Genome landscapes and bacteriophage codon usage

Across all kingdoms of biological life, protein-coding genes exhibit unequal usage of synonymous codons. Although alternative theories abound, translational selection has been accepted as an important mechanism that shapes the patterns of codon usage in prokaryotes and simple eukaryotes. Here we analyze patterns of codon usage across 74 diverse bacteriophages that infect E. coli, P. aeruginosa, and L. lactis as their primary host. We use the concept of a “genome landscape,” which helps reveal non-trivial, long-range patterns in codon usage across a genome. We develop a series of randomization tests that allow us to interrogate the significance of one aspect of codon usage, such as GC content, while controlling for another aspect, such as adaptation to host-preferred codons. We find that 33 phage genomes exhibit highly non-random patterns in their GC3-content, use of host-preferred codons, or both. We show that the head and tail proteins of these phages exhibit significant bias towards host-preferred codons, relative to the non-structural phage proteins. Our results support the hypothesis of translational selection on viral genes for host-preferred codons, over a broad range of bacteriophages.     

Viral adaption of staphylococcal phage: A genome-based analysis of the selective preference based on codon usage Bias

Given the high therapeutic value of the staphylococcal phage, the genome co-evolution of the phage and the host has gained great attention. Though the genome-wide AT richness in staphylococcal phages has been well-studied with nucleotide usage bias, here we proved that host factor, lifestyle and taxonomy are also important factors in understanding the phage nucleotide usages bias using information entropy formula. Such correlation is especially prominent when it comes to the synonymous codon usages of staphylococcal phages, despite the overall scattered codon usage pattern represented by principal component analysis. This strong relationship is explained by nucleotide skew which testified that the usage biases of nucleotide at different codon positions are acting on synonymous codons. Therefore, our study reveals a hidden relationship of genome evolution with host limitation and phagic phenotype, providing new insight into phage genome evolution at genetic level.     

Rapid divergence of codon usage patterns within the rice genome

Background

Synonymous codon usage varies widely between genomes, and also between genes within genomes. Although there is now a large body of data on variations in codon usage, it is still not clear if the observed patterns reflect the effects of positive Darwinian selection acting at the level of translational efficiency or whether these patterns are due simply to the effects of mutational bias. In this study, we have included both intra-genomic and inter-genomic comparisons of codon usage. This allows us to distinguish more efficiently between the effects of nucleotide bias and translational selection.

Results

We show that there is an extreme degree of heterogeneity in codon usage patterns within the rice genome, and that this heterogeneity is highly correlated with differences in nucleotide content (particularly GC content) between the genes. In contrast to the situation observed within the rice genome, Arabidopsis genes show relatively little variation in both codon usage and nucleotide content. By exploiting a combination of intra-genomic and inter-genomic comparisons, we provide evidence that the differences in codon usage among the rice genes reflect a relatively rapid evolutionary increase in the GC content of some rice genes. We also noted that the degree of codon bias was negatively correlated with gene length.

Conclusion

Our results show that mutational bias can cause a dramatic evolutionary divergence in codon usage patterns within a period of approximately two hundred million years.The heterogeneity of codon usage patterns within the rice genome can be explained by a balance between genome-wide mutational biases and negative selection against these biased mutations. The strength of the negative selection is proportional to the length of the coding sequences. Our results indicate that the large variations in synonymous codon usage are not related to selection acting on the translational efficiency of synonymous codons.

     

Synonymous codon usage in Thermosynechococcus elongatus (cyanobacteria) identifies the factors shaping codon usage variation

Analysis of synonymous codon usage pattern in the genome of a thermophilic cyanobacterium, Thermosynechococcus elongatus BP-1 using multivariate statistical analysis revealed a single major explanatory axis accounting for codon usage variation in the organism. This axis is correlated with the GC content at third base of synonymous codons (GC3s) in correspondence analysis taking T. elongatus genes. A negative correlation was observed between effective number of codons i.e. Nc and GC3s. Results suggested a mutational bias as the major factor in shaping codon usage in this cyanobacterium. In comparison to the lowly expressed genes, highly expressed genes of this organism possess significantly higher proportion of pyrimidine-ending codons suggesting that besides, mutational bias, translational selection also influenced codon usage variation in T. elongatus. Correspondence analysis of relative synonymous codon usage (RSCU) with A, T, G, C at third positions (A3s, T3s, G3s, C3s, respectively) also supported this fact and expression levels of genes and gene length also influenced codon usage. A role of translational accuracy was identified in dictating the codon usage variation of this genome. Results indicated that although mutational bias is the major factor in shaping codon usage in T. elongatus, factors like translational selection, translational accuracy and gene expression level also influenced codon usage variation.     

