Alternative Genetic Codes Research Articles

Reaching New Heights in Genetic Code Manipulation with High Throughput Screening.

The chemical and physical properties of proteins are limited by the 20 canonical amino acids. Genetic code manipulation allows for the incorporation of noncanonical amino acids (ncAAs) that enhance or alter protein functionality. This review explores advances in the three main strategies for introducing ncAAs into biosynthesized proteins, focusing on the role of high throughput screening in these advancements. The first section discusses engineering aminoacyl-tRNA synthetases (aaRSs) and tRNAs, emphasizing how novel selection methods improve characteristics including ncAA incorporation efficiency and selectivity. The second section examines high-throughput techniques for improving protein translation machinery, enabling accommodation of alternative genetic codes. This includes opportunities to enhance ncAA incorporation through engineering cellular components unrelated to translation. The final section highlights various discovery platforms for high-throughput screening of ncAA-containing proteins, showcasing innovative binding ligands and enzymes that are challenging to create with only canonical amino acids. These advances have led to promising drug leads and biocatalysts. Overall, the ability to discover unexpected functionalities through high-throughput methods significantly influences ncAA incorporation and its applications. Future innovations in experimental techniques, along with advancements in computational protein design and machine learning, are poised to further elevate this field.

Order of amino acid recruitment into the genetic code resolved by Last Universal Common Ancestor's protein domains.

The current "consensus" order in which amino acids were added to the genetic code is based on potentially biased criteria such as absence of sulfur-containing amino acids from the Urey-Miller experiment which lacked sulfur. Even if inferred perfectly, abiotic abundance might not reflect abundance in the organisms in which the genetic code evolved. Here, we instead exploit the fact that proteins that emerged prior to the genetic code's completion are likely enriched in early amino acids and depleted in late amino acids. We identify the most ancient protein-coding sequences born prior to the archaeal-bacterial split. Amino acid usage in protein sequences whose ancestors date back to a single homolog in the Last Universal Common Ancestor (LUCA) largely matches the consensus order. However, our findings indicate that metal-binding (cysteine and histidine) and sulfur-containing (cysteine and methionine) amino acids were added to the genetic code much earlier than previously thought. Surprisingly, even more ancient protein sequences - those that had already diversified into multiple distinct copies in LUCA - show a different pattern to single copy LUCA sequences: significantly less depleted in the late amino acids tryptophan and tyrosine, and enriched rather than depleted in phenylalanine. This is compatible with at least some of these sequences predating the current genetic code. Their distinct enrichment patterns thus provide hints about earlier, alternative genetic codes.

MRNA context and translation factors determine decoding in alternative nuclear genetic codes.

The genetic code is a set of instructions that determine how the information in our genetic material is translated into amino acids. In general, it is universal for all organisms, from viruses and bacteria to humans. However, in the last few decades, exceptions to this rule have been identified both in pro- and eukaryotes. In this review, we discuss the 16 described alternative eukaryotic nuclear genetic codes and observe theories of their appearance in evolution. We consider possible molecular mechanisms that allow codon reassignment. Most reassignments in nuclear genetic codes are observed for stop codons. Moreover, in several organisms, stop codons can simultaneously encode amino acids and serve as termination signals. In this case, the meaning of the codon is determined by the additional factors besides the triplets. A comprehensive review of various non-standard coding events in the nuclear genomes provides a new insight into the translation mechanism in eukaryotes.

Decoding huge phage diversity: a taxonomic classification of Lak megaphages.

High-throughput sequencing for uncultivated viruses has accelerated the understanding of global viral diversity and uncovered viral genomes substantially larger than any that have so far been cultured. Notably, the Lak phages are an enigmatic group of viruses that present some of the largest known phage genomes identified in human and animal microbiomes, and are dissimilar to any cultivated viruses. Despite the wealth of viral diversity that exists within sequencing datasets, uncultivated viruses have rarely been used for taxonomic classification. We investigated the evolutionary relationships of 23 Lak phages and propose a taxonomy for their classification. Predicted protein analysis revealed the Lak phages formed a deeply branching monophyletic clade within the class Caudoviricetes which contained no other phage genomes. One of the interesting features of this clade is that all current members are characterised by an alternative genetic code. We propose the Lak phages belong to a new order, the 'Grandevirales'. Protein and nucleotide-based analyses support the creation of two families, three sub-families, and four genera within the order 'Grandevirales'. We anticipate that the proposed taxonomy of Lak megaphages will simplify the future classification of related viral genomes as they are uncovered. Continued efforts to classify divergent viruses are crucial to aid common analyses of viral genomes and metagenomes.

