Comparative proteomics uncovers low asparagine content in Plasmodium tRip-KO proteins.

tRNAs are not only essential for decoding the genetic code, but their abundance also has a strong impact on the rate of protein production, folding, and on the stability of the translated messenger RNAs. Plasmodium expresses a unique surface protein called tRip, involved in the import of exogenous tRNAs into the parasite. Comparative proteomic analysis of the blood stage of wild-type and tRip-KO variant of P. berghei parasites revealed that downregulated proteins in the mutant parasite are distinguished by a bias in their asparagine content. Furthermore, the demonstration of the possibility of charging host tRNAs with Plasmodium aminoacyl-tRNA synthetases led us to propose that imported host tRNAs participate in parasite protein synthesis. These results also suggest a novel mechanism of translational control in which import of host tRNAs emerge as regulators of gene expression in the Plasmodium developmental cycle and pathogenesis, by enabling the synthesis of asparagine-rich regulatory proteins that efficiently and selectively control the parasite infectivity.

Underlying selection for the diversity of spike protein sequences of SARS-CoV-2.

The global spread of SARS-CoV-2 is fast moving and has caused a worldwide public health crisis. In the present article, we analyzed spike protein sequences of SARS-CoV-2 genomes to assess the impact of mutational diversity. We observed from amino acid usage patterns that spike proteins are associated with a diversity of mutational changes and most important underlying cause of variation of amino acid usage is the changes in hydrophobicity of spike proteins. The changing patterns of hydrophobicity of spike proteins over time and its influence on the receptor binding affinity provides crucial information on the SARS-CoV-2 interaction with human receptor. Our results also show that spike proteins have evolved to prefer more hydrophobic residues over time. The present study provides a comprehensive analysis of molecular sequence data to consider that mutational variants might play a crucial role in modulating the virulence and spread of the virus and has immediate implications for therapeutic strategies.

Structural insights into the amino acid usage variations in the profilin gene family.

Profilin protein is present ubiquitously in all forms of life and is allied with allergic responses among atopic individuals. In addition to this, profilins from various food sources are also associated with IgE cross-reactivity and are thus classified as pan-allergens. The present study unravels the physicochemical basis of differential amino acid usage patterns observed in the profilin gene family. Correspondence analysis based on amino acid usage of allergen and non-allergen profilins revealed discrete clusters among them, signifying differential patterns of amino acid usage. The amino acids, namely methionine, proline, histidine, glutamine, glutamic acid, tryptophan and glycine were found to be more frequently utilised by the allergen profilins compared to the non-allergens. Correlation analysis revealed that physicochemical features like protein disorder, trypsin digestion and solubility differed significantly among the allergen and non-allergen profilins, thus supporting the observations from correspondence analysis. In addition, comprehensive sequence analysis revealed that the allergen profilins possess conserved motifs which may correlate with their distinct physicochemical features. An in-depth structural analysis revealed that the over-represented amino acids in allergen profilins have a propensity of being exposed on the surface, which may be attributed to their distinct allergenic characteristics. The distinguished physicochemical features observed among allergens and non-allergens can be employed as descriptors to develop machine learning-based allergenicity prediction models.

Codon usage signatures in the genus Cryptococcus: A complex interplay of gene expression, translational selection and compositional bias.

The fungal genus Cryptococcus comprises of several diverse species. The pathogens forming Cryptococcus neoformans/ Cryptococcus gatti species complex are of immense clinical significance owing to the high frequency of infections and deaths globally. Three closely related non-pathogenic species namely, Cryptococcus amylolentus, Cryptococcus wingfieldii and Cryptococcus depauperatus are the non-pathogenic ancestral species from which pathogenic lineages have diverged. In the current study, a comprehensive analysis of factors influencing the codon and amino acid usage bias in six pathogenic and three non-pathogenic species was performed. Our results revealed that though compositional bias played a crucial role, translational selection and gene expression were the key determinants of codon usage variations. Analysis of relative dinucleotide abundance and codon context signatures revealed strict avoidance of TpA dinucleotide across genomes. Multivariate statistical analysis based on codon usage data resulted in discrete clustering of pathogens and non-pathogens which correlated with previous reports on their phylogenetic distribution.

Estimating the Influence of Physicochemical and Biochemical Property Indexes on Selection for Amino Acids Usage in Eukaryotic Cells.

