Imported, not invented, genes prevail among Escherichia coli ORFans

Bacterial genomes contain numerous ORFans-genes lacking homologs outside the species in which they are found. The source of these genes remains enigmatic because the major mechanism by which new genes originate-by duplication and divergence-is rare in bacteria. The proposed explanations for the birth of ORFan genes include horizontal transfer from sources unrepresented in the databases and rapid divergence from preexisting sequences; however, the lack of direct homology-based evidence has left this issue unresolved. We curated a high-confident set of Escherichia coli-specific ORFans whose distributions were then charted across the species' pangenome. Based on their patterns of occurrence, ORFan genes could be assigned to one of two modes of origin. The majority were recently acquired via horizontal transfer, with phage transduction making a significant contribution. A smaller fraction of genes emerged via sequence divergence from resident coding genes or de novo from noncoding sequences. Those acquired horizontally are chiefly of unknown function, whereas those arising from resident sequences are primarily involved in defense and membrane-associated activities. This phylogeny-informed approach demystifies the origins of ORFan genes and offers a route toward establishing their source across bacterial taxa.