The Cellular Abundance of Chemoreceptors, Chemosensory Signaling Proteins, Sensor Histidine Kinases, and Solute Binding Proteins of Pseudomonas aeruginosa Provides Insight into Sensory Preferences and Signaling Mechanisms.

Chemosensory pathways and two-component systems are important bacterial signal transduction systems. In the human pathogen Pseudomonas aeruginosa, these systems control many virulence traits. Previous studies showed that inorganic phosphate (Pi) deficiency induces virulence. We report here the abundance of chemosensory and two-component signaling proteins of P. aeruginosa grown in Pi deficient and sufficient media. The cellular abundance of chemoreceptors differed greatly, since a 2400-fold difference between the most and least abundant receptors was observed. For many chemoreceptors, their amount varied with the growth condition. The amount of chemoreceptors did not correlate with the magnitude of chemotaxis to their cognate chemoeffectors. Of the four chemosensory pathways, proteins of the Che chemotaxis pathway were most abundant and showed little variation in different growth conditions. The abundance of chemoreceptors and solute binding proteins indicates a sensing preference for amino acids and polyamines. There was an excess of response regulators over sensor histidine kinases in two-component systems. In contrast, ratios of the response regulators CheY and CheB to the histidine kinase CheA of the Che pathway were all below 1, indicative of different signaling mechanisms. This study will serve as a reference for exploring sensing preferences and signaling mechanisms of other bacteria.

The pH Robustness of Bacterial Sensing.

Bacteria have evolved many different signal transduction systems to sense and respond to changing environmental conditions. Signal integration is mainly achieved by signal recognition at extracytosolic ligand-binding domains (LBDs) of receptors. Hundreds of different LBDs have been reported, and our understanding of their sensing properties is growing. Receptors must function over a range of environmental pH values, but there is little information available on the robustness of sensing as a function of pH. Here, we have used isothermal titration calorimetry to determine the pH dependence of ligand recognition by nine LBDs that cover all major LBD superfamilies, of periplasmic solute-binding proteins, and cytosolic LBDs. We show that periplasmic LBDs recognize ligands over a very broad pH range, frequently stretching over eight pH units. This wide pH range contrasts with a much narrower pH response range of the cytosolic LBDs analyzed. Many LBDs must be dimeric to bind ligands, and analytical ultracentrifugation studies showed that the LBD of the Tar chemoreceptor forms dimers over the entire pH range tested. The pH dependences of Pseudomonas aeruginosa motility and chemotaxis were bell-shaped and centered at pH 7.0. Evidence for pH robustness of signaling in vivo was obtained by Förster Resonance Energy Transfer (FRET) measurements of the chemotaxis pathway responses in Escherichia coli. Bacteria have evolved several strategies to cope with extreme pH, such as periplasmic chaperones for protein refolding. The intrinsic pH resistance of periplasmic LBDs appears to be another strategy that permits bacteria to survive under adverse conditions. IMPORTANCE Demonstration of the pH robustness of extracytoplasmic sensing reveals a previously undescribed evolutionary mechanism that enables bacteria to monitor environmental changes under changing conditions. This mechanism includes the maintenance of the dimeric state of four-helixbundle ligand-binding domains (LBDs). The construction of biosensors is a rapidly growing field of research, and their use to monitor the progression of the COVID-19 pandemic has impressively demonstrated their usefulness. LBDs represent an enormous reservoir of binding modules that can be used to create novel biosensors. Among ligands recognized by LBDs are neurotransmitters, hormones, and quorum-sensing signals. The demonstration that extracytosolic LBDs bind their signals over a wide range of pH values will facilitate the design of biosensors that function under highly variable conditions of acidity and alkalinity.

Chemotaxis of the Human Pathogen Pseudomonas aeruginosa to the Neurotransmitter Acetylcholine.

