Helicobacter pylori cheV1 mutants recover semisolid agar migration due to loss of a previously uncharacterized Type IV filament membrane alignment complex homolog.

The bacterial chemotaxis system is a well-understood signaling pathway that promotes bacterial success. Chemotaxis systems comprise chemoreceptors and the CheA kinase, linked by CheW or CheV scaffold proteins. Scaffold proteins provide connections between chemoreceptors and CheA and also between chemoreceptors to create macromolecular arrays. Chemotaxis is required for host colonization by many microbes, including the stomach pathogen Helicobacter pylori. This bacterium builds chemoreceptor-CheA contacts with two distinct scaffold proteins, CheW and CheV1. H. pylori cheW or cheV1 deletion mutants both lose chemoreceptor array formation, but show differing semisolid agar chemotaxis assay behaviors: ∆cheW mutants exhibit total migration failure, whereas ∆cheV1::cat mutants display a 50% reduction. On investigating these varied responses, we found that both mutants initially struggle with migration. However, over time, ∆cheV1::cat mutants develop a stable, enhanced migration capability, termed "migration-able" (Mig+). Whole-genome sequencing analysis of four distinct ∆cheV1::cat Mig+ strains identified single-nucleotide polymorphisms (SNPs) in hpg27_252 (hp0273) that were predicted to truncate the encoded protein. Computational analysis of the hpg27_252-encoded protein revealed it encoded a hypothetical protein that was a remote homolog of the PilO Type IV filament membrane alignment complex protein. Although H. pylori lacks Type IV filaments, our analysis showed it retains an operon of genes for homologs of PilO, PilN, and PilM. Deleting hpg27_252 in the ∆cheV1::cat or wild type strain resulted in enhanced migration in semisolid agar. Our study thus reveals that while cheV1 mutants initially have significant migration defects, they can recover the migration ability through genetic suppressors, highlighting a complex regulatory mechanism in bacterial migration. IMPORTANCE: Chemotactic motility, present in over half of bacteria, depends on chemotaxis signaling systems comprising receptors, kinases, and scaffold proteins. In Helicobacter pylori, a stomach pathogen, chemotaxis is crucial for colonization, with CheV1 and CheW as key scaffold proteins. While both scaffolds are essential for building chemoreceptor complexes, their roles vary in other assays. Our research reexamines cheV1 mutants' behavior in semisolid agar, a standard chemotaxis test. Initially, cheV1 mutants exhibited defects similar to those of cheW mutants, but they evolved genetic suppressors that enhanced migration. These suppressors involve mutations in a previously uncharacterized gene, unknown in motility behavior. Our findings highlight the significant chemotaxis defects in cheV1 mutants and identify new elements influencing bacterial motility.

Signal integration in chemoreceptor complexes.

Motile bacteria use large receptor arrays to detect chemical and physical stimuli in their environment, process this complex information, and accordingly bias their swimming in a direction they deem favorable. The chemoreceptor molecules form tripod-like trimers of receptor dimers through direct contacts between their cytoplasmic tips. A pair of trimers, together with a dedicated kinase enzyme, form a core signaling complex. Hundreds of core complexes network to form extended arrays. While considerable progress has been made in revealing the hierarchical structure of the array, the molecular properties underlying signal processing in these structures remain largely unclear. Here we analyzed the signaling properties of nonnetworked core complexes in live cells by following both conformational and kinase control responses to attractant stimuli and to output-biasing lesions at various locations in the receptor molecule. Contrary to the prevailing view that individual receptors are binary two-state devices, we demonstrate that conformational coupling between the ligand binding and the kinase-control receptor domains is, in fact, only moderate. In addition, we demonstrate communication between neighboring receptors through their trimer-contact domains that biases them to adopt similar signaling states. Taken together, these data suggest a view of signaling in receptor trimers that allows significant signal integration to occur within individual core complexes.

A nonequilibrium allosteric model for receptor-kinase complexes: The role of energy dissipation in chemotaxis signaling.

