Campylobacter jejuni is an intestinal commensal of birds and animals and a leading cause of bacterial diarrheal disease in humans. In hosts, C. jejuni primarily resides in the mucus layer atop the lower intestinal epithelium. Persistence in this niche requires a single flagellar motor at both C. jejuni poles that generates high torque for flagellar rotation to facilitate motility and high swimming velocities. Unlike many bacterial flagellates, C. jejuni swimming velocity increases as external viscosity increases. We identified a complex formed by FlgV, VidA, and VidC (Cjj81176_1732) positioned near the MS-ring-rotor junction in the flagellar motor we annotated as the V-ring. Viscosity-influenced growth, modulation of swimming velocity, and transcription of iron/heme acquisition, respiratory, and energy-generating systems were dependent on the V-ring. C. jejuni Δ flgV and Δ vidC populations lacking a complete V-ring were motile, but could not optimally modulate swimming velocity. Like nonmotile flagellar stator or filament mutants, motile V-ring mutants had in vivo and in vitro growth and viability defects and dysregulated transcription of genes likely impacting physiology. Because the V-ring mutants behaved similarly as nonmotile mutants that experience little to no viscous drag on the filament, we propose C. jejuni V-ring mutants cannot detect viscous drag on their rotating filaments. We propose the V-ring evolved in C. jejuni and potentially other bacteria producing high torque flagellar motors to monitor external viscosity information via viscous drag on the rotating flagellar filament to adjust swimming velocity, transcription, and physiology for optimal fitness in different host lower intestinal niches.

Campylobacter jejuni is currently the most common cause of bacterial gastroenteritis worldwide. However, its genome provides few clues about how it interacts with the host. Moreover, infection screens have often been limited to classical cell culture or animal models. To identify C. jejuni genes involved in host cell interactions, we applied transposon sequencing in a humanized 3D intestinal infection model based on tissue engineering. This revealed key proteins required for host cell adherence and/or internalization, including an Rrf2 family transcriptional regulator as well as three so far uncharacterized genes ( pflC / Cj1643 , pflD / Cj0892c , pflE / Cj0978c ), which we demonstrate to encode factors essential for motility. Deletion mutants of pflC / D / E are non-motile but retain intact, paralysed flagella filaments. We demonstrate that two of these newly identified motility proteins, PflC and PflD, are components of the C. jejuni 's periplasmic disk structures of the high torque motor. The third gene, pflE , encodes a small protein of only 57 aa. Using CryoET imaging we uncovered that the small protein has a striking effect on motor biogenesis, leading to a complete loss of the flagellar disk and motor structures upon its deletion. While PflE does not appear to be a structural component of the motor itself, our data suggests that it is a lipoprotein and supports localization of the main basal disk protein FlgP, which is the first assembly step of the flagellar disk structure. Despite being annotated as a lipoprotein, we find that C. jejuni FlgP instead relies on PflE for its association with the outer membrane. Overall, our genome-wide screen revealed novel C. jejuni host interaction factors including a transcriptional regulator as well as two structural components and a small protein crucial for biogenesis of the C. jejuni high torque flagella motor. Since the flagella machinery is a critical virulence determining factor for C. jejuni , our work demonstrates how such a small protein can, quite literally, bring a bacterial pathogen to a halt.

