Mutational features of chromids and chromosomes in provide new insights into the evolution of secondary replicons.

The genomes of multi-replicon bacteria are composed of a primary replicon (the chromosome) and secondary replicons (chromids). Currently, there is a lack of understanding of the mutation features and evolutionary patterns of these different replicons. Specifically, in the genus , the chromids of multi-replicon species exhibit both unidirectional and bidirectional replication. Here, we investigated the similarities and differences between chromosomes and chromids in sequence composition and gene synteny of species by comparative genomic analysis, as well as the spontaneous mutation features of different replicons by mutation accumulation (MA) experiments combined with whole-genome sequencing strategy (MA-WGS). MA-WGS analysis revealed that there was no significant difference between chromids and chromosomes in the mutation rate or mutation spectrum of . sp. LC0214 (where the chromid is unidirectional in replication) and . sp. JCM12884 (where the chromid is bidirectional in replication). In addition, the context-dependence and variation pattern of the base-pair substitutions (BPSs) rates of the entire replicons exhibited differences that may be caused by the different replication directions of the chromids. The results of this study provide a new theoretical foundation for an in-depth understanding of the origin and evolution of chromids in multi-replicon bacterial species and facilitate further exploration of the complex mechanisms of bacterial diversity.IMPORTANCE mutations are a critical driving force in species evolution. Currently, there is a lack of sufficient research on the influence of replicon types on the occurrence of genomic mutations in bacteria. Moreover, the scarcity in systematic analysis and comparison of spontaneous mutation features between different replicons results in the limited information on the evolutionary dynamics of multi-replicon species. The diversity of replication direction in the multi-replicon species of the genus provides a unique opportunity for studying the impact of replication direction on the patterns of mutation. In addition to the composition characteristics between chromosomes and chromids, the spontaneous mutation rates in the context-dependence and variation pattern of the base-pair substitutions (BPSs) across different replicons within species revealed in this study provide valuable insights into the evolutionary dynamics of bacterial secondary replicons.