FrzS acts as a polar beacon to recruit SgmX, a central activator of type IV pili during Myxococcus xanthus motility.

In rod-shaped bacteria, type IV pili (Tfp) promote twitching motility by assembling and retracting at the cell pole. In Myxococcus xanthus, a bacterium that moves in highly coordinated cell groups, Tfp are activated by a polar activator protein, SgmX. However, while it is known that the Ras-like protein MglA is required for unipolar targeting, how SgmX accesses the cell pole to activate Tfp is unknown. Here, we demonstrate that a polar beacon protein, FrzS, recruits SgmX at the cell pole. We identified two main functional domains, including a Tfp-activating domain and a polar-binding domain. Within the latter, we show that the direct binding of MglA-GTP unveils a hidden motif that binds directly to the FrzS N-terminal response regulator (CheY). Structural analyses reveal that this binding occurs through a novel binding interface for response regulator domains. In conclusion, the findings unveil the protein interaction network leading to the spatial activation of Tfp at the cell pole. This tripartite system is at the root of complex collective behaviours in this predatory bacterium.

The switching mechanism of the bacterial rotary motor combines tight regulation with inherent flexibility.

Regulatory switches are wide spread in many biological systems. Uniquely among them, the switch of the bacterial flagellar motor is not an on/off switch but rather controls the motor's direction of rotation in response to binding of the signaling protein CheY. Despite its extensive study, the molecular mechanism underlying this switch has remained largely unclear. Here, we resolved the functions of each of the three CheY-binding sites at the switch in E. coli, as well as their different dependencies on phosphorylation and acetylation of CheY. Based on this, we propose that CheY motor switching activity is potentiated upon binding to the first site. Binding of potentiated CheY to the second site produces unstable switching and at the same time enables CheY binding to the third site, an event that stabilizes the switched state. Thereby, this mechanism exemplifies a unique combination of tight motor regulation with inherent switching flexibility.

PATAN-domain regulators interact with the Type IV pilus motor to control phototactic orientation in the cyanobacterium Synechocystis.

Many prokaryotes show complex behaviors that require the intricate spatial and temporal organization of cellular protein machineries, leading to asymmetrical protein distribution and cell polarity. One such behavior is cyanobacterial phototaxis which relies on the dynamic localization of the Type IV pilus motor proteins in response to light. In the cyanobacterium Synechocystis, various signaling systems encompassing chemotaxis-related CheY- and PatA-like response regulators are critical players in switching between positive and negative phototaxis depending on the light intensity and wavelength. In this study, we show that PatA-type regulators evolved from chemosensory systems. Using fluorescence microscopy and yeast two-hybrid analysis, we demonstrate that they localize to the inner membrane, where they interact with the N-terminal cytoplasmic domain of PilC and the pilus assembly ATPase PilB1. By separately expressing the subdomains of the response regulator PixE, we confirm that only the N-terminal PATAN domain interacts with PilB1, localizes to the membrane, and is sufficient to reverse phototactic orientation. These experiments established that the PATAN domain is the principal output domain of PatA-type regulators which we presume to modulate pilus extension by binding to the pilus motor components.

The Chemoreceptor Sensory Adaptation System Produces Coordinated Reversals of the Flagellar Motors on an Escherichia coli Cell.