Identification of HDV-like theta ribozymes involved in tRNA-based recoding of gut bacteriophages

Trillions of microorganisms, collectively known as the microbiome, inhabit our bodies with the gut microbiome being of particular interest in biomedical research. Bacteriophages, the dominant virome constituents, can utilize suppressor tRNAs to switch to alternative genetic codes (e.g., the UAG stop-codon is reassigned to glutamine) while infecting hosts with the standard bacterial code. However, what triggers this switch and how the bacteriophage manipulates its host is poorly understood. Here, we report the discovery of a subgroup of minimal hepatitis delta virus (HDV)-like ribozymes – theta ribozymes – potentially involved in the code switch leading to the expression of recoded lysis and structural phage genes. We demonstrate their HDV-like self-scission behavior in vitro and find them in an unreported context often located with their cleavage site adjacent to tRNAs, indicating a role in viral tRNA maturation and/or regulation. Every fifth associated tRNA is a suppressor tRNA, further strengthening our hypothesis. The vast abundance of tRNA-associated theta ribozymes – we provide 1753 unique examples – highlights the importance of small ribozymes as an alternative to large enzymes that usually process tRNA 3’-ends. Our discovery expands the short list of biological functions of small HDV-like ribozymes and introduces a previously unknown player likely involved in the code switch of certain recoded gut bacteriophages.

Create, Analyze, and Visualize Phylogenomic Datasets Using PhyloFisher.

PhyloFisher is a software package written primarily in Python3 that can be used for the creation, analysis, and visualization of phylogenomic datasets that consist of protein sequences from eukaryotic organisms. Unlike many existing phylogenomic pipelines, PhyloFisher comes with a manually curated database of 240 protein-coding genes, a subset of a previous phylogenetic dataset sampled from 304 eukaryotic taxa. The software package can also utilize a user-created database of eukaryotic proteins, which may be more appropriate for shallow evolutionary questions. PhyloFisher is also equipped with a set of utilities to aid in running routine analyses, such as the prediction of alternative genetic codes, removal of genes and/or taxa based on occupancy/completeness of the dataset, testing for amino acid compositional heterogeneity among sequences, removal of heterotachious and/or fast-evolving sites, removal of fast-evolving taxa, supermatrix creation from randomly resampled genes, and supermatrix creation from nucleotide sequences. © 2024 Wiley Periodicals LLC. Basic Protocol 1: Constructing a phylogenomic dataset Basic Protocol 2: Performing phylogenomic analyses Support Protocol 1: Installing PhyloFisher Support Protocol 2: Creating a custom phylogenomic database.

Single cell transcriptomics reveals UAR codon reassignment in Palmarella salina (Metopida, Armophorea) and confirms Armophorida belongs to APM clade

Anaerobes have emerged in several major lineages of ciliates, but the number of independent transitions to anaerobiosis among ciliates is unknown. The APM clade (Armophorea, Muranotrichea, Parablepharismea) represents the largest clade of obligate anaerobes among ciliates and contains free-living marine and freshwater representatives as well as gut endobionts of animals. The evolution of APM group has only recently started getting attention, and our knowledge on its phylogeny and genetics is still limited to a fraction of taxa. While ciliates portray a wide array of alternatives to the standard genetic code across numerous classes, the APM ciliates were considered to be the largest group using exclusively standard nuclear genetic code. In this study, we present a pan-ciliate phylogenomic analysis with emphasis on the APM clade, bringing the first phylogenomic analysis of the family Tropidoatractidae (Armophorea) and confirming the position of Armophorida within Armophorea. We include five newly sequenced single cell transcriptomes from marine, freshwater, and endobiotic APM ciliates – Palmarella salina, Anteclevelandella constricta, Nyctotherus sp., Caenomorpha medusula, and Thigmothrix strigosa. We report the first discovery of an alternative nuclear genetic code among APM ciliates, used by Palmarella salina (Tropidoatractidae, Armophorea), but not by its close relative, Tropidoatractus sp., and provide a comparative analysis of stop codon identity and frequency indicating the precedency to the UAG codon loss/reassignment over the UAA codon reassignment in the specific ancestor of Palmarella. Comparative genomic and proteomic studies of this group may help explain the constraints that underlie UAR stop-to-sense reassignment, the most frequent type of alternative nuclear genetic code, not only in ciliates, but eukaryotes in general.