Proteins can evolve by accumulating changes on amino acid sequences. These changes are mainly caused by missense mutations on its DNA coding sequences. Mutations with neutral or positive effects on fitness can be maintained while deleterious mutations tend to be eliminated by natural selection. Amino acid changes are influenced by the biophysical, chemical, and biological properties of amino acids. There is a multiplicity of amino acid properties that can influence the function and expression of proteins. Amino acid properties can be expressed into numerical indexes, which can help to predict functional and structural aspects of proteins and allow statistical inferences of selection pressure on amino acid usage. The accuracy of these analyses may be compromised by the existence of several numerical indexes that measure the same amino acid property, and the lack of objective parameters to determine the most accurate and biologically relevant index. In the present study, the gradient consistency test was used in order to estimate the magnitude of directional selection imparted by amino acid biochemical and biophysical properties on protein evolution.

Reconstruction and Characterization of Thermally Stable and Catalytically Active Proteins Comprising an Alphabet of ~ 13 Amino Acids.

While extant organisms synthesize proteins using approximately 20 kinds of genetically coded amino acids, the earliest protein synthesis system is likely to have been much simpler, utilizing a reduced set of amino acids. However, which types of building blocks were involved in primordial protein synthesis remains unclear. Herein, we reconstructed three convergent sequences of an ancestral nucleoside diphosphate kinase, each comprising a 10 amino acid "alphabet," and found that two of these variants folded into soluble and stable tertiary structures. Therefore, an alphabet consisting of 10 amino acids contains sufficient information for creating stable proteins. Furthermore, re-incorporation of a few more amino acid types into the active site of the 10 amino acid variants improved the catalytic activity, although the specific activity was not as high as that of extant proteins. Collectively, our results provide experimental support for the idea that robust protein scaffolds can be built with a subset of the current 20 amino acids that might have existed abundantly in the prebiotic environment, while the other amino acids, especially those with functional sidechains, evolved to contribute to efficient enzyme catalysis.

Insights into the riddles of codon usage patterns and codon context signatures in fungal genus Puccinia, a persistent threat to global agriculture.

The fungal genus Puccinia, comprising of several menacing pathogens, has been a persistent peril to global agriculture. Genome sequencing of various members of Puccinia offers a scope to excavate their genomic riddles. The present study has been addressed at exploring the complex niceties of codon and amino acid usage patterns and subsequent elucidation of the determinants that drive such behavior. Multivariate statistical analysis revealed a complex interplay of natural selection for translation and compositional bias to be operational on the codon usage patterns. Gene expression level was observed to be the most competent factor governing codon usage behavior of the genus. In spite of subtle AT richness of the genus, potential highly expressed gene sets were found to preferentially employ GC rich optimal codons. Estimation of relative dinucleotide abundance revealed preference toward the employment of GpA, CpA, TpC, and TpG dinucleotides and restraint from using TpA dinucleotide among the members of the genus. Extensive codon context analysis revealed that codon pairs with GpA, CpA, TpC, and TpG dinucleotides were over-represented and codon pairs with TpA dinucleotide were extensively avoided at the codon-codon (cP3-cA1) junctions. Amino acid usage signatures of the genus were found to be influenced considerably by several imperative factors like aromatic and hydrophobic character of the encoded gene products, genomic compositional constraint, and gene expressivity. Detailed know-how of the potential highly expressed gene sets and associated optimal codons in the genus promise to be informative for the scientific community engaged in combating Puccinia pathogenesis.

Contrasted evolutionary constraints on carbohydrate active enzymes (CAZymes) in selected Frankia strains.

Carbohydrate active enzymes (CAZymes) are capable of breaking complex polysaccharides into simpler form. In plant-host-associated microorganisms CAZymes are known to be involved in plant cell wall degradation. However, the biology and evolution of Frankia CAZymes are largely unknown. In the present study, we took a genomic approach to evaluate the presence and putative roles of CAZymes in Frankia. The CAZymes were found to be potentially highly expressed (PHX) proteins and contained more aromatic amino acids, which increased their biosynthetic energy cost. These energy rich amino acids were present in the active sites of CAZymes aiding in their carbohydrate binding capacity. Phylogenetic and evolutionary analyses showed that, in Frankia strains with the capacity to nodulate host plants, CAZymes were evolving slower than the other PHX genes, whereas similar genes from non-nodulating (or ineffectively nodulating) Frankia strains showed little variation in their evolutionary constraints compared to other PHX genes. Thus, the present study revealed the persistence of a strong purifying selection on CAZymes of Frankia indicating their crucial role.