The bacterial flagellum is a complex nanomachine essential for motility, environmental sensing, and host colonization. While many of its core components have been well characterized, the relevance of proteins such as FliO, which are inconsistently annotated and poorly conserved at the sequence level, has remained ambiguous in their evolutionary and functional status. Here, we present a comprehensive phylogenomic and structural analysis of FliO across >30,000 representative genomes spanning >100 bacterial phyla. Additionally, during this analysis, we found that approximately 40% of bacterial genomes contain flagellar genes-significantly fewer than previously reported. Using a custom pipeline combining low-threshold Hidden Markov models searches, operon context analysis, and structural information, we demonstrate that FliO is present in ~95% of genomes encoding the core flagellar components FliP, FliQ, and FliR. This suggests that FliO is a nearly ubiquitous and ancestral core component of the flagellar type III secretion system (fT3SS). FliO exhibits considerable structural diversity, including lineage-specific acquisitions of LysM and AMIN domains. We identify FliO homologs not only in canonical flagellar systems but also in some virulence-associated T3SS and even some nonflagellated organisms, suggesting functional repurposing and highlighting its functional plasticity. Functional studies in Campylobacter jejuni reveal that FliO and its AMIN domain are critical for efficient amphitrichous flagellation, membrane stability of the export gate component FlhB, and colonization of the host. These findings establish FliO as a core, yet evolutionarily dynamic, component of flagella and provide insights into the evolution and diversification of bacterial secretion systems.

Canonical bacterial two-component signal transduction systems (TCSs) detect cues by sensors with opposing kinase and phosphatase activities to alter the level of phosphorylation of cognate response regulators to mediate responses. We previously identified the Campylobacter jejuni BumSR TCS as the founding member of a bacterial TCS family in which the sensor solely functions as a phosphatase. Sensing specific intestinal metabolites inhibits BumS dephosphorylation of phospho-BumR (P-BumR) to impact BumR as a transcriptional regulator of genes influencing host colonization. Since BumS lacks kinase activity, BumR must depend upon a noncognate phosphodonor in the bacterium to form P-BumR. Through a genetic screen and selection, physiological assays, and biochemical analysis, we identified acetyl phosphate (AcP) and carbamoyl phosphate (CP) as natural in vivo phosphodonors for BumR. In C. jejuni, AcP and CP are products of metabolic pathways fueled by amino acids favored by the bacterium as carbon sources for growth. Producing and utilizing AcP and CP as bona fide BumR phosphodonors allows BumSR to integrate different types of inputs for signal transduction. Microbiota-generated gut metabolites are cues for BumS to control its phosphatase activity for P-BumR and inform about the spatial location of C. jejuni in host intestines. In contrast, AcP and CP are cues for BumR that directly influence P-BumR levels and activity and inform about the richness of favored carbon sources in intestinal niches for optimal energy generation and metabolism. Our study reveals how a bacterial TCS strategically integrates information from multiple cues through both essential components for optimal signal transduction.

Many bacteria produce flagella to swim and navigate through environments. Depending on the species, flagella are beneficial for other activities such as sensing surfaces to initiate biofilm development, secretion of specific proteins, and interactions with hosts to promote colonization or pathogenesis of disease. The extracellular region of the flagellum consists mostly of the filament, which is required for many flagellar-dependent activities. The length of the filament varies across bacterial flagellates and even stochastically within a clonal population, but it is unknown whether or how altering filament length impacts flagellar function for motility and other activities. We recently discovered a mechanism by which FlaG of Campylobacter jejuni and other polarly flagellated bacteria controls flagellar filament length so that these species produce shorter filaments than their peritrichous counterparts. We exploited a set of isogenic C. jejuni mutants producing significantly longer and shorter flagellar filaments than WT to assess the impact of filament length on activities of C. jejuni necessary for commensal colonization of a natural host. We found that production of shorter flagellar filaments slows swimming velocity and hinders intestinal colonization of chickens. Although production of shorter and longer flagellar filaments differently impacted cellular autoagglutination, only a subtle impact on biofilm formation was observed. We also acquired evidence that FlaG itself may impact biofilm development, suggesting that FlaG may have another function besides controlling flagellar filament length. Overall, our data suggest that shortening flagellar filaments has a greater impact on flagellar-dependent activities of C. jejuni than producing overly elongated filaments. IMPORTANCE:Flagella and flagellar motility are important to propel many bacteria through environments, biofilm formation, and infection of respective hosts. Most often, the importance of flagella for a species has been assessed with mutants lacking flagella altogether or only the extracellular filament while retaining the basal body. We addressed whether altering filament length impacts flagellar-dependent activities for host colonization. We exploited isogenic Campylobacter jejuni mutants that synthesize longer and shorter filaments on average relative to WT to find that production of shorter filaments had greater negative impacts on motility, autoagglutination, and commensal colonization compared to bacteria that produce elongated filaments. Our findings suggest that polar flagellates may produce a flagellar filament of minimal length to achieve flagellar functions and preserve fitness.