In isotropic environments, an Escherichia coli cell exhibits coordinated rotational switching of its flagellar motors, produced by fluctuations in the intracellular concentration of phosphorylated CheY (CheY-P) emanating from chemoreceptor signaling arrays. In this study, we show that these CheY-P fluctuations arise through modifications of chemoreceptors by two sensory adaptation enzymes: the methyltransferase CheR and the methylesterase CheB. A cell containing CheR, CheB, and the serine chemoreceptor Tsr exhibited motor synchrony, whereas a cell lacking CheR and CheB or containing enzymatically inactive forms did not. Tsr variants with different combinations of methylation-mimicking Q residues at the adaptation sites also failed to show coordinated motor switching in cells lacking CheR and CheB. Cells containing CheR, CheB, and Tsr [NDND], a variant in which the adaptation site residues are not substrates for CheR or CheB modifications, also lacked motor synchrony. TsrΔNWETF, which lacks a C-terminal pentapeptide-binding site for CheR and CheB, and the ribose-galactose receptor Trg, which natively lacks this motif, failed to produce coordinated motor switching, despite the presence of CheR and CheB. However, addition of the NWETF sequence to Trg enabled Trg-NWETF to produce motor synchrony, as the sole receptor type in cells containing CheR and CheB. Finally, CheBc, the catalytic domain of CheB, supported motor coordination in combination with CheR and Tsr. These results indicate that the coordination of motor switching requires CheR/CheB-mediated changes in receptor modification state. We conclude that the opposing receptor substrate-site preferences of CheR and CheB produce spontaneous blinking of the chemoreceptor array's output activity. IMPORTANCE Under steady-state conditions with no external stimuli, an Escherichia coli cell coordinately switches the rotational direction of its flagellar motors. Here, we demonstrate that the CheR and CheB enzymes of the chemoreceptor sensory adaptation system mediate this coordination. Stochastic fluctuations in receptor adaptation states trigger changes in signal output from the receptor array, and this array blinking generates fluctuations in CheY-P concentration that coordinate directional switching of the flagellar motors. Thus, in the absence of chemoeffector gradients, the sensory adaptation system controls run-tumble swimming of the cell, its optimal foraging strategy.

ZomB is essential for chemotaxis of Vibrio alginolyticus by the rotational direction control of the polar flagellar motor.

Bacteria exhibit chemotaxis by controlling flagellar rotation to move toward preferred places or away from nonpreferred places. The change in rotation is triggered by the binding of the chemotaxis signaling protein CheY-phosphate (CheY-P) to the C-ring in the flagellar motor. Some specific bacteria, including Vibrio spp. and Shewanella spp., have a single transmembrane protein called ZomB. ZomB is essential for controlling the flagellar rotational direction in Shewanella putrefaciens and Vibrio parahaemolyticus. In this study, we confirmed that the zomB deletion results only in the counterclockwise (CCW) rotation of the motor in Vibrio alginolyticus as previously reported in other bacteria. We found that ZomB is not required for a clockwise-locked phenotype caused by mutations in fliG and fliM, and that ZomB is essential for CW rotation induced by overproduction of CheY-P. Purified ZomB proteins form multimers, suggesting that ZomB may function as a homo-oligomer. These observations imply that ZomB interacts with protein(s) involved in either flagellar motor rotation, chemotaxis, or both. We provide the evidence that ZomB is a new player in chemotaxis and is required for the rotational control in addition to CheY in Vibrio alginolyticus.

Campylobacter Virulence Factors and Molecular Host-Pathogen Interactions.

Campylobacter jejuni and Campylobacter coli can be frequently isolated from poultry and poultry-derived products, and in combination these two species cause a large portion of human bacterial gastroenteritis cases. While birds are typically colonized by these Campylobacter species without clinical symptoms, in humans they cause (foodborne) infections at high frequencies, estimated to cost billions of dollars worldwide every year. The clinical outcome of Campylobacter infections comprises malaise, diarrhea, abdominal pain and fever. Symptoms may continue for up to two weeks and are generally self-limiting, though occasionally the disease can be more severe or result in post-infection sequelae. The virulence properties of these pathogens have been best-characterized for C. jejuni, and their actions are reviewed here. Various virulence-associated bacterial determinants include the flagellum, numerous flagellar secreted factors, protein adhesins, cytolethal distending toxin (CDT), lipooligosaccharide (LOS), serine protease HtrA and others. These factors are involved in several pathogenicity-linked properties that can be divided into bacterial chemotaxis, motility, attachment, invasion, survival, cellular transmigration and spread to deeper tissue. All of these steps require intimate interactions between bacteria and host cells (including immune cells), enabled by the collection of bacterial and host factors that have already been identified. The assortment of pathogenicity-associated factors now recognized for C. jejuni, their function and the proposed host cell factors that are involved in crucial steps leading to disease are discussed in detail.

In situ structure of the Caulobacter crescentus flagellar motor and visualization of binding of a CheY-homolog.