Acetylcholine is a central biological signal molecule present in all kingdoms of life. In humans, acetylcholine is the primary neurotransmitter of the peripheral nervous system; it mediates signal transmission at neuromuscular junctions. Here, we show that the opportunistic human pathogen Pseudomonas aeruginosa exhibits chemoattraction toward acetylcholine over a concentration range of 1 µM to 100 mM. The maximal magnitude of the response was superior to that of many other P. aeruginosa chemoeffectors. We demonstrate that this chemoattraction is mediated by the PctD (PA4633) chemoreceptor. Using microcalorimetry, we show that the PctD ligand-binding domain (LBD) binds acetylcholine with a equilibrium dissociation constant (KD) of 23 µM. It also binds choline and with lower affinity betaine. Highly sensitive responses to acetylcholine and choline, and less sensitive responses to betaine and l-carnitine, were observed in Escherichia coli expressing a chimeric receptor comprising the PctD-LBD fused to the Tar chemoreceptor signaling domain. We also identified the PacA (ECA_RS10935) chemoreceptor of the phytopathogen Pectobacterium atrosepticum, which binds choline and betaine but fails to recognize acetylcholine. To identify the molecular determinants for acetylcholine recognition, we report high-resolution structures of PctD-LBD (with bound acetylcholine and choline) and PacA-LBD (with bound betaine). We identified an amino acid motif in PctD-LBD that interacts with the acetylcholine tail. This motif is absent in PacA-LBD. Significant acetylcholine chemotaxis was also detected in the plant pathogens Agrobacterium tumefaciens and Dickeya solani. To the best of our knowledge, this is the first report of acetylcholine chemotaxis and extends the range of host signals perceived by bacterial chemoreceptors. IMPORTANCE P. aeruginosa causes a significant number of deaths annually worldwide. For many pathogens, chemotaxis plays an import role in the initial stages of infection, and deciphering the key chomoeffectors and their cognate chemoreceptors may permit the development of strategies to inhibit this process. Genome analyses have shown that many bacteria possess a large number of chemoreceptors. The chemoeffectors recognized by the large majority of chemoreceptors are unknown. However, identifying these chemoeffectors is crucial for deciphering the evolutionary forces that have shaped chemosensory signaling mechanisms in bacteria with different lifestyles. Our current understanding of the relationship between bacterial lifestyle and chemoreceptor repertoire is limited, and this work contributes to closing this gap in our knowledge. By expanding the list of known chemoeffectors and chemoreceptors, progress is made toward identifying functional receptor homologs in other bacteria.

A catalogue of signal molecules that interact with sensor kinases, chemoreceptors and transcriptional regulators.

Bacteria have evolved many different signal transduction systems that sense signals and generate a variety of responses. Generally, most abundant are transcriptional regulators, sensor histidine kinases and chemoreceptors. Typically, these systems recognize their signal molecules with dedicated ligand-binding domains (LBDs), which, in turn, generate a molecular stimulus that modulates the activity of the output module. There are an enormous number of different LBDs that recognize a similarly diverse set of signals. To give a global perspective of the signals that interact with transcriptional regulators, sensor kinases and chemoreceptors, we manually retrieved information on the protein-ligand interaction from about 1,200 publications and 3D structures. The resulting 811 proteins were classified according to the Pfam family into 127 groups. These data permit a delineation of the signal profiles of individual LBD families as well as distinguishing between families that recognize signals in a promiscuous manner and those that possess a well-defined ligand range. A major bottleneck in the field is the fact that the signal input of many signaling systems is unknown. The signal repertoire reported here will help the scientific community design experimental strategies to identify the signaling molecules for uncharacterised sensor proteins.

Reduction of alternative electron acceptors drives biofilm formation in Shewanella algae.

Shewanella spp. possess a broad respiratory versatility, which contributes to the occupation of hypoxic and anoxic environmental or host-associated niches. Here, we observe a strain-specific induction of biofilm formation in response to supplementation with the anaerobic electron acceptors dimethyl sulfoxide (DMSO) and nitrate in a panel of Shewanella algae isolates. The respiration-driven biofilm response is not observed in DMSO and nitrate reductase deletion mutants of the type strain S. algae CECT 5071, and can be restored upon complementation with the corresponding reductase operon(s) but not by an operon containing a catalytically inactive nitrate reductase. The distinct transcriptional changes, proportional to the effect of these compounds on biofilm formation, include cyclic di-GMP (c-di-GMP) turnover genes. In support, ectopic expression of the c-di-GMP phosphodiesterase YhjH of Salmonella Typhimurium but not its catalytically inactive variant decreased biofilm formation. The respiration-dependent biofilm response of S. algae may permit differential colonization of environmental or host niches.

