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

Peter M Burnham; William P Kolar; David R Hendrixson

doi:10.1128/mBio.03107-19

. 2020 Mar 3;11(2):e03107-19. doi: 10.1128/mBio.03107-19

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

Peter M Burnham ^a, William P Kolar ^a, David R Hendrixson ^a,^✉

Editors: Barbara I Kazmierczak^b, M Stephen Trent^c

PMCID: PMC7064773 PMID: 32127455

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.

KEYWORDS: Campylobacter jejuni, FlhF, FlhG, MS ring, Pseudomonas aeruginosa, T3SS, Vibrio cholerae, flagellar biogenesis, motility, polar flagellation, two-component signal transduction

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.

INTRODUCTION

Many bacteria synthesize flagella for swimming motility. Each species produces a specific flagellation pattern defined by the spatial arrangement and number of flagella presented on the cell surface. Peritrichous flagellates construct many flagella across the surface, whereas polar flagellates generate flagella only at polar regions. Polar flagellates are further categorized by the number of flagella per cell: monotrichous (one flagellum at one pole), amphitrichous (one flagellum at each pole), and lophotrichous (a tuft of a few flagella at one pole).

Flagellar placement and number in many polar flagellates are controlled by the FlhF GTPase and FlhG/FleN ATPase (1, 2). FlhF is hypothesized to function in some polar flagellates in a GTP-bound “on” state to perform an unknown essential step for flagellar biogenesis at a pole. Transitioning to a GDP-bound “off” state upon GTP hydrolysis may limit the production of additional polar flagella. In some polar flagellates, FlhG stimulates FlhF GTPase activity in vitro, which has been proposed to influence flagellum numbers by controlling in vivo FlhF-dependent polar flagellation activities (3 –6). However, FlhG orthologs in other species control polar flagellar number by repressing the activity or expression of a specific master transcriptional regulator so that an ideal level of flagellar genes sufficient to produce the correct number of flagella are expressed (7 –9). Many molecular details for how FlhF and FlhG control polar flagellation remain elusive. It is anticipated that FlhF and FlhG activities vary among species, resulting in different flagellation patterns in polar flagellates.

Despite different flagellation patterns, many peritrichous and polar flagellates possess some conserved strategies to coordinate flagellar gene transcription with stages of flagellar assembly (10 –13). These strategies allow for tight regulation of ordered flagellar protein secretion that is conducive to flagellar motor biogenesis. Stages of flagellar assembly can be marked by distinct cues or regulatory checkpoints that are detected by different mechanisms to stimulate gene transcription and protein production to complete the next stage of assembly. Flagellar biogenesis begins by activating the transcription of genes encoding components essential for the initial steps in assembly, which include the flagellar type III secretion system (fT3SS), MS ring, and C ring rotor and switch proteins (14 –20). MS and C ring formation around the fT3SS core completes biogenesis of a competent fT3SS for export and assembly of rod and hook components (21 –25). Up to this point, the alternative σ factor σ²⁸, which is required for transcription of flagellins and other motility genes, is inhibited by the anti-σ factor FlgM (11, 13, 26 –29). Hook biogenesis completes a regulatory checkpoint that facilitates an fT3SS substrate specificity switch to secrete FlgM out of the cell via the fT3SS (11, 13, 30). Derepression of σ²⁸ allows for transcription of genes that complete flagellar filament polymerization and motor assembly.

We previously explored how the amphitrichous polar flagellate Campylobacter jejuni coordinates the transcription of flagellar genes with flagellar assembly (Fig. 1) (31 –33). We discovered that fT3SS core proteins (FlhA, FlhB, FliP, FliQ, and FliR), the FliF MS ring, and FliG rotor of the C ring assemble into the MS ring-rotor-fT3SS complex to form a regulatory checkpoint monitored by C. jejuni. We found the flagellum-associated FlgSR two-component signal transduction system (TCS) detects MS ring-rotor-fT3SS formation to directly activate σ⁵⁴-dependent flagellar rod and hook gene expression (Fig. 1) (31 –33). As a means of signal detection, we observed that the cytoplasmic FlgS sensor kinase of the FlgSR TCS physically interacted with FliF and FliG only after these proteins multimerized into the MS ring and rotor around the fT3SS core (Fig. 1) (32). In mutants defective for MS ring-rotor-fT3SS complex assembly, FlgS did not interact with FliF and FliG. Currently, it is not known whether FlgS interacts with surfaces of adjacent FliF subunits, FliG subunits, or FliF-FliG complexes of the MS ring-rotor structure surrounding the fT3SS core. Detection of this regulatory checkpoint by an orthologous FlgSR TCS also may occur in Helicobacter pylori, a lophotrichous epsilonproteobacterium closely related to C. jejuni, for transcription of σ⁵⁴-dependent rod and hook genes (34 –37). Genetic analyses indicate that H. pylori FlgSR TCS activity for rod and hook gene transcription is also dependent on fT3SS, MS ring, and C ring proteins, suggesting that this TCS in H. pylori also senses the formation of a competent fT3SS (38 –43).

FIG 1 — Defined and unknown regulatory steps of the polar flagellar transcriptional program of *C. jejuni*, *V. cholerae*, and *P. aeruginosa*. Simplified models of transcriptional regulation of different classes of flagellar genes in the Gram-negative polar flagellates *C. jejuni*, *V. cholerae*, and *P. aeruginosa*. *V. cholerae* and *P. aeruginosa* (right) initiate flagellar gene transcription via master transcriptional regulators (FlrA and FleQ, respectively) to transcribe an initial class of flagellar genes; *C. jejuni* lacks a master transcriptional regulator and expression of this class of flagellar genes appears to be constitutive (left). These initial flagellar genes encode the fT3SS, MS ring, C ring, and a flagellum-associated TCS (FlgSR in *C. jejuni*, FlrBC in *V. cholerae*, and FleSR in *P. aeruginosa*). Expression of the *C. jejuni* flagellar rod, ring, and hook genes is dependent upon σ⁵⁴, the FlgSR TCS, and the fT3SS, FliF MS ring, and FliG C ring rotor proteins (31 –33). The formation of the MS ring-rotor-fT3SS complex is an early regulatory checkpoint sensed by the FlgS sensor kinase to initiate phosphotransfer to the FlgR response regulator, which allows FlgR to function with σ⁵⁴ for rod and hook gene expression. (Right) Exploration of factors that may form a similar regulatory checkpoint during flagellar assembly that are required for the activity of the *V. cholerae* FlrBC TCS and the *P. aeruginosa* FleSR TCS addressed in this work. A regulatory checkpoint during late flagellar assembly occurs upon formation of the flagellar rod and hook in all of these organisms and many other peritrichous and polar flagellates. Completion of rod and hook assembly promotes a substrate specificity switch that facilitates secretion of the anti-σ factor FlgM from the cytoplasm and derepression of σ²⁸ for transcription of flagellins and other proteins that complete flagellar biogenesis for motor function. *C. jejuni* also contains *fed* genes that are dependent on σ²⁸ for expression and are not involved in motility but are required for the colonization of avian species and some virulence processes (92, 93).

Vibrio cholerae and Pseudomonas aeruginosa are monotrichous polar flagellates that also produce a flagellum-associated TCS. The V. cholerae FlrBC and P. aeruginosa FleSR TCSs, like the C. jejuni FlgSR TCS, are directly required with σ⁵⁴ for flagellar rod and hook gene expression (17 –19, 44, 45). As with C. jejuni FlgSR, signal transduction through V. cholerae and P. aeruginosa TCSs results in phosphorylation of the cognate FlrC and FleR response regulators to promote their binding to rod and hook gene promoters and directly assist σ⁵⁴ in activating transcription of rod and hook genes (44 –47). Although transcription of the V. cholerae and P. aeruginosa FlrBC and FleSR TCSs occurs simultaneously with fT3SS, MS ring, and C ring genes by a master transcriptional regulator (18, 19), it is not known whether these TCSs sense cues associated with flagellar assembly or flagellum-independent cellular cues to initiate and coordinate flagellar rod and hook gene expression with a stage of flagellar assembly (Fig. 1). Transcription of rod and hook genes after expression of MS ring, C ring, and fT3SS components appears to comprise a polar flagellar transcriptional program not observed in peritrichous flagellates.

Noticeably, many peritrichous flagellates appear to ignore the formation of the MS ring-rotor-fT3SS complex as a regulatory checkpoint that we discovered in C. jejuni and is likely present in H. pylori. The model peritrichous bacteria Escherichia coli and Salmonella lack a flagellum-associated TCS and do not employ σ⁵⁴ for flagellar gene transcription. These bacteria also do not require the MS ring, C ring, and fT3SS proteins for rod and hook gene expression (10, 15, 48). Instead, these bacteria express fT3SS, MS ring, and C ring proteins simultaneously with flagellar rod and hook genes, which we designate, for the purposes of this report, the peritrichous flagellar transcriptional program. Hence, a competent fT3SS is not required for expression of rod and hook genes in peritrichous flagellates, in contrast to C. jejuni and H. pylori. These differences raise intriguing questions for flagellar biogenesis in polar flagellates. (i) How common is it for polar flagellates to possess flagellum-associated TCSs? (ii) Do polar flagellates with flagellum-associated TCSs broadly require MS ring, C ring, and fT3SS proteins for activity to result in rod and hook gene expression? (iii) Do polar flagellates employ the respective sensor kinases to detect a regulatory checkpoint formed by the MS ring, C ring, and/or fT3SS? (iv) Do some TCSs detect flagellum-independent cues to activate rod and hook gene expression? (v) Is the polar flagellar transcriptional program that separates production of fT3SS, MS ring, and C ring proteins from rod and hook proteins required for or beneficial to a specific process in polar flagellates to build flagella?

