ABSTRACT Background Knowing the molecules encoded by bacterial pathogens and how their expression is regulated is essential to understand how they survive, colonize, and cause disease. RNA-seq technologies that map transcriptomes have revealed a wealth of new transcripts in bacterial pathogens. However, they do not provide direct evidence or coordinates for coding potential. In particular, they miss small proteins (≤ 50-100 amino acids) translated from small open reading frames (sORFs). However, this still poorly annotated component of bacterial genomes shows emerging roles in bacterial physiology and virulence. Results Here, we present an integrated approach based on complementary ribosome profiling (Ribo-seq) techniques to map the “translatome” of Campylobacter jejuni , the most common cause of bacterial gastroenteritis. Besides conventional Ribo-seq, we employed translation initiation site (TIS) profiling to map start codons and reveal internal sORFs. We also developed a Ribo-seq approach for mapping of translation termination sites (TTS), which revealed stop codons not apparent from the reference genome in virulence-associated loci. Our translatome map confirms translation of leaderless ORFs and leader peptides, re-annotates start or stop codons of 35 genes, and reveals isoforms generated by internal start sites. It also adds 42 novel sORFs in diverse contexts to the C. jejuni annotation, such as within small RNAs, in 5′ untranslated regions (UTRs), or internal/out-of-frame/antisense in larger ORFs. Using epitope tagging/western blot and mass spectrometry we validated expression of almost 60 annotated and novel sORFs, including cioY , which we show encodes a conserved, 34 amino acid component of the CioAB terminal oxidase. Conclusions Overall, we provide a blueprint for integrating several Ribo-seq approaches to refine and enrich bacterial annotations.

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