The Escherichia coli chemotaxis signaling pathway has served as a model system for the adaptive sensing of environmental signals by large protein complexes. The chemoreceptors control the kinase activity of CheA in response to the extracellular ligand concentration and adapt across a wide concentration range by undergoing methylation and demethylation. Methylation shifts the kinase response curve by orders of magnitude in ligand concentration while incurring a much smaller change in the ligand binding curve. Here, we show that the disproportionate shift in binding and kinase response is inconsistent with equilibrium allosteric models. To resolve this inconsistency, we present a nonequilibrium allosteric model that explicitly includes the dissipative reaction cycles driven by adenosine triphosphate (ATP) hydrolysis. The model successfully explains all existing joint measurements of ligand binding, receptor conformation, and kinase activity for both aspartate and serine receptors. Our results suggest that the receptor complex acts as an enzyme: Receptor methylation modulates the ON-state kinetics of the kinase (e.g., phosphorylation rate), while ligand binding controls the equilibrium balance between kinase ON/OFF states. Furthermore, sufficient energy dissipation is responsible for maintaining and enhancing the sensitivity range and amplitude of the kinase response. We demonstrate that the nonequilibrium allosteric model is broadly applicable to other sensor-kinase systems by successfully fitting previously unexplained data from the DosP bacterial oxygen-sensing system. Overall, this work provides a nonequilibrium physics perspective on cooperative sensing by large protein complexes and opens up research directions for understanding their microscopic mechanisms through simultaneous measurements and modeling of ligand binding and downstream responses.

Bacterial chemoreceptor signaling complexes control kinase activity by stabilizing the catalytic domain of CheA.

Motile bacteria have a chemotaxis system that enables them to sense their environment and direct their swimming toward favorable conditions. Chemotaxis involves a signaling process in which ligand binding to the extracellular domain of the chemoreceptor alters the activity of the histidine kinase, CheA, bound ~300 Å away to the distal cytoplasmic tip of the receptor, to initiate a phosphorylation cascade that controls flagellar rotation. The cytoplasmic domain of the receptor is thought to propagate this signal via changes in dynamics and/or stability, but it is unclear how these changes modulate the kinase activity of CheA. To address this question, we have used hydrogen deuterium exchange mass spectrometry to probe the structure and dynamics of CheA within functional signaling complexes of the Escherichia coli aspartate receptor cytoplasmic fragment, CheA, and CheW. Our results reveal that stabilization of the P4 catalytic domain of CheA correlates with kinase activation. Furthermore, differences in activation of the kinase that occur during sensory adaptation depend on receptor destabilization of the P3 dimerization domain of CheA. Finally, hydrogen exchange properties of the P1 domain that bears the phosphorylated histidine identify the dimer interface of P1/P1' in the CheA dimer and support an ordered sequential binding mechanism of catalysis, in which dimeric P1/P1' has productive interactions with P4 only upon nucleotide binding. Thus stabilization/destabilization of domains is a key element of the mechanism of modulating CheA kinase activity in chemotaxis, and may play a role in the control of other kinases.

The structural analysis of the periplasmic domain of Sinorhizobium meliloti chemoreceptor McpZ reveals a novel fold and suggests a complex mechanism of transmembrane signaling.

Chemotaxis is a fundamental process whereby bacteria seek out nutrient sources and avoid harmful chemicals. For the symbiotic soil bacterium Sinorhizobium meliloti, the chemotaxis system also plays an essential role in the interaction with its legume host. The chemotactic signaling cascade is initiated through interactions of an attractant or repellent compound with chemoreceptors or methyl-accepting chemotaxis proteins (MCPs). S. meliloti possesses eight chemoreceptors to mediate chemotaxis. Six of these receptors are transmembrane proteins with periplasmic ligand-binding domains (LBDs). The specific functions of McpW and McpZ are still unknown. Here, we report the crystal structure of the periplasmic domain of McpZ (McpZPD) at 2.7 Å resolution. McpZPD assumes a novel fold consisting of three concatenated four-helix bundle modules. Through phylogenetic analyses, we discovered that this helical tri-modular domain fold arose within the Rhizobiaceae family and is still evolving rapidly. The structure, offering a rare view of a ligand-free dimeric MCP-LBD, reveals a novel dimerization interface. Molecular dynamics calculations suggest ligand binding will induce conformational changes that result in large horizontal helix movements within the membrane-proximal domains of the McpZPD dimer that are accompanied by a 5 Å vertical shift of the terminal helix toward the inner cell membrane. These results suggest a mechanism of transmembrane signaling for this family of MCPs that entails both piston-type and scissoring movements. The predicted movements terminate in a conformation that closely mirrors those observed in related ligand-bound MCP-LBDs.