Host translation machinery is not a barrier to phages that interact with both CPR and non-CPR bacteria.

Here, we profiled putative phages of Saccharibacteria, which are of particular importance as Saccharibacteria influence some human oral diseases. We additionally profiled putative phages of Gracilibacteria and Absconditabacteria, two Candidate Phyla Radiation (CPR) lineages of interest given their use of an alternative genetic code. Among the phages identified in this study, some are targeted by spacers from both CPR and non-CPR bacteria and others by both bacteria that use the standard genetic code as well as bacteria that use an alternative genetic code. These findings represent new insights into possible phage replication strategies and have relevance for phage therapies that seek to manipulate microbiomes containing CPR bacteria.

Genomes of a Novel Group of Phages That Use Alternative Genetic Code Found in Human Gut Viromes.

Metagenomics provides detection of phage genome sequences in various microbial communities. However, the use of alternative genetic codes by some phages precludes the correct analysis of their genomes. In this study, the unusual phage genome (phAss-1, 135,976 bp) was found after the de novo assembly of the human gut virome. Genome analysis revealed the presence of the TAG stop codons in 41 ORFs, including characteristic phage ORFs, and three genes of suppressor tRNA. Comparative analysis indicated that no phages with similar genomes were described. However, two phage genomes (BK046881_ctckW2 and BK025033_ct6IQ4) with substantial similarity to phAss-1 were extracted from the human gut metagenome data. These two complete genomes demonstrated 82.7% and 86.4% of nucleotide identity, respectively, similar genome synteny to phAss-1, the presence of suppressor tRNA genes and suppressor TAG stop codons in many characteristic phage ORFs. These data indicated that phAss-1, BK046881_ctckW2, and BK025033_ct6IQ4 are distinct species within the proposed Phassvirus genus. Moreover, a monophyletic group of divergent phage genomes containing the proposed Phassvirus genus was found among metagenome data. Several phage genomes from the group also contain ORFs with suppressor TAG stop codons, indicating the need to use various translation tables when depositing phage genomes in GenBank.

Evolution of an Alternative Genetic Code in the Providencia Symbiont of the Hematophagous Leech Haementeria acuecueyetzin.

Strict blood-feeding animals are confronted with a strong B-vitamin deficiency. Blood-feeding leeches from the Glossiphoniidae family, similarly to hematophagous insects, have evolved specialized organs called bacteriomes to harbor symbiotic bacteria. Leeches of the Haementeria genus have two pairs of globular bacteriomes attached to the esophagus which house intracellular "Candidatus Providencia siddallii" bacteria. Previous work analyzing a draft genome of the Providencia symbiont of the Mexican leech Haementeria officinalis showed that, in this species, the bacteria hold a reduced genome capable of synthesizing B vitamins. In this work, we aimed to expand our knowledge on the diversity and evolution of Providencia symbionts of Haementeria. For this purpose, we sequenced the symbiont genomes of three selected leech species. We found that all genomes are highly syntenic and have kept a stable genetic repertoire, mirroring ancient insect endosymbionts. Additionally, we found B-vitamin pathways to be conserved among these symbionts, pointing to a conserved symbiotic role. Lastly and most notably, we found that the symbiont of H. acuecueyetzin has evolved an alternative genetic code, affecting a portion of its proteome and showing evidence of a lineage-specific and likely intermediate stage of genetic code reassignment.

