Artificially ambiguous genetic code confers growth yield advantage

Abstract

A primitive genetic code is thought to have encoded statistical, ambiguous proteins in which more than one amino acid was inserted at a given codon. The relative vitality of organisms bearing ambiguous proteins and the kinds of pressures that forced development of the highly specific modern genetic code are unknown. Previous work demonstrated that, in the absence of selective pressure, enforced ambiguity in cells leads to death or to sequence reversion to eliminate the ambiguous phenotype. Here, we report the creation of a nonreverting strain of bacteria that produced statistical proteins. Ablating the editing activity of isoleucyl-tRNA synthetase resulted in an ambiguous code in which, through supplementation of a limited supply of isoleucine with an alternative amino acid that was noncoding, the mutant generating statistical proteins was favored over the wild-type isogenic strain. Such organisms harboring statistical proteins could have had an enhanced adaptive capacity and could have played an important role in the early development of living systems.

Fig. 1.

Misacylation in vitro and in vivo. (A) Production of Val-tRNA^Ile by IleRS_Ala. (B) The cysteine precursor S-carbamoyl-cysteine was loaded in a central well after dilution of overnight cultures of strains PS7108 (Left) and PS7096 (Right) were spread and dried on minimal glucose plates containing thymidine (0.3 mM). The halo of growth around the central well is marked with an arrow for the strain containing ileS_Ala. (C) Isoleucine analogs were loaded in a central well on plates spread with strains PS2066 and PS6231. The diameter (in cm) of the toxicity halos was measured after 24 h. The standard deviation of three independent experiments is given.

Fig. 2.

Norvaline misincorporation at Ile codons. The His-tagged protein AlaXp was expressed in Δilv strains containing the WT ileS or the mutant ileS_Ala allele, in minimal medium containing limiting Ile (0.02 mM) and excess Nor (0.5 mM). AlaXp was purified and analyzed by matrix-assisted laser desorption ionization and μ-liquid chromatography tandem MS (32). The spectrum for peptide Gln-313–Arg-320 containing an Ile residue at the second position is shown in Upper (WT cells, peptide mass = 1027.61 g/mol). It is resolved into two components when isolated from cells bearing the ileS_Ala allele (Lower). The second component has a mass of 1,013.59, exactly 14 mass units less than the “WT peptide.” Multiple peaks correspond to ¹³C isotopic forms.

Fig. 3.

Growth yields in different conditions. Strains β1412 (ΔilvGE ileS_Ala) and PS7068 (ΔilvGE) were grown overnight in minimal glucose supplemented with Ile (100 μM), Leu (100 μM), and various concentrations of valine (A) or norvaline (B) in an ELX808 Microplate Reader as described (27). The final OD at 595 nm was measured after 20 h to quantitate yield. Experiments were performed in triplicate. Error bars correspond to the standard deviations.

Fig. 4.

Increases in growth yield at a given norvaline concentration. Growth and viable counts of strains β1412 (ΔilvGE ileS_Ala) and PS7068 (ΔilvGE) in minimal glucose supplemented with Ile (100 μM), Leu (300 μM), or Val (300 μM) with or without Nor (200 μM). Experiments were performed with 12 independent cultures for each condition (error bars correspond to the standard deviations). (A) A representative growth curve for each condition is shown. (B) At stationary phase, viable counts were measured by making two independent dilutions for each culture and spotting, three times, 10 μl of a 10^–5 dilution on an LB plate.