Timescale separation in the coordinated switching of bacterial flagellar motors.

The output of the bacterial chemotaxis signaling pathway, the level of the intracellular regulator CheY-P, modulates the rotation direction of the flagellar motor, thereby regulating bacterial run-and-tumble behavior. The multiple flagellar motors on anE. colicell are controlled by a common cytoplasmic pool of CheY-P. Fluctuation of the CheY-P level was thought to be able to coordinate the switching of multiple motors. Here, we measured the correlation of rotation directions between two motors on a cell, finding that it surprisingly exhibits two well separated timescales. We found that the slow timescale (∼6 s) can be explained by the slow fluctuation of the CheY-P level due to stochastic activity of the chemotactic adaptation enzymes, whereas the fast timescale (∼0.3 s) can be explained by the random pulse-like fluctuation of the CheY-P level, due probably to the activity of the chemoreceptor clusters. We extracted information on the properties of the fast CheY-P pulses based on the correlation measurements. The two well-separated timescales in the fluctuation of CheY-P level help to coordinate multiple motors on a cell and to enhance bacterial chemotactic performance.

Precise Measurement of the Stoichiometry of the Adaptive Bacterial Flagellar Switch.

The cytoplasmic ring (C-ring) of the bacterial flagellar motor controls the motor rotation direction, thereby controlling bacterial run-and-tumble behavior. The C-ring has been shown to undergo adaptive remodeling in response to changes in motor directional bias. However, the stoichiometry and arrangement of the C-ring is still unclear due to contradiction between the results from fluorescence studies and cryo-electron microscopy (cryo-EM) structural analysis. Here, by using the copy number of FliG molecules (34) in the C-ring as a reference, we precisely measured the copy numbers of FliM molecules in motors rotating exclusively counterclockwise (CCW) and clockwise (CW). We surprisingly found that there are on average 45 and 58 FliM molecules in CW and CCW rotating motors, respectively, which are much higher than previous estimates. Our results suggested a new mechanism of C-ring adaptation, that is, extra FliM molecules could be bound to the primary C-ring with probability depending on the motor rotational direction. We further confirmed that all of the FliM molecules in the C-ring function in chemotaxis signaling transduction because all of them could be bound by the chemotactic response regulator CheY-P. Our measurements provided new insights into the structure and arrangement of the flagellar switch. IMPORTANCE The bacterial flagellar switch can undergo adaptive remodeling in response to changes in motor rotation direction, thereby shifting its operating point to match the output of the chemotaxis signaling pathway. However, it remains unclear how the flagellar switch accomplishes this adaptive remodeling. Here, via precise fluorescence studies, we measured the absolute copy numbers of the critical component in the switch for motors rotating counterclockwise and clockwise, obtaining much larger numbers than previous relative estimates. Our results suggested a new mechanism of adaptive remodeling of the flagellar switch and provided new insights for updating the conformation spread model of the switch.

Chemotaxis Coupling Protein CheW2 Is Not Required for the Chemotaxis but Contributes to the Full Pathogenicity of Borreliella burgdorferi.

The bacterial chemotaxis regulatory circuit mainly consists of coupling protein CheW, sensor histidine kinase CheA, and response regulator CheY. Most bacteria, such as Escherichia coli, have a single gene encoding each of these proteins. Interestingly, the Lyme disease pathogen, Borreliella burgdorferi, has multiple chemotaxis proteins, e.g., two CheA, three CheW, and three CheY proteins. The genes encoding these proteins mainly reside in two operons: cheW2-cheA1-cheB2-cheY2 (A-I) and cheA2-cheW3-cheX-cheY3 (A-II). Previous studies demonstrate that all the genes in A-II are essential for the chemotaxis of B. burgdorferi; however, the role of those genes in A-I remains unknown. This study aimed to fill this gap using the CheW2 gene, the first gene in A-I, as a surrogate. We first mapped the transcription start site of A-I upstream of cheW2 and identified a σ70-like promoter (PW2) and two binding sites (BS1 and BS2) of BosR, an unorthodox Fur/Per homolog. We then demonstrated that BosR binds to PW2 via BS1 and BS2 and that deletion of bosR significantly represses the expression of cheW2 and other genes in A-I, implying that BosR is a positive regulator of A-I. Deletion of cheW2 has no impact on the chemotaxis of B. burgdorferi in vitro but abrogates its ability to evade host adaptive immunity, because the mutant can establish systemic infection only in SCID mice and not in immunocompetent BALB/c mice. This report substantiates the previous proposition that A-I is not implicated in chemotaxis; rather, it may function as a signaling transduction pathway to regulate B. burgdorferi virulence gene expression.

