Identification of unannotated small genes in Salmonella. Salmonella enterica subsp. enterica serovar Typhimurium str. 14028S

Bacterial genomes serve as a blueprint in all aspects of biological research, and therefore accurate annotation is of paramount importance. However, increasing evidence suggests annotated bacterial genomes have missed many, if not all, small genes encoding proteins ≤60 amino acids. To uncover unannotated small genes in Salmonella enterica, we used a genomic technique ‘ribosome profiling’ that provides a snapshot of all mRNAs being translated (translatome). For comprehensive identification of the small genes, we obtained Salmonella translatomes in four different growth conditions including Luria-Bertani medium, MOPS rich defined medium, N-minimal medium with low Mg2+ (10 μM), and N-minimal medium at pH 5.7, respectively. Ribosome profiling data were analysed in combination with in silico predicted putative open reading frames and transcriptome profiles. As a result, we uncovered 127 previously unannotated genes, the majority of which were small genes encoding proteins ≤50 amino acids. We validated expression by western blot of the identified unannotated small genes, the smallest of which is 7-amino acid long. Because some unannotated small genes identified in this study are only expressed in the infection-relevant low Mg2+ conditions, they are likely involved in cellular processes required for Salmonella virulence. Our findings suggest that currently sequenced bacterial genomes are likely under-annotated with regard to small genes and need to be revised. Overall design: Ribosome profiling and RNA-seq were performed on Salmonella enterica 14028s cells grown in four different growth conditions.