A Regulatory Checkpoint during Flagellar Biogenesis in <named-content content-type="genus-species">Campylobacter jejuni</named-content> Initiates Signal Transduction To Activate Transcription of Flagellar Genes

ABSTRACT Many polarly flagellated bacteria require similar two-component regulatory systems (TCSs) and σ54 to activate transcription 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 T3SS mutants in FlgSR- and σ54-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 FliG multimerization into the MS ring and rotor. As a result, these flagellar structures form a cytoplasmic complex that interacts 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 activates σ54 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. jejuni, 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, Helicobacter, and Campylobacter species, require a similar TCS and σ54 for flagellar gene transcription, but the activating signals for these TCSs are unknown. We explored signals that activate the Campylobacter jejuni FlgSR TCS to initiate σ54-dependent flagellar gene transcription. Our discoveries suggest that the FlgS histidine kinase monitors the formation of the flagellar type III secretion system and the surrounding MS and C rings. The synthesis of these structures creates a regulatory checkpoint in flagellar biogenesis that is sensed by FlgS to ensure proper transcription of the next set of genes for subsequent steps in flagellation. Given the conservation of flagellar-associated TCSs and transcriptional cascades in polar flagellates, this regulatory checkpoint in flagellar biogenesis likely impacts flagellation in a broad range of bacteria