The bacterial flagellar motor, which spins a helical propeller for propulsion, has undergone evolutionary diversification across bacterial species, often involving the addition of structures associated with increasing torque for motility in viscous environments. Understanding how such structures function and have evolved is hampered by challenges in visualizing motors in situ. Here we developed a Campylobacter jejuni minicell system for in situ cryogenic electron microscopy imaging and single-particle analysis of its motor, one of the most complex flagellar motors known, to subnanometre resolution. Focusing on the large periplasmic structures which are essential for increasing torque, our structural data, interpreted with molecular models, show that the basal disk comprises concentric rings of FlgP. The medial disk is a lattice of PflC with PflD, while the proximal disk is a rim of PflB attached to spokes of PflA. PflAB dimerization is essential for proximal disk assembly, recruiting FliL to scaffold more stator complexes at a wider radius which increases torque. We also acquired insights into universal principles of flagellar torque generation. This in situ approach is broadly applicable to other membrane-residing bacterial molecular machines.

Bacteria power rotation of an extracellular flagellar filament for swimming motility. Thousands of flagellin subunits compose the flagellar filament, which extends several microns from the bacterial surface. It is unclear whether bacteria actively control filament length. Many polarly flagellated bacteria produce shorter flagellar filaments than peritrichous bacteria, and FlaG has been reported to limit flagellar filament length in polar flagellates. However, a mechanism for how FlaG may function is unknown. We observed that deletion of flaG in the polarly flagellated pathogens Vibrio cholerae, Pseudomonas aeruginosa, and Campylobacter jejuni caused extension of flagellar filaments to lengths comparable to peritrichous bacteria. Using C. jejuni as a model to understand how FlaG controls flagellar filament length, we found that FlaG and FliS chaperone-flagellin complexes antagonize each other for interactions with FlhA in the flagellar type III secretion system (fT3SS) export gate. FlaG interacted with an understudied region of FlhA, and this interaction appeared to be enhanced in ΔfliS and FlhA FliS-binding mutants. Our data support that FlaG evolved in polarly flagellated bacteria as an antagonist to interfere with the ability of FliS to interact with and deliver flagellins to FlhA in the fT3SS export gate to control flagellar filament length so that these bacteria produce relatively shorter flagella than peritrichous counterparts. This mechanism is similar to how some gatekeepers in injectisome T3SSs prevent chaperones from delivering effector proteins until completion of the T3SS and host contact occurs. Thus, flagellar and injectisome T3SSs have convergently evolved protein antagonists to negatively impact respective T3SSs to secrete their major terminal substrates.

