Identification of new genes on a whole genome scale using saturated reporter transposon mutagenesis

Abstract

AbstractSmall or overlapping genes are prevalent across all domains of life but are often overlooked for annotation and function because of challenges in their detection. The advent of high-density mutagenesis and data-mining studies suggest the existence of further coding potential within bacterial genomes. To overcome limitations in existing protein detection methods, we applied a genetics-based approach. We combined transposon insertion sequencing with a translation reporter to identify translated open reading frames throughout the genome at scale, independent of genome annotation. We applied our method to the well characterised speciesEscherichia coliand identified ∼200 putative novel protein coding sequences (CDS). These are mostly short CDSs (<50 amino acids) and in some cases highly conserved. We validate the expression of selected CDSs demonstrating the utility of this approach. Despite the extensive study ofE. coli, this method revealed proteins that have not been previously described, including proteins that are conserved and neighbouring functionally important genes, suggesting significant functional roles of small proteins that are still overlooked. We present this as a complementary method to whole cell proteomics and ribosome trapping for condition-dependent identification of protein CDSs. We anticipate this technique will be a starting point for future high-throughput genetics investigations to determine the existence of unannotated genes in multiple bacterial species.