Solving the riddle of codon usage preferences: a test for translational selection

Translational selection is responsible for the unequal usage of synonymous codons in protein coding genes in a wide variety of organisms. It is one of the most subtle and pervasive forces of molecular evolution, yet, establishing the underlying causes for its idiosyncratic behaviour across living kingdoms has proven elusive to researchers over the past 20 years. In this study, a statistical model for measuring translational selection in any given genome is developed, and the test is applied to 126 fully sequenced genomes, ranging from archaea to eukaryotes. It is shown that tRNA gene redundancy and genome size are interacting forces that ultimately determine the action of translational selection, and that an optimal genome size exists for which this kind of selection is maximal. Accordingly, genome size also presents upper and lower boundaries beyond which selection on codon usage is not possible. We propose a model where the coevolution of genome size and tRNA genes explains the observed patterns in translational selection in all living organisms. This model finally unifies our understanding of codon usage across prokaryotes and eukaryotes. Helicobacter pylori, Saccharomyces cerevisiae and Homo sapiens are codon usage paradigms that can be better understood under the proposed model.     

Stress induced MAPK genes show distinct pattern of codon usage in Arabidopsis thaliana,Glycine max and Oryza sativa

Mitogen activated protein kinase (MAPK) genes provide resistance to various biotic and abiotic stresses. Codon usage profiling of the genes reveals the characteristic features of the genes like nucleotide composition, gene expressivity, optimal codons etc. The present study is a comparative analysis of codon usage patterns for different MAPK genes in three organisms, viz. Arabidopsis thaliana, Glycine max (soybean) and Oryza sativa (rice). The study has revealed a high AT content in MAPK genes of Arabidopsis and soybean whereas in rice a balanced AT-GC content at the third synonymous position of codon. The genes show a low bias in codon usage profile as reflected in the higher values (50.83 to 56.55) of effective number of codons (N_c). The prediction of gene expression profile in the MAPK genes revealed that these genes might be under the selective pressure of translational optimization as reflected in the low codon adaptation index (CAI) values ranging from 0.147 to 0.208.     

Romulus and Remus,Two Phage Isolates Representing a Distinct Clade within the Twortlikevirus Genus,Display Suitable Properties for Phage Therapy Applications

The renewed interest in controlling Staphylococcus aureus infections using their natural enemies, bacteriophages, has led to the isolation of a limited number of virulent phages so far. These phages are all members of the Twortlikevirus, displaying little variance. We present two novel closely related (95.9% DNA homology) lytic myoviruses, Romulus and Remus, with double-stranded DNA (dsDNA) genomes of 131,333 bp and 134,643 bp, respectively. Despite their relatedness to Staphylococcus phages K, G1, ISP, and Twort and Listeria phages A511 and P100, Romulus and Remus can be proposed as isolates of a new species within the Twortlikevirus genus. A distinguishing feature for these phage genomes is the unique distribution of group I introns compared to that in other staphylococcal myoviruses. In addition, a hedgehog/intein domain was found within their DNA polymerase genes, and an insertion sequence-encoded transposase exhibits splicing behavior and produces a functional portal protein. From a phage therapy application perspective, Romulus and Remus infected approximately 70% of the tested S. aureus isolates and displayed promising lytic activity against these isolates. Furthermore, both phages showed a rapid initial adsorption and demonstrated biofilm-degrading capacity in a proof-of-concept experiment.     

Gradients in nucleotide and codon usage along Escherichia coli genes

The usage of codons and nucleotide combinations varies along genes and systematic variation causes gradients in usage. We have studied such gradients of nucleotides and nucleotide combinations and their immediate context in Escherichia coli. To distinguish mutational and selectional effects, the genes were subdivided into three groups with different codon usage bias and the gradients of nucleotide usage were studied in each group. Some combinations that can be associated with a propensity for processivity errors show strong negative gradients that become weaker in genes with low codon bias, consistent with a selection on translational efficiency. One of the strongest gradients is for third position G, which shows a pervasive positive gradient in usage in most contexts of surrounding bases.     