Bacterial flagellar motility is controlled by the binding of CheY proteins to the cytoplasmic switch complex of the flagellar motor, resulting in changes in swimming speed or direction. Despite its importance for motor function, structural information about the interaction between effector proteins and the motor are scarce. To address this gap in knowledge, we used electron cryotomography and subtomogram averaging to visualize such interactions inside Caulobacter crescentus cells. In C. crescentus, several CheY homologs regulate motor function for different aspects of the bacterial lifestyle. We used subtomogram averaging to image binding of the CheY family protein CleD to the cytoplasmic Cring switch complex, the control center of the flagellar motor. This unambiguously confirmed the orientation of the motor switch protein FliM and the binding of a member of the CheY protein family to the outside rim of the C ring. We also uncovered previously unknown structural elaborations of the alphaproteobacterial flagellar motor, including two novel periplasmic ring structures, and the stator ring harboring eleven stator units, adding to our growing catalog of bacterial flagellar diversity.

Comparative transcriptome analysis reveals that deletion of CheY influences gene expressions of ABC transports and metabolism in Haemophilus parasuis.

Haemophilus (Glaesserella) parasuis is a commensal bacterium that causes Glässer's disease (GD) in swine. As a global transcriptional factor, CheY regulates the expression of hundreds of genes in H. parasuis. In this study, we measured changes in gene expression at the whole transcriptome level using RNAseq. We identified 2058 co-expressed genes, and found 624 differentially expressed genes (q < 0.05) in ΔcheY and SC1401. Several important GO annotations and signaling pathways were identified. RNA-seq results were assembled according to the reference genome, compared with the annotated gene model, and 12 new transcriptional regions were found. Finally, q-PCR results validated the RNA-seq results with 8 randomly selected genes. The present study indicated that CheY is mainly involved in the regulation of ABC transport, oxidative phosphorylation, and ß-Lactam resistance. We draw the regulatory network of CheY, which offers greater insight into the regulatory mechanism of CheY in H.parasuis.

Cluster II che genes of Pseudomonas syringae pv. tabaci 6605, orthologs of cluster I in Pseudomonas aeruginosa, are required for chemotaxis and virulence.

Pseudomonas syringae pv. tabaci 6605 (Pta6605) is a causal agent of wildfire disease in host tobacco plants and is highly motile. Pta6605 has multiple clusters of chemotaxis genes including cheA, a gene encoding a histidine kinase, cheY, a gene encoding a response regulator, mcp, a gene for a methyl-accepting chemotaxis protein, as well as flagellar and pili biogenesis genes. However, only two major chemotaxis gene clusters, cluster I and cluster II, possess cheA and cheY. Deletion mutants of cheA or cheY were constructed to evaluate their possible role in Pta6605 chemotaxis and virulence. Motility tests and a chemotaxis assay to known attractant demonstrated that cheA2 and cheY2 mutants were unable to swarm and to perform chemotaxis, whereas cheA1 and cheY1 mutants retained chemotaxis ability almost equal to that of the wild-type (WT) strain. Although WT and cheY1 mutants of Pta6605 caused severe disease symptoms on host tobacco leaves, the cheA2 and cheY2 mutants did not, and symptom development with cheA1 depended on the inoculation method. These results indicate that chemotaxis genes located in cluster II are required for optimal chemotaxis and host plant infection by Pta6605 and that cluster I may partially contribute to these phenotypes.

Structure and function of the archaeal response regulator CheY.

Motility is a central feature of many microorganisms and provides an efficient strategy to respond to environmental changes. Bacteria and archaea have developed fundamentally different rotary motors enabling their motility, termed flagellum and archaellum, respectively. Bacterial motility along chemical gradients, called chemotaxis, critically relies on the response regulator CheY, which, when phosphorylated, inverses the rotational direction of the flagellum via a switch complex at the base of the motor. The structural difference between archaellum and flagellum and the presence of functional CheY in archaea raises the question of how the CheY protein changed to allow communication with the archaeal motility machinery. Here we show that archaeal CheY shares the overall structure and mechanism of magnesium-dependent phosphorylation with its bacterial counterpart. However, bacterial and archaeal CheY differ in the electrostatic potential of the helix α4. The helix α4 is important in bacteria for interaction with the flagellar switch complex, a structure that is absent in archaea. We demonstrated that phosphorylation-dependent activation, and conserved residues in the archaeal CheY helix α4, are important for interaction with the archaeal-specific adaptor protein CheF. This forms a bridge between the chemotaxis system and the archaeal motility machinery. Conclusively, archaeal CheY proteins conserved the central mechanistic features between bacteria and archaea, but differ in the helix α4 to allow binding to an archaellum-specific interaction partner.

