The Effect of Multiple Evolutionary Selections on Synonymous Codon Usage of Genes in the Mycoplasma bovis Genome

Mycoplasma bovis is a major pathogen causing arthritis, respiratory disease and mastitis in cattle. A better understanding of its genetic features and evolution might represent evidences of surviving host environments. In this study, multiple factors influencing synonymous codon usage patterns in M. bovis (three strains’ genomes) were analyzed. The overall nucleotide content of genes in the M. bovis genome is AT-rich. Although the G and C contents at the third codon position of genes in the leading strand differ from those in the lagging strand (p<0.05), the 59 synonymous codon usage patterns of genes in the leading strand are highly similar to those in the lagging strand. The over-represented codons and the under-represented codons were identified. A comparison of the synonymous codon usage pattern of M. bovis and cattle (susceptible host) indicated the independent formation of synonymous codon usage of M. bovis. Principal component analysis revealed that (i) strand-specific mutational bias fails to affect the synonymous codon usage pattern in the leading and lagging strands, (ii) mutation pressure from nucleotide content plays a role in shaping the overall codon usage, and (iii) the major trend of synonymous codon usage has a significant correlation with the gene expression level that is estimated by the codon adaptation index. The plot of the effective number of codons against the G+C content at the third codon position also reveals that mutation pressure undoubtedly contributes to the synonymous codon usage pattern of M. bovis. Additionally, the formation of the overall codon usage is determined by certain evolutionary selections for gene function classification (30S protein, 50S protein, transposase, membrane protein, and lipoprotein) and translation elongation region of genes in M. bovis. The information could be helpful in further investigations of evolutionary mechanisms of the Mycoplasma family and heterologous expression of its functionally important proteins.     

The Distribution of Synonymous Codon Choice in the Translation Initiation Region of Dengue Virus

Dengue is the most common arthropod-borne viral (Arboviral) illness in humans. The genetic features concerning the codon usage of dengue virus (DENV) were analyzed by the relative synonymous codon usage, the effective number of codons and the codon adaptation index. The evolutionary distance between DENV and the natural hosts (Homo sapiens, Pan troglodytes, Aedes albopictus and Aedes aegypti) was estimated by a novel formula. Finally, the synonymous codon usage preference for the translation initiation region of this virus was also analyzed. The result indicates that the general trend of the 59 synonymous codon usage of the four genotypes of DENV are similar to each other, and this pattern has no link with the geographic distribution of the virus. The effect of codon usage pattern of Aedes albopictus and Aedes aegypti on the formation of codon usage of DENV is stronger than that of the two primates. Turning to the codon usage preference of the translation initiation region of this virus, some codons pairing to low tRNA copy numbers in the two primates have a stronger tendency to exist in the translation initiation region than those in the open reading frame of DENV. Although DENV, like other RNA viruses, has a high mutation to adapt its hosts, the regulatory features about the synonymous codon usage have been ‘branded’ on the translation initiation region of this virus in order to hijack the translational mechanisms of the hosts.     

The Selective Advantage of Synonymous Codon Usage Bias in Salmonella

The genetic code in mRNA is redundant, with 61 sense codons translated into 20 different amino acids. Individual amino acids are encoded by up to six different codons but within codon families some are used more frequently than others. This phenomenon is referred to as synonymous codon usage bias. The genomes of free-living unicellular organisms such as bacteria have an extreme codon usage bias and the degree of bias differs between genes within the same genome. The strong positive correlation between codon usage bias and gene expression levels in many microorganisms is attributed to selection for translational efficiency. However, this putative selective advantage has never been measured in bacteria and theoretical estimates vary widely. By systematically exchanging optimal codons for synonymous codons in the tuf genes we quantified the selective advantage of biased codon usage in highly expressed genes to be in the range 0.2–4.2 x 10⁻⁴ per codon per generation. These data quantify for the first time the potential for selection on synonymous codon choice to drive genome-wide sequence evolution in bacteria, and in particular to optimize the sequences of highly expressed genes. This quantification may have predictive applications in the design of synthetic genes and for heterologous gene expression in biotechnology.     

On finding poorly translated codons based on their usage frequency

Long stretches of “rare” codons are known to severely inhibit the efficiency of translation. Understanding the distribution of such rare codons is of critical importance in improving the efficiency of heterologous gene expression systems. Accurate estimates of codon usage take the abundance of each protein into consideration. In this paper, we analyze the correlation between approximate measures of codon usage and the availability of tRNA at various growth rates in E coli. We show that the computationally derived estimates of tRNA isoacceptor concentration enable the finding of poorly translated codons.     