Improving the Safety of Staphylococcus aureus Polyvalent Phages by Their Production on a Staphylococcus xylosus Strain

Team1 (vB_SauM_Team1) is a polyvalent staphylococcal phage belonging to the Myoviridae family. Phage Team1 was propagated on a Staphylococcus aureus strain and a non-pathogenic Staphylococcus xylosus strain used in industrial meat fermentation. The two Team1 preparations were compared with respect to their microbiological and genomic properties. The burst sizes, latent periods, and host ranges of the two derivatives were identical as were their genome sequences. Phage Team1 has 140,903 bp of double stranded DNA encoding for 217 open reading frames and 4 tRNAs. Comparative genomic analysis revealed similarities to staphylococcal phages ISP (97%) and G1 (97%). The host range of Team1 was compared to the well-known polyvalent staphylococcal phages phi812 and K using a panel of 57 S. aureus strains collected from various sources. These bacterial strains were found to represent 18 sequence types (MLST) and 14 clonal complexes (eBURST). Altogether, the three phages propagated on S. xylosus lysed 52 out of 57 distinct strains of S. aureus. The identification of phage-insensitive strains underlines the importance of designing phage cocktails with broadly varying and overlapping host ranges. Taken altogether, our study suggests that some staphylococcal phages can be propagated on food-grade bacteria for biocontrol and safety purposes.     

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.     

Trends in the codon usage patterns of Chromohalobacter salexigens genes

Chromohalobacter salexigens, a Gammaproteobacterium belonging to the family Halomonadaceae, shows a broad salinity range for growth. In order to reveal the factors influencing architecture of protein coding genes in C. salexigens, pattern of synonymous codon usage bias has been investigated. Overall codon usage analysis of the microorganism revealed that C and G ending codons are predominantly used in all the genes which are indicative of mutational bias. Multivariate statistical analysis showed that the genes are separated along the first major explanatory axis according to their expression levels and their genomic GC content at the synonymous third positions of the codons. Both NC plot and correspondence analysis on Relative Synonymous Codon Usage (RSCU) indicates that the variation in codon usage among the genes may be due to mutational bias at the DNA level and natural selection acting at the level of mRNA translation. Gene length and the hydrophobicity of the encoded protein also influence the codon usage variation of genes to some extent. A comparison of the relative synonymous codon usage between 10% each of highly and lowly expressed genes determines 23 optimal codons, which are statistically over represented in the former group of genes and may provide useful information for salt-stressed gene prediction and gene-transformation. Furthermore, genes for regulatory functions; mobile and extrachromosomal element functions; and cell envelope are observed to be highly expressed. The study could provide insight into the gene expression response of halophilic bacteria and facilitate establishment of effective strategies to develop salt-tolerant crops of agronomic value.     

Novel organization of the site-specific integration and excision recombination functions of the Staphylococcus aureus serotype F virulence-converting phages φ13 and φ42

Functions required for site-specific integration and excision of the Staphylococcus aureus serotype F virulence-converting phages φ13 and φ42 were localized and characterized. Like other temperate phages, integration of φ13 and φ42 sequences was found to require the product of an int gene located close to the phage attP site. Both int genes are almost identical, express proteins possessing characteristic features of the Int (integrase) family of recombinases, but share very little homology with previously described int genes, including those of the serotype B S. aureus phages L54a and φ11. Nevertheless, all four S. aureus phages share an almost identical short sequence located immediately 5′ to these distinct int genes, suggesting a common mechanism of int gene regulation. Upstream from these common sequences, the sequences of φ13 and φ42 are quite distinct from each other, and from the corresponding regions of φ11 and L54a which encode the Xis proteins that are required with Int to mediate site-specific excision of the latter phages. Surprisingly, φ13 and φ42 sequences encompassing the attP sites and int genes, but lacking either an adjacent or more distant phage excision protein gene, were sufficient to mediate site-specific excision of integrated phage DNA sequences.     

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.     