Viruses in deep-sea cold seep sediments harbor diverse survival mechanisms and remain genetically conserved within species

Deep sea cold seep sediments have been discovered to harbor novel, abundant, and diverse bacterial and archaeal viruses. However, little is known about viral genetic features and evolutionary patterns in these environments. Here, we examined the evolutionary ecology of viruses across active and extinct seep stages in the area of Haima cold seeps in the South China Sea. A total of 338 viral operational taxonomic units are identified and linked to 36 bacterial and archaeal phyla. The dynamics of host-virus interactions are informed by diverse antiviral defense systems across 43 families found in 487 microbial genomes. Cold seep viruses are predicted to harbor diverse adaptive strategies to persist in this environment, including counter-defense systems, auxiliary metabolic genes, reverse transcriptases, and alternative genetic code assignments. Extremely low nucleotide diversity is observed in cold seep viral populations, being influenced by factors including microbial host, sediment depth, and cold seep stage. Most cold seep viral genes are under strong purifying selection with trajectories that differ depending on whether cold seeps are active or extinct. This work sheds light on the understanding of environmental adaptation mechanisms and evolutionary patterns of viruses in the sub-seafloor biosphere.

BLINCAR: a reusable bioluminescent and Cas9-based genetic toolset for repeatedly modifying wild-type Scheffersomyces stipitis.

Scheffersomyces stipitis is a yeast that robustly ferments the 5-carbon sugar xylose, making the yeast a valuable candidate for lignocellulosic ethanol fermentation. However, the non-canonical codon usage of S. stipitis is an obstacle for implementing molecular tools that were developed for other yeast species, thereby limiting the molecular toolset available for S. stipitis. Here, we developed a series of molecular tools for S. stipitis including BLINCAR, a Bio-Luminescent Indicator that is Nullified by Cas9-Actuated Recombination, which can be used repeatedly to add different exogenous DNA payloads to the wild-type S. stipitis genome or used repeatedly to remove multiple native S. stipitis genes from the wild-type genome. Through the use of BLINCAR tools, one first produces antibiotic-resistant, bioluminescent colonies of S. stipitis whose bioluminescence highlights those clones that have been genetically modified; then second, once candidate clones have been confirmed, one uses a transient Cas9-producing plasmid to nullify the antibiotic resistance and bioluminescent markers from the prior introduction, thereby producing non-bioluminescent colonies that highlight those clones which have been re-sensitized to the antibiotic and are therefore susceptible to another round of BLINCAR implementation. IMPORTANCE Cellulose and hemicellulose that comprise a large portion of sawdust, leaves, and grass can be valuable sources of fermentable sugars for ethanol production. However, some of the sugars liberated from hemicellulose (like xylose) are not easily fermented using conventional glucose-fermenting yeast like Saccharomyces cerevisiae, so engineering robust xylose-fermenting yeast that is not inhibited by other components liberated from cellulose/hemicellulose will be important for maximizing yield and making lignocellulosic ethanol fermentation cost efficient. The yeast Scheffersomyces stipitis is one such yeast that can ferment xylose; however, it possesses several barriers to genetic manipulation. It is difficult to transform, has only a few antibiotic resistance markers, and uses an alternative genetic code from most other organisms. We developed a genetic toolset for S. stipitis that lowers these barriers and allows a user to deliver and/or delete multiple genetic elements to/from the wild-type genome, thereby expanding S. stipitis's potential.

The Influence of the Selection at the Amino Acid Level on Synonymous Codon Usage from the Viewpoint of Alternative Genetic Codes

Synonymous codon usage can be influenced by mutations and/or selection, e.g., for speed of protein translation and correct folding. However, this codon bias can also be affected by a general selection at the amino acid level due to differences in the acceptance of the loss and generation of these codons. To assess the importance of this effect, we constructed a mutation-selection model model, in which we generated almost 90,000 stationary nucleotide distributions produced by mutational processes and applied a selection based on differences in physicochemical properties of amino acids. Under these conditions, we calculated the usage of fourfold degenerated (4FD) codons and compared it with the usage characteristic of the pure mutations. We considered both the standard genetic code (SGC) and alternative genetic codes (AGCs). The analyses showed that a majority of AGCs produced a greater 4FD codon bias than the SGC. The mutations producing more thymine or adenine than guanine and cytosine increased the differences in usage. On the other hand, the mutational pressures generating a lot of cytosine or guanine with a low content of adenine and thymine decreased this bias because the nucleotide content of most 4FD codons stayed in the compositional equilibrium with these pressures. The comparison of the theoretical results with those for real protein coding sequences showed that the influence of selection at the amino acid level on the synonymous codon usage cannot be neglected. The analyses indicate that the effect of amino acid selection cannot be disregarded and that it can interfere with other selection factors influencing codon usage, especially in AT-rich genomes, in which AGCs are usually used.

