Elevated rates and biased spectra of mutations in anaerobically cultured lactic acid bacteria

The rate, spectrum, and biases of mutations represent a fundamental force shaping biological evolution. Convention often attributes oxidative DNA damage as a major driver of spontaneous mutations. Yet, despite the contribution of oxygen to mutagenesis and the ecological, industrial, and biomedical importance of anaerobic organisms, relatively little is known about the mutation rates and spectra of anaerobic species. Here, we present the rates and spectra of spontaneous mutations assessed anaerobically over 1,000 generations for three fermentative lactic acid bacteria species with varying levels of aerotolerance: <i>Lactobacillus acidophilus</i>, <i>Lactobacillus crispatus</i>, and <i>Lactococcus lactis</i>. Our findings reveal highly elevated mutation rates compared to the average rates observed in aerobically respiring bacteria, with mutations strongly biased toward transitions, emphasizing the prevalence of spontaneous deamination in these anaerobic species and highlighting the inherent fragility of purines even under conditions that minimize oxidative stress. Beyond these overarching patterns, we identify several novel mutation dynamics: positional mutation bias around the origin of replication in <i>Lb. acidophilus</i>; a significant disparity between observed and equilibrium GC content in <i>Lc. lactis</i>; and repeated independent deletions of spacer sequences from within the clustered, regularly interspaced, short palindromic repeat locus in <i>Lb. crispatus,</i> providing mechanistic insights into the evolution of bacterial adaptive immunity. Overall, our study provides new insights into the mutational landscape of anaerobes, revealing how non-oxygenic factors shape mutation rates and influence genome evolution.IMPORTANCEDespite Earth's oxygen-free origins and the abundance of microorganisms that thrive without oxygen, little is known about the rates and patterns of mutations in anaerobic species. This study directly measures the mutation rates of three fermentative lactic acid bacteria across 1,000 generations in oxygen-free conditions, revealing elevated and highly biased genome-wide mutation rates in these species compared to oxygen-using bacteria. These results highlight how non-oxygen-related factors contribute to mutagenesis, shaping genome evolution in microbes with anaerobic life histories.