Comprehensive profiling of codon usage signatures and codon context variations in the genus Ustilago.

The fungal genus Ustilago consists of intimidating pathogens associated with disease manifestations in plants of agricultural importance and gravity. Rapid progress of genome sequencing has opened the floodgates for biological research. Availability of Ustilago genomes provides a scope to explore complex codon and amino acid usage patterns in the genus. An extensive scrutiny of the factors underlying the complex modalities of codon and amino acid usage in Ustilago has been executed in the present analysis. Multivariate statistical analysis revealed a dominant effect of natural selection pressure, aimed at translational accuracy, to be operative on the codon usage behavior. Subtle impact of GC compositional constraint was also evident on the codon usage patterns. Gene expressivity was inferred to be the most crucial determinant governing observed codon usage variations. Amino acid usage patterns were found to be significantly governed by aromatic and hydrophobic characters of the encoded proteins. GC content and length of protein coding sequences also had considerable influence on the amino acid usage signatures. Extensive analysis of codon context variations revealed that UpA dinucleotides were strictly avoided at the codon-codon junctions (cP3-cA1) which might be attributed to reduce the risk of nonsense mutations and subsequently, improve translational finesse. Identification of the optimal codons, employed preferentially among the genes with high expressivity, and estimation of preferred and avoided codon pairs in Ustilago promises to be useful pertaining to mutational experiments at the codonic level, targeted to thwart the growth of Ustilago and combat associated pathogenesis.

Three new Diplozoidae mitogenomes expose unusual compositional biases within the Monogenea class: implications for phylogenetic studies.

BACKGROUND: As the topologies produced by previous molecular and morphological studies were contradictory and unstable (polytomy), evolutionary relationships within the Diplozoidae family and the Monogenea class (controversial relationships among the Discocotylinea, Microcotylinea and Gastrocotylinea suborders) remain unresolved. Complete mitogenomes carry a relatively large amount of information, sufficient to provide a much higher phylogenetic resolution than traditionally used morphological traits and/or single molecular markers. However, their implementation is hampered by the scarcity of available monogenean mitogenomes. Therefore, we sequenced and characterized mitogenomes belonging to three Diplozoidae family species, and conducted comparative genomic and phylogenomic analyses for the entire Monogenea class. RESULTS: Taxonomic identification was inconclusive, so two of the species were identified merely to the genus level. The complete mitogenomes of Sindiplozoon sp. and Eudiplozoon sp. are 14,334 bp and 15,239 bp in size, respectively. Paradiplozoon opsariichthydis (15,385 bp) is incomplete: an approximately 2000 bp-long gap within a non-coding region could not be sequenced. Each genome contains the standard 36 genes (atp8 is missing). G + T content and the degree of GC- and AT-skews of these three mitogenome (and their individual elements) were higher than in other monogeneans. nad2, atp6 and nad6 were the most variable PCGs, whereas cox1, nad1 and cytb were the most conserved. Mitochondrial phylogenomics analysis, conducted using concatenated amino acid sequences of all PCGs, indicates that evolutionary relationships of the three genera are: (Eudiplozoon, (Paradiplozoon, Sindiplozoon)); and of the three suborders: (Discocotylinea, (Microcotylinea, Gastrocotylinea)). These intergeneric relationships were also supported by the skewness and principal component analyses. CONCLUSIONS: Our results show that nad2, atp6 and nad6 (fast-evolving) would be better candidates than cox1 (slow-evolving) for species identification and population genetics studies in Diplozoidae. Nucleotide bias and codon and amino acid usage patterns of the three diplozoid mitogenomes are more similar to cestodes and trematodes than to other monogenean flatworms. This unusual mutational bias was reflected in disproportionately long branches in the phylogram. Our study offsets the scarcity of molecular data for the subclass Polyopisthocotylea to some extent, and might provide important new insights into the evolutionary history of the three genera and three suborders.

Comparative genomic analysis for nucleotide, codon, and amino acid usage patterns of mycoplasmas.