We evaluated the conservation of regulatory systems that order gene expression for a polar flagellar transcriptional program in diverse polar flagellates. Our results, combined with previous findings in alphaproteobacteria, indicate a broad, common theme in polar flagellates, whereby different mechanisms are employed to coordinate rod and hook protein production with a stage of flagellar assembly involving the formation of a competent fT3SS. We found that a large subset of polar flagellates with FlhF/FlhG flagellar biogenesis regulatory systems encode orthologous flagellum-associated TCSs. Additional evidence combined with our previous findings suggests that these TCSs have a conserved function in detecting a similar regulatory checkpoint centered around MS ring-rotor-fT3SS complex formation. Our findings suggest that the polar flagellar transcriptional program, rather than a peritrichous one, allows polar flagellates to sustain flagellation and motility if FlhF and/or FlhG activity is altered and provide speculation into the evolution of polar flagellates. Our work provides insight into connections between flagellar transcriptional and biogenesis regulatory systems involved in polar flagellation.

RESULTS

A large subset of gram-negative polar flagellates possesses flagellum-associated TCSs.

We followed a bioinformatic strategy to evaluate the prevalence of flagellum-associated TCS in a broad range of Gram-negative polar flagellates. We limited our analysis to Gram-negative bacteria to enable comparisons with previous studies in bacteria that employ flagellum-associated TCSs for direct activation of σ⁵⁴-dependent flagellar rod and hook gene expression, such as C. jejuni, H. pylori, V. cholerae, and P. aeruginosa. For this approach, we performed reciprocal best hit sequence alignments with a set of 117 reference bacterial genomes available at NCBI Assembly to identify Gram-negative flagellates. Genomes included in this reference set were curated by NCBI to represent high-quality, community-standard bacterial genomes or genomes of medically relevant bacteria. Although not every sequenced bacterial genome is included in this reference set, it is sufficient to survey and acquire information regarding features present among phylogenetically diverse bacteria.

We first used E. coli FlgH as a marker for Gram-negative bacteria producing flagella (Fig. 2A). FlgH forms the L-ring required for external flagella to penetrate the outer membrane barrier in Gram-negative flagellates (49 –51). Since FlhF is involved in polar flagellation, we then used V. cholerae FlhF as a marker to predict polar flagellates within the Gram-negative flagellates (Fig. 2A) (1, 2, 7). Our results initially identified 23 of 47 putative Gram-negative flagellates within the reference set as polarly flagellated species (Fig. 2B), with two clear false positives: Bordetella bronchiseptica, which encodes a predicted FlhF (BN112_0372) but is a known peritrichous organism (52), and Burkholderia mallei, which harbors mutations in multiple flagellar genes and is likely undergoing reductive genome evolution (53). Twenty of 21 predicted Gram-negative polar flagellates encoded a predicted FlhG ortholog immediately downstream of flhF, indicating that an FlhF/FlhG flagellar biogenesis regulatory system was intact; only Rhodospirillum rubrum lacked an flhG ortholog organized with flhF. Of the 24 predicted Gram-negative flagellates lacking FlhF, 18 are known peritrichous organisms. The remaining six are actually polar flagellates with five species from alphaproteobacteria, best represented by Caulobacter crescentus, which employs factors other than FlhF for polar flagellation. Thus, FlgH and FlhF were relatively robust predictors of Gram-negative polar flagellates within the reference set except for alphaproteobacterial species.

From the 21 remaining predicted Gram-negative polar flagellates with FlhF orthologs, we performed reciprocal best hit sequence alignments using the V. cholerae FlrB sensor kinase of the flagellum-associated FlrBC TCS to identify 16 species with flagellum-associated TCSs (Fig. 2A and B). Although this approach identified putative flagellum-associated TCS sensor kinases in many polar flagellates (including FleS of the FleSR TCS of different Pseudomonas species), C. jejuni FlgS was not identified. We repeated our reciprocal best hit sequence alignment analysis using C. jejuni FlgS. C. jejuni FlgS did not identify FlrB or FleS as orthologs but did identify flagellum-associated sensor kinases in two of the four predicted polar flagellates without FlrB orthologs, H. pylori FlgS and Burkholderia pseudomallei BPSL0127 (Fig. 2A and B). These observations suggest that sensor kinases of flagellum-associated TCSs in polar flagellates are divided into distinct FlrB-like or FlgS-like groups. Overall, our bioinformatic analysis indicated that 19 of 21 predicted Gram-negative polar flagellates from the reference set encode both an FlhF/FlhG regulatory system and a putative flagellum-associated TCS. Thus, our results suggest a high degree of cooccurrence between the two regulatory systems exist in bacterial species that produce polar flagella. The only two Gram-negative FlhF/FlhG-positive species in the reference set that did not encode a flagellum-associated TCS were the known polar flagellate Xanthamonas campestris pv. campestris, previously noted to lack a respective TCS, and Thermanaerovibrio acidaminovorans, whose prediction as a polar flagellate may be dubious, as it produces lateral flagella on its concave surface (54, 55) (Fig. 2B).

To ensure that our FlrB homologs are flagellum-associated sensor kinases rather than conserved unrelated sensor kinases involved in other processes, we examined the correlation of V. cholerae sensor kinases with the presence of FlhF across genomes in the reference set (see Fig. S1 in the supplemental material). If non-flagellum-associated sensor kinases are within our predicted FlrB orthologs, we would expect a weak to negative correlation of these kinases with FlhF that is indistinguishable from the correlation of a random V. cholerae sensor kinase to FlhF. In contrast, if FlrB orthologs are flagellum-associated TCS kinases, we would expect V. cholerae FlrB to be one of the kinases most highly correlated with FlhF. We found that predicted FlrB orthologs had a stronger positive correlation to V. cholerae FlhF than all but two of 51 V. cholerae sensor kinases (Fig. S1). One of these kinases is V. cholerae CheA, which is the major sensor kinase in the chemotaxis system that influences flagellar rotation and motility in many bacterial species (56, 57). The other V. cholerae kinase is VCA0851, an uncharacterized kinase. These results support our flagellum-associated TCS predictions and further emphasize the correlation between the FlhF/FlhG flagellar biogenesis system and the flagellum-associated TCSs.

FIG S1

Correlation of V. cholerae sensor kinase orthologs in predicted polar flagellates to V. cholerae FlhF orthologs. The relative correlation of each set of kinase orthologs for the 51 V. cholerae sensor kinases taken from each of the reference genomes to FlhF is shown as a Pearson correlation coefficient. Values closer to 1 indicate that the sensor kinase orthologs are more positively correlated with FlhF orthologs. Values greater than 0.4 are considered significant positive correlations. The score for FlrB orthologs is indicated in red. Download FIG S1, TIF file, 1.1 MB^{(1.1MB, tif)}.

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Our bioinformatic analysis suggested that the sensor kinases in flagellum-associated TCS systems of Gram-negative polar flagellates belong to two or more unrelated groups and possess different features, since C. jejuni FlgS and V. cholerae FlrB did not identify each other as orthologs. Many of these sensor kinases are predicted to be cytoplasmic kinases lacking transmembrane domains. Representative kinases such as C. jejuni FlgS, H. pylori FlgS, V. cholerae FlrB, and P. aeruginosa FleS contain similar HisKA and HATPase_c domains that function in histidine autophosphorylation, dimerization, and ATPase activity but have divergent N-terminal sensor domains (Fig. 2C) (58, 59). Both V. cholerae FlrB and P. aeruginosa FleS contain a predicted PAS domain that is a common sensing domain in sensor kinases; FleS also has a predicted coiled-coil domain for potential protein interactions (Fig. 2C) (59). However, the only predicted domain within the C. jejuni FlgS sensor region is a coiled-coil domain, and no predicted structural domain was identified in H. pylori FlgS (Fig. 2C). These observations suggest that flagellum-associated sensor kinases within FlhF/FlhG-containing polar flagellates detect different cellular signals or detect similar signals by different mechanisms to initiate signal transduction for flagellar gene expression and polar flagellar biogenesis.

The V. cholerae FlrBC TCS requires the fT3SS, MS ring, and rotor for activity.

As described above, we previously discovered that C. jejuni FlgS detects MS ring-rotor-fT3SS assembly by a direct interaction as a regulatory checkpoint to initiate signal transduction for FlgSR- and σ⁵⁴-dependent transcription of rod and hook genes for the polar flagellar transcriptional program (Fig. 1) (32). The V. cholerae and P. aeruginosa flagellum-associated FlrBC/FleSR TCSs are expressed simultaneously with the fT3SS, MS ring, and C ring genes. However, investigations to identify what signals are detected by these TCSs to initiate signal transduction for TCS- and σ⁵⁴-dependent flagellar rod and hook gene expression that occurs in the subsequent tier of the polar flagellar transcriptional program are lacking (Fig. 1) (18, 19). Considering that the sensor domains of V. cholerae FlrB and P. aeruginosa FleS differ from that of C. jejuni FlgS, we hypothesized that the FlrB and FleS kinases detect a similar regulatory checkpoint formed by fT3SS, MS ring, and/or C ring components but by different means, or they detect a signal independent of flagellar biogenesis (Fig. 1). Thus, we first investigated whether disruption of the fT3SS, MS ring, and/or C ring impacted the activity of the V. cholerae FlrBC TCS for σ⁵⁴-dependent rod and hook gene expression.

