Many fungi restructured their proteomes through incorporation of serine (Ser) at thousands of protein sites coded by the leucine (Leu) CUG codon. How these fungi survived this potentially lethal genetic code alteration and its relevance for their biology are not understood. Interestingly, the human pathogen Candida albicans maintains variable Ser and Leu incorporation levels at CUG sites, suggesting that this atypical codon assignment flexibility provided an effective mechanism to alter the genetic code. To test this hypothesis, we have engineered C. albicans strains to misincorporate increasing levels of Leu at protein CUG sites. Tolerance to the misincorporations was very high, and one strain accommodated the complete reversion of CUG identity from Ser back to Leu. Increasing levels of Leu misincorporation decreased growth rate, but production of phenotypic diversity on a phenotypic array probing various metabolic networks, drug resistance, and host immune cell responses was impressive. Genome resequencing revealed an increasing number of genotype changes at polymorphic sites compared with the control strain, and 80% of Leu misincorporation resulted in complete loss of heterozygosity in a large region of chromosome V. The data unveil unanticipated links between gene translational fidelity, proteome instability and variability, genome diversification, and adaptive phenotypic diversity. They also explain the high heterozygosity of the C. albicans genome and open the door to produce microorganisms with genetic code alterations for basic and applied research.codon reassignment | evolution | tRNA N atural alterations to the standard genetic code have been discovered in Mycoplasma (1, 2), Micrococci (3), ciliates (4), fungi (5, 6), and mitochondria (7), modifying the hypothesis of a universal genetic code (8). Both neutral (9) and nonneutral theories (10) have been proposed to explain codon reassignments; however, experimental data to support or refute them are scarce, and genetic code alterations remain an intriguing biological puzzle. Despite this fact, it is becoming clear that genetic code alterations are associated with mutations in tRNAs and translation release factors that expand or restrict codon decoding capacity (7). In other words, alterations of translational factors have the potential to release the genetic code from its frozen state. This hypothesis is strongly supported by the widespread cotranslational incorporation of selenocysteine into the active site of selenoprotein (11) and pyrrolysine in the active site of the methyltransferases of several Metanosarcina species (12), Desulfitobacterium hafniense (13), and the gutless worm Olavius algarvensis (14). The selective advantages produced by these two amino acids are associated with evolution of proteins with unique catalytic properties.The flexibility of the genetic code is further highlighted by the in vivo incorporation of artificial amino acids into recombinant proteins of Escherichia coli, yeast, and mammalian cells using orthogonal pairs of tRNA...

BackgroundOrganisms use highly accurate molecular processes to transcribe their genes and a variety of mRNA quality control and ribosome proofreading mechanisms to maintain intact the fidelity of genetic information flow. Despite this, low level gene translational errors induced by mutations and environmental factors cause neurodegeneration and premature death in mice and mitochondrial disorders in humans. Paradoxically, such errors can generate advantageous phenotypic diversity in fungi and bacteria through poorly understood molecular processes.ResultsIn order to clarify the biological relevance of gene translational errors we have engineered codon misreading in yeast and used profiling of total and polysome-associated mRNAs, molecular and biochemical tools to characterize the recombinant cells. We demonstrate here that gene translational errors, which have negligible impact on yeast growth rate down-regulate protein synthesis, activate the unfolded protein response and environmental stress response pathways, and down-regulate chaperones linked to ribosomes.ConclusionsWe provide the first global view of transcriptional and post-transcriptional responses to global gene translational errors and we postulate that they cause gradual cell degeneration through synergistic effects of overloading protein quality control systems and deregulation of protein synthesis, but generate adaptive phenotypes in unicellular organisms through activation of stress cross-protection. We conclude that these genome wide gene translational infidelities can be degenerative or adaptive depending on cellular context and physiological condition.

