Adaptive evolution of oriC through in vitro propagation of a mini-chromosome in RCR

Diversification and selective propagation are the main driving forces of evolution, resulting in the emergence of organisms possessing various strategies. Here, we conducted in vitro evolution of the replication origin (oriC) under the pressure of an AT-rich mini-chromosome amplification using a reconstituted Escherichia coli replication-cycle reaction (RCR). Using next-generation sequencing, we identified that these evolved oriCs contain select mutations within the duplex unwinding element (DUE) and DnaA-binding site (DnaA box) regions, which are crucial for replication initiation. Real-time detection of RCR amplification (real-time RCR) revealed that the DUE mutations, which decreased the GC content, along with the introduction of a specific A/T sequence in DUE-M and a consecutive KAK (K = T or G) motif in DUE-R, enhanced the RCR amplification efficiency compared to the wild-type oriC (oriCwt). A competitive amplification assay also elucidated that the DnaA box mutations confer a competitive advantage over coexistent oriCwt. Although these DUE and DnaA box mutations were selected through the same amplification reaction, they exhibited distinct competitive amplification strategies. The DUE mutant represents a faster propagation strategy (r-strategy), while the DnaA box mutant represents an adaptive strategy with a competitive advantage (K-strategy), representing the r/K-selection theory in molecular evolution.