Genetically identical cells frequently display substantial heterogeneity in gene expression, cellular morphology and physiology. It has been suggested that by rapidly generating a subpopulation with novel phenotypic traits, phenotypic heterogeneity (or plasticity) accelerates the rate of adaptive evolution in populations facing extreme environmental challenges. This issue is important as cell-to-cell phenotypic heterogeneity may initiate key steps in microbial evolution of drug resistance and cancer progression. Here, we study how stochastic transitions between cellular states influence evolutionary adaptation to a stressful environment in yeast Saccharomyces cerevisiae. We developed inducible synthetic gene circuits that generate varying degrees of expression stochasticity of an antifungal resistance gene. We initiated laboratory evolutionary experiments with genotypes carrying different versions of the genetic circuit by exposing the corresponding populations to gradually increasing antifungal stress. Phenotypic heterogeneity altered the evolutionary dynamics by transforming the adaptive landscape that relates genotype to fitness. Specifically, it enhanced the adaptive value of beneficial mutations through synergism between cell-to-cell variability and genetic variation. Our work demonstrates that phenotypic heterogeneity is an evolving trait when populations face a chronic selection pressure. It shapes evolutionary trajectories at the genomic level and facilitates evolutionary rescue from a deteriorating environmental stress. Phenotypic heterogeneity of genetically identical cells can generate nonheritable variation in a population. Is this heterogeneity favorable for microbes? In a changing environment, the answer is a definite yes. While scholars have argued that stochastically generated variation precedes genetic changes and thereby facilitate the evolution of complex traits, this idea has remained disputed, not least because of the shortage of experimental studies. We address this long-standing and controversial issue by integrating synthetic biology, laboratory experimental evolution, and genomic analyses. We explicitly tested the mechanisms whereby phenotypic heterogeneity may promote evolvability. Our work demonstrates that phenotypic heterogeneity facilitates evolutionary rescue from deteriorating environmental stress by generating individuals with exceptionally high fitness. Remarkably, elevated phenotypic heterogeneity evolves as a direct response to stress and thereby it promotes evolution of rare combinations of mutations. These results indicate that phenotypic heterogeneity might have an important role in the evolution of key innovations.

Codon usage and codon-pair context are important gene primary structure features that influence mRNA decoding fidelity. In order to identify general rules that shape codon-pair context and minimize mRNA decoding error, we have carried out a large scale comparative codon-pair context analysis of 119 fully sequenced genomes. We have developed mathematical and software tools for large scale comparative codon-pair context analysis. These methodologies unveiled general and species specific codon-pair context rules that govern evolution of mRNAs in the 3 domains of life. We show that evolution of bacterial and archeal mRNA primary structure is mainly dependent on constraints imposed by the translational machinery, while in eukaryotes DNA methylation and tri-nucleotide repeats impose strong biases on codon-pair context. The data highlight fundamental differences between prokaryotic and eukaryotic mRNA decoding rules, which are partially independent of codon usage.

Background: Genetic code alterations have been reported in mitochondrial, prokaryotic, and eukaryotic cytoplasmic translation systems, but their evolution and how organisms cope and survive such dramatic genetic events are not understood. Results: Here we used an unusual decoding of leucine CUG codons as serine in the main human fungal pathogen Candida albicans to elucidate the global impact of genetic code alterations on the proteome. We show that C. albicans decodes CUG codons ambiguously and tolerates partial reversion of their identity from serine back to leucine on a genome-wide scale. Conclusion: Such codon ambiguity expands the proteome of this human pathogen exponentially and is used to generate important phenotypic diversity. This study highlights novel features of C. albicans biology and unanticipated roles for codon ambiguity in the evolution of the genetic code.

Genetic code alterations discovered over the last 40 years in bacteria and eukaryotes invalidate the hypothesis that the code is universal and frozen. Mitochondria of various yeast species translate the UGA stop codon as tryptophan (Trp) and leucine (Leu) CUN codons (N = any nucleotide) as threonine (Thr) and fungal CTG clade species reassigned Leu CUG codons to serine and translate them ambiguously in their cytoplasms. This unique sense-to-sense genetic code alteration is mediated by a Ser-tRNA containing a Leu 5’-CAG-3’anticodon (ser-tRNA _CAG ), which is recognized and charged with Ser (∼97%) by the seryl-tRNA synthetase (SerRS) and with Leu (∼3%) by the leucyl-tRNA synthetase (LeuRS). This unusual tRNA appeared 272 ± 25 million years ago and had a profound impact on the evolution of the CTG clade species. Here, we review the most recent results and concepts arising from the study of this codon reassignment and we highlight how its study is changing our views of the evolution of the genetic code.

Using the (near) complete genome sequences of the yeastsCandida albicans, Saccharomyces cerevisiae, andSchizosaccharomyces pombe, we address the evolution of a unique genetic code change, which involves decoding of the standard leucine-CTG codon as serine inCandidaspp. By using two complementary comparative genomics approaches, we have been able to shed new light on both the origin of the novelCandidaspp. Ser-tRNA_CAG, which has mediated CTG reassignment, and on the evolution of the CTG codon in the genomes ofC. albicans,S. cerevisiae, andS. pombe. Sequence analyses of newly identified tRNAs from theC. albicansgenome demonstrate that the Ser-tRNA_CAGis derived from a serine and not a leucine tRNA in the ancestor yeast species and that this codon reassignment occurred ∼170 million years ago, but the origin of the Ser-tRNA_CAGis more ancient, implying that the ancestral Leu-tRNA that decoded the CTG codon was lost after the appearance of the Ser-tRNA_CAG. Ambiguous CTG decoding by the Ser-tRNA_CAGcombined with biased AT pressure forced the evolution of CTG into TTR codons and have been major forces driving evolution of the CTN codon family inC. albicans. Remarkably, most of the CTG codons present in extantC. albicansgenes are encoded by serine and not leucine codons in homologousS. cerevisiaeandS. pombegenes, indicating that a significant number of serine TCN and AGY codons evolved into CTG codons either directly by simultaneous double mutations or indirectly through an intermediary codon. In either case, CTG reassignment had a major impact on the evolution of the coding component of theCandidaspp. genome.[Supplemental material is available online athttp://www.genome.organd athttp://www.bio.ua.pt/genomica/Lab/Genomedata.html. The following individuals kindly provided reagents, samples, or unpublished information as indicated in the paper: N. Federspiel.]