The amino acid composition (AAC) of proteomes differs greatly between microorganisms and is associated with the environmental
niche they inhabit, suggesting that these changes may be adaptive. Similarly, the oligonucleotide composition of genomes varies and
mayconferadvantagesattheDNA/RNAlevel.Theseinfluencesoverlapinprotein-codingsequences,makingitdifficulttogaugetheir
relative contributions. We disentangle these effects by systematically evaluating the correspondence between intergenic nucleotide
composition, where protein-level selection is absent, the AAC, and ecological parameters of 909 prokaryotes. We find that G + C
content, the most frequently used measure of genomic composition, cannot capture diversity in AAC and across ecological contexts.
However, di-/trinucleotide composition in intergenic DNA predicts amino acid frequencies of proteomes to the point where very little
cross-speciesvariabilityremainsunexplained(91%ofvarianceaccountedfor).Qualitativelysimilarresultswereobtainedfor49fungal
genomes, where 80% of the variability in AAC could be explained by the composition of introns and intergenic regions. Upon
factoring out oligonucleotide composition and phylogenetic inertia, the residual AAC is poorly predictive of the microbes’ ecological
preferences, in stark contrast with the original AAC. Moreover, highly expressed genes do not exhibit more prominent environmentrelated
AAC signatures than lowly expressed genes, despite contributing more to the effective proteome. Thus, evolutionary shifts in
overallAACappeartooccuralmostexclusivelythroughfactorsshapingtheglobaloligonucleotidecontentofthegenome.Wediscuss
these results in light of contravening evidence from biophysical data and further reading frame-specific analyses that suggest that
adaptation takes place at the protein level.