Analysis of CheW-like domains provides insights into organization of prokaryotic chemotaxis systems.

The ability to control locomotion in a dynamic environment provides a competitive advantage for microorganisms, thus driving the evolution of sophisticated regulatory systems. In total, 19 known categories of chemotaxis systems control motility mediated by flagella or Type IV pili, plus other cellular functions. A key feature that distinguishes chemotaxis systems from generic two-component regulatory systems is separation of receptor and kinase functions into distinct proteins, linked by CheW scaffold proteins. This arrangement allows for formation of varied arrays with remarkable signaling properties. We recently analyzed sequences of CheW-like domains found in CheA kinases and CheW and CheV scaffold proteins. In total, 16 Architectures of CheA, CheW, and CheV proteins contain ~94% of all CheW-like domains and form six Classes with likely functional specializations. We surveyed chemotaxis system categories and proteins containing CheW-like domains in ~1900 prokaryotic species, the most comprehensive analysis to date, revealing new insights. Co-occurrence analyses suggested that many chemotaxis systems occur in non-random combinations within species, implying synergy or antagonism. Furthermore, many Architectures of proteins containing CheW-like domains occurred predominantly with specific categories of chemotaxis systems, suggesting specialized functional interactions. We propose Class 1 (~80%) and Class 6 (~20%) CheW proteins exhibit preferences for distinct chemoreceptor structures. Furthermore, rare (~1%) Class 2 CheW proteins frequently co-occurred with methyl-accepting coiled coil proteins, which contain both receptor and kinase functions and so do not require connection via a CheW scaffold but may benefit from arrays. Last, rare multidomain CheW proteins may interact with different receptors than single-domain CheW proteins.

Mechanisms of E. coli chemotaxis signaling pathways visualized using cryoET and computational approaches.

Chemotaxis signaling pathways enable bacteria to sense and respond to their chemical environment and, in some species, are critical for lifestyle processes such as biofilm formation and pathogenesis. The signal transduction underlying chemotaxis behavior is mediated by large, highly ordered protein complexes known as chemosensory arrays. For nearly two decades, cryo-electron tomography (cryoET) has been used to image chemosensory arrays, providing an increasingly detailed understanding of their structure and function. In this mini-review, we provide an overview of the use of cryoET to study chemosensory arrays, including imaging strategies, key results, and outstanding questions. We further discuss the application of molecular modeling and simulation techniques to complement structure determination efforts and provide insight into signaling mechanisms. We close the review with a brief outlook, highlighting promising future directions for the field.

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.

Methylation-Independent Chemotaxis Systems Are the Norm for Gastric-Colonizing Helicobacter Species.

Many bacteria and archaea rely on chemotaxis signal transduction systems for optimal fitness. These complex, multiprotein signaling systems have core components found in all chemotactic microbes, as well as variable proteins found in only some species. We do not yet understand why these variations exist or whether there are specific niches that favor particular chemotaxis signaling organization. One variation is in the presence/absence of the chemotaxis methylation adaptation enzymes CheB and CheR. Genes for CheB and CheR are missing in the gastric pathogen Helicobacter pylori but present in related Helicobacter that colonize the liver or intestine. In this work, we asked whether there was a general pattern of CheB/CheR across multiple Helicobacter species. Helicobacter spp. all possess chemotactic behavior, based on the presence of genes for core signaling proteins CheA, CheW, and chemoreceptors. Genes for the CheB and CheR proteins, in contrast, were variably present. Niche mapping supported the idea that these genes were present in enterohepatic Helicobacter species and absent in gastric ones. We then analyzed whether there were differences between gastric and enterohepatic species in the CheB/CheR chemoreceptor target methylation sites. Indeed, these sites were less conserved in gastric species that lack CheB/CheR. Lastly, we determined that cheB and cheR could serve as markers to indicate whether an unknown Helicobacter species was of enterohepatic or gastric origin. Overall, these findings suggest the interesting idea that methylation-based adaptation is not required in specific environments, particularly the stomach. IMPORTANCE Chemotaxis signal transduction systems are common in the archaeal and bacterial world, but not all systems contain the same components. The rationale for this system variation remains unknown. In this report, comparative genomics analysis showed that the presence/absence of CheR and CheB is one main variation within the Helicobacter genus, and it is strongly associated with the niche of Helicobacter species: gastric Helicobacter species, which infect animal stomachs, have lost their CheB and CheR, while enterohepatic Helicobacter species, which infect the liver and intestine, retain them. This study not only provides an example that a chemotaxis system variant is associated with particular niches but also proposes that CheB and CheR are new markers distinguishing gastric from enterohepatic Helicobacter species.

