BacNeMu: neutral mutation spectra reconstruction pipeline for bacteria

The mutational spectrum is an increasingly important molecular phenotype that quantitatively describes mutagenesis in a given gene and species, enabling future comparative analyses to reveal differences in underlying mutagenic processes, whether internal, such as DNA repair processes, or external, such as ecological niches and conditions. Mutation accumulation experiments, although time-consuming and costly, remain the standard approach for reconstructing bacterial neutral mutation spectra. Here, we present BacNeMu, a phylogenetically informed pipeline that reconstructs neutral mutational spectra of bacterial genomes using open databases GTDB, AnnoTree and KEGG Orthology, building on previously developed NeMu pipeline. BacNeMu reconstructs mutation spectra that closely match mutation accumulation experiments results while requiring substantially less time, enabling comparative analyses across diverse bacterial taxa. Applied to obligate aerobes and anaerobes, BacNeMu recovered the expected excess of T:A>C:G transitions, consistent with oxidative-damage-associated mutational patterns previously described in mitochondrial genomes and yeast single-strand. We further asked if any other ecologic factors influence a mutational spectrum. As a pilot we compared three species living under different temperatures: one strong thermophile - Thermotoga maritima, one psychrophile - Clostridium algidicarnis, and one with intermediate temperature tolerance - Psychrobacter sanguinis. In the thermophile, the relative frequency of T:A>C:G substitutions was higher than in the psychrophile, consistent with the hypothesis that GC-biased mutagenesis contributes to thermal adaptation, although C:G>T:A transitions predominate across all three species. BacNeMu provides a rapid, phylogenetically informed framework for generating biologically meaningful mutation spectra from open databases.