ABSTRACT Enteric pathogens such as Salmonella enterica serovar Typhimurium experience spatial and temporal changes to the metabolic landscape throughout infection. Host reactive oxygen and nitrogen species non-enzymatically convert monosaccharides to alpha hydroxy acids, including L-tartrate. Salmonella utilizes L-tartrate early during infection to support fumarate respiration, while L-tartrate utilization ceases at later time points due to the increased availability of exogenous electron acceptors such as tetrathionate, nitrate, and oxygen. It remains unknown how Salmonella regulates its gene expression to metabolically adapt to changing nutritional environments. Here, we investigated how the transcriptional regulation for L-tartrate metabolism in Salmonella is influenced by infection-relevant cues. L-tartrate induces the transcription of ttdBAU , genes involved in L-tartrate utilization. L-tartrate metabolism is negatively regulated by two previously uncharacterized transcriptional regulators TtdV (STM3357) and TtdW (STM3358), and both TtdV and TtdW are required for the sensing of L-tartrate. The electron acceptors nitrate, tetrathionate, and oxygen repress ttdBAU transcription via the two-component system ArcAB. Furthermore, the regulation of L-tartrate metabolism is required for optimal fitness in a mouse model of Salmonella -induced colitis. TtdV, TtdW, and ArcAB allow for the integration of two cues, i.e., substrate availability and availability of exogenous electron acceptors, to control L-tartrate metabolism. Our findings provide novel insights into how Salmonella prioritizes the utilization of different electron acceptors for respiration as it experiences transitional nutrient availability throughout infection. IMPORTANCE Bacterial pathogens must adapt their gene expression profiles to cope with diverse environments encountered during infection. This coordinated process is carried out by the integration of cues that the pathogen senses to fine-tune gene expression in a spatiotemporal manner. Many studies have elucidated the regulatory mechanisms of how Salmonella sense metabolites in the gut to activate or repress its virulence program; however, our understanding of how Salmonella coordinates its gene expression to maximize the utilization of carbon and energy sources found in transitional nutrient niches is not well understood. In this study, we discovered how Salmonella integrates two infection-relevant cues, substrate availability and exogenous electron acceptors, to control L-tartrate metabolism. From our experiments, we propose a model for how L-tartrate metabolism is regulated in response to different metabolic cues in addition to characterizing two previously unknown transcriptional regulators. This study expands our understanding of how microbes combine metabolic cues to enhance fitness during infection.

One hurdle to understanding how molecular machines work, and how they evolve, is our inability to see their structures in situ. Here we describe a minicell system that enables in situ cryogenic electron microscopy imaging and single particle analysis to investigate the structure of an iconic molecular machine, the bacterial flagellar motor, which spins a helical propeller for propulsion. We determine the structure of the high-torque Campylobacter jejuni motor in situ, including the subnanometre-resolution structure of the periplasmic scaffold, an adaptation essential to high torque. Our structure enables identification of new proteins, and interpretation with molecular models highlights origins of new components, reveals modifications of the conserved motor core, and explain how these structures both template a wider ring of motor proteins, and buttress the motor during swimming reversals. We also acquire insights into universal principles of flagellar torque generation. This approach is broadly applicable to other membrane-residing bacterial molecular machines complexes.

ABSTRACT Enteric pathogens such as Salmonella enterica serovar Typhimurium experience spatial and temporal changes to the metabolic landscape throughout infection. Host reactive oxygen and nitrogen species non-enzymatically convert monosaccharides to alpha hydroxy acids, including L-tartrate. Salmonella utilizes L-tartrate early during infection to support fumarate respiration, while L-tartrate utilization ceases at later time points due to the increased availability of exogenous electron acceptors such as tetrathionate, nitrate, and oxygen. It remains unknown how Salmonella regulates its gene expression to metabolically adapt to changing nutritional environments. Here, we investigated how the transcriptional regulation for L-tartrate metabolism in Salmonella is influenced by infection-relevant cues. L-tartrate induces the transcription of ttdBAU , genes involved in L-tartrate utilization. L-tartrate metabolism is negatively regulated by two previously uncharacterized transcriptional regulators TtdV (STM3357) and TtdW (STM3358), and both TtdV and TtdW are required for sensing of L-tartrate. The electron acceptors nitrate, tetrathionate, and oxygen repress ttdBAU transcription via the two-component system ArcAB. Furthermore, regulation of L-tartrate metabolism is required for optimal fitness in a mouse model of Salmonella -induced colitis. TtdV, TtdW, and ArcAB allow for the integration of two cues, substrate availability and availability of exogenous electron acceptors, to control L-tartrate metabolism. Our findings provide novel insights into how Salmonella prioritizes utilization of different electron acceptors for respiration as it experiences transitional nutrient availability throughout infection. IMPORTANCE Bacterial pathogens must adapt their gene expression profiles to cope with diverse environments encountered during infection. This coordinated process is carried out by the integration of cues that the pathogen senses to fine-tune gene expression in a spatiotemporal manner. Many studies have elucidated the regulatory mechanisms on how Salmonella sense metabolites in the gut to activate or repress its virulence program, however our understanding of how Salmonella coordinates its gene expression to maximize the utilization of carbon and energy sources found in transitional nutrient niches is not well understood. In this study, we discovered how Salmonella integrates two infection-relevant cues, substrate availability and exogenous electron acceptors, to control L-tartrate metabolism. From our experiments, we propose a model for how L-tartrate metabolism is regulated in response to different metabolic cues in addition to characterizing two previously unknown transcriptional regulators. This study expands our understanding of how microbes combine metabolic cues to enhance fitness during infection.

