Aerobiosis is not associated with GC content and G to T mutations are not the signature of oxidative stress in prokaryotic evolution

Background: Among the four bases, guanine is the most susceptible to damage from oxidative stress. Replication of DNA containing damaged guanines result in G to T mutations. Therefore, the mutations resulting from oxidative DNA damage are generally expected to predominantly consist of G to T (and C to A when the damaged guanine is not in the reference strand) and result in decreased GC content. However, the opposite pattern was reported 16 years ago in a study of prokaryotic genomes. Although that result has been widely cited and confirmed by nine later studies with similar methods, the omission of the effect of shared ancestry requires a re-examination of the reliability of the results.\n\nResults: We retrieved 70 aerobe-anaerobe pairs of prokaryotes, and members of each pair were adjacent on the phylogenetic tree. Pairwise comparisons of either whole-genome GC content or the GC content at 4-fold degenerate sites of orthologous genes among these 70 pairs did not show significant differences between aerobes and anaerobes. The signature of guanine oxidation on GC content evolution has not been detected even after extensive controlling of other influencing factors. Furthermore, the anaerobes were not different from the aerobes in the rate of either G to T, C to A, or other directions of substitutions. The presence of the enzymes responsible for guanine oxidation in anaerobic prokaryotes provided additional evidence that guanine oxidation might be prevalent in anaerobic prokaryotes. In either aerobes or anaerobes, the rates of G:C to T:A mutations were not significantly higher than the reverse mutations.\n\nConclusions: The previous counterintuitive results on the relationship between oxygen requirement and GC content should be attributed to the methodological artefact resulting from phylogenetically non-independence among the analysed samples. Our results showed that aerobiosis does not increase or decrease GC content in evolution. Furthermore, our study challenged the widespread belief that abundant G:C to T:A transversions are the signature of oxidative stress in prokaryotic evolution.