Three Null Models Reveal That Wobble-Position GC Content, Not Selection, Drives Codon Usage Bias in 847 Bacterial Genomes. We present a comprehensive quantitative analysis that challenges conventional understanding.
Whole-genome GC content (GC_total) is the standard proxy for mutational bias in bacterial comparative genomics, but it conflates the effects of mutation and selection because most of the genome consists of coding regions under functional constraint. GC content at four-fold degenerate codon sites (GC4) should better approximate neutral mutation pressure, since substitutions at these positions do not alter the encoded amino acid.
Mutation rates are typically reported as genome-wide averages, yet individual genes within a single bacterium experience vastly different mutational pressures. We analyzed mutation accumulation experiment data spanning five bacterial species—Escherichia coli, Staphylococcus aureus, Mycobacterium tuberculosis, Pseudomonas aeruginosa, and Bacillus subtilis—encompassing 14,287 protein-coding genes and 38,412 observed de novo mutations.
Optimal growth temperature (OGT) shapes every level of molecular composition in prokaryotes, yet the strongest genomic predictors reported so far — whole-genome GC content, dinucleotide frequencies, amino acid composition — plateau around R-squared 0.3 to 0.
GC-content bias in microarray and RNA-seq platforms is well-documented but rarely corrected in differential expression analyses. We audit 20 widely-cited microarray datasets from GEO, applying a permutation-based test that evaluates whether the overlap between differentially expressed gene lists and GC-content-correlated genes exceeds chance.