Conditional lethal amber mutations in essential Escherichia coli genes

Abstract

The essential genes of microorganisms encode biological functions important for survival and thus tend to be of high scientific interest. Drugs that interfere with essential functions are likely to be interesting candidates for antimicrobials. However, these genes are hard to study genetically because knockout mutations in them are by definition inviable. We recently described a conditional mutation system in Escherichia coli that uses a plasmid to produce an amber suppressor tRNA regulated by the arabinose promoter. This suppressor was used here in the construction of amber mutations in seven essential E. coli genes. Amber stop codons were introduced as "tagalong" mutations in the flanking DNA of a downstream antibiotic resistance marker by lambda red recombination. The drug marker was removed by expression of I-SceI meganuclease, leaving a markerless mutation. We demonstrate the method with the genes frr, gcpE, lpxC, map, murA, ppa, and rpsA. We were unable to isolate an amber mutation in ftsZ. Kinetics of cell death and morphological changes were measured following removal of arabinose. As expected given the wide range of cellular mechanisms represented, different mutants showed widely different death curves. All of the mutations were bactericidal except the mutation in gcpE, which was bacteriostatic. The strain carrying an amber mutation in murA was by far the most sensitive, showing rapid killing in nonpermissive medium. The MurA protein is critical for peptidoglycan synthesis and is the target for the antibiotic fosfomycin. Such experiments may inexpensively provide valuable information for the identification and prioritization of targets for antibiotic development.

FIG. 1.

(A) Plasmids used in tagalong mutagenesis (not to scale). (B) Mutagenesis strategy. A linear DNA fragment is produced from WT template genomic DNA by overlap extension PCR. The positions of primers are indicated by one-sided arrows. The PCR fusion product is electroporated into cells, and integrations resulting from a double-recombination event are selected. After identification of a clone carrying the tagalong amber stop codon, the I-SceI gene is induced, resulting in removal of the gene encoding Cam^r. Recombination within a short duplicated region leads to the generation of an amber mutant that is otherwise scarless.

FIG. 2.

Growth in permissive and nonpermissive media. Overnight cultures were inoculated into glucose (diamonds) or arabinose (circles) medium at the optimal dilution ratios shown in the upper right corner of each panel. OD₆₀₀ was monitored in a 96-well plate in a shaking Spectramax plate reader at 37°C with periodic removal of samples for microscopy and plating on arabinose plates. The change in viability, given on the right axis (triangles), was calculated by using the concentration of CFU normalized by dividing by the OD₆₀₀. For each time point, this value was divided by the value at T = 0 and the log of that value is presented here. The numbers of CFU per milliliter per OD unit at T = 0 measured from the saturated inoculum and corrected for dilution were as follows: WT, 4.6 × 10⁹; frr mutant, 3.2 × 10⁹; gcpE mutant, 3.4 × 10⁹; lpxC mutant, 2.2 × 10⁹; map mutant, 2.9 × 10⁹; murA mutant, 8.7 × 10⁸; ppa mutant, 1.5 × 10⁹; rpsA mutant, 1.2 × 10⁹. OD is shown on a linear rather than on a logarithmic scale to best show the dynamics of the faltering cultures, and the scale of the right axis differs for lpxC and map to prevent the trend lines from overlapping. For the last two time points of the murA measurement, no colonies grew.

FIG. 3.

Inoculation effects. Overnight cultures of seven amber mutants (listed by gene name) were inoculated into glucose medium at three different dilution ratios as indicated. OD₆₀₀ was monitored in a 96-well plate in a shaking Spectramax plate reader at 37°C, and the maximum OD that each culture reached is given.