethoprim resistance mechanism resulting from the induced synthesis of a large molecular weight dihydrofolate reductase which is only slightly resistant to trimethoprim. The possible origins of the type IV enzyme are discussed.     

Construction of plasmid vectors for cloning of promoters using the dihydrofolate reductase gene of Escherichia coli K-12

Vectors for cloning promoter-DNA fragments were derived from pTP 30-5 which was constructed in our previous work (M. Iwakura et al. (1982) J. Biochem. 91, 1205). The selection was based on the expression of trimethoprim resistance of transformed bacteria in which the enhancement of dihydrofolate reductase production was directed by the cloned promoter. A linear relationship between the content of dihydrofolate reductase and the strength of trimethoprim resistance was observed. It is suggested that the promoter activities can be estimated by trimethoprim resistance without measuring the enzyme activities.     

Rapid Decline of Ceftazidime Resistance in Antibiotic-Free and Sublethal Environments Is Contingent on Genetic Background

Trade-offs of antibiotic resistance evolution, such as fitness cost and collateral sensitivity (CS), could be exploited to drive evolution toward antibiotic susceptibility. Decline of resistance may occur when resistance to other drug leads to CS to the first one and when compensatory mutations, or genetic reversion of the original ones, reduce fitness cost. Here we describe the impact of antibiotic-free and sublethal environments on declining ceftazidime resistance in different Pseudomonas aeruginosa resistant mutants. We determined that decline of ceftazidime resistance occurs within 450 generations, which is caused by newly acquired mutations and not by reversion of the original ones, and that the original CS of these mutants is preserved. In addition, we observed that the frequency and degree of this decline is contingent on genetic background. Our results are relevant to implement evolution-based therapeutic approaches, as well as to redefine global policies of antibiotic use, such as drug cycling.     

Mapping of transfer and H pilus coding regions of the IncHII plasmid pHH1508a

The IncHII plasmid pHH1508a (208 kilobases) encodes resistance to potassium tellurite, trimethoprim, and streptomycin. Conjugative pili encoded by pHH1508a were isolated, purified, and used for preparation of anti-H pilus antiserum. Immuno-gold labelling experiments using H pilus specific antiserum showed that antigenic determinants were located along the entire length of the H pilus. Immuno-gold labelling and lysis studies using pilH alpha, a bacteriophage specific for H pili, were used to investigate transfer-deficient mutants of pHH1508a obtained by Tn5 mutagenesis and an in vitro constructed derivative of 96 kilobases, pDT1178, which also conferred resistance to potassium tellurite, trimethoprim, and streptomycin. The transfer-deficient mutants did not specify H pili, whereas pDT1178, which transferred at low frequency (1 x 10(-4) transconjugants per recipient), specified a small number of H pili. A naturally occurring plasmid, pMG110, was found to encode the production of H pili, but was completely transfer deficient (less than 1 x 10(-7) transconjugants per recipient). This study suggests that genes required for H pilus production and assembly as well as low level transfer are located separately within the 96-kilobase fragment of pDT1178 and that other genes, located outside this region, are essential for the regulation and full expression of conjugative transfer.     

Genetic analysis of chromosomal resistance to trimethoprim derived from clinical isolates of Escherichia coli

Chromosomal genes conferring resistance to trimethoprim were transferred from three independently isolated thy+ clinical strains of Escherichia coli to Escherichia coli K12 by using P1 transduction. Trimethoprim-resistant transductants were obtained less frequently than transductants of other chromosomal markers, suggesting that there were problems related to the expression of the trimethoprim resistance genes in E. coli K12. Mapping studies revealed that one of the resistance determinants was located at a similar position on the chromosome (1 min) to the fol-type mutations previously described in E. coli K12. The two remaining resistance determinants mapped at separate positions between 2.5 and 3 min on the chromosome. The presence of one of these determinants reduced the efficiency with which either donor or recipient cells carrying it could participate in conjugation mediated by the sex factor F and also resulted in phenotypic interaction with the azi gene. The mechanisms of trimethoprim resistance in the three clinical E. coli isolates studied were more complex and diverse than was expected from previous studies of E. coli K12 mutants.     