Transcriptional fusions of the promoters of four V. cholerae FlrBC- and σ⁵⁴-dependent operons, flgBCDE, flgFGHIJ, flgKL, and flaA, to a promoterless lacZ gene were created in WT V. cholerae and isogenic flagellar mutants. The former three operons encode rod and hook genes, whereas flaA encodes the major flagellin. V. cholerae is different from many other flagellates in that the major flagellin is not expressed from a σ²⁸-dependent promoter; instead, minor flagellins are expressed from a σ²⁸-dependent promoter in this bacterium (60). Of note, the flgFGHIJ rod operon was previously proposed to be cotranscribed with the flgBCDE rod and hook operon from the flgB promoter (18). However, we identified a putative σ⁵⁴ binding site between flgE and flgF and constructed an flgFp-lacZ transcriptional fusion for analysis. A cheVp-lacZ transcriptional fusion served as a control, as cheV expression is independent of FlrBC TCS and σ⁵⁴ (18).

We verified previous findings that V. cholerae ΔflrA, Δσ⁵⁴ ΔflrB, and ΔflrC (but not Δσ²⁸) mutants were defective for flaAp-, flgBp-, and flgKp-lacZ expression (Fig. 3A) (18, 45); expression of these reporters was reduced 6- to >50-fold in these mutants relative to that of WT V. cholerae. The defect in ΔflrA is due to FlrA functioning as a master transcriptional regulator required for flrBC expression (Fig. 1) (17, 18). We also confirmed that an FlrBC- and σ⁵⁴-dependent promoter upstream of flgF drives expression of flgFGHIJ independently of flgBp; flgFp-lacZ expression was decreased 50- to 100-fold in FlrBC TCS or σ⁵⁴ mutants (Fig. 3A).

FIG 3 — Analysis of TCS- and σ⁵⁴-dependent flagellar gene expression in flagellar mutants of *V. cholerae*. (A) Expression of flagellar rod and hook operons in WT *V. cholerae* C6706 and isogenic flagellar mutants. *flgBp*-, *flgFp*-, *flgKp*-, *flaAp*-, and *cheVp*-*lacZ* transcriptional reporters were maintained on plasmids in *V. cholerae* strains. The level of expression of each transcriptional reporter in each mutant is relative to the level of expression in WT *V. cholerae*, which was set to 100 U. MR, master regulator; TCS, two-component signal transduction system. (B) Expression of *flaAp*-*lacZ* transcriptional fusion in WT *V. cholerae* and an isogenic mutant containing vector alone (solid blue bars) or vectors to express genes from a constitutive promoter for complementation (hatched blue bars). The level of expression of transcriptional reporters in each mutant is relative to the level of expression in WT *V. cholerae* with vector alone, which was set to 100 U. For panels A and B, results are from a representative assay with each sample analyzed in triplicate. Error bars indicate standard deviations of the average level of expression from three samples. An asterisk indicates significant difference in expression from the WT (A) or WT containing vector alone (B) (P < 0.05). Two asterisks indicate significant increase in expression from the respective mutant containing vector only (P < 0.05). (C) Immunoblot analysis of the FlrB sensor kinase levels in whole-cell lysates of WT *V. cholerae* and isogenic mutants. Specific antiserum to FlrB was used to detect the protein. Detection of RpoA served as a control to ensure equal loading of proteins across strains.

We next discovered that V. cholerae mutants lacking fT3SS core proteins (including FlhA, FlhB, FliP, FliQ, and FliR) and the FliF MS ring protein were defective for FlrBC activity and rod and hook transcription, as expression of the respective transcriptional reporters was reduced 3- to 20-fold (Fig. 3A). The C ring FliG rotor protein was also required for FlrBC- and σ⁵⁴-dependent flagellar gene expression, but the C ring FliM and FliN switch proteins were not (Fig. 3A). These results are similar to our findings for requirements for C. jejuni FlgSR TCS directly activating σ⁵⁴-dependent flagellar rod and hook gene expression (Fig. S2) (31 –33). Either no reductions or only modest reductions in cheVp-lacZ expression were observed in these flagellar mutants. In trans complementation of the V. cholerae fT3SS, fliF, and fliG mutants with the respective gene restored expression of flaAp-lacZ to a significantly higher level than that of each mutant with vector alone (and to at least 60% of the level observed in the WT; Fig. 3B). We were unable to construct a complementing vector for the fliP mutant due to toxicity during attempted construction. flaAp-lacZ expression was partially or fully restored in flrA, flrB, and flrC mutants with complementation relative to the mutants with vector alone. Additionally, we verified that FlrB production was unaffected in mutants lacking the fT3SS, FliF, or FliG, which eliminated the possibility that the FlrBC TCS was unstable or not expressed in these mutants as an explanation of their reduction in rod and hook gene expression (Fig. 3C). The finding that a lack of individual MS ring, rotor, and fT3SS proteins abolishes FlrBC-dependent gene expression is consistent with our hypothesis that V. cholerae flagellar components form a regulatory checkpoint, possibly involving functional fT3SS assembly, required for FlrBC to activate rod and hook gene expression, as we previously demonstrated in C. jejuni (31 –33).

FIG S2

C. jejuni FlgSR TCS activity for σ⁵⁴-dependent flagellar gene expression in WT and flagellar mutants. Expression of the ΔflaB::astA transcriptional reporter in WT C. jejuni strain 81-176 and isogenic flagellar mutants is shown and comparable to our previously published analysis (32). The level of flaB::astA expression in each mutant is relative to the level of expression in WT C. jejuni, which was set to 100 U. Results from a representative assay with each sample analyzed in triplicate are shown. Error bars indicate standard deviations of the average levels of expression from three samples. An asterisk indicates the mutant had significantly increased or decreased reporter expression relative to that of the WT strain (P < 0.05). Download FIG S2, TIF file, 1.1 MB^{(1.1MB, tif)}.

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The P. aeruginosa FleSR TCS requires the fT3SS, MS ring, and rotor for activity.

We next investigated whether P. aeruginosa FleSR-dependent expression of flagellar rod and hook genes was influenced by fT3SS, MS ring, and C ring protein production and possibly assembly into an MS ring-rotor-fT3SS complex. We generated complete or partial in-frame deletion mutants of flagellar genes in P. aeruginosa PA14 and then integrated transcriptional fusions of the promoters of the flgB rod and hook operon and fliA (encoding σ²⁸) to lacZ in the att site on the chromosome. We verified that transcription of the P. aeruginosa flgB rod and hook operon is dependent on σ⁵⁴ and the FleSR TCS, in addition to the FleQ master transcriptional regulator that is required for fleSR transcription (Fig. 1 and 4) (19, 20). The fliAp-lacZ reporter served as a control, as fliA expression is independent of σ⁵⁴ or the FleSR TCS (19). Deletion of σ²⁸ did not affect expression of either reporter. We discovered that FleSR- and σ⁵⁴-dependent expression of flgBp-lacZ in P. aeruginosa required the fT3SS, FliF MS ring, and the FliG C ring proteins, but not FliM or FliN, for full expression, similar to our analysis of the V. cholerae FlrBC and C. jejuni FlgSR TCSs (Fig. 3A and 4 and Fig. S2) (31 –33). Expression of fliAp-lacZ was unaffected in these mutants. Thus, our data continue to support that many polar flagellates with the FlhF/FlhG flagellar biogenesis regulatory system contain flagellum-associated TCSs that require flagellar-dependent cues to stimulate expression of σ⁵⁴-dependent flagellar rod and hook genes as a discrete step for the polar flagellar transcriptional program.

FIG 4 — Analysis of TCS- and σ⁵⁴-dependent flagellar gene expression in flagellar mutants of *P. aeruginosa*. Expression of the flagellar rod and hook operon in WT *P. aeruginosa* PA14 and isogenic flagellar mutants is shown. *flgBp*-*lacZ* and fliAp-*lacZ* transcriptional reporters were integrated at the *att* site on the chromosome of *P. aeruginosa* strains. The level of expression of transcriptional reporters in each mutant is relative to the level of expression in WT *P. aeruginosa*, which was set to 100 U. Results are from a representative assay, with each sample analyzed in triplicate. Error bars indicate standard deviations of the average level of expression from three samples. An asterisk indicates the mutant had a significantly increased or decreased reporter expression relative to that of the WT strain (P < 0.05). MR, master regulator; TCS, two-component signal transduction system.

Requirements of FliF and FliG for flagellum-associated TCS activity.

Having established that deletion of individual MS ring, rotor, and fT3SS proteins impeded FlrBC/FleSR TCS activity, we assessed whether these proteins alone or as a part of the MS ring-rotor-fT3SS complex were required for the activity of V. cholerae and P. aeruginosa TCSs to stimulate flagellar rod and hook gene transcription. Because FlrB and FleS are predicted to be cytoplasmic kinases, we hypothesized that they detect signals within the cytoplasm, similar to C. jejuni FlgS sensing formation of the MS ring and rotor by FliF and FliG via a direct interaction with the cytoplasmic domains of these structures (32). FliF is predicted to contain two transmembrane domains with a large central periplasmic domain and smaller N- and C-terminal cytoplasmic domains. A conserved periplasmic ASVXV motif in FliF is required for flagellation in Salmonella (61). This motif has been hypothesized to promote recruitment of FliF to the fT3SS core via interactions with FlhA and/or FliF multimerization into the MS ring around the fT3SS core. Alteration of the C. jejuni FliF ASVXV motif eliminated FlgS interactions with FliF and FliG and abolished FlgSR TCS signal transduction for rod and hook gene expression (32), supporting the hypothesis that FliF multimerization into the MS ring (and simultaneous rotor formation by FliG) around the complete fT3SS core is required to form a signal directly detected by FlgS.