Modulation of Response Regulator CheY Reaction Kinetics by Two Variable Residues That Affect Conformation.

Microorganisms and plants utilize two-component systems to regulate adaptive responses to changing environmental conditions. Sensor kinases detect stimuli and alter their autophosphorylation activity accordingly. Signal propagation occurs via the transfer of phosphoryl groups from upstream kinases to downstream response regulator proteins. Removal of phosphoryl groups from the response regulator typically resets the system. Members of the same protein family may catalyze phosphorylation and dephosphorylation reactions with different efficiencies, exhibiting rate constants spanning many orders of magnitude to accommodate response time scales from milliseconds to days. We previously found that variable positions one or two residues to the C-terminal side of the conserved Asp phosphorylation site (D+2) or Thr/Ser (T+1/T+2) in response regulators alter reaction kinetics by direct interaction with phosphodonor or phosphoacceptor molecules. Here, we explore the kinetic effects of amino acid substitutions at the two positions immediately C-terminal to the conserved Lys (K+1/K+2) in the model Escherichia coli response regulator CheY. We measured CheY autophosphorylation and autodephosphorylation rate constants for 27 pairs of K+1/K+2 residues that represent 84% of naturally occurring response regulators. Effects on autodephosphorylation were modest, but autophosphorylation rate constants varied by 2 orders of magnitude, suggesting that the K+1/K+2 positions influence reaction kinetics by altering the conformational spectrum sampled by CheY at equilibrium. Additional evidence supporting this indirect mechanism includes the following: the effect on autophosphorylation rate constants is independent of the phosphodonor, the autophosphorylation rate constants and dissociation constants for the phosphoryl group analog BeF3- are inversely correlated, and the K+1/K+2 positions are distant from the phosphorylation site.IMPORTANCE We have identified five variable positions in response regulators that allow the rate constants of autophosphorylation and autodephosporylation reactions each to be altered over 3 orders of magnitude in CheY. The distributions of variable residue combinations across response regulator subfamilies suggest that distinct mechanisms associated with different variable positions allow reaction rates to be tuned independently during evolution for diverse biological purposes. This knowledge could be used in synthetic-biology applications to adjust the properties (e.g., background noise and response duration) of biosensors and may allow prediction of response regulator reaction kinetics from the primary amino acid sequence.

CheY1 and CheY2 of Azorhizobium caulinodans ORS571 Regulate Chemotaxis and Competitive Colonization with the Host Plant.

The genome of Azorhizobium caulinodans ORS571 encodes two chemotaxis response regulators: CheY1 and CheY2. cheY1 is located in a chemotaxis cluster (cheAWY1BR), while cheY2 is located 37 kb upstream of the cheAWY1BR cluster. To determine the contributions of CheY1 and CheY2, we compared the wild type (WT) and mutants in the free-living state and in symbiosis with the host Sesbania rostrata Swim plate tests and capillary assays revealed that both CheY1 and CheY2 play roles in chemotaxis, with CheY2 having a more prominent role than CheY1. In an analysis of the swimming paths of free-swimming cells, the ΔcheY1 mutant exhibited decreased frequency of direction reversal, whereas the ΔcheY2 mutant appeared to change direction much more frequently than the WT. Exopolysaccharide (EPS) production in the ΔcheY1 and ΔcheY2 mutants was lower than that in the WT, but the ΔcheY2 mutant had more obvious EPS defects that were similar to those of the ΔcheY1 ΔcheY2 and Δeps1 mutants. During symbiosis, the levels of competitiveness for root colonization and nodule occupation of ΔcheY1 and ΔcheY2 mutants were impaired compared to those of the WT. Moreover, the competitive colonization ability of the ΔcheY2 mutant was severely impaired compared to that of the ΔcheY1 mutant. Taken together, the ΔcheY2 phenotypes are more severe than the ΔcheY1 phenotype in free-living and symbiotic states, and that of the double mutant resembles the ΔcheY2 single-mutant phenotype. These defects of ΔcheY1 and ΔcheY2 mutants were restored to the WT phenotype by complementation. These results suggest that there are different regulatory mechanisms of CheY1 and CheY2 and that CheY2 is a key chemotaxis regulator under free-living and symbiosis conditions.IMPORTANCEAzorhizobium caulinodans ORS571 is a motile soil bacterium that has the dual capacity to fix nitrogen both under free-living conditions and in symbiosis with Sesbania rostrata, forming nitrogen-fixing root and stem nodules. Bacterial chemotaxis to chemoattractants derived from host roots promotes infection and subsequent nodule formation by directing rhizobia to appropriate sites of infection. In this work, we identified and demonstrated that CheY2, a chemotactic response regulator encoded by a gene outside the chemotaxis cluster, is required for chemotaxis and multiple other cell phenotypes. CheY1, encoded by a gene in the chemotaxis cluster, also plays a role in chemotaxis. Two response regulators mediate bacterial chemotaxis and motility in different ways. This work extends the understanding of the role of multiple response regulators in Gram-negative bacteria.