Thymidine phosphorylase activity and its relationship to trimethoprim in initial strains and thymidine-, thymine-dependent and trimethoprim-resistant mutants of plague microbe]

The comparative study revealed thymidine phosphorylase activity in the initial strains of a plague microbe of the field variety and in thymidine-, thymine-dependent and trimethoprim-resistant mutants of the plague microbe of other varieties. The data fully conformed to the results of the microbiological investigation of the strains' ability to grow on the nutrient media with trimethoprim in the presence of thymine and thymidine. On the basis of these results it appeared possible to divide the initial and mutant strains of the plague microbe into four arbitrary groups: initial strains of the plague microbe of all the varieties except the field ones sensitive to trimethoprim under any temperature conditions of incubation on any medium with any supplements; initial strains of the plague microbe of the field variety resistant to trimethoprim at 28 degrees C in the presence of thymine or thymidine alone; Tmpr mutants whose resistance to trimethoprim at 28 degrees C did not depend on the presence of thymine or thymidine, purine and vitamins, but depended on the presence of these substances at a temperature of 37 degrees C.     

The transition of human estrogen sulfotransferase from generalist to specialist using directed enzyme evolution

Broad specificity is believed to be a property of primordial enzymes that diverged during natural protein evolution to produce highly specific and efficient enzymes. Human estrogen sulfotransferase (SULT1E1) is a broad-specificity enzyme that detoxifies a variety of chemicals, including estrogens, by the transfer of sulfate. To study the molecular basis for the broad specificity of this enzyme and to investigate the process of SULT1E1 specialization, we have adopted a directed enzyme evolution approach. Using two iterative rounds of evolution, we generated SULT1E1 mutants with increased thermostability and narrower specificity from the broadly specific wild-type enzyme. To identify mutants with enhanced specificity, we developed an unbiased screening assay to assess sulfate transfer to three different acceptors in parallel. Such an assay enabled the isolation of SULT1E1 mutants with enhanced or wild-type activity toward an estrogen acceptor and significantly reduced activity for phenol or coumarin type of acceptors, leading to up to 3 orders of magnitude increase in specificity. We found that mutations conferring novel specificity are located in the vicinity of the active site and thus may play a direct role in reshaping the acceptor-binding site. Finally, such mutations resulted in reduced SULT1E1 thermostability, revealing a trade-off between SULT1E1 thermostability and acquisition of novel function.     

Evolution of an antibiotic resistance enzyme constrained by stability and activity trade-offs

Pressured by antibiotic use, resistance enzymes have been evolving new activities. Does such evolution have a cost? To investigate this question at the molecular level, clinically isolated mutants of the beta-lactamase TEM-1 were studied. When purified, mutant enzymes had increased activity against cephalosporin antibiotics but lost both thermodynamic stability and kinetic activity against their ancestral targets, penicillins. The X-ray crystallographic structures of three mutant enzymes were determined. These structures suggest that activity gain and stability loss is related to an enlarged active site cavity in the mutant enzymes. In several clinically isolated mutant enzymes, a secondary substitution is observed far from the active site (Met182-->Thr). This substitution had little effect on enzyme activity but restored stability lost by substitutions near the active site. This regained stability conferred an advantage in vivo. This pattern of stability loss and restoration may be common in the evolution of new enzyme activity.     

Substrate and inhibitor specificity of Mycobacterium avium dihydrofolate reductase

Dihydrofolate reductase (EC 1.5.1.3) is a key enzyme in the folate biosynthetic pathway. Information regarding key residues in the dihydrofolate-binding site of Mycobacterium avium dihydrofolate reductase is lacking. On the basis of previous information, Asp31 and Leu32 were selected as residues that are potentially important in interactions with dihydrofolate and antifolates (e.g. trimethoprim), respectively. Asp31 and Leu32 were modified by site-directed mutagenesis, giving the mutants D31A, D31E, D31Q, D31N and D31L, and L32A, L32F and L32D. Mutated proteins were expressed in Escherichia coli BL21(DE3)pLysS and purified using His-Bind resin; functionality was assessed in comparison with the recombinant wild type by a standard enzyme assay, and growth complementation and kinetic parameters were evaluated. All Asp31 substitutions affected enzyme function; D31E, D31Q and D31N reduced activity by 80-90%, and D31A and D31L by > 90%. All D31 mutants had modified kinetics, ranging from three-fold (D31N) to 283-fold (D31L) increases in K(m) for dihydrofolate, and 12-fold (D31N) to 223 077-fold (D31L) decreases in k(cat)/K(m). Of the Leu32 substitutions, only L32D caused reduced enzyme activity (67%) and kinetic differences from the wild type (seven-fold increase in K(m); 21-fold decrease in k(cat)/K(m)). Only minor variations in the K(m) for NADPH were observed for all substitutions. Whereas the L32F mutant retained similar trimethoprim affinity as the wild type, the L32A mutation resulted in a 12-fold decrease in affinity and the L32D mutation resulted in a seven-fold increase in affinity for trimethoprim. These findings support the hypotheses that Asp31 plays a functional role in binding of the substrate and Leu32 plays a functional role in binding of trimethoprim.     

