Unveiling the small protein landscape of uropathogenic E. coli

Uropathogenic Escherichia coli (UPEC) is the predominant causative agent of urinary tract infections. The UPEC strain, CFT073, has a mosaic genome containing 60 genomic islands enriched in virulence genes. In this study, we explore the small proteome of CFT073 using ribosome profiling (Ribo-seq) coupled with RNA sequencing (RNA-seq), and reveal 338 translated, previously unannotated, putative small open reading frames (sORFs) with less than 100 codons. Among them, 78 sORFs are located in intergenic regions. Fifteen intergenic sORFs encode a predicted transmembrane helix (smemORFs), of which seven are hypothetical or have not been characterized in other E. coli strains. The majority of the remaining putative sORFs encodes uncharacterized or novel soluble proteins (ssolORFs). Considering the evolution of many sORFs is likely to be recent, functional sORFs are more likely to have conserved gene neighborhood in other genomes. Searching into the genomes of 10 other E. coli strains and four enterobacteria species, synteny analysis reveals that 10 of 15 putative smemORFs, and 27 of 63 putative ssolORFs, have a homologous sequence in a similar genomic context in at least one other genome, supporting their likelihood of encoding functional proteins. Further validation of putative sORFs using proteomics, cell-free protein synthesis, and mNeonGreen tagging provide strong evidence for the translation of nine predicted intergenic sORFs. Notably, four previously uncharacterized small membrane proteins have increased abundance under oxidative stress, suggesting a potential role in the stress response. Together, our findings reveal the small proteome of an extraintestinal pathogenic E. coli and provide a catalog of putative sORFs for further investigation.