The V. cholerae FliF MS ring protein contains a motif in its periplasmic region (ASASVXL from residues 200 to 206) similar to that of Salmonella and C. jejuni FliF. We expressed WT FliF, FliF_ΔAS200-201, and FliF_ΔAS202-203 from plasmids in V. cholerae ΔfliF and monitored expression of flaAp-lacZ to assess FlrBC TCS activity. WT FliF restored flaAp-lacZ expression to the ΔfliF mutant, but FliF_ΔAS200-201 and FliF_ΔAS202-203 did not (Fig. 5A). Consistent with these observations, WT FliF restored flagellation and motility, but the mutant FliF proteins did not (data not shown). Immunoblot analysis verified that the WT FliF and FliF mutant proteins were produced at similar levels, as were the FliG rotor proteins in these strains (Fig. 5B). Of note, we did observe that FliG is dependent upon FliF for stability, as FliG levels were reduced in the V. cholerae ΔfliF mutant with or without empty vector (Fig. 5B). If the ASVXV motif in V. cholerae FliF is required for recruitment to the fT3SS core and/or FliF multimerization into the MS ring around the fT3SS core, as in other flagellates, these data, along with our analysis of mutants lacking FliF, FliG, and fT3SS core proteins, support a model that the formation of the V. cholerae MS ring-rotor-T3SS complex, rather than the production of unassembled fT3SS, MS ring, and rotor proteins, is a cue and regulatory checkpoint influencing FlrBC activity. We were unable to perform a similar analysis in P. aeruginosa, as we could not construct mutations in the FliF ASVXV motif.

We next explored whether differences in the requirements of FliG rotor domains for activity of the V. cholerae, P. aeruginosa, and C. jejuni flagellum-associated TCSs existed. Typical FliG proteins possess three major domains: an N-terminal domain (NTD) that interacts with the cytoplasmic C-terminal domain (CTD) of FliF and is required for multimerization of the MS ring and rotor; a middle domain (MD) to interact with FliM for assembly of the switch complex and the lower portion of the C ring; and a CTD to interact with stator proteins that generate torque for flagellar rotation (62 –64). In C. jejuni, only the FliG NTD was required with FliF to form a signal detected by the FlgSR TCS for flagellar rod and hook gene expression (32). We generated V. cholerae and P. aeruginosa fliG mutants with deletions of the NTD, MD, or CTD and then assessed TCS- and σ⁵⁴-dependent flagellar gene expression.

FliG mutants lacking the NTD or CTD were unable to restore FlrBC- and σ⁵⁴-dependent flagellar gene expression in V. cholerae (Fig. 5C). However, these proteins were produced at reduced levels compared to those of WT FliG (Fig. 5D), tempering interpretations that these domains are essential for FlrBC TCS activity. In contrast, the expression of FliG_ΔMD restored gene expression to ∼70% of the WT level (Fig. 5C), suggesting that at least the middle domain of FliG is not required for FlrBC TCS-dependent flagellar gene expression.

For analysis of P. aeruginosa FliG, WT fliG was replaced on the chromosome with fliG mutants encoding in-frame deletions of the NTD, MD, or CTD. FliG levels were reduced only modestly with removal of the NTD, but levels of FliG_ΔMD and FliG_ΔCTD were comparable to those of WT FliG (Fig. 5F). Expression of the FleSR- and σ⁵⁴-dependent flgB-lacZ reporter was reduced to the same level in the fliG_ΔNTD and fliG_ΔCTD mutants as in the ΔfliG mutant (Fig. 5E). However, a 2.5-fold increase in P. aeruginosa FleSR activity and flagellar gene expression was observed with FliG_ΔMD. These observations suggest that at least the P. aeruginosa FliG CTD is required for FleSR TCS activity, whereas the requirement for the NTD is less clear. Considering that only the C. jejuni FliG NTD was required to activate flagellar gene expression (32), our findings indicated different FliG domains, along with FliF, are required in P. aeruginosa (and possibly V. cholerae) for flagellum-associated TCS activity. While we did not analyze MS ring-rotor-fT3SS complex formation directly in V. cholerae and P. aeruginosa, these differences in FliF and FliG domains required for activity of the flagellar TCSs suggest requirements for the assembly of functional fT3SSs vary in these organisms. Additionally, the different sensor domains within FleS and FlrB relative to C. jejuni FlgS may be needed to detect the distinctive signal composed by the different FliF and FliG domains from the respective bacteria.

A functional link between the polar flagellar transcriptional program and the FlhF/FlhG flagellar biogenesis regulatory system.

Our analysis presented above suggested a connection with many polar flagellates possessing (i) a FlhF/FlhG regulatory system, for spatial and numerical control of polar flagellar biogenesis; (ii) a flagellum-associated TCS whose activity is dependent on MS ring, rotor, and fT3SS proteins; and (iii) a polar flagellar transcriptional program that requires MS ring-rotor-fT3SS protein production and possibly assembly for subsequent flagellar rod and hook gene expression. The FlhF/FlhG flagellar biogenesis regulatory systems and flagellum-associated TCSs are absent from peritrichous bacteria (with Bacillus subtilis as an exception for FlhF and FlhG). In these peritrichous bacteria, a master transcriptional regulator promotes the peritrichous flagellar transcriptional program by expressing MS ring, C ring, rod, and hook genes simultaneously to result in efficient creation of multiple flagella across the surface (Fig. S3) (10, 12, 14, 48). The cooccurrence of the FlhF/FlhG system, flagellum-associated TCSs, and a transcriptional program that separates MS ring-C ring-fT3SS complex gene expression from that of rod and hook genes raises interesting questions. (i) Do polar flagellates require the specific polar flagellar transcriptional program to build flagella in general or to specifically construct polar flagella? (ii) Is ordering rod and hook gene transcription after MS ring-C ring-fT3SS assembly required for an FlhF/FlhG-dependent activity for flagellation? (iii) Can polar flagellates produce flagella (polar or otherwise) if reprogrammed to transcribe flagellar genes similarly to a peritrichous organism in the presence or absence of the FlhF/FlhG flagellar biogenesis regulatory system?

FIG S3

Construction of V. cholerae mutants for altering a flagellar transcriptional program. The normal peritrichous flagellar transcriptional program for Salmonella (left) and the normal polar flagellar transcriptional program for WT V. cholerae (middle) are shown. Note the WT V. cholerae polar flagellar transcriptional program (middle) includes both FlrA-dependent transcription of initial flagellar genes, the regulatory checkpoint associated with MS ring, rotor, and fT3SS core proteins discovered in this work, and FlrBC- and σ⁵⁴-dependent transcription of flagellar rod and hook genes. Full V. cholerae operons for FlrA- or FlrBC-/σ⁵⁴-dependent operons are shown. For creation of transcriptional reprogramming mutants (right), the native σ⁵⁴- and FlrBC TCS-dependent promoters for the flgB, flgF, and flgK operons encoding flagellar rod and hook genes were replaced with the FlrA-dependent fliE promoter (fliEp). Promoter mutations were made individually or in different combinations to create a full array of V. cholerae mutants with up to all three flagellar rod and hook operons dependent on FlrA for transcription (right), rather than on FlrBC TCS and σ⁵⁴ (middle), to resemble a peritrichous flagellar transcriptional program (left). Download FIG S3, TIF file, 1.1 MB^{(1.1MB, tif)}.

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For these analyses, we developed a transcriptional reprogramming strategy in V. cholerae so that expression of one, two, or all three FlrBC- and σ⁵⁴-dependent flagellar rod and hook operons (flgBCDE, flgFGHIJ, and flgKL) were under the control of the FlrA master transcriptional regulator that normally only controls expression of MS ring, C ring, fT3SS, and FlrBC TCS genes (Fig. S3). By replacing the FlrBC- and σ⁵⁴-dependent flgB, flgF, and flgK promoters with the FlrA-dependent fliE promoter at the native locations on the V. cholerae chromosome (Fig. S3) (18), the requirement to detect the regulatory checkpoint centered around MS ring-rotor-fT3SS protein production for rod and hook gene expression would be bypassed. Thus, V. cholerae would produce some or all rod and hook proteins earlier than normal and at the same time as the MS ring, C ring, and fT3SS proteins, which shifts the normal V. cholerae polar flagellar transcriptional program to one more closely following the peritrichous transcriptional program that normally exists in E. coli and Salmonella species (Fig. S3) (10, 15).

In these experiments, we retained flaA expression under the control of its natural FlrBC- and σ⁵⁴-dependent promoter. By doing so, the FlaA major flagellin in these V. cholerae transcriptional reprogramming mutants was produced either simultaneously with some rod and hook proteins as normal (if the mutant contained only one or two promoter alterations) or after all rod and hook proteins (if the mutant contained all three promoter alterations) (Fig. S3). Transcription of flaA after rod and hook protein production in these V. cholerae mutants would be temporally similar to how most polar and peritrichous flagellates naturally express major flagellins from a σ²⁸-dependent promoter after formation of the flagellar rod and hook (Fig. 1 and Fig. S3). As shown below, maintaining flaA expression under its natural FlrBC- and σ⁵⁴-dependent promoter allowed for sufficient flagellin production for filament assembly and motility in many transcriptionally reprogrammed mutants.

We also deleted flhF and flhG from these V. cholerae transcriptional reprogramming mutants to examine any potential link between the activity of the FlhF/FlhG regulatory system for monotrichous flagellation in V. cholerae and alteration in the timing of rod and hook gene transcription relative to MS ring, C ring, and fT3SS expression. As reported previously, FlhF is required for flagellar biogenesis in V. cholerae, and we confirmed that V. cholerae ΔflhF lacked flagella (7 and data not shown); thus, we could not analyze flagellation in our transcriptional reprogramming mutants in the ΔflhF background. However, deletion of flhG from the classical V. cholerae O395 strain allowed for the production of polar flagella but with hyperflagellation due to the lack of proper numerical control of flagellar biogenesis (7). Hyperflagellation was proposed to be due to increased flrA expression to cause overexpression of all flagellar genes, resulting in multiple flagella. However, this hyperflagellated phenotype was unstable in V. cholerae O395 ΔflhG; after subsequent in vitro passaging, flagellar gene expression was reduced and the monotrichous phenotype returned (7).

