Signal Transduction Pathways That Impact Polar Flagellar Biogenesis




Burnham, Peter Michael

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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, rotor, and flagellar type III secretion system (fT3SS) core proteins. This thesis describes two different ways MS ring-rotor-fT3SS assembly regulates flagellar gene expression. MS ring-rotor-fT3SS assembly stimulates expression of the next stage of flagellar genes establishing a unique polar flagellar transcriptional program. Conserved regulatory mechanisms in diverse polar flagellates to create this polar flagellar transcriptional program centered on MS ring-rotor-fT3SS assembly have not been thoroughly examined. Using in silico and genetic analyses and our previous findings in Campylobacter jejuni as a foundation, we observed that a large subset of Gram-negative bacteria with the FlhF/FlhG regulatory system for polar flagellation also possess flagellum-associated two-component signal transduction systems (TCS). I present data supporting a general theme in polar flagellates where MS ring, rotor, and fT3SS proteins contribute to a regulatory checkpoint during polar flagellar biogenesis. I demonstrated that Vibrio cholerae and Pseudomonas aeruginosa require the formation of this regulatory checkpoint for the TCS 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, I discovered a link between the polar flagellar transcription program and the activity of FlhF and 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. I discovered a second mechanism by which the MS ring-rotor-fT3SS regulates polar flagellar gene expression as V. cholerae MS ring-rotor-fT3SS mutants increased expression of flrB, the sensor kinase of flagellar FlrBC TCS in V. cholerae. This suggested that MS ring-rotor-fT3SS formation may act as a feedback inhibition mechanism to repress the activity of the master flagellar regulator, FlrA. I examined if this effect was on flrA transcription or FlrA activity and found that early flagellar formation appears to impact V. cholerae FlrA activity. I hypothesized that early flagellar formation may repress FlrA activity through c-di-GMP in a FlhG-independent or dependent manner. I then examined the effect of DGC and PDE mutants that either 1) increased c-di-GMP levels in a FlhA mutant or 2) were known to affect V. cholerae motility to identify DGC or PDE that may link early flagellar formation to FlrA activity. I found evidence for two different early flagellar formation feedback inhibition mechanisms: a possibly c-di-GMP-independent mechanism through FlhA, and a c-di-GMP-related mechanism through FlhG, CdgE, and RocS. Although more characterization is needed, our data suggests a complex previously undescribed feedback inhibition mechanism that links completion of the MS ring-rotor-fT3SS complex to both repress FlrA activity and stimulate flagella-associated TCS.

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