Promoter recruitment drives the emergence of proto-genes in a long-term evolution experiment with Escherichia coli

The phenomenon of de novo gene birth-the emergence of genes from non-genic sequences-has received considerable attention due to the widespread occurrence of genes that are unique to particular species or genomes. Most instances of de novo gene birth have been recognized through comparative analyses of genome sequences in eukaryotes, despite the abundance of novel, lineage-specific genes in bacteria and the relative ease with which bacteria can be studied in an experimental context. Here, we explore the genetic record of the Escherichia coli long-term evolution experiment (LTEE) for changes indicative of "proto-genic" phases of new gene birth in which non-genic sequences evolve stable transcription and/or translation. Over the time span of the LTEE, non-genic regions are frequently transcribed, translated and differentially expressed, with levels of transcription across low-expressed regions increasing in later generations of the experiment. Proto-genes formed downstream of new mutations result either from insertion element activity or chromosomal translocations that fused preexisting regulatory sequences to regions that were not expressed in the LTEE ancestor. Additionally, we identified instances of proto-gene emergence in which a previously unexpressed sequence was transcribed after formation of an upstream promoter, although such cases were rare compared to those caused by recruitment of preexisting promoters. Tracing the origin of the causative mutations, we discovered that most occurred early in the history of the LTEE, often within the first 20,000 generations, and became fixed soon after emergence. Our findings show that proto-genes emerge frequently within evolving populations, can persist stably, and can serve as potential substrates for new gene formation.