Frameshift and wild-type proteins are often highly similar because the genetic code and genomes were optimized for frameshift tolerance

Frameshift mutations have been considered of significant importance for the molecular evolution of proteins and their coding genes, while frameshift protein sequences encoded in the alternative reading frames of coding genes have been considered to be meaningless. However, functional frameshifts have been found widely existing. It was puzzling how a frameshift protein kept its structure and functionality while substantial changes occurred in its primary amino-acid sequence. This study shows that the similarities among frameshifts and wild types are higher than random similarities and are determined at different levels. Frameshift substitutions are more conservative than random substitutions in the standard genetic code (SGC). The frameshift substitutions score of SGC ranks in the top 2.0-3.5% of alternative genetic codes, showing that SGC is nearly optimal for frameshift tolerance. In many genes and certain genomes, frameshift-resistant codons and codon pairs appear more frequently than expected, suggesting that frameshift tolerance is achieved through not only the optimality of the genetic code but, more importantly, the further optimization of a specific gene or genome through the usages of codons/codon pairs, which sheds light on the role of frameshift mutations in molecular and genomic evolution.

Widespread stop-codon recoding in bacteriophages may regulate translation of lytic genes.

Bacteriophages (phages) are obligate parasites that use host bacterial translation machinery to produce viral proteins. However, some phages have alternative genetic codes with reassigned stop codons that are predicted to be incompatible with bacterial translation systems. We analysed 9,422 phage genomes and found that stop-codon recoding has evolved in diverse clades of phages that infect bacteria present in both human and animal gut microbiota. Recoded stop codons are particularly over-represented in phage structural and lysis genes. We propose that recoded stop codons might function to prevent premature production of late-stage proteins. Stop-codon recoding has evolved several times in closely related lineages, which suggests that adaptive recoding can occur over very short evolutionary timescales.

Evidence for an Independent Hydrogenosome-to-Mitosome Transition in the CL3 Lineage of Fornicates

Fornicata, a lineage of a broader and ancient anaerobic eukaryotic clade Metamonada, contains diverse taxa that are ideally suited for evolutionary studies addressing various fundamental biological questions, such as the evolutionary trajectory of mitochondrion-related organelles (MROs), the transition between free-living and endobiotic lifestyles, and the derivation of alternative genetic codes. To this end, we conducted detailed microscopic and transcriptome analyses in a poorly documented strain of an anaerobic free-living marine flagellate, PCS, in the so-called CL3 fornicate lineage. Fortuitously, we discovered that the original culture contained two morphologically similar and closely related CL3 representatives, which doubles the taxon representation within this lineage. We obtained a monoeukaryotic culture of one of them and formally describe it as a new member of the family Caviomonadidae, Euthynema mutabile gen. et sp. nov. In contrast to previously studied caviomonads, the endobiotic Caviomonas mobilis and Iotanema spirale, E. mutabile possesses an ultrastructurally discernible MRO. We sequenced and assembled the transcriptome of E. mutabile, and by sequence subtraction, obtained transcriptome data from the other CL3 clade representative present in the original PCS culture, denoted PCS-ghost. Transcriptome analyses showed that the reassignment of only one of the UAR stop codons to encode Gln previously reported from I. spirale does not extend to its free-living relatives and is likely due to a unique amino acid substitution in I. spirale’s eRF1 protein domain responsible for termination codon recognition. The backbone fornicate phylogeny was robustly resolved in a phylogenomic analysis, with the CL3 clade amongst the earliest branching lineages. Metabolic and MRO functional reconstructions of CL3 clade members revealed that all three, including I. spirale, encode homologs of key components of the mitochondrial protein import apparatus and the ISC pathway, indicating the presence of a MRO in all of them. In silico evidence indicates that the organelles of E. mutabile and PCS-ghost host ATP and H2 production, unlike the cryptic MRO of I. spirale. These data suggest that the CL3 clade has experienced a hydrogenosome-to-mitosome transition independent from that previously documented for the lineage leading to Giardia.

BioKIT: a versatile toolkit for processing and analyzing diverse types of sequence data.