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.

The use of isothermal titration calorimetry to unravel chemotactic signalling mechanisms.

Chemotaxis is based on the action of chemosensory pathways and is typically initiated by the recognition of chemoeffectors at chemoreceptor ligand-binding domains (LBD). Chemosensory signalling is highly complex; aspect that is not only reflected in the intricate interaction between many signalling proteins but also in the fact that bacteria frequently possess multiple chemosensory pathways and often a large number of chemoreceptors, which are mostly of unknown function. We review here the usefulness of isothermal titration calorimetry (ITC) to study this complexity. ITC is the gold standard for studying binding processes due to its precision and sensitivity, as well as its capability to determine simultaneously the association equilibrium constant, enthalpy change and stoichiometry of binding. There is now evidence that members of all major LBD families can be produced as individual recombinant proteins that maintain their ligand-binding properties. High-throughput screening of these proteins using thermal shift assays offer interesting initial information on chemoreceptor ligands, providing the basis for microcalorimetric analyses and microbiological experimentation. ITC has permitted the identification and characterization of many chemoreceptors with novel specificities. This ITC-based approach can also be used to identify signal molecules that stimulate members of other families of sensor proteins.

Determination of Ligand Profiles for Pseudomonas aeruginosa Solute Binding Proteins.

Solute binding proteins (SBPs) form a heterogeneous protein family that is found in all kingdoms of life. In bacteria, the ligand-loaded forms bind to transmembrane transporters providing the substrate. We present here the SBP repertoire of Pseudomonas aeruginosa PAO1 that is composed of 98 proteins. Bioinformatic predictions indicate that many of these proteins have a redundant ligand profile such as 27 SBPs for proteinogenic amino acids, 13 proteins for spermidine/putrescine, or 9 proteins for quaternary amines. To assess the precision of these bioinformatic predictions, we have purified 17 SBPs that were subsequently submitted to high-throughput ligand screening approaches followed by isothermal titration calorimetry studies, resulting in the identification of ligands for 15 of them. Experimentation revealed that PA0222 was specific for γ-aminobutyrate (GABA), DppA2 for tripeptides, DppA3 for dipeptides, CysP for thiosulphate, OpuCC for betaine, and AotJ for arginine. Furthermore, RbsB bound D-ribose and D-allose, ModA bound molybdate, tungstate, and chromate, whereas AatJ recognized aspartate and glutamate. The majority of experimentally identified ligands were found to be chemoattractants. Data show that the ligand class recognized by SPBs can be predicted with confidence using bioinformatic methods, but experimental work is necessary to identify the precise ligand profile.

The Molecular Mechanism of Nitrate Chemotaxis via Direct Ligand Binding to the PilJ Domain of McpN.

Chemotaxis and energy taxis permit directed bacterial movements in gradients of environmental cues. Nitrate is a final electron acceptor for anaerobic respiration and can also serve as a nitrogen source for aerobic growth. Previous studies indicated that bacterial nitrate taxis is mediated by energy taxis mechanisms, which are based on the cytosolic detection of consequences of nitrate metabolism. Here we show that Pseudomonas aeruginosa PAO1 mediates nitrate chemotaxis on the basis of specific nitrate sensing by the periplasmic PilJ domain of the PA2788/McpN chemoreceptor. The presence of nitrate reduced mcpN transcript levels, and McpN-mediated taxis occurred only under nitrate starvation conditions. In contrast to the NarX and NarQ sensor kinases, McpN bound nitrate specifically and showed no affinity for other ligands such as nitrite. We report the three-dimensional structure of the McpN ligand binding domain (LBD) at 1.3-Å resolution in complex with nitrate. Although structurally similar to 4-helix bundle domains, the ligand binding mode differs since a single nitrate molecule is bound to a site on the dimer symmetry axis. As for 4-helix bundle domains, ligand binding stabilized the McpN-LBD dimer. McpN homologues showed a wide phylogenetic distribution, indicating that nitrate chemotaxis is a widespread phenotype. These homologues were particularly abundant in bacteria that couple sulfide/sulfur oxidation with nitrate reduction. This work expands the range of known chemotaxis effectors and forms the basis for the exploration of nitrate chemotaxis in other bacteria and for the study of its physiological role.IMPORTANCE Nitrate is of central importance in bacterial physiology. Previous studies indicated that movements toward nitrate are due to energy taxis, which is based on the cytosolic sensing of consequences of nitrate metabolism. Here we present the first report on nitrate chemotaxis. This process is initiated by specific nitrate binding to the periplasmic ligand binding domain (LBD) of McpN. Nitrate chemotaxis is highly regulated and occurred only under nitrate starvation conditions, which is helpful information to explore nitrate chemotaxis in other bacteria. We present the three-dimensional structure of the McpN-LBD in complex with nitrate, which is the first structure of a chemoreceptor PilJ-type domain. This structure reveals striking similarities to that of the abundant 4-helix bundle domain but employs a different sensing mechanism. Since McpN homologues show a wide phylogenetic distribution, nitrate chemotaxis is likely a widespread phenomenon with importance for the life cycle of ecologically diverse bacteria.

