Question. The presence of overlapping genes (OLGs) with substantial coding overlaps beyond translational coupling challenges and transforms our understanding of genomic complexity. The prevalence of overlapping genes, either fully embedded or with extended overlaps, is likely considerably underreported. Currently, only a handful of these genes have been thoroughly confirmed, but the number of prokaryotic overlapping genes is steadily expanding. Validating the presence of genes within alternate reading frames has the potential to elucidate taxon-specific evolutionary innovations.

Methods. mRNA sequencing, ribosome profiling, phenotype testing of strand-specific knockouts, and in some cases mass spectrometry was used to verify protein-coding overlapping genes. Genome-wide analysis of transcription start sites was conducted in an exemplary organism. Bioinformatic comparisons were applied to number of different datasets for published omics-data, to identify serendipitously discovered overlapping genes.

Results. Overlapping genes were identified through RNA sequencing and ribosome profiling. Bioinformatics confirmed that the identified candidate genes possess characteristic structural elements allowing their expression. Clear phenotypic effects were observed in strand-specific knock-out mutants. For some proteins, we successfully used mass spectrometry identifying expressed proteins. Specific overlapping genes are often constrained to specific taxonomic groups, but overlapping genes have been unintentionally identified in a number of different taxa.

Conclusions. Overlapping genes are more prevalent in bacterial genomes (outside prophages) than anticipated. They may contribute evolutionarily novel functionalities and could be an important contribution of evolutionary novelties in bacterial genomes.