Organization of the Flagellar Switch Complex of Bacillus subtilis.

While the protein complex responsible for controlling the direction (clockwise [CW] or counterclockwise [CCW]) of flagellar rotation has been fairly well studied in Escherichia coli and Salmonella, less is known about the switch complex in Bacillus subtilis or other Gram-positive species. Two component proteins (FliG and FliM) are shared between E. coli and B. subtilis, but in place of the protein FliN found in E. coli, the B. subtilis complex contains the larger protein FliY. Notably, in B. subtilis the signaling protein CheY-phosphate induces a switch from CW to CCW rotation, opposite to its action in E. coli Here, we have examined the architecture and function of the switch complex in B. subtilis using targeted cross-linking, bacterial two-hybrid protein interaction experiments, and characterization of mutant phenotypes. In major respects, the B. subtilis switch complex appears to be organized similarly to that in E. coli The complex is organized around a ring built from the large middle domain of FliM; this ring supports an array of FliG subunits organized in a similar way to that of E. coli, with the FliG C-terminal domain functioning in the generation of torque via conserved charged residues. Key differences from E. coli involve the middle domain of FliY, which forms an additional, more outboard array, and the C-terminal domains of FliM and FliY, which are organized into both FliY homodimers and FliM heterodimers. Together, the results suggest that the CW and CCW conformational states are similar in the Gram-negative and Gram-positive switches but that CheY-phosphate drives oppositely directed movements in the two cases.IMPORTANCE Flagellar motility plays key roles in the survival of many bacteria and in the harmful action of many pathogens. Bacterial flagella rotate; the direction of flagellar rotation is controlled by a multisubunit protein complex termed the switch complex. This complex has been extensively studied in Gram-negative model species, but little is known about the complex in Bacillus subtilis or other Gram-positive species. Notably, the switch complex in Gram-positive species responds to its effector CheY-phosphate (CheY-P) by switching to CCW rotation, whereas in E. coli or Salmonella CheY-P acts in the opposite way, promoting CW rotation. In the work here, the architecture of the B. subtilis switch complex has been probed using cross-linking, protein interaction measurements, and mutational approaches. The results cast light on the organization of the complex and provide a framework for understanding the mechanism of flagellar direction control in B. subtilis and other Gram-positive species.

Borrelia burgdorferi CheY2 Is Dispensable for Chemotaxis or Motility but Crucial for the Infectious Life Cycle of the Spirochete.

The requirements for bacterial chemotaxis and motility range from dispensable to crucial for host colonization. Even though more than 50% of all sequenced prokaryotic genomes possess at least one chemotaxis signaling system, many of those genomes contain multiple copies of a chemotaxis gene. However, the functions of most of those additional genes are unknown. Most motile bacteria possess at least one CheY response regulator that is typically dedicated to the control of motility and which is usually essential for virulence. Borrelia burgdorferi appears to be notably different, in that it has three cheY genes, and our current studies on cheY2 suggests that it has varied effects on different aspects of the natural infection cycle. Mutants deficient in this protein exhibit normal motility and chemotaxis in vitro but show reduced virulence in mice. Specifically, the cheY2 mutants were severely attenuated in murine infection and dissemination to distant tissues after needle inoculation. Moreover, while ΔcheY2 spirochetes are able to survive normally in the Ixodes ticks, mice fed upon by the ΔcheY2-infected ticks did not develop a persistent infection in the murine host. Our data suggest that CheY2, despite resembling a typical response regulator, functions distinctively from most other chemotaxis CheY proteins. We propose that CheY2 serves as a regulator for a B. burgdorferi virulence determinant that is required for productive infection within vertebrate, but not tick, hosts.