Expression and secretion of human bone morphogenetic protein-7 in pichia pastoris

The synonymous codons are used in a highly nonrandom manner in hosts of widely divergent species, which is termed ‘codon usage bias’. Several reports suggest that codon usage bias sometimes obstructs attempts to express high levels of exogenous genes. In this study, an attempt was made to express mature peptide of human bone morphogenetic protein-7 with optimized codons in P. pastoris expression system. Three low-usage ARG codons (CGG or CGA) in hBmp7 mature domain have been successfully transformed into P. pastoris-preferred ARG codons (AGA) with overlap extension PCR-based multiple-site-directed mutagenesis for a high level expression of hBMP7 mature peptide. The results of this study showed that the production level (25.45 mg/L) of a codon-optimized strain increased 4.6-fold in comparison with that (5.5 mg/L) of noncodon-optimized strain. A strain harboring multicopy of codon-optimized hbmp7 expression cassette showed an even higher expression level, which was about 2-fold compared with that of the single-copy one. These recombinant hBMP7 mature peptides were produced as 18-kD monomer proteins and were easily purified from culture supernatants using ion-exchange chromatography. Functional assay demonstrated that rhBMP7 could induce ectopic cartilage formation, although its inductive ability was much less active than that of CHO cell-derived hBMP7.     

Secretory signal sequence non-optimal codons are required for expression and export of beta-lactamase

In this study we altered the codon usage in the signal sequence of the bla gene, encoding β-lactamase in Escherichia coli. Changing all of the thirteen non-optimal codons to optimal lowered expression 4-fold as measured by minimum inhibitory concentration (MIC) to the β-lactam antibiotic ampicillin. The difference in ampicillin resistance was reduced at 28 °C compared to expression at 37 °C, suggesting that the optimised bla allele is misfolded and degraded by heat-shock regulated proteases. A screen was carried out, designed specifically to identify revertants with changes in codon usage resulting in higher MIC to ampicillin. The nine revertants revealed by this method all had optimal to non-optimal codon changes in the signal sequence. These results, and those of our previous study with maltose binding protein model system, confirm that non-optimal codons are important for expression and export of secretory proteins via both the SecB-dependent and -independent pathways.     

Characterization of synonymous codon usage bias in the pseudorabies virus US1 gene

In the present study, we examined the codon usage bias between pseudorabies virus (PRV) US1 gene and the US1-like genes of 20 reference alphaherpesviruses. Comparative analysis showed noticeable disparities of the synonymous codon usage bias in the 21 alphaherpesviruses, indicated by codon adaptation index, effective number of codons (ENc) and GC3s value. The codon usage pattern of PRV US1 gene was phylogenetically conserved and similar to that of the US1-like genes of the genus Varicellovirus of alphaherpesvirus, with a strong bias towards the codons with C and G at the third codon position. Cluster analysis of codon usage pattern of PRV US1 gene with its reference alphaherpesviruses demonstrated that the codon usage bias of US1-like genes of 21 alphaherpesviruses had a very close relation with their gene functions. ENc-plot revealed that the genetic heterogeneity in PRV US1 gene and the 20 reference alphaherpesviruses was constrained by G+C content, as well as the gene length. In addition, comparison of codon preferences in the US1 gene of PRV with those of E. coli, yeast and human revealed that there were 50 codons showing distinct usage differences between PRV and yeast, 49 between PRV and human, but 48 between PRV and E. coli. Although there were slightly fewer differences in codon usages between E.coli and PRV, the difference is unlikely to be statistically significant, and experimental studies are necessary to establish the most suitable expression system for PRV US1. In conclusion, these results may improve our understanding of the evolution, pathogenesis and functional studies of PRV, as well as contributing to the area of herpesvirus research or even studies with other viruses.     

Differential Responses of CO2 Assimilation,Carbohydrate Allocation and Gene Expression to NaCl Stress in Perennial Ryegrass with Different Salt Tolerance

Little is known about the effects of NaCl stress on perennial ryegrass (Lolium perenne L.) photosynthesis and carbohydrate flux. The objective of this study was to understand the carbohydrate metabolism and identify the gene expression affected by salinity stress. Seventy-four days old seedlings of two perennial ryegrass accessions (salt-sensitive ‘PI 538976’ and salt-tolerant ‘Overdrive’) were subjected to three levels of salinity stress for 5 days. Turf quality in all tissues (leaves, stems and roots) of both grass accessions negatively and significantly correlated with GFS (Glu+Fru+Suc) content, except for ‘Overdrive’ stems. Relative growth rate (RGR) in leaves negatively and significantly correlated with GFS content in ‘Overdrive’ (P<0.01) and ‘PI 538976’ (P<0.05) under salt stress. ‘Overdrive’ had higher CO₂ assimilation and F_v/F_m than ‘PI 538976’. Intercellular CO₂ concentration, however, was higher in ‘PI 538976’ treated with 400 mM NaCl relative to that with 200 mM NaCl. GFS content negatively and significantly correlated with RGR in ‘Overdrive’ and ‘PI 538976’ leaves and in ‘PI 538976’ stems and roots under salt stress. In leaves, carbohydrate allocation negatively and significantly correlated with RGR (r² = 0.83, P<0.01) and turf quality (r² = 0.88, P<0.01) in salt-tolerant ‘Overdrive’, however, the opposite trend for salt-sensitive ‘PI 538976’ (r² = 0.71, P<0.05 for RGR; r² = 0.62, P>0.05 for turf quality). A greater up-regulation in the expression of SPS, SS, SI, 6-SFT gene was observed in ‘Overdrive’ than ‘PI 538976’. A higher level of SPS and SS expression in leaves was found in ‘PI 538976’ relative to ‘Overdrive’. Accumulation of hexoses in roots, stems and leaves can induce a feedback repression to photosynthesis in salt-stressed perennial ryegrass and the salt tolerance may be changed with the carbohydrate allocation in leaves and stems.     

