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).