R-factor trimethoprim resistance mechanism: an insusceptible target site

R-factor R388 increases the resistance of $\text{Escherichia coli}$ to trimethoprim by 10,000 fold, and mediates the synthesis of an addional dihydrofolate reductase that is less susceptible to trimethoprim by a similar order of magnitude. The dihydrofolate reductase conferred by the R-factor was of a larger molecular weight than the wild-type enzyme and exhibited a different pattern of response to trimethoprim inhibition. This is thought to be the first example of an R-factor conferring an altered target site mechanism of resistance to a chemotherapeutic agent.     

Isolation and characterization of thymidylate synthetase mutants of Xanthomonas maltophilia

Thymidylate synthetase mutants of Xanthomonas maltophilia ATCC 13270 were isolated on a solid minimal medium containing 50 mg/l thymidine and a high concentration of trimethoprim (500 mg/l). It was found that a high concentration of trimethoprim was required to prevent background growth of the wild-type strain. The isolated mutants could grow on thymidine or dTMP at a concentration of 50 mg/l while they were unable to grow on 1000 mg/l thymine or 50 mg/l deoxyridine. Thymidylate synthetase activity was assayed in the wild-type cells and in the mutant cells but only the wild-type cells contained measurable enzyme activity.     

Trimethoprim, failure to penetrate into Pseudomonas aeruginosa cells

Abstract Permeability mutants of Escherichia coli and Pseudomonas aeruginosa demonstrated a significantly higher susceptibility for trimethoprim (TMP). Although the target enzyme of this drug, dihydrofolic acid reductase, expressed about the same activity, it was comparably inhibited by TMP in permeability mutants and in wild-type strains. The weak activity in wild-type strains depends on the decreased uptake.     

Regulatory changes in the formation of chromosomal dihydrofolate reductase causing resistance to trimethoprim

High resistance to trimethoprim mediated by the several hundredfold overproduction of the drug target enzyme, dihyrofolate reductase, in a clinically isolated Escherichia coli strain, 1810, was cloned onto several vector plasmids and seemed to be comprised of a single dihydrofolate reductase gene, which by DNA-DNA hybridization and restriction enzyme digestion mapping was very similar to the corresponding gene of E. coli K-12. Determination of mRNA formation in the originally isolated resistant strain and strains with cloned trimethoprim resistance determinant demonstrated an about 15-fold increase in production of dihydrofolate reductase mRNA compared with that in E. coli K-12. This was explained by the occurrence of a promoter up mutation in the resistant isolate accompanied by changes in the restriction enzyme digestion pattern found by comparison with the corresponding pattern from E. coli K-12.     

Sexual reproduction reshapes the genetic architecture of digital organisms

Modularity and epistasis, as well as other aspects of genetic architecture, have emerged as central themes in evolutionary biology. Theory suggests that modularity promotes evolvability, and that aggravating (synergistic) epistasis among deleterious mutations facilitates the evolution of sex. Here, by contrast, we investigate the evolution of different genetic architectures using digital organisms, which are computer programs that self-replicate, mutate, compete and evolve. Specifically, we investigate how genetic architecture is shaped by reproductive mode. We allowed 200 populations of digital organisms to evolve for over 10 000 generations while reproducing either asexually or sexually. For 10 randomly chosen organisms from each population, we constructed and analysed all possible single mutants as well as one million mutants at each mutational distance from 2 to 10. The genomes of sexual organisms were more modular than asexual ones; sites encoding different functional traits had less overlap and sites encoding a particular trait were more tightly clustered. Net directional epistasis was alleviating (antagonistic) in both groups, although the overall strength of this epistasis was weaker in sexual than in asexual organisms. Our results show that sexual reproduction profoundly influences the evolution of the genetic architecture.     

Presence of a small plasmid in clinical isolates of Streptococcus pneumoniae

Twenty-four of 63 enteric Gram-negative organisms (38.1%) which were isolated from 35 apparently healthy Nigerian students were found to have low trimethoprim resistance (MIC less than 1000 mg/l). These isolates were also found to be resistant to several other antibiotics and trimethoprim resistance was found to be transferable from 15 (62.5%) of the trimethoprim resistant organisms into E. coli EC 1005. It is likely that the high percentage of trimethoprim resistance encountered in this study is related to the high rate of resistance transfer which was observed.