Integrating Circadian Activity and Gene Expression Profiles to Predict Chronotoxicity of Drosophila suzukii Response to Insecticides

Native to Southeast Asia, Drosophila suzukii (Matsumura) is a recent invader that infests intact ripe and ripening fruit, leading to significant crop losses in the U.S., Canada, and Europe. Since current D. suzukii management strategies rely heavily on insecticide usage and insecticide detoxification gene expression is under circadian regulation in the closely related Drosophila melanogaster, we set out to determine if integrative analysis of daily activity patterns and detoxification gene expression can predict chronotoxicity of D. suzukii to insecticides. Locomotor assays were performed under conditions that approximate a typical summer or winter day in Watsonville, California, where D. suzukii was first detected in North America. As expected, daily activity patterns of D. suzukii appeared quite different between ‘summer’ and ‘winter’ conditions due to differences in photoperiod and temperature. In the ‘summer’, D. suzukii assumed a more bimodal activity pattern, with maximum activity occurring at dawn and dusk. In the ‘winter’, activity was unimodal and restricted to the warmest part of the circadian cycle. Expression analysis of six detoxification genes and acute contact bioassays were performed at multiple circadian times, but only in conditions approximating Watsonville summer, the cropping season, when most insecticide applications occur. Five of the genes tested exhibited rhythmic expression, with the majority showing peak expression at dawn (ZT0, 6am). We observed significant differences in the chronotoxicity of D. suzukii towards malathion, with highest susceptibility at ZT0 (6am), corresponding to peak expression of cytochrome P450s that may be involved in bioactivation of malathion. High activity levels were not found to correlate with high insecticide susceptibility as initially hypothesized. Chronobiology and chronotoxicity of D. suzukii provide valuable insights for monitoring and control efforts, because insect activity as well as insecticide timing and efficacy are crucial considerations for pest management. However, field research is necessary for extrapolation to agricultural settings.     

Mitotic checkpoint gene expression is tuned by codon usage bias

The mitotic checkpoint (also called spindle assembly checkpoint, SAC) is a signaling pathway that safeguards proper chromosome segregation. Correct functioning of the SAC depends on adequate protein concentrations and appropriate stoichiometries between SAC proteins. Yet very little is known about the regulation of SAC gene expression. Here, we show in the fission yeast Schizosaccharomyces pombe that a combination of short mRNA half‐lives and long protein half‐lives supports stable SAC protein levels. For the SAC genes mad2 ⁺ and mad3 ⁺, their short mRNA half‐lives are caused, in part, by a high frequency of nonoptimal codons. In contrast, mad1 ⁺ mRNA has a short half‐life despite a higher frequency of optimal codons, and despite the lack of known RNA‐destabilizing motifs. Hence, different SAC genes employ different strategies of expression. We further show that Mad1 homodimers form co‐translationally, which may necessitate a certain codon usage pattern. Taken together, we propose that the codon usage of SAC genes is fine‐tuned to ensure proper SAC function. Our work shines light on gene expression features that promote spindle assembly checkpoint function and suggests that synonymous mutations may weaken the checkpoint.     

Convergence and constraint in eukaryotic release factor 1 (eRF1) domain 1: the evolution of stop codon specificity

Class 1 release factor in eukaryotes (eRF1) recognizes stop codons and promotes peptide release from the ribosome. The ‘molecular mimicry’ hypothesis suggests that domain 1 of eRF1 is analogous to the tRNA anticodon stem–loop. Recent studies strongly support this hypothesis and several models for specific interactions between stop codons and residues in domain 1 have been proposed. In this study we have sequenced and identified novel eRF1 sequences across a wide diversity of eukaryotes and re-evaluated the codon-binding site by bioinformatic analyses of a large eRF1 dataset. Analyses of the eRF1 structure combined with estimates of evolutionary rates at amino acid sites allow us to define the residues that are under structural (i.e. those involved in intramolecular interactions) versus non-structural selective constraints. Furthermore, we have re-assessed convergent substitutions in the ciliate variant code eRF1s using maximum likelihood-based phylogenetic approaches. Our results favor the model proposed by Bertram et al. that stop codons bind to three ‘cavities’ on the protein surface, although we suggest that the stop codon may bind in the opposite orientation to the original model. We assess the feasibility of this alternative binding orientation with a triplet stop codon and the eRF1 domain 1 structures using molecular modeling techniques.     