The flagellar motors of Campylobacter jejuni (C. jejuni) and related Campylobacterota (previously epsilonproteobacteria) feature 100-nm-wide periplasmic "basal disks" that have been implicated in scaffolding a wider ring of additional motor proteins to increase torque, but the size of these disks is excessive for a role solely in scaffolding motor proteins. Here, we show that the basal disk is a flange that braces the flagellar motor during disentanglement of its flagellar filament from interactions with the cell body and other filaments. We show that motor output is unaffected when we shrink or displace the basal disk, and suppressor mutations of debilitated motors occur in flagellar-filament or cell-surface glycosylation pathways, thus sidestepping the need for a flange to overcome the interactions between two flagellar filaments and between flagellar filaments and the cell body. Our results identify unanticipated co-dependencies in the evolution of flagellar motor structure and cell-surface properties in the Campylobacterota.

ABSTRACT Two-component signal transduction systems (TCSs) are nearly ubiquitous across bacterial species and enable bacteria to sense and respond to specific cues for environmental adaptation. The Campylobacter jejuni BumSR TCS is unusual in that the BumS sensor exclusively functions as a phosphatase rather than a kinase to control phosphorylated levels of its cognate BumR response regulator (P-BumR). We previously found that BumSR directs a response to the short-chain fatty acid butyrate generated by resident microbiota so that C. jejuni identifies ideal lower intestinal niches in avian and human hosts for colonization. However, butyrate is an indirect cue for BumS and did not inhibit in vitro BumS phosphatase activity for P-BumR. In this work, we expanded the repertoire of lower intestinal metabolites that are cues sensed by BumS that modulate the expression of genes required for colonization to include the branched short-chain fatty acids isobutyrate and isovalerate. Unlike butyrate, isobutyrate and isovalerate inhibited in vitro BumS phosphatase activity for P-BumR, indicating that these metabolites are direct cues for BumS. Isobutyrate and isovalerate reduced the thermostability of BumS and caused a reorganization of protein structure to suggest how sensing these cues inhibits phosphatase activity. We also identified residues in the BumS sensory domain required to detect isobutyrate, isovalerate, and butyrate and for optimal colonization of hosts to reveal how gut bacteria can recognize these intestinal metabolites. Our work reveals how this unusual bacterial sensor phosphatase senses a repertoire of intestinal metabolites and how cues alter BumSR signal transduction to influence C. jejuni colonization of hosts. IMPORTANCE TCSs are prevalent in many bacteria, but the cues sensed by each are not actually known for many of these systems. Microbiota-generated butyrate in human and avian hosts is detected by the Campylobacter jejuni BumS sensor phosphatase so that the bacterium identifies ideal lower intestinal niches for colonization. However, BumS only indirectly senses butyrate to inhibit dephosphorylation of its cognate BumR response regulator. Here, we expanded the repertoire of cues sensed by BumS to the branched-short chain fatty acids isobutyrate and isovalerate that are also abundant in the lower intestines. Both isobutyrate and isovalerate are potent, direct cues for BumS, whereas butyrate is an indirect cue. Leveraging isobutyrate and isovalerate as direct cues, we reveal BumS structure is altered upon cue detection to inhibit its phosphatase activity. We provide an understanding of the mechanics of an unusual mode of signal transduction executed by BumSR and other bacterial sensor phosphatase-driven TCSs.