Bioinformatic analysis-such as genome assembly quality assessment, alignment summary statistics, relative synonymous codon usage, file format conversion, and processing and analysis-is integrated into diverse disciplines in the biological sciences. Several command-line pieces of software have been developed to conduct some of these individual analyses, but unified toolkits that conduct all these analyses are lacking. To address this gap, we introduce BioKIT, a versatile command line toolkit that has, upon publication, 42 functions, several of which were community-sourced, that conduct routine and novel processing and analysis of genome assemblies, multiple sequence alignments, coding sequences, sequencing data, and more. To demonstrate the utility of BioKIT, we conducted a comprehensive examination of relative synonymous codon usage across 171 fungal genomes that use alternative genetic codes, showed that the novel metric of gene-wise relative synonymous codon usage can accurately estimate gene-wise codon optimization, evaluated the quality and characteristics of 901 eukaryotic genome assemblies, and calculated alignment summary statistics for 10 phylogenomic data matrices. BioKIT will be helpful in facilitating and streamlining sequence analysis workflows. BioKIT is freely available under the MIT license from GitHub (https://github.com/JLSteenwyk/BioKIT), PyPi (https://pypi.org/project/jlsteenwyk-biokit/), and the Anaconda Cloud (https://anaconda.org/jlsteenwyk/jlsteenwyk-biokit). Documentation, user tutorials, and instructions for requesting new features are available online (https://jlsteenwyk.com/BioKIT).

Six Newly Sequenced Chloroplast Genomes From Trentepohliales: The Inflated Genomes, Alternative Genetic Code and Dynamic Evolution.

Cephaleuros is often known as an algal pathogen with 19 taxonomically valid species, some of which are responsible for red rust and algal spot diseases in vascular plants. No chloroplast genomes have yet been reported in this genus, and the limited genetic information is an obstacle to understanding the evolution of this genus. In this study, we sequenced six new Trentepohliales chloroplast genomes, including four Cephaleuros and two Trentepohlia. The chloroplast genomes of Trentepohliales are large compared to most green algae, ranging from 216 to 408 kbp. They encode between 93 and 98 genes and have a GC content of 26–36%. All new chloroplast genomes were circular-mapping and lacked a quadripartite structure, in contrast to the previously sequenced Trentepohlia odorata, which does have an inverted repeat. The duplicated trnD-GTC, petD, and atpA genes in C. karstenii may be remnants of the IR region and shed light on its reduction. Chloroplast genes of Trentepohliales show elevated rates of evolution, strong rearrangement dynamics and several genes display an alternative genetic code with reassignment of the UGA/UAG codon presumably coding for arginine. Our results present the first whole chloroplast genome of the genus Cephaleuros and enrich the chloroplast genome resources of Trentepohliales.

A computational screen for alternative genetic codes in over 250,000 genomes.

The genetic code has been proposed to be a 'frozen accident,' but the discovery of alternative genetic codes over the past four decades has shown that it can evolve to some degree. Since most examples were found anecdotally, it is difficult to draw general conclusions about the evolutionary trajectories of codon reassignment and why some codons are affected more frequently. To fill in the diversity of genetic codes, we developed Codetta, a computational method to predict the amino acid decoding of each codon from nucleotide sequence data. We surveyed the genetic code usage of over 250,000 bacterial and archaeal genome sequences in GenBank and discovered five new reassignments of arginine codons (AGG, CGA, and CGG), representing the first sense codon changes in bacteria. In a clade of uncultivated Bacilli, the reassignment of AGG to become the dominant methionine codon likely evolved by a change in the amino acid charging of an arginine tRNA. The reassignments of CGA and/or CGG were found in genomes with low GC content, an evolutionary force that likely helped drive these codons to low frequency and enable their reassignment.

The Unexpected Strategies behind Alternative Genetic Codes

This article proposes a number of codon reassignment strategies. These strategies are inferred by comparing the alternative (non-standard) genetic codes to the standard genetic code. Codon reassignments show strong and non-random connection to the standard code. The strategies interconnect all genetic codes and model a selected pathway through the heterotrophic part of life. The article also shows that the codon reassignment strategies model the evolution from the standard to the vertebrate mitochondrial code. Furthermore, it introduces a genetic code table based on third position synonyms that compares the vertebrate mitochondrial code to the standard code.