Enhancing the recombinant protein expression of halohydrin dehalogenase HheA in Escherichia coli by applying a codon optimization strategy

Halohydrin dehalogenases are attractive biocatalysts in producing a series of important chiral building blocks. Recombinant expression of halohydrin dehalogenase from Arthrobacter sp. AD2 (HheA) in Escherichia coli using T7 promoter-based pGEF(+) system revealed much lower expression level than that of the well-studied halohydrin dehalogenase from Agrobacterium radiobacter AD1 (HheC). In this study, we changed the codon usage in the 5′-end of hheA gene to improve the expression yield of HheA. Our results showed that the expression of HheA could be largely improved by the replacement of G-rich +2 codon (adjacent to the start codon) with less G-containing codons. The expression of one of the resulting mutants HheA-D1 (replaced +2 codon GTG with CCA) was about 4-fold higher and purified yields about 8-fold greater than that of the wild-type HheA. Moreover, the expression level of the resulting HheA variants correlated well with the minimal folding free energy (ΔG) of the mRNA secondary structure surrounding the 5′-end region of the genes. These findings suggested that the G-rich +2 codon of hheA gene might be the main suppressive factor for limiting the recombinant expression of HheA and that +2 codon optimization strategy could be used as a general tool in modulating recombinant protein production in E. coli.     

Translational Features of Human Alpha 2b Interferon Production in Escherichia coli

The yield of human alpha 2b interferon in Escherichia coli was optimized by replacement of low-usage arginine codons located in the mRNA 5′ end. The differences observed among the various gene variants suggest that codon usage, Shine-Dalgarno-like sequences, and mRNA secondary structure contribute to the performance of E. coli translation machinery.     

Comparative genome sequence analysis of Sulfolobus acidocaldarius and 9 other isolates of its genus for factors influencing codon and amino acid usage

In the present study, major constraints for codon and amino acid usage of Sulfolobus acidocaldarius, Sulfolobus solfataricus, Sulfolobus tokodali, Sulfolobus islandis and 6 other isolates from islandicus species of genus Sulfolobus were investigated. Correspondence analysis revealed high significant correlation between the major trend of synonymous codon usage and gene expression level, as assessed by the “Codon Adaptation Index” (CAI). There is a significant negative correlation between Nc (Effective number of codons) and CAI demonstrating role of codon bias as an important determinant of codon usage. The significant correlation between major trend of synonymous codon usage and GC3s (G + C at third synonymous position) indicated dominant role of mutational bias in codon usage pattern. The result was further supported from SCUO (synonymous codon usage order) analysis. The amino acid usage was found to be significantly influenced by aromaticity and hydrophobicity of proteins. However, translational selection which causes a preference for codons that are most rapidly translated by current tRNA with multiple copy numbers was not found to be highly dominating for all studied isolates. Notably, 26 codons that were found to be optimally used by genes of S. acidocaldarius at higher expression level and its comparative analysis with 9 other isolates may provide some useful clues for further in vivo genetic studies on this genus.     

Versatile Dual Reporter Gene Systems for Investigating Stop Codon Readthrough in Plants

Background

Translation is most often terminated when a ribosome encounters the first in-frame stop codon (UAA, UAG or UGA) in an mRNA. However, many viruses (and some cellular mRNAs) contain “stop” codons that cause a proportion of ribosomes to terminate and others to incorporate an amino acid and continue to synthesize a “readthrough”, or C-terminally extended, protein. This dynamic redefinition of codon meaning is dependent on specific sequence context.

Methodology

We describe two versatile dual reporter systems which facilitate investigation of stop codon readthrough in vivo in intact plants, and identification of the amino acid incorporated at the decoded stop codon. The first is based on the reporter enzymes NAN and GUS for which sensitive fluorogenic and histochemical substrates are available; the second on GST and GFP.

Conclusions

We show that the NAN-GUS system can be used for direct in planta measurements of readthrough efficiency following transient expression of reporter constructs in leaves, and moreover, that the system is sufficiently sensitive to permit measurement of readthrough in stably transformed plants. We further show that the GST-GFP system can be used to affinity purify readthrough products for mass spectrometric analysis and provide the first definitive evidence that tyrosine alone is specified in vivo by a ‘leaky’ UAG codon, and tyrosine and tryptophan, respectively, at decoded UAA, and UGA codons in the Tobacco mosaic virus (TMV) readthrough context.