Internal genes in Enterobacteria

• Applicant: Dr. Janka Joline Schmidt
• Subject Area: Metabolism, Biochemistry and Genetics of Microorganisms
• Term: since 2025
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 557142412

Conventional gene annotation assumes that each coding sequence corresponds to a single protein product. However, the genetic code of bacteria is highly plastic, allowing the expansion of the proteome without increasing their genome size, through “internal genes”. Internal genes coexist within the confines of a primary gene, leading to the translation of alternative open reading frames (ORFs), collectively referred to as “internal translatome”. Alternative translation is mediated by frameshifting, stop codon readthrough or through in-frame or out-of-frame internal translation initiation sites (iTIS). However, little is known about the factors contributing to alternative translation, the number of internal genes present in an organism and their role in bacterial physiology. This project aims to comprehensively identify, characterize, and understand the role of internal genes in enterobacteria (Escherichia coli, Klebsiella pneumoniae, and Salmonella enterica serovar Typhimurium). Enterobacteria are a diverse family of Gram-negative bacteria that include many clinically relevant pathogens, making them critical targets for understanding gene function and evolution. Investigating these three organisms will provide insights into the evolutionary origins and functional redundancy of internal genes across species. Initially, ribosome profiling (RIBO-seq) will be used to identify alternative open reading frames (ORFs) on a genome-wide scale. By selectively stalling ribosomes at translation initiation or termination sites using specific drugs, such as the pleuromutilin retapamulin or the PrAMP antimicrobial peptide apidaecin, the RIBO-seq enables mapping of these sites. Specifically, ribosomes protect mRNA fragments from degradation by RNAse, generating ribosome footprints that can be isolated and subjected to high-throughput sequencing. To uncover condition-dependent internal genes, each organism will be cultivated under different environmental conditions, such as heat shock, nutrient-rich environments, or oxidative stress, representing diverse cellular responses. Following the global analysis, the active translation of selected candidate internal genes will be confirmed both in vivo and by mass spectrometry-based approaches, including bottom-up or top-down mass spectrometry. If active translation is confirmed, the function of the internal gene is either adjacent to or completely distinct from the primary gene, necessitating their genetic separation to study their functional dependence and further refine their roles. Ultimately, this project aims to establish a robust and transferable pipeline for the identification and characterization of internal genes, with potential applications in other organisms.

• DFG Programme: WBP Fellowship
• International Connection: USA
• Host: Professorin Gisela Storz, Ph.D.