A regulatory checkpoint during flagellar biogenesis in Campylobacter jejuni initiates signal transduction to activate transcription of flagellar genes

ABSTRACT Many polarly flagellated bacteria require similar two-component regulatory systems (TCSs) and54 to activate tran-scription of genes essential for flagellar motility. Herein, we discovered that in addition to the flagellar type III secretion system (T3SS), the Campylobacter jejuni flagellar MS ring and rotor are required to activate the FlgSR TCS. Mutants lacking the FliF MS ring and FliG C ring rotor proteins were as defective as T3SSmutants in FlgSR- and54-dependent flagellar gene expression. Also, FliF and FliG required each other for stability, which is mediated by atypical extensions to the proteins. A FliF mutant that presumably does not interact with the T3SS protein FlhA did not support flagellar gene transcription, suggesting that FliF-T3SS interactions are essential to generate a signal sensed by the cytoplasmic FlgS histidine kinase. Furthermore, the flagellar T3SS was required for FlgS to immunoprecipitate with FliF and FliG.We propose a model whereby the flagellar T3SS facilitates FliF and FliGmultimerization into theMS ring and rotor. As a result, these flagellar structures form a cytoplasmic complex that in-teracts with and is sensed by FlgS. The synthesis of these structures appears to be a regulatory checkpoint in flagellar biogenesis that the FlgS kinase monitors to initiate signal transduction that activates54 and expression of genes required for the next stage of flagellation. Given that other polar flagellates have flagellar transcriptional hierarchies that are organized similarly as in C. je-juni, this regulatory checkpoint may exist in a broad range of bacteria to influence similar TCSs and flagellar gene transcription. IMPORTANCE Despite the presence of numerous two-component regulatory systems (TCSs) in bacteria, direct signals sensed by TCSs to activate signal transduction are known for only a minority. Polar flagellates, including Pseudomonas, Vibrio,Helicobac