Functional Annotation of Bacterial Signal Transduction Systems: Progress and Challenges.

Bacteria possess a large number of signal transduction systems that sense and respond to different environmental cues. Most frequently these are transcriptional regulators, two-component systems and chemosensory pathways. A major bottleneck in the field of signal transduction is the lack of information on signal molecules that modulate the activity of the large majority of these systems. We review here the progress made in the functional annotation of sensor proteins using high-throughput ligand screening approaches of purified sensor proteins or individual ligand binding domains. In these assays, the alteration in protein thermal stability following ligand binding is monitored using Differential Scanning Fluorimetry. We illustrate on several examples how the identification of the sensor protein ligand has facilitated the elucidation of the molecular mechanism of the regulatory process. We will also discuss the use of virtual ligand screening approaches to identify sensor protein ligands. Both approaches have been successfully applied to functionally annotate a significant number of bacterial sensor proteins but can also be used to study proteins from other kingdoms. The major challenge consists in the study of sensor proteins that do not recognize signal molecules directly, but that are activated by signal molecule-loaded binding proteins.

High-Affinity Chemotaxis to Histamine Mediated by the TlpQ Chemoreceptor of the Human Pathogen Pseudomonas aeruginosa.

Histamine is a key biological signaling molecule. It acts as a neurotransmitter in the central and peripheral nervous systems and coordinates local inflammatory responses by modulating the activity of different immune cells. During inflammatory processes, including bacterial infections, neutrophils stimulate the production and release of histamine. Here, we report that the opportunistic human pathogen Pseudomonas aeruginosa exhibits chemotaxis toward histamine. This chemotactic response is mediated by the concerted action of the TlpQ, PctA, and PctC chemoreceptors, which display differing sensitivities to histamine. Low concentrations of histamine were sufficient to activate TlpQ, which binds histamine with an affinity of 639 nM. To explore this binding, we resolved the high-resolution structure of the TlpQ ligand binding domain in complex with histamine. It has an unusually large dCACHE domain and binds histamine through a highly negatively charged pocket at its membrane distal module. Chemotaxis to histamine may play a role in the virulence of P. aeruginosa by recruiting cells at the infection site and consequently modulating the expression of quorum-sensing-dependent virulence genes. TlpQ is the first bacterial histamine receptor to be described and greatly differs from human histamine receptors, indicating that eukaryotes and bacteria have pursued different strategies for histamine recognition.IMPORTANCE Genome analyses indicate that many bacteria possess an elevated number of chemoreceptors, suggesting that these species are able to perform chemotaxis to a wide variety of compounds. The scientific community is now only beginning to explore this diversity and to elucidate the corresponding physiological relevance. The discovery of histamine chemotaxis in the human pathogen Pseudomonas aeruginosa provides insight into tactic movements that occur within the host. Since histamine is released in response to bacterial pathogens, histamine chemotaxis may permit bacterial migration and accumulation at infection sites, potentially modulating, in turn, quorum-sensing-mediated processes and the expression of virulence genes. As a consequence, the modulation of histamine chemotaxis by signal analogues may result in alterations of the bacterial virulence. As the first report of bacterial histamine chemotaxis, this study lays the foundation for the exploration of the physiological relevance of histamine chemotaxis and its role in pathogenicity.