The evolutionary factors in influencing the genetic characteristics of nucleotide, synonymous codon, and amino acid usage of 18 mycoplasma species were analyzed. The nucleotide usage at the 1st and 2nd codon position which determines amino acid composition of proteins has a significant correlation with the total nucleotide composition of gene population of these mycoplasma species, however, the nucleotide usage at the 3rd codon position which affects synonymous codon usage patterns has a slight correlation with either the total nucleotide composition or the nucleotide usage at the 1st and 2nd codon position. Other evolutionary factors join in the evolutionary process of mycoplasma apart from mutation pressure caused by nucleotide usage constraint based on the relationships between effective number of codons/codon adaptation index and nucleotide usage at the 3rd codon position. Although nucleotide usage of gene population in mycoplasma dominates in forming the overall codon usage trends, the relative abundance of codon with nucleotide context and amino acid usage pattern show that translation selection involved in translation accuracy and efficiency play an important role in synonymous codon usage patterns. In addition, synonymous codon usage patterns of gene population have a bigger power to represent genetic diversity among different species than amino acid usage. These results suggest that although the mycoplasmas reduce its genome size during the evolutionary process and shape the form, which is opposite to their hosts, of AT usages at high levels, this kind organism still depends on nucleotide usage at the 1st and 2nd codon positions to control syntheses of the requested proteins for surviving in their hosts and nucleotide usage at the 3rd codon position to develop genetic diversity of different mycoplasma species. This systemic analysis with 18 mycoplasma species may provide useful clues for further in vivo genetic studies on the related species.

Adaptation of Borrelia burgdorferi to its natural hosts by synonymous codon and amino acid usage.

Lyme disease, caused by Borrelia burgdorferi, is a focally endemic tick-transmitted zoonotic infection. In this study, the major factors underlying synonymous codon-related amino acid usage in the B. burgdorferi genome and bias in synonymous codon usage of the translation initiation region of coding sequences were analyzed. Additionally, adaptation of B. burgdorferi to several of its hosts was analyzed in the context of synonymous codon usage. Principal component analysis (PCA) revealed that nucleotide content at the third synonymous position of a codon influenced the synonymous codon usage pattern, but the strand-specific factor did not influence the synonymous codon usage pattern of B. burgdorferi. In terms of the low GC content of B. burgdorferi coding sequences, the effective number of codons (ENC) showed a significant correlation with GC3 content (at the synonymous position). For the amino acid usage pattern for B. burgdorferi, PCA showed that the strand-specific factor did not contribute to this pattern, while the properties (aromaticity and hydrophobicity) of the amino acids themselves showed strong correlations with this pattern. Under-represented codons, which were frequently selected in the translation initiation region, possibly play roles in regulating gene expression in B. burgdorferi. In terms of co-evolution and synonymous codon usage patterns, adaptation of B. burgdorferi to different intermediate hosts was apparent to different degrees, and the degree of adaptation of this spirochete to wild animals was stronger than that of humans or mice.

Synonymous Codon Usages as an Evolutionary Dynamic for Chlamydiaceae.

The family of Chlamydiaceae contains a group of obligate intracellular bacteria that can infect a wide range of hosts. The evolutionary trend of members in this family is a hot topic, which benefits our understanding of the cross-infection of these pathogens. In this study, 14 whole genomes of 12 Chlamydia species were used to investigate the nucleotide, codon, and amino acid usage bias by synonymous codon usage value and information entropy method. The results showed that all the studied Chlamydia spp. had A/T rich genes with over-represented A or T at the third positions and G or C under-represented at these positions, suggesting that nucleotide usages influenced synonymous codon usages. The overall codon usage trend from synonymous codon usage variations divides the Chlamydia spp. into four separate clusters, while amino acid usage divides the Chlamydia spp. into two clusters with some exceptions, which reflected the genetic diversity of the Chlamydiaceae family members. The overall codon usage pattern represented by the effective number of codons (ENC) was significantly positively correlated to gene GC3 content. A negative correlation exists between ENC and the codon adaptation index for some Chlamydia species. These results suggested that mutation pressure caused by nucleotide composition constraint played an important role in shaping synonymous codon usage patterns. Furthermore, codon usage of T3ss and Pmps gene families adapted to that of the corresponding genome. Taken together, analyses help our understanding of evolutionary interactions between nucleotide, synonymous codon, and amino acid usages in genes of Chlamydiaceae family members.

Plasmodium, the Apicomplexa Outlier When It Comes to Protein Synthesis.