We verified the hyperflagellation phenotype of a ΔflhG mutant in V. cholerae C6706 (the strain used throughout this study). In our analysis, 54% of the WT population produced exclusively monotrichous flagella, with 46% lacking a flagellum (Table 1 and Fig. 6A). Only a small minority of the WT flagellated population was hyperflagellated (1.4%). V. cholerae ΔflhG cells produced flagella in a higher percentage of the population (67.5% versus 54.2% for the WT). Furthermore, 61% of flagellated ΔflhG cells were hyperflagellated by producing 2 to 7 polar flagella at a single pole (Table 1 and Fig. 6A). The flagella of V. cholerae ΔflhG cells occasionally appeared thinner in structure with possible defects in flagellar sheath formation compared to the monotrichous flagellum of WT V. cholerae. Despite hyperflagellation, the ΔflhG mutant was motile, although modestly less so than the WT (Fig. 6B). Contrary to a previous report, hyperflagellation was stable in the V. cholerae C6706 ΔflhG strain (7). Expression of lacZ transcriptional reporters linked to promoters from different classes of flagellar genes were generally altered in the ΔflhG strain but with either increased or decreased expression depending on the promoter examined (Fig. 7A). Thus, we could not link hyperflagellation to gross overexpression of flagellar genes in the ΔflhG strain, as previously hypothesized (7), suggesting that FlhG regulates flagellar number by another means, such as controlling in vivo FlhF activity by modulating its GTP-binding state, as postulated for Vibrio alginolyticus and C. jejuni (3 –5). In summary, the V. cholerae ΔflhG mutant with a WT polar flagellar transcriptional program efficiently produced polar flagella (possibly due to dysregulated FlhF activity), as observed by an increase in polarly flagellated cells and in the number of polar flagella per cell (hyperflagellation).

TABLE 1.

Measurement of flagellation in WT V. cholerae, V. cholerae ΔflhG mutant, and transcriptional reprogramming mutants

Strain	% Of population^a		% Hyperflagellated	Strain	% Of population^a		% Hyperflagellated^b
Strain	Flagellated	Aflagellated	% Hyperflagellated	Strain	Flagellated	Aflagellated	% Hyperflagellated^b
WT	54.2 ± 2.5	45.9 ± 2.5	1.4 ± 2.5	ΔflhG	67.5 ± 11.1	32.5 ± 11.1	61.4 ± 11.6
fliEp-flgB operon	56.6 ± 4.3	43.4 ± 4.3	0 ± 0	ΔflhG fliEp-flgB operon	8.3 ± 4.1^c	91.7 ± 4.1^c	0 ± 0^c
fliEp-flgF operon	48.2 ± 4.0	51.8 ± 4.0	0.8 ± 1.3	ΔflhG fliEp-flgF operon	0 ± 0^c	100 ± 0^c	0 ± 0^c
fliEp-flgK operon	53.4 ± 5.6	46.6 ± 5.6	0.9 ± 0.9	ΔflhG fliEp-flgK operon	0 ± 0^c	100 ± 0^c	0 ± 0^c
fliEp-flgB operon	48.1 ± 6.3	51.9 ± 6.3	0.6 ± 0.5	ΔflhG fliEp-flgB operon	13.9 ± 2.1^c	86.1 ± 2.1^c	9.5 ± 4.8^c
fliEp-flgF operon				fliEp-flgF operon
fliEp-flgB operon	55.6 ± 2.0	44.4 ± 2.0	2.0 ± 0.6	ΔflhG fliEp-flgB operon	0 ± 0^c	100 ± 0^c	0 ± 0^c
fliEp-flgK operon				fliEp-flgK operon
fliEp-flgF operon	50.9 ± 6.4	49.1 ± 6.4	4.4 ± 6.3	ΔflhG fliEp-flgF operon	0.4 ± 0.6^c	99.6 ± 0.6^c	0 ± 0^c
fliEp-flgK operon				fliEp-flgK operon
fliEp-flgB operon	53.0 ± 3.5	47.0 ± 3.5	2.1 ± 1.9	ΔflhG fliEp-flgB operon	0 ± 0^c	100 ± 0^c	0 ± 0^c
fliEp-flgF operon				fliEp-flgF operon
fliEp-flgK operon				fliEp-flgK operon

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For each strain, three independent samples with at least 100 bacterial cells per sample were analyzed. Values are presented as averages ± standard deviations.

The values for the hyperflagellated population represent the percentages of the flagellated population that had at least two or more flagella at one pole of the cell.

The level of flagellation, aflagellation, or hyperflagellation of individual V. cholerae ΔflhG transcriptional reprogramming mutants was statistically significantly different from the V. cholerae ΔflhG mutant with normal flagellar gene transcription (P < 0.05). No significant differences were noted among WT V. cholerae and the respective transcriptional reprogramming mutants.

FIG 6 — Effect of transcriptional reprogramming of flagellar genes on *V. cholerae* flagellation. Shown are electron micrographs (A) and motility phenotypes (B) of WT *V. cholerae*, the *V. cholerae* Δ*flhG* mutant, or isogenic transcriptional reprogramming mutants in which one or more operons with an FlrBC TCS- and σ⁵⁴-dependent promoter was replaced with the *fliE* promoter (*fliEp*), as indicated, to transition flagellar gene transcription toward the peritrichous flagellar program. All combinations of transcriptional reprogramming mutants were made in *V. cholerae* Δ*flhG* and WT *V. cholerae* strains (left and right columns, respectively). In panel A, the bar represents 1 μm. In panel B, motility was assessed after inoculating overnight cultures in LB with 0.3% agar and incubation at 37°C for 8 h. The box on the left in panel B is a map depicting how the Δ*flhG* or WT strain and their corresponding transcriptionally reprogrammed mutants were inoculated into motility agar in the center and right panels.

FIG 7 — Activity of flagellar promoters in WT *V. cholerae* and Δ*flhG* mutant strains. (A) Expression of *lacZ* transcriptional fusions from different flagellar promoters in WT *V. cholerae* C6706 and an isogenic Δ*flhG* mutant. *flrAp-, flrBp-, fliEp-, flgBp-, flgF-, flgKp-, flaAp-,* and *cheVp*-*lacZ* transcriptional reporters were maintained on plasmids in *V. cholerae* strains. The level of expression of each transcriptional reporter in each mutant is relative to the level of expression in WT *V. cholerae*, which was set to 100 U. Results from a representative assay with each sample analyzed in triplicate are shown. Error bars indicate standard deviations of the average level of expression from three samples. An asterisk indicates significant difference in expression from the WT containing vector alone (P < 0.05). (B) *fliE* promoter activity in WT *V. cholerae* and the Δ*flhG* mutant with either a normal polar flagellar transcriptional program or transcriptionally reprogrammed toward a peritrichous pattern. An *fliEp*-*lacZ* transcriptional reporter was introduced on a plasmid in WT *V. cholerae* or the Δ*flhG* mutant with a normal polar flagellar transcriptional program (indicated by a dash) and select transcriptional reprogramming mutants. In the transcriptional reprogramming mutants analyzed, the promoter for one or more rod and hook operons that was replaced with the *fliE* promoter is indicated. The level of expression of the *fliEp*-*lacZ* transcriptional reporter in each mutant is relative to the level of expression in WT *V. cholerae*, which was set to 100 U. Results from a representative assay, with each sample analyzed in triplicate, are shown. Error bars indicate standard deviations of the average level of expression from three samples. An asterisk indicates significant difference in expression relative to that of WT *V. cholerae* (P < 0.05).

Upon alteration of the polar flagellar transcriptional program in the V. cholerae ΔflhG mutant to more closely resemble a peritrichous transcriptional program by replacing the FlrBC- and σ⁵⁴-dependent promoter for a single rod and hook operon with the FlrA-dependent fliE promoter (Fig. S3), we observed almost complete elimination of polar flagellar biogenesis and the prominent ΔflhG hyperflagellation phenotype (Table 1 and Fig. 6A). Further shifts toward the peritrichous transcriptional program in which two or all three rod and hook promoters were replaced with fliEp were also mostly or completely aflagellated. The only ΔflhG transcriptional reprogramming mutants that were flagellated were those with fliEp expressing the flgB operon alone or both the flgB and flgF operons, but a significantly smaller population of cells were flagellated than those of the ΔflhG mutant with the normal polar flagellar transcriptional program (8.3% to 13.9%) (Table 1 and Fig. 6A). These mutants also were severely reduced for motility relative to V. cholerae WT and ΔflhG strains (Fig. 6B). The flagella of the ΔflhG fliEp-flgBCDE mutant that were produced tended to be shorter than those of WT V. cholerae and the ΔflhG mutant (Fig. 6A).

In select ΔflhG transcriptional reprogramming mutants, we did not observe gross decreases in FlrA activity, which was driving rod and hook gene expression in these mutants, as monitored by fliEp-lacZ expression relative to that of WT V. cholerae (Fig. 7B). Instead, FlrA activity was comparable to or modestly greater in ΔflhG than in WT strains. Thus, reduced or absent flagellation in the ΔflhG transcriptional reprogramming mutants was not due to impaired FlrA activity and expression of rod and hook genes from the fliE promoter. Our data indicate that polar flagellar biogenesis efficiently occurs in V. cholerae with altered activity of the FlhF/FlhG flagellar biogenesis system (as in a ΔflhG mutant), albeit with hyperflagellation, as long as the WT polar flagellar transcriptional program is maintained by the FlrBC TCS to order rod and hook gene transcription after MS ring-C ring-fT3SS production. Any alteration toward a peritrichous flagellar transcriptional program without an intact FlhF/FlhG polar flagellar regulatory system leads to severe reduction or loss of flagellation.