Structural Basis for Polyamine Binding at the dCACHE Domain of the McpU Chemoreceptor from Pseudomonas putida.

Many bacteria can move chemotactically to a variety of compounds and the recognition of chemoeffectors by the chemoreceptor ligand binding domain (LBD) defines the specificity of response. Many chemoreceptors were found to recognize different amino and organic acids, but the McpU chemoreceptor from Pseudomonas putida was identified as the first chemoreceptor that bound specifically polyamines. We report here the three-dimensional structure of McpU-LBD in complex with putrescine at a resolution of 2.4 Å, which fitted well a solution structure generated by small-angle X-ray scattering. Putrescine bound to a negatively charged pocket in the membrane distal module of McpU-LBD. Similarities exist in the binding of putrescine to McpU-LBD and taurine to the LBD of the Mlp37 chemoreceptor of Vibrio cholerae. In both structures, the primary amino group of the respective ligand is recognized by hydrogen bonds established by two aspartate and a tyrosine side chain. This feature may be used to predict the ligands of chemoreceptors with unknown function. Analytical ultracentrifugation revealed that McpU-LBD is monomeric in solution and that ligand binding does not alter this oligomeric state. This sensing mode thus differs from that of the well-characterised four-helix bundle domains where ligands bind to two sites at the LBD dimer interface. Although there appear to be different sensing modes, results are discussed in the context of data, indicating that chemoreceptors employ the same mechanism of transmembrane signaling. This work enhances our understanding of CACHE domains, which are the most abundant sensor domains in bacterial chemoreceptors and sensor kinases.

The activity of the C4-dicarboxylic acid chemoreceptor of Pseudomonas aeruginosa is controlled by chemoattractants and antagonists.

Chemotaxis toward organic acids has been associated with colonization fitness and virulence and the opportunistic pathogen Pseudomonas aeruginosa exhibits taxis toward several tricarboxylic acid intermediates. In this study, we used high-throughput ligand screening and isothermal titration calorimetry to demonstrate that the ligand binding domain (LBD) of the chemoreceptor PA2652 directly recognizes five C4-dicarboxylic acids with KD values ranging from 23 µM to 1.24 mM. In vivo experimentation showed that three of the identified ligands act as chemoattractants whereas two of them behave as antagonists by inhibiting the downstream chemotaxis signalling cascade. In vitro and in vivo competition assays showed that antagonists compete with chemoattractants for binding to PA2652-LBD, thereby decreasing the affinity for chemoattractants and the subsequent chemotactic response. Two chemosensory pathways encoded in the genome of P. aeruginosa, che and che2, have been associated to chemotaxis but we found that only the che pathway is involved in PA2652-mediated taxis. The receptor PA2652 is predicted to contain a sCACHE LBD and analytical ultracentrifugation analyses showed that PA2652-LBD is dimeric in the presence and the absence of ligands. Our results indicate the feasibility of using antagonists to interfere specifically with chemotaxis, which may be an alternative strategy to fight bacterial pathogens.

High-Throughput Screening to Identify Chemoreceptor Ligands.

The majority of bacterial chemoreceptors remain functionally un-annotated. The knowledge of chemoreceptor function, however, is indispensable to understanding the evolution of the chemotaxis system in bacteria with different lifestyles. Significant progress in the annotation of chemoreceptor function has been made using experimental strategies that are based on the individual, genetically engineered ligand binding domain (LBD) of chemoreceptors. There is now evidence that all major classes of LBDs can be produced as individual domains that retain their ligand binding activity. Here, we provide a protocol for the combined use of high-throughput ligand screening using Differential Scanning Fluorimetry followed by Isothermal Titration Calorimetry to identify and characterize ligands that bind to recombinant chemoreceptor LBDs. This approach has been shown to be very efficient for determining the function of novel chemoreceptors.

Identification of a Chemoreceptor in Pseudomonas aeruginosa That Specifically Mediates Chemotaxis Toward α-Ketoglutarate.