Universal function-specificity of codon usage

Synonymous codon usage has long been known as a factor that affects average expression level of proteins in fast-growing microorganisms, but neither its role in dynamic changes of expression in response to environmental changes nor selective factors shaping it in the genomes of higher eukaryotes have been fully understood. Here, we propose that codon usage is ubiquitously selected to synchronize the translation efficiency with the dynamic alteration of protein expression in response to environmental and physiological changes. Our analysis reveals that codon usage is universally correlated with gene function, suggesting its potential contribution to synchronized regulation of genes with similar functions. We directly show that coexpressed genes have similar synonymous codon usages within the genomes of human, yeast, Caenorhabditis elegans and Escherichia coli. We also demonstrate that perturbing the codon usage directly affects the level or even direction of changes in protein expression in response to environmental stimuli. Perturbing tRNA composition also has tangible phenotypic effects on the cell. By showing that codon usage is universally function-specific, our results expand, to almost all organisms, the notion that cells may need to dynamically alter their intracellular tRNA composition in order to adapt to their new environment or physiological role.     

Evidence from simulation studies for selective constraints on the codon usage of the Angiosperm psbA gene

The codon usage of the Angiosperm psbA gene is atypical for flowering plant chloroplast genes but similar to the codon usage observed in highly expressed plastid genes from some other Plantae, particularly Chlorobionta, lineages. The pattern of codon bias in these genes is suggestive of selection for a set of translationally optimal codons but the degree of bias towards these optimal codons is much weaker in the flowering plant psbA gene than in high expression plastid genes from lineages such as certain green algal groups. Two scenarios have been proposed to explain these observations. One is that the flowering plant psbA gene is currently under weak selective constraints for translation efficiency, the other is that there are no current selective constraints and we are observing the remnants of an ancestral codon adaptation that is decaying under mutational pressure. We test these two models using simulations studies that incorporate the context-dependent mutational properties of plant chloroplast DNA. We first reconstruct ancestral sequences and then simulate their evolution in the absence of selection on codon usage by using mutation dynamics estimated from intergenic regions. The results show that psbA has a significantly higher level of codon adaptation than expected while other chloroplast genes are within the range predicted by the simulations. These results suggest that there have been selective constraints on the codon usage of the flowering plant psbA gene during Angiosperm evolution.     

Analysis of synonymous codon usage patterns in the genus Rhizobium

The codon usage patterns of rhizobia have received increasing attention. However, little information is available regarding the conserved features of the codon usage patterns in a typical rhizobial genus. The codon usage patterns of six completely sequenced strains belonging to the genus Rhizobium were analysed as model rhizobia in the present study. The relative neutrality plot showed that selection pressure played a role in codon usage in the genus Rhizobium. Spearman’s rank correlation analysis combined with correspondence analysis (COA) showed that the codon adaptation index and the effective number of codons (ENC) had strong correlation with the first axis of the COA, which indicated the important role of gene expression level and the ENC in the codon usage patterns in this genus. The relative synonymous codon usage of Cys codons had the strongest correlation with the second axis of the COA. Accordingly, the usage of Cys codons was another important factor that shaped the codon usage patterns in Rhizobium genomes and was a conserved feature of the genus. Moreover, the comparison of codon usage between highly and lowly expressed genes showed that 20 unique preferred codons were shared among Rhizobium genomes, revealing another conserved feature of the genus. This is the first report of the codon usage patterns in the genus Rhizobium.     

Unusual codon usage bias in low expression genes of Vibrio cholerae

Positive correlation between gene expression and synonymous codon usage bias is well documented in the literature. However, in the present study of Vibrio cholerae genome, we have identified a group of genes having unusually high codon usage bias despite being low potential expressivity. Our results suggest that codon usage in lowly expressed genes might also be selected on to preferably use non-optimal codons to maintain a low cellular concentration of the proteins that they encode. This would predict that lowly expressed genes are also biased in codon usage, but in a way that is opposite to the bias of highly expressed genes.     

Compositional dynamics and codon usage pattern of BRCA1 gene across nine mammalian species

The BRCA1 gene is located on the human chromosome 17q21.31 and plays important role in biological processes. The aminoacyl-tRNA synthetases (AARS) are a family of heterogenous enzymes responsible protein synthesis and whose secondary functions include a role in autoimmune myositis. Our findings reveal that the compositional constraint and the preference of more A/T –ending codons determine the codon usage patterns in BRCA1 gene while more G/C-ending codons influence the codon usage pattern of AARS gene among mammals. The codon usage bias in BRCA1 and AARS genes is low. The codon CGC encoding arginine amino acid and the codon TTA encoding leucine were uniformly distributed in BRCA1 and AARS genes, respectively in mammals including human. Natural selection might have played a major role while mutation pressure might have played a minor role in shaping the codon usage pattern of BRCA1 and AARS genes.