Synonymous codon usage is subject to selection in thermophilic bacteria

Abstract

The patterns of synonymous codon usage, both within and among genomes, have been extensively studied over the past two decades. Despite the accumulating evidence that natural selection can shape codon usage, it has not been possible to link a particular pattern of codon usage to a specific external selective force. Here, we have analyzed the patterns of synonymous codon usage in 40 completely sequenced prokaryotic genomes. By combining the genes from several genomes (more than 80 000 genes in all) into a single dataset for this analysis, we were able to investigate variations in codon usage, both within and between genomes. The results show that synonymous codon usage is affected by two major factors: (i) the overall G+C content of the genome and (ii) growth at high temperature. This study focused on the relationship between synonymous codon usage and the ability to grow at high temperature. We have been able to eliminate both phylogenetic history and lateral gene transfer as possible explanations for the characteristic pattern of codon usage among the thermophiles. Thus, these results demonstrate a clear link between a particular pattern of codon usage and an external selective force.

Figure 1

Correspondence analysis of the relative synonymous codon usage in 83 985 genes from 40 bacterial genomes (see Table 1). (A) Genes from thermophilic bacteria are shown in red while those from mesophilic bacteria are colored blue. For each of these two sub-groups, means and standard deviations are shown. Note that there is extensive overlap between thermophilic and mesophilic genes along the horizontal axis, whereas there is relatively little overlap between the two groups along the vertical axis. The difference between mesophiles and thermophiles along this latter axis is highly significant (P < 0.0001). (B) The distribution of synonymous codons along the first and second axes of the correspondence analysis. Here, we see that the GC-ending codons (shown in red) cluster to the right and the AT-ending codons (shown in green) cluster to the left.

Figure 1

Correspondence analysis of the relative synonymous codon usage in 83 985 genes from 40 bacterial genomes (see Table 1). (A) Genes from thermophilic bacteria are shown in red while those from mesophilic bacteria are colored blue. For each of these two sub-groups, means and standard deviations are shown. Note that there is extensive overlap between thermophilic and mesophilic genes along the horizontal axis, whereas there is relatively little overlap between the two groups along the vertical axis. The difference between mesophiles and thermophiles along this latter axis is highly significant (P < 0.0001). (B) The distribution of synonymous codons along the first and second axes of the correspondence analysis. Here, we see that the GC-ending codons (shown in red) cluster to the right and the AT-ending codons (shown in green) cluster to the left.

Figure 2

Variation in codon usage within and between genomes. Genes shown are identified by genome and the means (±99.99% confidence intervals) for each genome are shown (the abbreviations for each organism are shown in Table 1). From this plot it is clear that the among-genome variation far exceeds the within-genome variation along both axes. Amongst the thermophiles we have circled the two eubacterial genomes (T.maritima and A.aeolicus). We have also circled the single mesophilic archaeal species (Halobacterium sp.).

Figure 3

Evidence for selection on synonymous codon usage. Synonymous codon bias in highly expressed genes. Each arrow represents one genome, with the base of the arrow at the mean position for the whole genome and the arrowhead ending at the mean for the ribosomal genes within that genome. Note that the arrows tend to point away from the division between the thermophiles and the mesophiles; in other words, the ribosomal protein genes have a greater degree of synonymous codon bias than the genomes as a whole.

Figure 4

Summary of phylogenetic analyses based on the concatenated sequences of 10 ribosomal protein genes from each of the 40 genomes used in this study. Two possible phylogenetic groupings were compared. The grouping shown on the left side of the figure represents the accepted organismal tree. The tips of the branches are color coded (red for thermophiles and blue for mesophiles); it shows that both thermophiles and mesophiles are polyphyletic. The alternative hypothesis (shown on the right) describes the horizontal gene transfer hypothesis, whereby the thermophilic Eubacteria (A.aeolicus and T.maritima) would have acquired portions of their genomes, including the ribosomal protein genes, from thermophilic Archaea. In this case, the tree based on the ribosomal protein sequences would indicate that all thermophilic species would be monophyletic (again, as indicated by the color coding on the tips of the branches). Our results, using both distance-based and maximum likelihood methods of phylogenetic reconstruction (J. Felsenstein, PHYLIP v.3.6a2.1; http://evolution.genetics.washington.edu/phylip.html), greatly favor the first grouping (left) over the second (right), with 100% bootstrapping support.

Figure 5

Frequency distributions of synonymous codon usage among thermophilic (red) and the mesophilic genes (blue) along the second axis of inertia. We have also plotted the A.aeolicus gene frequencies as a proportion of all thermophilic genes (shown in green). This genome, despite being eubacterial, shows a unimodal distribution that fits the distribution pattern of the other thermophilic genomes. The ‘shoulder’ in each curve is due to the AT-rich genomes in each category.