We next addressed whether flagellar biogenesis was affected in a V. cholerae hybrid that contained an intact FlhF/FlhG flagellar biogenesis regulatory system but with alterations to follow more closely a peritrichous flagellar transcriptional program. Contrary to the V. cholerae ΔflhG transcriptional reprogramming mutants, the level of monotrichous flagellation in any population of transcriptional reprogramming mutants with a WT FlhF/FlhG regulatory system did not change relative to that of WT V. cholerae (Table 1 and Fig. 6A), even in the V. cholerae mutant with all three fliE promoter substitutions (fliEp-flgB, fliEp-flgF, and fliEp-flgK), to most closely resemble a peritrichous flagellar transcriptional program. We also did not detect differences in flagellar filament length, appearance, or function in motility in vitro (Fig. 6A and B). Thus, V. cholerae with a peritrichous flagellar transcriptional program produces polar flagella normally during in vitro growth, as long as the FlhF/FlhG flagellar biogenesis regulatory system is fully operational and intact. Disruption of the FlhF/FlhG system in such a transcriptionally reprogrammed V. cholerae cell abolishes or greatly reduces flagellation and motility. Thus, we propose that flagellum-associated TCSs of polar flagellates mediate the polar flagellar transcriptional program, characterized by the ordered transcription of flagellar rod and hook genes after the MS ring-rotor-fT3SS regulatory checkpoint during flagellar assembly, to allow polar flagellates flexibility in producing flagella and retaining motility (even with hyperflagellation) when the FlhF/FlhG system may not function properly.

DISCUSSION

Polarly flagellated bacteria are present in a wide range of proteobacterial classes. To achieve species-specific flagellation patterns for optimal motility, each polar flagellate must have transcriptional mechanisms to correctly control flagellar gene expression and biogenesis regulators to create specific flagellation patterns composed of the correct number of flagella at one or both poles. However, little is known regarding how broadly conserved these transcriptional and biosynthetic regulatory mechanisms are and how they may be intertwined for correct biogenesis of polar flagella and polar flagellation patterns.

To explore what may contribute to the conserved polar flagellar transcriptional program for creating a specific ordering of transcription of different classes of flagellar genes, we showed by in silico analysis that Gram-negative polar flagellates can be divided into two distinct groups. One group produces an FlhF/FlhG flagellar biogenesis regulatory system with a flagellum-associated TCS, and another is composed of alphaproteobacteria that lack an FlhF/FlhG system. We found that FlhF/FlhG- and flagellum-associated TCS-positive polar flagellates may be subdivided into two or more groups based on the sensory domains of the TCS kinases. The different kinases are best represented by C. jejuni FlgS, which contains a predicted coiled-coil domain in the sensory region, and V. cholerae FlrB/P. aeruginosa FleS, which contain PAS domains.

Despite their identification years ago, the actual signals detected by the V. cholerae FlrB and P. aeruginosa FleS sensor kinases had not been analyzed. We previously discovered that the amphitrichous polar flagellate C. jejuni detects a regulatory checkpoint formed by the MS ring and rotor assembling around the fT3SS core by FlgS of its flagellum-associated FlgSR TCS (32). In our current work, we found that MS ring, rotor, and fT3SS proteins are broadly required for the activity of the V. cholerae FlrB and P. aeruginosa FleS sensor kinases to result in rod and hook gene transcription. Additionally, we found that V. cholerae FliF mutants that likely fail to form an MS ring were defective in FlrBC TCS activity. These findings are similar to our previous studies in C. jejuni and what others have reported in H. pylori (32, 40, 41, 43). Furthermore, we accumulated evidence that for at least P. aeruginosa (and possibly V. cholerae), more domains of the FliG rotor were required for flagellum-associated TCS activity than in C. jejuni. These combined data build support for a general conserved mechanism in which flagellum-associated TCSs of polar flagellates broadly detect a regulatory checkpoint centered around MS ring-rotor-fT3SS assembly as a signal to facilitate the polar flagellar transcriptional program that orders rod and hook gene expression as a subsequent step for the progression of flagellar biogenesis. We suspect that the FliF MS ring, FliG rotor, and fT3SS core proteins (FlhA, FlhB, FliP, FliQ, and FliR) would be required for the activity of flagellum-associated TCSs for rod and hook gene expression in many other polar flagellates that have yet to be explored.

A caveat to our work presented here is that we have not yet been able to detect a direct interaction between the FlrB or FleS kinase with the FliF MS ring or FliG rotor proteins, like we observed with C. jejuni FlgS, as a mechanism to monitor formation of the MS ring and rotor around the fT3SS core (32). This indicates that the interactions between the kinases and flagellar proteins are weaker or more transient than in C. jejuni or that FlrB and FleS monitor fT3SS assembly indirectly through an unidentified factor. Differences in FliG domains potentially required for signal formation and detection, the abilities of the kinases to interact with MS ring and rotor components, and potential sensory domains within the kinases may reflect varied mechanisms for how these flagellum-associated TCSs monitor the formation of the regulatory checkpoint. Regardless, our findings continue to support that bacteria have mechanisms to monitor the formation of intracellular macromolecular structures and to relay information that influences behavior. Verification and deeper investigation of how each flagellum-associated TCS might detect MS ring-rotor-fT3SS assembly will be insightful for how these regulatory systems function.

When also considering the alphaproteobacterial polar flagellates that lack the FlhF/FlhG flagellar biogenesis regulatory system, monitoring formation of a competent fT3SS by different systems to influence subsequent rod and hook gene expression emerges as a common strategy across Gram-negative polar flagellates for the development of the conserved polar flagellar transcriptional program. Of the alphaproteobacterial polar flagellates in the reference collection, the flagellar system of C. crescentus is the best characterized (reviewed in references 65). C. crescentus executes the polar flagellar transcriptional program so that transcription of MS ring, C ring, and fT3SS genes occurs prior to σ⁵⁴-dependent transcription of rod and hook genes (66). C. crescentus σ⁵⁴ requires FlbD, an enhancer-binding protein similar to the C. jejuni FlgR, V. cholerae FlrC, and P. aeruginosa FleR response regulators of the flagellum-associated TCSs, to activate flagellar rod and hook gene expression (67 –71). MS ring, C ring, and fT3SS proteins are also required for FlbD activity and FlbD- and σ⁵⁴-dependent rod and hook gene expression (69, 71). Thus, FlbD activity is linked to MS ring-C ring-fT3SS complex formation, yet FlbD lacks a cognate sensor kinase like FlgS, FlrB, or FleS to monitor flagellar assembly and control its activity. The FliX transactivating factor has been identified as the link that relays the status of fT3SS assembly to positively or negatively control the activity of FlbD to bind to target flagellar rod and hook promoters (72 –75). Although its regulatory mechanism is not understood, FliX does not function as a kinase to transduce signals regarding fT3SS assembly.

As diverse polar flagellates have evolved different mechanisms to create and maintain the polar flagellar transcriptional program so that rod and hook gene expression occurs after formation of the regulatory checkpoint at fT3SS assembly, we hypothesized that this program is beneficial for biogenesis of polar flagella. E. coli and Salmonella, as models with the peritrichous flagellar transcriptional program, do not recognize this checkpoint and transcribe most basal, rod, and hook genes simultaneously to efficiently build peritrichous flagella (10, 14, 48). Since, in this study, we show a V. cholerae cell containing the WT FlhF/FlhG regulatory system engineered with a peritrichous flagellar transcriptional program produced a monotrichous flagellum efficiently, the type of flagellar transcriptional program itself does not seem to determine the peritrichous or polar flagellation pattern of the species.

Instead, we discovered that possessing a polar transcriptional program and the FlhF/FlhG flagellar biogenesis regulatory system allows polar flagellation while retaining motility to a modest extent when perturbations to FlhF and FlhG activity occur. For example, the V. cholerae ΔflhG mutant with a polar flagellar transcriptional program was hyperflagellated, indicating high proficiency in producing flagella. However, the ΔflhG mutant was less motile than the WT monotrichous strain, likely due to the inability of the ΔflhG mutant to coordinate multiple rotating polar flagella for optimal swimming motility. In contrast, flagellation was severely diminished or even abolished in V. cholerae mutants that more closely resembled most peritrichous bacteria by lacking a properly functioning FlhF/FlhG flagellar biogenesis system (through deletion of flhG) and engineered with a peritrichous flagellar transcriptional program. Thus, the peritrichous transcriptional program is much more affected by alterations in FlhF/FlhG activity in a polar flagellate, resulting in greatly decreased flagellation, motility, and, likely, fitness in nature. Currently, it is unknown whether FlhF/FlhG activity is naturally regulated or altered by extrinsic factors or metabolic capacity. We have observed hyperflagellation in WT polarly flagellated systems in a small minority of cells (in V. cholerae in this work and previously in C. jejuni [3, 76]). Thus, the FlhF/FlhG flagellar biogenesis regulatory system is likely affected by stochastic influences on a cell-to-cell basis that may at least alter FlhG activity and likely its ability to regulate FlhF. It remains to be determined whether the polar flagellar transcriptional program also provides an advantage to C. crescentus that has a different collection of determinants to produce polar flagella during developmental stages and asymmetrical division (77 –80).