Plasmodium is an obligate intracellular parasite that has numerous interactions with different hosts during its elaborate life cycle. This is also the case for the other parasites belonging to the same phylum Apicomplexa. In this study, we bioinformatically identified the components of the multi-synthetase complexes (MSCs) of several Apicomplexa parasites and modelled their assembly using AlphaFold2. It appears that none of these MSCs resemble the two MSCs that we have identified and characterized in Plasmodium. Indeed, tRip, the central protein involved in the association of the two Plasmodium MSCs is different from its homologues, suggesting also that the tRip-dependent import of exogenous tRNAs is not conserved in other apicomplexan parasites. Based on this observation, we searched for obvious differences that could explain the singularity of Plasmodium protein synthesis by comparing tRNA genes and amino acid usage in the different genomes. We noted a contradiction between the large number of asparagine residues used in Plasmodium proteomes and the single gene encoding the tRNA that inserts them into proteins. This observation remains true for all the Plasmodia strains studied, even those that do not contain long asparagine homorepeats.

Emergence of Genomic Diversity in the Spike Protein of the "Omicron" Variant.

SARS-CoV-2 (Severe Acute Respiratory Syndrome Coronavirus) has constantly been evolving into different forms throughout its spread in the population. Emerging SARS-CoV-2 variants, predominantly the variants of concern (VOCs), could have an impact on the virus spread, pathogenicity, and diagnosis. The recently emerged "Omicron" variant has exhibited rapid transmission and divergence. The spike protein of SARS-CoV-2 has consistently been appearing as the mutational hotspot of all these VOCs. In order to determine a deeper understanding of the recently emerged and extremely divergent "Omicron", a study of amino acid usage patterns and their substitution patterns was performed and compared with those of the other four successful variants of concern ("Alpha", "Beta", "Gamma", and "Delta"). We observed that the amino acid usage of "Omicron" has a distinct pattern that distinguishes it from other VOCs and is significantly correlated with the increased hydrophobicity in spike proteins. We observed an increase in the non-synonymous substitution rate compared with the other four VOCs. Considering the phylogenetic relationship, we hypothesized about the functional interdependence between recombination and the mutation rate that might have resulted in a shift in the optimum of the mutation rate for the evolution of the "Omicron" variant. The results suggest that for improved disease prevention and control, more attention should be given to the significant genetic differentiation and diversity of newly emerging variants.

A Comprehensive Phylogenetic Analysis of SARS-CoV-2: Utilizing a Novel and Convenient In-House RT-PCR Method for Characterization without Virus Culture and BSL-3 Facilities.

We developed a convenient method for amplifying the complete SARS-CoV-2 sequence using in-house RT-PCR without virus culture. Forty-one stored throat swabs and blood specimens were collected from eight SARS-CoV-2 infections at multiple time points. Total RNA was extracted using the QIAamp viral RNA mini kit and pooled for higher RNA levels. Only those positive specimens by commercial real-time RT-PCR (RT-qPCR) were selected and amplified by in-house RT-PCR for complete sequences, followed by sequencing. Phylogenetic trees and exploratory analyses were performed using MEGA 11 and Simplot 3.5.1 software. Swab samples had significantly higher total RNA concentrations than plasma (p < 0.01). Positive results were found mainly in swabs, but one was found in plasma. Successful gene amplification depended on Ct values (Ct < 38). A non-synonymous substitution was found in ORF1ab/Nsp3 (at NC045512.2 position 6312, C to A) and most spike protein mutations occurred in the S1 subunit (residues 14-685). The proposed method is time-saving and reliable for rapid genomic analysis. Increasing sample volume and pooling them for RNA extraction increases RNA concentration without culture. Combining nucleotide sequences from specific variable regions of the genome is more efficient than conventional methods.

Convergent Usage of Amino Acids in Human Cancers as A Reversed Process of Tissue Development.

Genome- and transcriptome-wide amino acid usage preference across different species is a well-studied phenomenon in molecular evolution, but its characteristics and implication in cancer evolution and therapy remain largely unexplored. Here, we analyzed large-scale transcriptome/proteome profiles, such as The Cancer Genome Atlas (TCGA), the Genotype-Tissue Expression (GTEx), and the Clinical Proteomic Tumor Analysis Consortium (CPTAC), and found that compared to normal tissues, different cancer types showed a convergent pattern toward using biosynthetically low-cost amino acids. Such a pattern can be accurately captured by a single index based on the average biosynthetic energy cost of amino acids, termed energy cost per amino acid (ECPA). With this index, we further compared the trends of amino acid usage and the contributing genes in cancer and tissue development, and revealed their reversed patterns. Finally, focusing on the liver, a tissue with a dramatic increase in ECPA during development, we found that ECPA represents a powerful biomarker that could distinguish liver tumors from normal liver samples consistently across 11 independent patient cohorts and outperforms any index based on single genes. Our study reveals an important principle underlying cancer evolution and suggests the global amino acid usage as a system-level biomarker for cancer diagnosis.