ABSTRACTCampylobacter jejuni colonizes the thick mucus layer lining the lower intestinal epithelium of hosts to promote commensalism in birds and animals or diarrheal disease in humans. C. jejuni displays higher swimming velocity as external viscosity increases, which likely benefits colonization and persistence in the intestinal mucus layer of hosts. C. jejuni produces one of the most structurally complex bacterial flagellar motors, which has been proposed to generate high torque for high swimming velocity. However, it is unknown how this flagellar motor alters output to impact swimming velocities in a viscosity-dependent manner. In this work, we identified viscosity-dependent determinant A (VidA) (Cjj81176_0996) and VidB (Cjj81176_1107) in modulating swimming velocity primarily in low-viscosity environments. C. jejuni ΔvidA cells were non-motile or swam slowly in low-viscosity media, but swam with high velocity similar to wild-type cells in both Newtonian and non-Newtonian media with high viscosity. These data indicate that VidA is required for swimming in low-viscosity environments. Suppressor mutants suggested that the lower swimming velocity of ΔvidA in low viscosity was due to unregulated activity of VidB, which we propose has a brake- or clutch-like activity to reduce swimming velocity in low viscosity. Unlike other bacterial flagellar brakes or clutches, we found no evidence for cyclic diguanylate monophosphate influencing VidB activity. Our analyses suggest that the mechanics of the C. jejuni flagellar motor has naturally evolved for high-velocity swimming in milieus with high viscosity and requires the unique combination of VidA and VidB to modulate its activity to slow swimming velocity in low-viscosity environments.IMPORTANCEBacteria can adapt flagellar motor output in response to the load that the extracellular milieu imparts on the flagellar filament to enable propulsion. Bacteria can adapt flagellar motor output in response to the load that the extracellular milieu imparts on the flagellar filament to enable propulsion through diverse environments. These changes may involve increasing power and torque in high-viscosity environments or reducing power and flagellar rotation upon contact with a surface. C. jejuni swimming velocity in low-viscosity environments is comparable to other bacterial flagellates and increases significantly as external viscosity increases. In this work, we provide evidence that the mechanics of the C. jejuni flagellar motor has evolved to naturally promote high swimming velocity in high-viscosity environments. We found that C. jejuni produces VidA and VidB as auxiliary proteins to specifically affect flagellar motor activity in low viscosity to reduce swimming velocity. Our findings provide some of the first insights into different mechanisms that exist in bacteria to alter the mechanics of a flagellar motor, depending on the viscosity of extracellular environments.

Spirochetes can be distinguished from other bacteria by their spiral-shaped morphology and subpolar periplasmic flagella. This study focused on FlhF and FlhG, which control the spatial and numerical regulation of flagella in many exoflagellated bacteria, in the spirochete Leptospira. In contrast to flhF which seems to be essential in Leptospira, we demonstrated that flhG(-) mutants in both the saprophyte L. biflexa and the pathogen L. interrogans were less motile than the wild-type strains in gel-like environments but not hyperflagellated as reported previously in other bacteria. Cryo-electron tomography revealed that the distance between the flagellar basal body and the tip of the cell decreased significantly in the flhG(-) mutant in comparison to wild-type and complemented strains. Additionally, comparative transcriptome analyses of L. biflexa flhG(-) and wild-type strains showed that FlhG acts as a negative regulator of transcription of some flagellar genes. We found that the L. interrogans flhG(-) mutant was attenuated for virulence in the hamster model. Cross-species complementation also showed that flhG is not interchangeable between species. Our results indicate that FlhF and FlhG in Leptospira contribute to governing cell motility but our data support the hypothesis that FlhF and FlhG function differently in each bacterial species, including among spirochetes.