The Agrobacterium tumefaciens CheY-like protein ClaR regulates biofilm formation.

The switch from a motile, planktonic existence to an attached biofilm is a major bacterial lifestyle transition that is often mediated by complex regulatory pathways. In this report, we describe a CheY-like protein required for control of the motile-to-sessile switch in the plant pathogen Agrobacterium tumefaciens. This regulator, which we have designated ClaR, possesses two distinct CheY-like receiver (REC) domains and is involved in the negative regulation of biofilm formation, through production of the unipolar polysaccharide (UPP) adhesin and cellulose. The ClaR REC domains share predicted structural homology with characterized REC domains and contain the majority of active site residues known to be essential for protein phosphorylation. REC1 is missing the conserved aspartate (N72) residue and although present in REC 2 (D193), it is not required for ClaR-dependent regulation suggesting that phosphorylation, which modulates the activity of many CheY-like proteins, appears not to be essential for ClaR activity. We also show that ClaR-dependent negative regulation of attachment is diminished significantly in mutants for PruA and PruR, proteins known to be involved in a pterin-mediated attachment regulation pathway. In A. tumefaciens, pterins are required for control of the intracellular signal cyclic diguanylate monophosphate through the DcpA regulator, but our findings suggest that pterin-dependent ClaR control of attachment can function independently from DcpA, including dampening of c-di-GMP levels. This report of a novel CheY-type biofilm regulator in A. tumefaciens thus also adds significant details to the role of pterin-mediated signalling.

Secondary bacterial flagellar system improves bacterial spreading by increasing the directional persistence of swimming.

As numerous bacterial species, Shewanella putrefaciens CN-32 possesses a complete secondary flagellar system. A significant subpopulation of CN-32 cells induces expression of the secondary system under planktonic conditions, resulting in formation of one, sometimes two, filaments at lateral positions in addition to the primary polar flagellum. Mutant analysis revealed that the single chemotaxis system primarily or even exclusively addresses the main polar flagellar system. Cells with secondary filaments outperformed their monopolarly flagellated counterparts in spreading on soft-agar plates and through medium-filled channels despite having lower swimming speed. While mutant cells with only polar flagella navigate by a "run-reverse-flick" mechanism resulting in effective cell realignments of about 90°, wild-type cells with secondary filaments exhibited a range of realignment angles with an average value of smaller than 90°. Mathematical modeling and computer simulations demonstrated that the smaller realignment angle of wild-type cells results in the higher directional persistence, increasing spreading efficiency both with and without a chemical gradient. Taken together, we propose that in S. putrefaciens CN-32, cell propulsion and directional switches are mainly mediated by the polar flagellar system, while the secondary filament increases the directional persistence of swimming and thus of spreading in the environment.

Modulation of frustration in folding by sequence permutation.

Folding of globular proteins can be envisioned as the contraction of a random coil unfolded state toward the native state on an energy surface rough with local minima trapping frustrated species. These substructures impede productive folding and can serve as nucleation sites for aggregation reactions. However, little is known about the relationship between frustration and its underlying sequence determinants. Chemotaxis response regulator Y (CheY), a 129-amino acid bacterial protein, has been shown previously to populate an off-pathway kinetic trap in the microsecond time range. The frustration has been ascribed to premature docking of the N- and C-terminal subdomains or, alternatively, to the formation of an unproductive local-in-sequence cluster of branched aliphatic side chains, isoleucine, leucine, and valine (ILV). The roles of the subdomains and ILV clusters in frustration were tested by altering the sequence connectivity using circular permutations. Surprisingly, the stability and buried surface area of the intermediate could be increased or decreased depending on the location of the termini. Comparison with the results of small-angle X-ray-scattering experiments and simulations points to the accelerated formation of a more compact, on-pathway species for the more stable intermediate. The effect of chain connectivity in modulating the structures and stabilities of the early kinetic traps in CheY is better understood in terms of the ILV cluster model. However, the subdomain model captures the requirement for an intact N-terminal domain to access the native conformation. Chain entropy and aliphatic-rich sequences play crucial roles in biasing the early events leading to frustration in the folding of CheY.

