Potential role of phenotypic mutations in the evolution of protein expression and stability

Abstract

Phenotypic mutations (errors occurring during protein synthesis) are orders of magnitude more frequent than genetic mutations. Consequently, the sequences of individual protein molecules transcribed and translated from the same gene can differ. To test the effects of such mutations, we established a bacterial system in which an antibiotic resistance gene (TEM-1 beta-lactamase) was transcribed by either a high-fidelity RNA polymerase or its error-prone mutant. This setup enabled the analysis of individual mRNA transcripts that were synthesized under normal or error-prone conditions. We found that an increase of approximately 20-fold in the frequency of transcription errors promoted the evolution of higher TEM-1 expression levels and of more stable enzyme variants. The stabilized variants exhibited a distinct advantage under error-prone transcription, although under normal transcription they conferred resistance similar to wild-type TEM-1. They did so, primarily, by increasing TEM-1's tolerance to destabilizing deleterious mutations that arise from transcriptional errors. The stabilized TEM-1 variants also showed increased tolerance to genetic mutations. Thus, although phenotypic mutations are not individually subjected to inheritance and natural selection, as are genetic mutations, they collectively exert a direct and immediate effect on protein fitness. They may therefore play a role in shaping protein traits such as expression levels, stability, and tolerance to genetic mutations.

Fig. 1.

A schematic description of the experimental system. TEM-1 β-lactamase was transcribed from plasmid TEM-1-pET27 under a T7 promoter. To enable the transcription of TEM-1, wild-type T7 RNA polymerase, or a mutant that exhibits ≈20-fold higher rate of transcriptional errors (mutator T7 RNAP) were expressed from plasmid pACYC-T7. TEM-1 transcripts carrying mutations resulted in mutated enzyme molecules, and reduced levels of ampicillin resistance. The reproduction of single transcripts and their encoded TEM-1 molecules was achieved by reverse transcription and PCR amplification (RT/PCR) to obtain cDNAs that were subsequently cloned and individually analyzed.

Fig. 2.

Ampicillin resistance of cells expressing TEM-1 cDNA libraries. From each library, 80 randomly chosen cDNA clones were assayed for their ability to confer ampicillin resistance. Each line represents an average of 2 independently generated and assayed cDNA libraries. (A) A comparison of wild-type TEM-1 gene as control (not a cDNA library) (red squares) and cDNA libraries generated from the same TEM-1 gene using either wild-type T7 RNAP (black triangles) or mutator-T7 RNAP (green circles). (B) A comparison of cDNA libraries of wild-type TEM-1 (green circles) and cDNAs of evolved TEM-1 variants R120G-M182T (brown squares) and E147K-M182T (purple diamonds) all transcribed by the mutator-T7 RNAP.

Fig. 3.

Active enzyme levels of wild type TEM-1 and evolved variants. Wild-type TEM-1 β-lactamase and its evolved variants harboring either signal peptide mutations (Q6R, H7R) or promoter deletion mutations (ΔC-117, ΔG-123) were expressed in cells using either wild-type T7 RNAP (black bars), or mutator T7 RNAP (gray bars). After IPTG induction, the cells were incubated with nitrocefin (a synthetic, chromogenic, β-lactamase substrate) and the initial rates of nitrocefin breakdown, proportional to the periplasmic levels of active β-lactamase were monitored. TEM-1 levels were confirmed by a Western blot analysis (Fig. S2c).

Fig. 4.

Ampicillin resistance of cells expressing TEM-1 variants. For each TEM-1 variant, 80 randomly chosen clones were assayed for their ability to confer resistance at different ampicillin concentrations. Each line represents the average for 2 independent measurements. (A) Wild-type TEM-1, and its double mutants E147K-M182T and R120G-M182T, transcribed by mutator-T7 RNAP. Single TEM-1 mutants: E147K, R120G and M182T similarly transcribed, are shown for comparison. (B) The same genes transcribed by wild-type T7 RNAP.