Interplay of two small RNAs fine-tunes hierarchical flagellar gene expression in the foodborne pathogen Campylobacter jejuni

Like for many enteric bacteria, flagella are a crucial virulence factor for the foodborne pathogen Campylobacter jejuni, allowing the bacteria to move through the viscous mucus of the human intestine. Assembly of the complex flagellar machinery and filament requires hierarchical regulation via transcriptional control of each component. In C. jejuni, class I genes are transcribed from σ70-dependent promoters and class II/III genes with the help of the alternative sigma factors RpoN (σ54) and FliA (σ28). In contrast to transcriptional control, less is known about post-transcriptional regulation of flagellar biosynthesis cascades via small regulatory RNAs (sRNAs). Here, we characterized two sRNAs with opposing effects on the cascade that fine-tune C. jejuni flagellar filament assembly and thereby impact motility. We demonstrate that the highly conserved Campylobacter sRNA CJnc230 (FlmE, flagellar length and motility enhancer), encoded downstream of the flagellar hook structural protein FlgE, is dependent on RpoN and that RNase III processes CJnc230 from the flgE mRNA, while RNase Y and PNPase mature its 3’ end. We identify mRNAs encoding a regulator of flagella-flagella interactions and the anti-σ28 factor FlgM as direct targets of CJnc230 repression. Overexpression of CJnc230 de-represses FliA activity and upregulates class III flagellar genes, such as the major flagellin flaA, culminating in longer flagella and increased motility. In contrast, overexpression of the FliA-dependent sRNA CJnc170 (FlmR, flagellar length and motility repressor) reduces flagellar length and motility. Overall, our study demonstrates sRNA-mediated post-transcriptional regulation fine-tunes C. jejuni flagellar biosynthesis through balancing of the hierarchically expressed components.