Campylobacter jejuni rotates a flagellum at each pole to swim through the viscous mucosa of its hosts' gastrointestinal tracts. Despite their importance for host colonization, however, how C. jejuni coordinates rotation of these two opposing flagella is unclear. As well as their polar placement, C. jejuni's flagella deviate from the norm of Enterobacteriaceae in other ways: their flagellar motors produce much higher torque and their flagellar filament is made of two different zones of two different flagellins. To understand how C. jejuni's opposed motors coordinate, and what contribution these factors play in C. jejuni motility, we developed strains with flagella that could be fluorescently labeled, and observed them by high-speed video microscopy. We found that C. jejuni coordinates its dual flagella by wrapping the leading filament around the cell body during swimming in high-viscosity media and that its differentiated flagellar filament and helical body have evolved to facilitate this wrapped-mode swimming.

Campylobacter jejuni monitors intestinal metabolites produced by the host and microbiota to initiate intestinal colonization of avian and animal hosts for commensalism and infection of humans for diarrheal disease. We previously discovered that C. jejuni has the capacity to spatially discern different intestinal regions by sensing lactate and the short-chain fatty acids acetate and butyrate and then alter transcription of colonization factors appropriately for in vivo growth. In this study, we identified the C. jejuni butyrate-modulated regulon and discovered that the BumSR two-component signal transduction system (TCS) directs a response to butyrate by identifying mutants in a genetic screen defective for butyrate-modulated transcription. The BumSR TCS, which is important for infection of humans and optimal colonization of avian hosts, senses butyrate likely by indirect means to alter transcription of genes encoding important colonization determinants. Unlike many canonical TCSs, the predicted cytoplasmic sensor kinase BumS lacked in vitro autokinase activity, which would normally lead to phosphorylation of the cognate BumR response regulator. Instead, BumS has likely evolved mutations to naturally function as a phosphatase whose activity is influenced by exogenous butyrate to control the level of endogenous phosphorylation of BumR and its ability to alter transcription of target genes. To our knowledge, the BumSR TCS is the only bacterial signal transduction system identified so far that mediates responses to the microbiota-generated intestinal metabolite butyrate, an important factor for host intestinal health and homeostasis. Our findings suggest that butyrate sensing by this system is vital for C. jejuni colonization of multiple hosts.

Many bacterial pathogens display glycosylated surface structures that contribute to virulence, and targeting these structures is a viable strategy for pathogen control. The foodborne pathogen Campylobacter jejuni expresses a vast diversity of flagellar glycans, and flagellar glycosylation is essential for its virulence. Little is known about why C. jejuni encodes such a diverse set of flagellar glycans, but it has been hypothesized that evolutionary pressure from bacteriophages (phages) may have contributed to this diversity. However, interactions between Campylobacter phages and host flagellar glycans have not been characterized in detail. Previously, we observed that Gp047 (now renamed FlaGrab), a conserved Campylobacter phage protein, binds to C. jejuni flagella displaying the nine-carbon monosaccharide 7-acetamidino-pseudaminic acid, and that this binding partially inhibits cell growth. However, the mechanism of this growth inhibition, as well as how C. jejuni might resist this activity, are not well-understood. Here we use RNA-Seq to show that FlaGrab exposure leads C. jejuni 11168 cells to downregulate expression of energy metabolism genes, and that FlaGrab-induced growth inhibition is dependent on motile flagella. Our results are consistent with a model whereby FlaGrab binding transmits a signal through flagella that leads to retarded cell growth. To evaluate mechanisms of FlaGrab resistance in C. jejuni, we characterized the flagellar glycans and flagellar glycosylation loci of two C. jejuni strains naturally resistant to FlaGrab binding. Our results point toward flagellar glycan diversity as the mechanism of resistance to FlaGrab. Overall, we have further characterized the interaction between this phage-encoded flagellar glycan-binding protein and C. jejuni, both in terms of mechanism of action and mechanism of resistance. Our results suggest that C. jejuni encodes as-yet unidentified mechanisms for generating flagellar glycan diversity, and point to phage proteins as exciting lenses through which to study bacterial surface glycans.