Prenatal exposure to ethanol leads to a myriad of developmental disorders known as fetal alcohol spectrum disorder, often characterized by growth and mental retardation, central nervous system damage, and specific craniofacial dysmorphic features. The mechanisms of ethanol toxicity are not fully understood, but exposure during development affects the expression of several genes involved in cell cycle control, apoptosis, and transcriptional regulation. MicroRNAs (miRNAs) are implicated in some of these processes, however, it is not yet clear if they are involved in ethanol-induced toxicity. In order to clarify this question, we have exposed zebrafish embryos to ethanol and evaluated whether a miRNA deregulation signature could be obtained. Zebrafish embryos were exposed to 1 and 1.5% of ethanol from 4 h postfertilization (hpf) to 24 hpf. The miRNA expression profiles obtained reveal significant miRNA deregulation and show that both ethanol concentrations upregulate miR-153a, miR-725, miR-30d, let-7k, miR-100, miR-738, and miR-732. Putative gene targets of deregulated miRNAs are involved in cell cycle control, apoptosis, and transcription, which are the main processes affected by ethanol toxicity. The conservation of affected mechanisms among vertebrates leads us to postulate that similar miRNA deregulation occurs in humans, highlighting a relevant role of miRNAs in ethanol toxicology.

In winemaking, non-Saccharomyces yeast species contribute important organoleptic complexity. Current interest focuses on abundant and dominant strains characteristically present in the early phase of spontaneous alcoholic fermentations. Non-Saccharomyces species are particularly relevant in Port wine production such that the fermentation is prematurely stopped, after the metabolism of only one half of the available sugar, through fortification with aguardente. This work aimed to isolate, identify and characterize non-Saccharomyces species present in spontaneously fermenting Port. To accomplish these goals, yeasts were isolated from a selection of frozen must samples (2012–2016 harvests), using a pre-screening process choosing only the best candidates based on the organoleptic quality of the corresponding fortified wine. From five hundred non-Saccharomyces isolates, twelve species were identified. The three most abundant species, Hanseniaspora uvarum, Lachancea thermotolerans, and Metschnikowia pulcherrima, representing 89% of the isolates, exhibited particularly high diversity with high growth performance variability when exposed to typical stress conditions associated with common enological parameters. Less abundant species included Issatchenkia orientalis, Torulaspora delbrueckii, Hanseniaspora vineae, Hanseniaspora osmophila, Candida zemplinina, Rhodotorula mucilaginosa, Hanseniaspora guilliermondii, Issatchenkia occidentalis, and Zygosaccharomyces bisporus. This is the first study providing insights into the identification and characterization of non-Saccharomyces species responsible for spontaneous Port wine production.

The ascomycete Candida albicans is a normal resident of the gastrointestinal tract of humans and other warm-blooded animals. It occurs in a broad range of body sites and has high capacity to survive and proliferate in adverse environments with drastic changes in oxygen, carbon dioxide, pH, osmolarity, nutrients, and temperature. Its biology is unique due to flexible reassignment of the leucine CUG codon to serine and synthesis of statistical proteins. Under standard growth conditions, CUG sites incorporate leucine (3% of the times) and serine (97% of the times) on a proteome wide scale, but leucine incorporation fluctuates in response to environmental stressors and can be artificially increased up to 98%. In order to determine whether such flexibility also exists at other codons, we have constructed several serine tRNAs that decode various non-cognate codons. Expression of these tRNAs had minor effects on fitness, but growth of the mistranslating strains at different temperatures, in medium with different pH and nutrients composition was often enhanced relatively to the wild type (WT) strain, supporting our previous data on adaptive roles of CUG ambiguity in variable growth conditions. Parallel evolution of the recombinant strains (100 generations) followed by full genome resequencing identified various strain specific single nucleotide polymorphisms (SNP) and one SNP in the deneddylase (JAB1) gene in all strains. Since JAB1 is a subunit of the COP9 signalosome complex, which interacts with cullin (Cdc53p) to mediate degradation of a variety of cellular proteins, our data suggest that neddylation plays a key role in tolerance and adaptation to codon ambiguity in C. albicans.