A cyanobacterial chemotaxis-like system controls phototactic orientation via phosphorylation of two antagonistic response regulators.

Photosynthetic cyanobacteria exhibit phototaxis, utilizing type IV pili (T4P) to navigate either toward or away from a light source. The Tax1 system is a chemotaxis-like signal transduction pathway that controls the switch in cell polarity, which is crucial for positive phototaxis in Synechocystis sp. PCC 6803. The system consists of the blue/green light sensor PixJ, which controls the histidine kinase PixL and two CheY-like response regulators, PixG and PixH. However, the molecular mechanism by which Tax1 regulates T4P activity and polarity is poorly understood. Here, we investigated the phosphotransfer between PixL and its cognate response regulators in vitro and analyzed the localization and function of wild-type and phosphorylation-deficient PixG and PixH during phototaxis. We found that both PixG and PixH are phosphorylated by PixL but have different roles in phototaxis regulation. Only phosphorylated PixG interacts with the T4P motor protein PilB1 and localizes to the leading cell pole under directional light, thereby promoting positive phototaxis. In contrast, PixH is a negative regulator of PixG phosphorylation and inhibits positive phototaxis. We also demonstrated that the C-terminal receiver domain of PixL is essential for positive phototaxis, and modulates the kinase activity of PixL. Our findings reveal the molecular basis of positive phototaxis regulation by the Tax1 system and provide insights into the division of labor between PatA-type and CheY-like response regulators in cyanobacterial chemotaxis-like systems. Furthermore, these findings highlight similarities in the regulation of movement direction during twitching motility in phototactic and chemotactic bacteria.

Structure-function analysis of PorXFj, the PorX homolog from Flavobacterium johnsioniae, suggests a role of the CheY-like domain in type IX secretion motor activity.

The type IX secretion system (T9SS) is a large multi-protein transenvelope complex distributed into the Bacteroidetes phylum and responsible for the secretion of proteins involved in pathogenesis, carbohydrate utilization or gliding motility. In Porphyromonas gingivalis, the two-component system PorY sensor and response regulator PorX participate to T9SS gene regulation. Here, we present the crystal structure of PorXFj, the Flavobacterium johnsoniae PorX homolog. As for PorX, the PorXFj structure is comprised of a CheY-like N-terminal domain and an alkaline phosphatase-like C-terminal domain separated by a three-helix bundle central domain. While not activated and monomeric in solution, PorXFj crystallized as a dimer identical to active PorX. The CheY-like domain of PorXFj is in an active-like conformation, and PorXFj possesses phosphodiesterase activity, in agreement with the observation that the active site of its phosphatase-like domain is highly conserved with PorX.

Bacterial Proprioception: Can a Bacterium Sense Its Movement?

The evolution of the bacterial flagellum gave rise to motility and repurposing of a signaling network, now termed the chemotaxis network, enabled biasing of cell movements. This made it possible for the bacterium to seek out favorable chemical environments. To enable chemotaxis, the chemotaxis network sensitively detects extracellular chemical stimuli and appropriately modulates flagellar functions. Additionally, the flagellar motor itself is capable of detecting mechanical stimuli and adapts its structure and function in response, likely triggering a transition from planktonic to surface-associated lifestyles. Recent work has shown a link between the flagellar motor's response to mechanical stimuli and the chemotactic output. Here, we elaborate on this link and discuss how it likely helps the cell sense and adapt to changes in its swimming speeds in different environments. We discuss the mechanism whereby the motor precisely tunes its chemotaxis output under different mechanical loads, analogous to proprioception in higher order organisms. We speculate on the roles bacterial proprioception might play in a variety of phenomena including the transition to surface-associated lifestyles such as swarming and biofilms.