Complementary Ribo-seq approaches map the translatome and provide a small protein census in the foodborne pathogen Campylobacter jejuni

Kathrin Froschauer, Sarah L. Svensson, Rick Gelhausen, Elisabetta Fiore, Philipp Kible, Alicia Klaude, Martin Kucklick, Stephan Fuchs, Florian Eggenhofer, Chao Yang, Daniel Falush, Susanne Engelmann, Rolf Backofen and Cynthia M. Sharma ()
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Kathrin Froschauer: Department of Molecular Infection Biology II
Sarah L. Svensson: Department of Molecular Infection Biology II
Rick Gelhausen: University of Freiburg
Elisabetta Fiore: Department of Molecular Infection Biology II
Philipp Kible: Department of Molecular Infection Biology II
Alicia Klaude: Institute for Microbiology
Martin Kucklick: Institute for Microbiology
Stephan Fuchs: Methodenentwicklung und Forschungsinfrastruktur (MF)
Florian Eggenhofer: University of Freiburg
Chao Yang: Chinese Academy of Sciences
Daniel Falush: Chinese Academy of Sciences
Susanne Engelmann: Institute for Microbiology
Rolf Backofen: University of Freiburg
Cynthia M. Sharma: Department of Molecular Infection Biology II

Nature Communications, 2025, vol. 16, issue 1, 1-19

Abstract: Abstract In contrast to transcriptome maps, bacterial small protein (≤50-100 aa) coding landscapes, including overlapping genes, are poorly characterized. However, an emerging number of small proteins have crucial roles in bacterial physiology and virulence. Here, we present a Ribo-seq-based high-resolution translatome map for the major foodborne pathogen Campylobacter jejuni. Besides conventional Ribo-seq, we employed translation initiation site (TIS) profiling to map start codons and also developed a translation termination site (TTS) profiling approach, which revealed stop codons not apparent from the reference genome in virulence loci. Our integrated approach combined with independent validation expanded the small proteome by two-fold, including CioY, a new 34 aa component of the CioAB oxidase. Overall, our study generates a high-resolution annotation of the C. jejuni coding landscape, provided in an interactive browser, and showcases a strategy for applying integrated Ribo-seq to other species to enrich our understanding of small proteomes.

Date: 2025
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DOI: 10.1038/s41467-025-58329-w

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