Signal binding at both modules of its dCache domain enables the McpA chemoreceptor of Bacillus velezensis to sense different ligands.

Bacteria have evolved multiple signal transduction systems that permit an adaptation to changing environmental conditions. Chemoreceptor-based signaling cascades are very abundant in bacteria and are among the most complex signaling systems. Currently, our knowledge on the molecular features that determine signal recognition at chemoreceptors is limited. Chemoreceptor McpA of Bacillus velezensis SQR9 has been shown to mediate chemotaxis to a broad range of different ligands. Here we show that its ligand binding domain binds directly 13 chemoattractants. We provide support that organic acids and amino acids bind to the membrane-distal and membrane-proximal module of the dCache domain, respectively, whereas binding of sugars/sugar alcohols occurred at both modules. Structural biology studies combined with site-directed mutagenesis experiments have permitted to identify 10 amino acid residues that play key roles in the recognition of multiple ligands. Residues in membrane-distal and membrane-proximal regions were central for sensing organic acids and amimo acids, respectively, whereas all residues participated in sugars/sugar alcohol sensing. Most characterized chemoreceptors possess a narrow and well-defined ligand spectrum. We propose here a sensing mechanism involving both dCache modules that allows the integration of very diverse signals by a single chemoreceptor.

Structural signatures of Escherichia coli chemoreceptor signaling states revealed by cellular crosslinking.

The chemotaxis machinery of Escherichia coli has served as a model for exploring the molecular signaling mechanisms of transmembrane chemoreceptors known as methyl-accepting chemotaxis proteins (MCPs). Yet, fundamental questions about signal transmission through MCP molecules remain unanswered. Our work with the E. coli serine chemoreceptor Tsr has developed in vivo reporters that distinguish kinase-OFF and kinase-ON structures in the cytoplasmic methylation helix (MH) cap, which receives stimulus signals from an adjoining, membrane-proximal histidine kinase, adenylyl cyclases, MCPs, and phosphatases (HAMP) domain. The cytoplasmic helices of the Tsr homodimer interact mainly through packing interactions of hydrophobic residues at a and d heptad positions. We investigated the in vivo crosslinking properties of Tsr molecules bearing cysteine replacements at functionally tolerant g heptad positions in the N-terminal and C-terminal cap helices. Upon treatment of cells with bismaleimidoethane (BMOE), a bifunctional thiol-reagent, Tsr-G273C/Q504C readily formed a doubly crosslinked product in the presence of serine but not in its absence. Moreover, a serine stimulus combined with BMOE treatment during in vivo Förster resonance energy transfer-based kinase assays locked Tsr-G273C/Q504C in kinase-OFF output. An OFF-shifting lesion in MH1 (D269P) promoted the formation of the doubly crosslinked species in the absence of serine, whereas an ON-shifting lesion (G268P) suppressed the formation of the doubly crosslinked species. Tsr-G273C/Q504C also showed output-dependent crosslinking patterns in combination with ON-shifting and OFF-shifting adaptational modifications. Our results are consistent with a helix breathing-axial rotation-bundle repacking signaling mechanism and imply that in vivo crosslinking tools could serve to probe helix-packing transitions and their output consequences in other regions of the receptor molecule.

Structural insights into the mechanism of archaellar rotational switching.

