A Polar Flagellar Transcriptional Program Mediated by Diverse Two-Component Signal Transduction Systems and Basal Flagellar Proteins Is Broadly Conserved in Polar Flagellates

Abstract

Bacterial flagella are rotating nanomachines required for motility. Flagellar gene expression and protein secretion are coordinated for efficient flagellar biogenesis. Polar flagellates, unlike peritrichous bacteria, commonly order flagellar rod and hook gene transcription as a separate step after production of the MS ring, C ring, and flagellar type III secretion system (fT3SS) core proteins that form a competent fT3SS. Conserved regulatory mechanisms in diverse polar flagellates to create this polar flagellar transcriptional program have not been thoroughly assimilated. Using in silico and genetic analyses and our previous findings in Campylobacter jejuni as a foundation, we observed a large subset of Gram-negative bacteria with the FlhF/FlhG regulatory system for polar flagellation to possess flagellum-associated two-component signal transduction systems (TCSs). We present data supporting a general theme in polar flagellates whereby MS ring, rotor, and fT3SS proteins contribute to a regulatory checkpoint during polar flagellar biogenesis. We demonstrate that Vibrio cholerae and Pseudomonas aeruginosa require the formation of this regulatory checkpoint for the TCSs to directly activate subsequent rod and hook gene transcription, which are hallmarks of the polar flagellar transcriptional program. By reprogramming transcription in V. cholerae to more closely follow the peritrichous flagellar transcriptional program, we discovered a link between the polar flagellar transcription program and the activity of FlhF/FlhG flagellar biogenesis regulators in which the transcriptional program allows polar flagellates to continue to produce flagella for motility when FlhF or FlhG activity may be altered. Our findings integrate flagellar transcriptional and biogenesis regulatory processes involved in polar flagellation in many species. IMPORTANCE Relative to peritrichous bacteria, polar flagellates possess regulatory systems that order flagellar gene transcription differently and produce flagella in specific numbers only at poles. How transcriptional and flagellar biogenesis regulatory systems are interlinked to promote the correct synthesis of polar flagella in diverse species has largely been unexplored. We found evidence for many Gram-negative polar flagellates encoding two-component signal transduction systems with activity linked to the formation of flagellar type III secretion systems to enable production of flagellar rod and hook proteins at a discrete, subsequent stage during flagellar assembly. This polar flagellar transcriptional program assists, in some manner, the FlhF/FlhG flagellar biogenesis regulatory system, which forms specific flagellation patterns in polar flagellates in maintaining flagellation and motility when activity of FlhF or FlhG might be altered. Our work provides insight into the multiple regulatory processes required for polar flagellation.

Transcriptional organization and regulation of thePseudomonas putidaflagellar system

“…An additional tier, Class III, activated by the FleSR (also known as FlrBC) twocomponent system is required in P. aeruginosa, V. cholerae, V. parahemolyticus and A. hydrolitica to complete the assembly of the components of the hook-basal body (HBB) complex (Dasgupta et al, 2003;Khan et al, 2020;Wilhelms et al, 2011). Activation of FleSR/FlrBC was recently shown to respond to the assembly of the EBB in V. cholerae and P. aeruginosa (Burnham et al, 2020), as previously observed with the Epsilonproteobacteria Campylobacter jejuni and Helicobacter pylori (Boll and Hendrixson, 2013;Tsang and Hoover, 2015). Sensing of the assembly status may be mediated by FlrD, a membrane protein containing a HAMP domain that stimulates FlrBC-dependent activation in V. cholerae (Moisi et al, 2009).…”

Experimental Evolution of Campylobacter jejuni Leads to Loss of Motility, rpoN (σ54) Deletion and Genome Reduction

“…Sigma factor 70 controls biosynthesis of the flagellar export apparatus, FlhF, and Rpo N (σ54). Current models of C. jejuni flagellar biosynthesis show that the type III flagellar secretion system and its assembly with the inner membrane anchor and motor protein complexes, together with FlhF, influence the activity of the FlgSR two-component signal transduction pathway; FlgR in turn is required to stimulate activity of the alternative sigma factor, σ54 ( Rpo N) ( Gilbreath et al, 2011 ; Burnham et al, 2020 ). The σ54 regulon is almost exclusively composed of flagellar structural genes in C. jejuni , and expression of these genes generates essential components of flagella, including the basal body, hook and minor flagellin subunits ( Chaudhuri et al, 2011 ).…”

Pseudomonas Flagella: Generalities and Specificities

“…Furthermore, polar flagellated bacteria that contain the FlhF/FlhG regulatory system mostly possess TCSs, such as FleS/R in P. aeruginosa , FlgS/R in C. jejuni , or FlrB/C in V. cholerae . Such TCSs serve as an additional check point in flagellar biogenesis and are essential to allow rod and hook subunit expression, which are sigma 54 dependant, after correct assembly of the MS-ring/rotor/fT3SS complex [ 67 ]. Peritrichous bacteria do not possess a similar regulatory mechanism because rod and hook subunits are co-expressed with basal body components.…”

“…In P. aeruginosa , FleS is a cytoplasmic protein that senses intracellular signals and transfers its phosphate to FleR. However, the interaction between FleS and FliG/M/fT3SS has not yet been shown, but Burham et al demonstrated the importance of primary assembly in the activation of these TCSs [ 67 ]. Although this mechanism has not yet been elucidated in Pseudomonas , FlgS has been shown to physically interact with FliF and FliG in Campylobacter jejuni only if multimerized around the fT3SS.…”