A ribosome-dependent small-ORF switch controls flagellar gene expression and virulence

RNA structures are active regulators of bacterial translation. Here, we identified a naturally occurring translation-enhancing small open reading frame (tesORF) that controls the expression of the flagellar operon fliFGHIJK in Salmonella enterica and other motile Enterobacterales. Nucleotide-resolution ribosome profiling revealed this previously unannotated 54-nucleotide ORF immediately upstream of fliF. Genetic, transcriptomic, and biochemical analyses support a model in which tesORF translation counteracts an inhibitory RNA structure, exposing the fliF ribosome-binding site and promoting translation initiation independently of the encoded peptide sequence. Disruption of tesORF translation reduced expression of the downstream operon and impaired motility and virulence-associated phenotypes in Escherichia coli, Salmonella, and Yersinia. Together, our findings reveal how translation of a conserved small ORF regulates an entire flagellar operon through RNA structural remodelling, with consequences expending to the wider flagellar regulon, linking RNA structural dynamics to bacterial motility and virulence.