A Plasmid-Derived Machinery Drives Chromosomal Replication Initiation in the Colorectal Cancer-Associated Bacterium Fusobacterium nucleatum and Its Close Relatives

Abstract

Chromosome replication initiation in bacterial genomes typically occurs at a single origin, called oriC, through the binding of the DnaA initiator protein to specific chromosomal repeats known as DnaA-boxes. Here, relying on comparative genomics, we show that the family Fusobacteriaceae, which belongs to the order Fusobacteriales alongside the family Leptotrichiaceae, deviates from this paradigm. Our analysis revealed a stark contrast between the two lineages: while dnaA flanks oriC in Leptotrichiaceae, it is notably missing from Fusobacteriaceae genomes. Adjacent to oriC in Fusobacteriaceae, we identified a gene encoding replication initiator protein A (RPA). We characterized RPA as a Rep-type initiator involved in plasmid replication that likely originated from an iteron-containing plasmid, iterons being the plasmid analogues of DnaA-boxes. Phylogenetic reconstruction indicates that the gene encoding RPA was acquired together with its associated iterons and subsequently domesticated by the Fusobacteriaceae ancestor, successfully displacing the ancestral chromosomal dnaA gene at the origin of replication. This shift to a plasmid-derived replication system may represent an adaptive trait of the Fusobacteriaceae, potentially offering new insights into the biology of Fusobacterium nucleatum, a member of this family known to be a major driver of colorectal cancer progression within the tumor microenvironment.