Figure 9. Nc-plot of the average codon usage bias in the Hsp60 genes of 17 phyla. The plot space was divided into six quadrants using the ENC and GC3 thresholds. The ENC thresholds, reflecting the level of the Hsp60 gene expression, were as follows: ENC < 40 for genes with high expression; 40 < ENC ≤ 55 for moderately expressed genes; ENC > 55 for low expressed genes. The GC3 thresholds reflecting the direction of the mutational pressure were as follows: GC3 < 0.5 represents the AT-mutation pressure; GC3> 0.5 represents the GC-mutation pressure. The average ENC values were grouped according to the obtained quadrants: Group 1 (Apicomplexa, Firmicutes, and Bacteroidetes); Group 2 (Chlamydiae, Streptophyta, Nematoda, Mollusca, Cyanobacteria, and Chordata); Group 3 (Euryarchaeota, Arthropoda, and Euglenozoa); Group 4 (Ascomycota, Proteobacteria, Basidiomycota, Chlorophyta, and Actinobacteria). The black bell-shaped curve represents the expected effective number of codons (ENCexp ), i.e. predicted ENC values if the codon bias is influenced only by the GC content at the third synonymous position of codons (GC3) in the Hsp60 gene. The horizontal dashed lines (ENC=40 and ENC=55) indicate ENC thresholds for determining the codon usage bias and gene expression level. The vertical dashed line indicates the GC3 content of 0.5. The position of the ENC value regarding this line indicates the direction of the mutation pressure affecting the Hsp60 genes (depicted by arrows).
The maximum and minimum average ENC values were observed for Streptophyta (ENC 51.6±3.2) and Actinobacteria (ENC 31.6±5.3), respectively. Using the convention of 40 < ENC ≤ 55 to determine the degree of codon usage bias60,66,67, it can be assumed that Hsp60 genes of 17 phyla are highly and moderately biased. The Hsp60 genes from Group 1 (Apicomplexa, Firmicutes, and Bacteroidetes) with AT-mutation pressure and Group 4 (Ascomycota, Proteobacteria, Basidiomycota, Chlorophyta, and Actinobacteria) with GC-mutation pressure have a high level of expression. In turn, a moderate level of Hsp60 gene expression is characteristic of Group 2 (Chlamydiae, Streptophyta, Nematoda, Mollusca, Cyanobacteria, Chordata) with AT-mutation pressure and Group 3 (Euryarchaeota, Arthropoda, and Euglenozoa) with GC-mutation pressure.
However, it should be noted that the individual ENC values of the Hsp60 genes from Streptophyta, Mollusca, Chordata, Euryarchaeota, Arthropoda, Basidiomycota exceed 55 (Figure 7). In addition, there was a high statistical variance in the average ENC values ranging from 3.2 (Streptophyta) to 8.8 (Apicomplexa) (Supplementary, ENC) for Hsp60 genes of 17 phyla, which may indicate that overall ENC values are not conserved for these genes within the phylum. Therefore, the ENC values were compared using Student’s t-test (Figure 10; Supplementary, ENC).