HIV long-term non-progressors share similar features with simian immunodeficiency virus infection of chimpanzees.

HIV-1 infection in human beings has been an outcome of cross-species transmission event of simian immunodeficiency virus from chimpanzees (SIVcpz). Present study reveals differential features of envelope genes representing different categories of HIV-1 disease progression in human beings, namely, rapid progressors (RP), slow progressors (SP) and long-term non-progressors (LTNP) with respect to SIVcpz, based on their amino acid usage patterns. It was evident that SP, LTNP and SIVcpz envelope genes displayed similar patterns of amino acid usage which strongly contrasted with the features exhibited by the envelope genes representing RP category. Robust analysis revealed that selection constraint of human host on SP and LTNP associated envelope genes and chimpanzee host on SIVcpz envelope genes were more severe compared to selection pressure operational on RP associated envelope genes. Evolutionary forces of selection appeared to be comparatively more relaxed on the RP envelope genes in contrast to SP, LTNP and SIVcpz types. Better binding of RP envelope glycoprotein 120 (gp120) compared to envelope gp120 representing SP, LTNP and SIVcpz with host cellular receptor CD4, as inferred employing molecular docking approaches, promises to confer meaningful insights into the event of speedy progression of HIV in rapid progressors. It was interesting to note that envelope glycoprotein exhibited a tendency of hindering proper interaction of host (human/chimpanzee) CD4 and major histocompatibility complex II (MHC II), with a better efficacy in rapid progressors, thus, facilitating highest degrees of immune suppression. Proper identification of the contrasting features might confer a scope to modulate rapid progression of HIV to a long-term non-progressive controlled case, as observed in LTNP and SIVcpz infection, simultaneously aiding therapeutic research against AIDS targeted at drug and vaccine development.Communicated by Ramaswamy H. Sarma.

Compositional Analysis of Flatworm Genomes Shows Strong Codon Usage Biases Across All Classes.

In the present work, we performed a comparative genome-wide analysis of 22 species representative of the main clades and lifestyles of the phylum Platyhelminthes. We selected a set of 700 orthologous genes conserved in all species, measuring changes in GC content, codon, and amino acid usage in orthologous positions. Values of 3rd codon position GC spanned over a wide range, allowing to discriminate two distinctive clusters within freshwater turbellarians, Cestodes and Trematodes respectively. Furthermore, a hierarchical clustering of codon usage data differs remarkably from the phylogenetic tree. Additionally, we detected a synonymous codon usage bias that was more dramatic in extreme GC-poor or GC-rich genomes, i.e., GC-poor Schistosomes preferred to use AT-rich terminated synonymous codons, while GC-rich M. lignano showed the opposite behavior. Interestingly, these biases impacted the amino acidic usage, with preferred amino acids encoded by codons following the GC content trend. These are associated with non-synonymous substitutions at orthologous positions. The detailed analysis of the synonymous and non-synonymous changes provides evidence for a two-hit mechanism where both mutation and selection forces drive the diverse coding strategies of flatworms.

Translational Selection for Speed Is Not Sufficient to Explain Variation in Bacterial Codon Usage Bias.

Increasing growth rate across bacteria strengthens selection for faster translation, concomitantly increasing the total number of tRNA genes and codon usage bias (CUB: enrichment of specific synonymous codons in highly expressed genes). Typically, enriched codons are translated by tRNAs with higher gene copy numbers (GCN). A model of tRNA-CUB coevolution based on fast growth-associated selection on translational speed recapitulates these patterns. A key untested implication of the coevolution model is that translational selection should favor higher tRNA GCN for more frequently used amino acids, potentially weakening the effect of growth-associated selection on CUB. Surprisingly, we find that CUB saturates with increasing growth rate across γ-proteobacteria, even as the number of tRNA genes continues to increase. As predicted, amino acid-specific tRNA GCN is positively correlated with the usage of corresponding amino acids, but there is no correlation between growth rate associated changes in CUB and amino acid usage. Instead, we find that some amino acids-cysteine and those in the NNA/G codon family-show weak CUB that does not increase with growth rate, despite large variation in the corresponding tRNA GCN. We suggest that amino acid-specific variation in CUB is not explained by tRNA GCN because GCN does not influence the difference between translation times of synonymous codons as expected. Thus, selection on translational speed alone cannot fully explain quantitative variation in overall or amino acid-specific CUB, suggesting a significant role for other functional constraints and amino acid-specific codon features.