Phase variation in Mannheimia haemolytica challenges the static genome paradigm

Mannheimia haemolytica is a key agent in bovine respiratory disease (BRD), driving antibiotic use in feedyard cattle. As a facultative anaerobe commonly found in the upper respiratory tracts of cattle, its role under anaerobic conditions in BRD has not been extensively studied despite the known importance of anaerobiosis in human respiratory infections. Utilizing a combined omics approach, we refuted the null hypothesis that the M. haemolytica genome sequence is independent of the environment. This finding provides the rationale for research exploring how anaerobiosis-driven genome plasticity might contribute to a transition from a commensal to a virulent state. Genome sequencing of colony morphology variants from aerobic and anaerobic cultures revealed phase variation via homologous recombination between ribosomal RNA (rRNA) operons and slipped strand mispairing at simple sequence repeats (SSRs), frameshifting genes "on" and "off." Homologous recombination was exclusive to anaerobic conditions. SSR length variation in a DNA methyltransferase gene, targeting 5'-GACAT, correlated with methylation status. We observed statistically significant differences in the fraction of methylated motifs in isolates derived from anaerobic and aerobic cultures, as well as in the transcript abundances of genes in a nitrate reduction pathway and in a ribosomal protein class among anaerobic culture-derived colonial variants. We propose that instead of representing a M. haemolytica isolate's genome as a single static sequence, dynamic genome models should be developed. These models should account for the stochastic changes in genome sequence induced by phase variation, represented as a probability-weighted mixture of homologous recombinants and SSR switches.IMPORTANCEThe anaerobic growth of Mannheimia haemolytica generates phase variants through homologous recombination and slipped strand mispairing at simple sequence repeats. Homologous recombination between ribosomal RNA operons occurred exclusively in anaerobic conditions, likely similar to those present in infected lung tissue. Phase variation may yield variants better adapted for persistence in pulmonary tissues, potentially impacting antimicrobial persistence, as observed in persisters of other species. The genomic diversity resulting from phase variation complicates analyses based on static genome sequences, highlighting the need for dynamic genome models that reflect the stochastic nature of phase variation to understand M. haemolytica's role in host disease biology.