Our analysis of V. cholerae flagellar transcriptional reprogramming mutants provided some intriguing observations, but many questions remain. One such question is what advantage exactly does the polar flagellar transcriptional program provide for the FlhF/FlhG regulatory system to enable efficient flagellation when FlhF or FlhG activity is altered that the peritrichous flagellar transcriptional program does not provide. It is currently unclear how FlhG controls flagellum numbers in V. cholerae. FlhG orthologs regulate flagellum number by at least two different processes, including influencing the activity of FlhF or a master regulator of flagellar gene transcription, such as FlrA (1 –5, 7 –9, 81). In contrast to a previous report, we did not observe a broad increase in transcription across classes of flagellar genes in the V. cholerae ΔflhG mutant that would explain the consistent hyperflagellation phenotype we observed (7). Thus, hyperflagellation in the V. cholerae ΔflhG mutant may be due to a dysregulated, hyperactive FlhF, which has been proposed in C. jejuni and other Vibrio species (3 –5).

The molecular mechanism by which the FlhF GTPase influences polar flagellation has not been determined in many bacteria. One hypothesis includes that FlhF localizes MS ring, C ring, and fT3SS core proteins at a pole or facilitates interactions between these proteins to create a new flagellum (1, 2, 82). FlhG presumably functions in some polar flagellates to transition FlhF from a GTP-bound “on” state competent for a function to initiate flagellation to a GDP-bound “off” state. Tight control of FlhF by FlhG may be required so that FlhF can organize flagellar proteins properly for MS ring-C ring-fT3SS assembly either with the stepwise production of MS ring, C ring, fT3SS, rod, and hook proteins provided by the polar flagellar transcriptional program or with their simultaneous production facilitated by the peritrichous flagellar transcriptional program. However, a dysregulated FlhF in the ΔflhG mutant may be unable to perform its natural function in flagellation when MS ring, C ring, fT3SS, rod, and hook proteins are produced at the same time. Consistent with this, we observed a great reduction or abolishment of flagellation when some or all rod and hook proteins were simultaneously produced with MS ring, C ring, and fT3SS proteins in the ΔflhG mutant engineered to follow more closely a peritrichous transcriptional flagellar program. Our findings may point toward a more expansive role for FlhF: in addition to its hypothesized role in assisting polar assembly of the MS ring-C ring-fT3SS complex, FlhF may also organize flagellar proteins, such as the rod and hook proteins, for secretion via the fT3SS. If so, production of multiple FlhF-interacting proteins (fT3SS complex proteins and their secretion substrates) simultaneously may overwhelm a dysregulated FlhF so that flagellar biogenesis does not occur. Other possibilities exist, including that the peritrichous flagellar transcriptional program in a ΔflhG mutant disrupts flagellar protein stoichiometry. In this case, there may not be enough rod and hook proteins produced for the multiple fT3SSs that may form in the ΔflhG mutant. Undoubtedly, there are functions for FlhF and FlhG that are not yet adequately understood to reveal how the polar flagellar transcriptional program contributes to the FlhF/FlhG flagellar biogenesis system for efficient polar biogenesis. Continued exploration will likely further reveal how transcriptional and biosynthetic processes are integrated in polar flagellates to construct the ideal number and positioning of these macromolecular machines for motility in bacterial cells.

Our findings raise some questions regarding how different flagellar transcriptional programs formed across flagellated species. One prominent question is whether polar and peritrichous flagellar transcriptional programs developed independently of each other or if one evolved from the progenitor of another. It is clear that flagellar structural components are largely conserved across bacterial species. Even the mechanism to detect rod and hook formation as a late regulatory checkpoint required for activation of σ²⁸ and expression of terminal flagellar genes is widely conserved (11, 13, 30). However, regulatory factors and mechanisms required for expression of flagellar components required for formation of the fT3SS, rod, and hook differ in peritrichous and polar flagellates. Most peritrichous bacteria (albeit with B. subtilis as a Gram-positive exception) have a seemingly less complex flagellar transcriptional program so that MS ring, C ring, fT3SS core, rod, and hook proteins are produced simultaneously by the activity of a flagellar master regulator, and these bacteria do not require FlhF or FlhG to efficiently construct multiple flagella across their surfaces.

Polar flagellates may have originated from a peritrichous progenitor but also could have developed independently. Comparisons between C. crescentus and many other Gram-negative polar flagellates as discussed above clearly show that different polar flagellar biogenesis systems exist in the presence of somewhat conserved regulatory mechanisms to facilitate the polar flagellar transcriptional program, indicating convergent evolution of polar flagellates. Regardless, our findings suggest that a species needs to acquire a polar flagellar biogenesis system (such as the FlhF/FlhG system) and a mechanism to order flagellar genes for the polar flagellar transcriptional program (such as a flagellum-associated TCS) to become an efficient polar flagellate. Possessing only the FlhF/FlhG system with the peritrichous program does not guarantee optimal flagellation and motility if FlhF/FlhG activity is affected by extrinsic, stochastic factors.

It is unknown which came first in a polar flagellate, the FlhF/FlhG polar flagellar biogenesis system or the flagellum-associated TCSs, to drive the polar flagellar transcriptional program. Both the FlhF GTPase and FlhG ATPase are members of the SIMIBI class of nucleotide-binding proteins that commonly function in cellular organization and protein targeting (83, 84). FlhF is related to the Ffh GTPase of the signal recognition particle system, whereas FlhG is closely associated with the MinD and ParA ATPases that generally perform partitioning functions related to division and DNA segregation (6, 83 –85). Development of the FlhF/FlhG flagellar biogenesis regulatory system, perhaps from Ffh and MinD/ParA superfamilies, could have caused the emergence of a polar flagellate in a Gram-negative organism. The motile, monotrichous V. cholerae strain we engineered with an intact FlhF/FlhG system and a peritrichous flagellar transcriptional program might resemble this ancestor. As revealed in this work, this bacterium is heavily reliant on a precisely functioning FlhF/FlhG system to form any flagella and retain some level of motility; perturbations to FlhF or FlhG activity severely reduce or completely abolish flagellation. By possessing a mechanism mediated by the flagellum-associated TCSs to order rod and hook gene transcription after production of MS ring, rotor, and fT3SS proteins (and possibly assembly of a functional fT3SS), a bacterium can produce polar flagella with some alterations to FlhF/FlhG activity. In this bacterium, an optimally functioning FlhF/FlhG system allows for the correct number and placement of polar flagella and WT motility; an impaired FlhF/FlhG system (at least by altering FlhG) results in polar flagellation with extra flagella produced and at least modest motility. This hyperflagellated bacterium has an advantage over one with the FlhF/FlhG system and a peritrichous program that cannot maintain flagellation and motility with perturbations to the FlhF/FlhG system.

Modulations in FlhF and FlhG activity in different species with flagellum-associated TCSs to maintain the polar flagellar transcriptional program and flagellar biogenesis may have facilitated the emergence of different polar flagellation patterns, amphitrichous, lophotrichous, and monotrichous. An example of this is C. jejuni and H. pylori, which, while closely related, produce amphitrichous and lophotrichous flagella, respectively, yet have the FlhF/FlhG flagellar biogenesis regulatory system and similar flagellum-associated FlgSR TCSs. A study comparing FlhF and FlhG biochemical activity and biological function between these two bacterial species has not been conducted. Although many details remain to be discovered for how FlhF and FlhG function in polar flagellates, our results indicate regulatory links between the FlhF/FlhG flagellar biogenesis regulatory systems and the order of flagellar protein production controlled by the flagellum-associated TCSs for polar flagellar biogenesis.

MATERIALS AND METHODS

General growth and storage conditions of bacteria.

C. jejuni 81-176 strains were stored at −80°C as frozen stocks in a solution of 85% Mueller-Hinton (MH) broth and 15% glycerol. All C. jejuni strains were grown from frozen stocks on MH agar for 48 h under microaerobic conditions (10% CO₂, 5% O₂, and 85% N₂) at 37°C and then restreaked on MH agar and grown for another 16 h under microaerobic conditions at 37°C. As required, antibiotics were added to MH medium at the following concentrations: 10 μg/ml trimethoprim (TMP), 15 μg/ml chloramphenicol, 50 or 100 μg/ml kanamycin, or 0.5, 1, 2, or 5 mg/ml streptomycin.

V. cholerae C6706 lacZ, a spontaneous lacZ derivative of the WT El Tor C6706 strain, and isogenic mutants were used for all analyses involving V. cholerae strains (86). Pseudomonas aeruginosa PA14 and isogenic mutants were used for all analyses involving P. aeruginosa strains (87, 88). E. coli, V. cholerae, and P. aeruginosa strains were routinely grown in LB broth at 37 or 30°C and stored as frozen stocks at −80°C in a solution of 80% LB and 20% glycerol. As required, antibiotics or growth inhibitors were added to LB broth or agar at the following concentrations: 100 μg/ml ampicillin, 10 μg/ml chloramphenicol, 100 μg/ml kanamycin, 100 μg/ml streptomycin, 15 μg/ml gentamicin, 12.5 μg/ml tetracycline, and 10% sucrose.

Bacterial strains and plasmid construction.

All methodologies to construct plasmids and C. jejuni, V. cholerae, and P. aeruginosa mutants are described in Text S1 of the supplemental material. All bacterial strains and plasmids used in this study are listed in Tables S1 and S2.

TEXT S1

Additional materials and methods. Download Text S1, PDF file, 0.2 MB^{(158.6KB, pdf)}.

This content is distributed under the terms of the Creative Commons Attribution 4.0 International license.

TABLE S1

Bacterial strains used in this study. Download Table S1, PDF file, 0.2 MB^{(178.2KB, pdf)}.

This content is distributed under the terms of the Creative Commons Attribution 4.0 International license.

