The relationship between change in genomic GC content and protein evolution in bacteria was studied by simple correlational analysis (at the genus level) and by Felsenstein's (1985) independent contrast test. We first used the dnaA gene in bacteria as an example to show (1) that the amino acid composition of a protein can be dramatically affected by mutational pressure (the genomic GC content), (2) that surprisingly, deleting relatively closely-related genera may increase rather than decrease the correlation between genomic GC content and amino acid composition, and (3) that most unexpectedly, as the genomic GC content increases, both strongly hydrophobic and strongly hydrophilic amino acids tend to change to ambivalent amino acids, suggesting that the majority of these amino acid substitutions are not caused by positive Darwinian selection. These patterns were then also shown to hold for the 14 other genes studied, indicating their generality for the evolution of bacterial proteins. As directional mutation pressure can affect the amino acid composition of proteins, it may mislead phylogenetic inference, even if protein instead of DNA sequences are used.

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