Bacterial flagella are reversible rotary motors that rotate external filaments for bacterial propulsion. Some flagellar motors have diversified by recruiting additional components that influence torque and rotation, but little is known about the possible diversification and evolution of core motor components. The mechanistic core of flagella is the cytoplasmic C ring, which functions as a rotor, directional switch, and assembly platform for the flagellar type III secretion system (fT3SS) ATPase. The C ring is composed of a ring of FliG proteins and a helical ring of surface presentation of antigen (SPOA) domains from the switch proteins FliM and one of two usually mutually exclusive paralogs, FliN or FliY. We investigated the composition, architecture, and function of the C ring of Campylobacter jejuni, which encodes FliG, FliM, and both FliY and FliN by a variety of interrogative approaches. We discovered a diversified C. jejuni C ring containing FliG, FliM, and both FliY, which functions as a classical FliN-like protein for flagellar assembly, and FliN, which has neofunctionalized into a structural role. Specific protein interactions drive the formation of a more complex heterooligomeric C. jejuni C-ring structure. We discovered that this complex C ring has additional cellular functions in polarly localizing FlhG for numerical regulation of flagellar biogenesis and spatial regulation of division. Furthermore, mutation of the C. jejuni C ring revealed a T3SS that was less dependent on its ATPase complex for assembly than were other systems. Our results highlight considerable evolved flagellar diversity that impacts motor output, biogenesis, and cellular processes in different species. IMPORTANCE The conserved core of bacterial flagellar motors reflects a shared evolutionary history that preserves the mechanisms essential for flagellar assembly, rotation, and directional switching. In this work, we describe an expanded and diversified set of core components in the Campylobacter jejuni flagellar C ring, the mechanistic core of the motor. Our work provides insight into how usually conserved core components may have diversified by gene duplication, enabling a division of labor of the ancestral protein between the two new proteins, acquisition of new roles in flagellar assembly and motility, and expansion of the function of the flagellum beyond motility, including spatial regulation of cell division and numerical control of flagellar biogenesis in C. jejuni. Our results highlight that relatively small changes, such as gene duplications, can have substantial ramifications on the cellular roles of a molecular machine.

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.

The stator units of the flagellum supply power to the flagellar motor via ion transport across the cytoplasmic membrane and generate torque on the rotor for rotation. Flagellar motors across bacterial species have evolved adaptations that impact and enhance stator function to meet the demands of each species, including producing stator units using different fuel types or various stator units for different motility modalities. Campylobacter jejuni produces one of the most complex and powerful flagellar motors by positioning 17 stator units at a greater radial distance than in most other bacteria to increase power and torque for high velocity of motility. We report another evolutionary adaptation impacting flagellar stators by identifying FlgX as a chaperone for C. jejuni stator units to ensure sufficient power and torque for flagellar rotation and motility. We discovered that FlgX maintains MotA and MotB stator protein integrity likely through a direct interaction with MotA that prevents their degradation. Suppressor analysis suggested that the physiology of C. jejuni drives the requirement for FlgX to protect stator units from proteolysis by the FtsH protease complex. C. jejuni ΔflgX was strongly attenuated for colonization of the natural avian host, but colonization capacity was greatly restored by a single mutation in MotA. These findings suggest that the likely sole function of FlgX is to preserve stator unit integrity for the motility required for host interactions. Our findings demonstrate another evolved adaptation in motile bacteria to ensure the equipment of the flagellar motor with sufficient power to generate torque for motility.IMPORTANCE The bacterial flagellum is a reversible rotating motor powered by ion transport through stator units, which also exert torque on the rotor component to turn the flagellum for motility. Species-specific adaptations to flagellar motors impact stator function to meet the demands of each species to sufficiently power flagellar rotation. We identified another evolutionary adaptation by discovering that FlgX of Campylobacter jejuni preserves the integrity of stator units by functioning as a chaperone to protect stator proteins from degradation by the FtsH protease complex due to the physiology of the bacterium. FlgX is required to maintain a level of stator units sufficient to power the naturally high-torque flagellar motor of C. jejuni for motility in intestinal mucosal layers to colonize hosts. Our work continues to identify an increasing number of adaptations to flagellar motors across bacterial species that provide the mechanics necessary for producing an effective rotating nanomachine for motility.