TABLE S2

Plasmids used in this study. Download Table S2, PDF file, 0.1 MB^{(134KB, pdf)}.

This content is distributed under the terms of the Creative Commons Attribution 4.0 International license.

Bioinformatic analyses.

Complete reference bacterial genomes were acquired from www.ncbi.nlm.nih.gov/assembly to form a database containing 117 genomes. tBLASTn was run against the genome database to identify the top-scoring hit for each genome in the database for the following protein sequences in FASTA format from the UniProt database: E. coli FlgH (P0A6S0), V. cholerae FlhF (C3LP19) or C. jejuni FlhF (A0A0H3P9N0), and V. cholerae FlrB (C3LPE1) or C. jejuni FlgS (A0A0H3PDD6). To perform a reciprocal best hit sequence alignment, we performed another tBLASTn search with each top-scoring hit from each genome against the genome containing each protein query and only considered positive hits as those that were able to identify the protein query as the top-scoring hit in the respective genome. We additionally acquired 51 sensor histidine kinase sequences from the V. cholerae N16961 proteome from UniProt. We performed reciprocal best hit sequence alignments against the reference bacterial genome database with these 51 V. cholerae sensor kinases and calculated the Pearson correlation coefficient between each of the 51 sets of sensor kinase hits and V. cholerae FlhF hits in the reference bacterial genomes. For completion of all bioinformatics analysis, the following software was used: tBLASTn (www.ncbi.nlm.nih.gov) for reciprocal best hit sequence alignments, Biopython (www.biopython.org) to read XML files and prepare command lines, Python 2.7.6 (www.python.org) to run scripts, and NumPy (www.numpy.org) to calculate Pearson correlation coefficients.

Arylsulfatase assays.

Arylsulfatase assays were used to measure the level of expression of the flaB::astA transcriptional fusion on the chromosome of C. jejuni ΔastA strains as previously described (31, 89, 90). Each strain was analyzed in triplicate, and each assay was performed three times. The level of expression of the transcriptional fusion in each strain was calculated relative to the expression in the wild-type C. jejuni ΔastA strain, which was set to 100 U.

β-Galactosidase assays.

The level of gene expression in V. cholerae and P. aeruginosa strains was compared by monitoring the β-galactosidase activity of strains harboring lacZ transcriptional fusions to specific promoters by standard procedures (91). Strains were grown in LB at 37°C with shaking to an optical density at 600 nm (OD₆₀₀) of approximately 0.8 prior to the start of the assays. Each strain was analyzed in triplicate, and each assay was performed three times. The level of expression of the transcriptional fusion in each strain was calculated relative to the expression in wild-type V. cholerae C6706 lacZ or PA14, which was set to 100 U.

Antiserum production.

All use of animals in experimentation has been approved by the IACUC at the University of Texas Southwestern Medical Center. Recombinant protein for antiserum production was produced by first cloning the coding sequences from codon 2 to the stop codon of V. cholerae flrB, fliG, and rpoA into the SmaI site, the BamHI site, or the BamHI and SalI sites of pGEX4T-2 to create N-terminal fusions of glutathione S-transferase. For recombinant V. cholerae FliF, the region of fliF encoding the predicted periplasmic domain, from codons 45 to 473, was cloned into the BamHI and SalI sites of pQE30 to create an N-terminal fusion of 6× His tag. For recombinant P. aeruginosa FliG, the coding sequence from codon 2 to the stop codon was cloned into the BamHI and SmaI sites of pQE30 to create an N-terminal fusion of a 6× His tag. Resultant plasmids were transformed into BL21(DE3) or XL1-Blue and then induced in LB broth with 1 mM isopropyl-β-d-thiogalactopyranoside (IPTG). Recombinant protein was purified from the soluble fractions by affinity chromatography according to the manufacturer’s instructions. Purified recombinant protein was used to immunize guinea pigs by standard procedures for antiserum generation via a commercial vendor (Cocalico Biologicals).

Immunoblot analysis.

Whole-cell lysates (WCLs) of V. cholerae and P. aeruginosa strains for immunoblot analysis were prepared by first inoculating 5 ml LB with a 1:50 dilution of overnight cultures. Cultures were grown at 37°C with shaking to an OD₆₀₀ of 0.8. One-milliliter aliquots of each culture were recovered by centrifugation in microcentrifuge tubes, washed once with phosphate-buffered saline (PBS), and then resuspended in 50 μl of 1× SDS-loading buffer. Samples were boiled for 5 min prior to separation by SDS-PAGE and transferred to membranes for immunoblotting by standard procedures. For specific detection of proteins in WCLs, 10 μl of WCLs was analyzed to detect FliF, FliG, and RpoA, and 25 μl of WCLs was analyzed to detect FlrB. Proteins were detected with specific guinea pig antisera generated as described above. Primary antisera were applied to immunoblots for 1 to 2 h and used at the following concentrations: V. cholerae FliF UTGP151 (1:1,000), V. cholerae FliG UTGP198 (1:1,000), V. cholerae FlrB UTGP151 (1:2,000), V. cholerae RpoA UTGP197 (1:2,000), and P. aeruginosa FliG UTGP145 (1:1,000). A 1:10,000 dilution of horseradish peroxidase-conjugated goat anti-guinea pig antibody was then applied for detection of proteins.

Electron microscopy analysis.

Overnight cultures of V. cholerae strains were inoculated into 5 ml LB at a 1:50 dilution and grown at 37°C with shaking to an OD₆₀₀ of approximately 0.8. One milliliter of each culture was pelleted for 3 min at 13,200 rpm in a microcentrifuge, resuspended in 2% glutaraldehyde in 0.1 M cacodylate, and then incubated on ice for 1 h. Copper-coated Formvar grids were negatively glow discharged, and bacterial samples then were applied to the grids. The samples were stained with 2% uranyl acetate and visualized with an FEI Technai G2 Spirit Bio TWIN transmission electron microscope. Flagellum numbers were counted from at least 100 individual cells and averaged from three biological replicates to determine the proportion of bacterial populations producing different flagellation phenotypes: hyperflagellated (producing at two or more flagella at least at one pole), wild-type (producing a single flagellum at one pole), or aflagellated (lacking a flagellum). After averaging, the standard deviations for each population were calculated.

Motility analysis.

V. cholerae strains were grown from freezer stocks in 5 ml LB overnight at 37°C with shaking. After growth, each strain was inoculated into LB motility agar (containing 0.3% agar) with an inoculation needle. Agar plates then were incubated for 8 h at 37°C.

Statistical analysis.

Tests for significance in differences in expression of transcriptional reporter assays were conducted using the Student's t test (two-tailed distribution with two-sample, equal variance calculations). For analysis of flagellation of V. cholerae populations, a Student's t test (two-tailed distribution with two-sample, equal variance calculations) was used to evaluate statistical significance of monotrichous flagellation, aflagellation, or hyperflagellation between WT V. cholerae and transcriptional reprogramming mutants in the WT background and between the V. cholerae ΔflhG strains and transcriptional reprogramming mutants in the ΔflhG background. As indicated in the tables, figures, or figure legends, statistically significant differences between relevant strains possessed P values of <0.05.

Data availability.

All data and methodologies are available upon request.

ACKNOWLEDGMENTS

We thank Kimberly Reynolds and Andrew Schober for discussions and assistance with in silico analysis performed in this work. We thank Marvin Whiteley, Jyl Matson, and Jeff Withey for helpful advice on experimentation to complete research activities.

This work was supported by Public Health NIH grants R01AI065539 and R21AI115362.

The funders had no role in study design, data collection and interpretation, or decision to submit the work for publication.

Footnotes

Citation Burnham PM, Kolar WP, Hendrixson DR. 2020. A polar flagellar transcriptional program mediated by diverse two-component signal transduction systems and basal flagellar proteins is broadly conserved in polar flagellates. mBio 11:e03107-19. https://doi.org/10.1128/mBio.03107-19.

Contributor Information

Barbara I. Kazmierczak, Yale University School of Medicine.

M. Stephen Trent, University of Georgia.

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TEXT S1

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TABLE S1

Bacterial strains used in this study. Download Table S1, PDF file, 0.2 MB^{(178.2KB, pdf)}.

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TABLE S2

Plasmids used in this study. Download Table S2, PDF file, 0.1 MB^{(134KB, pdf)}.

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Data Availability Statement

All data and methodologies are available upon request.

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PERMALINK

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

Peter M Burnham

William P Kolar

David R Hendrixson

Roles

ABSTRACT

INTRODUCTION

FIG 1.

RESULTS

A large subset of gram-negative polar flagellates possesses flagellum-associated TCSs.

FIG 2.

The V. cholerae FlrBC TCS requires the fT3SS, MS ring, and rotor for activity.

FIG 3.

The P. aeruginosa FleSR TCS requires the fT3SS, MS ring, and rotor for activity.

FIG 4.

Requirements of FliF and FliG for flagellum-associated TCS activity.

FIG 5.

A functional link between the polar flagellar transcriptional program and the FlhF/FlhG flagellar biogenesis regulatory system.

TABLE 1.

FIG 6.

FIG 7.

DISCUSSION

MATERIALS AND METHODS

General growth and storage conditions of bacteria.

Bacterial strains and plasmid construction.

Bioinformatic analyses.

Arylsulfatase assays.

β-Galactosidase assays.

Antiserum production.

Immunoblot analysis.

Electron microscopy analysis.

Motility analysis.

Statistical analysis.

Data availability.

ACKNOWLEDGMENTS

Footnotes

Contributor Information

REFERENCES

Associated Data

Supplementary Materials

Data Availability Statement

ACTIONS

PERMALINK

RESOURCES

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