Signal transduction via phosphorylated CheY towards the flagellum and the archaellum involves a conserved mechanism of CheY phosphorylation and subsequent conformational changes within CheY. This mechanism is conserved among bacteria and archaea, despite substantial differences in the composition and architecture of archaellum and flagellum, respectively. Phosphorylated CheY has higher affinity towards the bacterial C-ring and its binding leads to conformational changes in the flagellar motor and subsequent rotational switching of the flagellum. In archaea, the adaptor protein CheF resides at the cytoplasmic face of the archaeal C-ring formed by the proteins ArlCDE and interacts with phosphorylated CheY. While the mechanism of CheY binding to the C-ring is well-studied in bacteria, the role of CheF in archaea remains enigmatic and mechanistic insights are absent. Here, we have determined the atomic structures of CheF alone and in complex with activated CheY by X-ray crystallography. CheF forms an elongated dimer with a twisted architecture. We show that CheY binds to the C-terminal tail domain of CheF leading to slight conformational changes within CheF. Our structural, biochemical and genetic analyses reveal the mechanistic basis for CheY binding to CheF and allow us to propose a model for rotational switching of the archaellum.

Noncanonical Sensing Mechanisms for Bacillus subtilis Chemoreceptors.

Bodhankar et al. reported a noncanonical sensing mechanism that involves signal interaction with the McpA chemoreceptor signaling domain resulting in a chemorepellence response of Bacillus subtilis. The identified repellent binding site is analogous to that for attractant binding in McpB, another B. subtilis chemoreceptor.

Studying bacterial chemosensory array with CryoEM.

Bacteria direct their movement in respond to gradients of nutrients and other stimuli in the environment through the chemosensory system. The behavior is mediated by chemosensory arrays that are made up of thousands of proteins to form an organized array near the cell pole. In this review, we briefly introduce the architecture and function of the chemosensory array and its core signaling unit. We describe the in vivo and in vitro systems that have been used for structural studies of chemosensory array by cryoEM, including reconstituted lipid nanodiscs, 2D lipid monolayer arrays, lysed bacterial ghosts, bacterial minicells and native bacteria cells. Lastly, we review recent advances in structural analysis of chemosensory arrays using state-of-the-art cryoEM and cryoET methodologies, focusing on the latest developments and insights with a perspective on current challenges and future directions.

Mechanosensitive recruitment of stator units promotes binding of the response regulator CheY-P to the flagellar motor.

Reversible switching of the bacterial flagellar motor between clockwise (CW) and counterclockwise (CCW) rotation is necessary for chemotaxis, which enables cells to swim towards favorable chemical habitats. Increase in the viscous resistance to the rotation of the motor (mechanical load) inhibits switching. However, cells must maintain homeostasis in switching to navigate within environments of different viscosities. The mechanism by which the cell maintains optimal chemotactic function under varying loads is not understood. Here, we show that the flagellar motor allosterically controls the binding affinity of the chemotaxis response regulator, CheY-P, to the flagellar switch complex by modulating the mechanical forces acting on the rotor. Mechanosensitive CheY-P binding compensates for the load-induced loss of switching by precisely adapting the switch response to a mechanical stimulus. The interplay between mechanical forces and CheY-P binding tunes the chemotactic function to match the load. This adaptive response of the chemotaxis output to mechanical stimuli resembles the proprioceptive feedback in the neuromuscular systems of insects and vertebrates.

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.

Pseudomonas aeruginosa as a Model To Study Chemosensory Pathway Signaling.

Bacteria have evolved a variety of signal transduction mechanisms that generate different outputs in response to external stimuli. Chemosensory pathways are widespread in bacteria and are among the most complex signaling mechanisms, requiring the participation of at least six proteins. These pathways mediate flagellar chemotaxis, in addition to controlling alternative functions such as second messenger levels or twitching motility. The human pathogen Pseudomonas aeruginosa has four different chemosensory pathways that carry out different functions and are stimulated by signal binding to 26 chemoreceptors. Recent research employing a diverse range of experimental approaches has advanced enormously our knowledge on these four pathways, establishing P. aeruginosa as a primary model organism in this field. In the first part of this article, we review data on the function and physiological relevance of chemosensory pathways as well as their involvement in virulence, whereas the different transcriptional and posttranscriptional regulatory mechanisms that govern pathway function are summarized in the second part. The information presented will be of help to advance the understanding of pathway function in other organisms.