Pseudomonas aeruginosa is an ubiquitous pathogen able to infect humans, animals, and plants. Chemotaxis was found to be associated with the virulence of this and other pathogens. Although established as a model for chemotaxis research, the majority of the 26 P. aeruginosa chemoreceptors remain functionally un-annotated. We report here the identification of PA5072 (named McpK) as chemoreceptor for α-ketoglutarate (αKG). High-throughput thermal shift assays and isothermal titration calorimetry studies (ITC) of the recombinant McpK ligand binding domain (LBD) showed that it recognizes exclusively α-ketoglutarate. The ITC analysis indicated that the ligand bound with positive cooperativity (Kd1 = 301 µM, Kd2 = 81 µM). McpK is predicted to possess a helical bimodular (HBM) type of LBD and this and other studies suggest that this domain type may be associated with the recognition of organic acids. Analytical ultracentrifugation (AUC) studies revealed that McpK-LBD is present in monomer-dimer equilibrium. Alpha-KG binding stabilized the dimer and dimer self-dissociation constants of 55 µM and 5.9 µM were derived for ligand-free and αKG-bound forms of McpK-LBD, respectively. Ligand-induced LBD dimer stabilization has been observed for other HBM domain containing receptors and may correspond to a general mechanism of this protein family. Quantitative capillary chemotaxis assays demonstrated that P. aeruginosa showed chemotaxis to a broad range of αKG concentrations with maximal responses at 500 µM. Deletion of the mcpK gene reduced chemotaxis over the entire concentration range to close to background levels and wild type like chemotaxis was recovered following complementation. Real-time PCR studies indicated that the presence of αKG does not modulate mcpK expression. Since αKG is present in plant root exudates it was investigated whether the deletion of mcpK altered maize root colonization. However, no significant changes with respect to the wild type strain were observed. The existence of a chemoreceptor specific for αKG may be due to its central metabolic role as well as to its function as signaling molecule. This work expands the range of known chemoreceptor types and underlines the important physiological role of chemotaxis toward tricarboxylic acid cycle intermediates.

McpQ is a specific citrate chemoreceptor that responds preferentially to citrate/metal ion complexes.

Chemoreceptors are at the beginning of chemosensory pathways that mediate chemotaxis. Pseudomonas putida KT2440 is predicted to have 27 chemoreceptors, most of which uncharacterized. We have previously identified McpS as chemoreceptor for Krebs cycle intermediates. Citrate is primarily present in the environment as metal complex, which, however, is not recognized by McpS. We show here that the McpS paralogue McpQ recognizes specifically citrate and citrate/metal2+ complexes. The McpQ ligand binding domain (McpQ-LBD) binds citrate/metal2+ complexes with higher affinity than citrate. McpQ-LBD is present in a monomer-dimer equilibrium and citrate and particularly citrate/Mg2+ binding stabilize the dimer. The bacterium showed much stronger responses to citrate/Mg2+ than to citrate and mcpQ inactivation caused a dramatic reduction in chemotaxis. Responses to Krebs cycle intermediates are thus mediated by the broad range McpS and McpQ that responds specifically to an intermediate not recognized by McpS. Interesting parallels exist to the paralogous amino acid chemoreceptors of Pseudomonas aeruginosa and Bacillus subtilis. Whereas one paralogue recognizes most amino acids, the remaining paralogue binds specifically one of the few acids not recognized by the broad range receptors. Therefore, chemotaxis to compound families by the concerted action of broad and narrow range receptors may represent a general mechanism.

Identification of ligands for bacterial sensor proteins.

Bacteria have evolved a variety of different signal transduction mechanisms. However, the cognate signal molecule for the very large amount of corresponding sensor proteins is unknown and their functional annotation represents a major bottleneck in the field of signal transduction. The knowledge of the signal molecule is an essential prerequisite to understand the signalling mechanisms. Recently, the identification of signal molecules by the high-throughput protein screening of commercially available ligand collections using differential scanning fluorimetry has shown promise to resolve this bottleneck. Based on the analysis of a significant number of different ligand binding domains (LBDs) in our laboratory, we identified two issues that need to be taken into account in the experimental design. Since a number of LBDs require the dimeric state for ligand recognition, it has to be assured that the protein analysed is indeed in the dimeric form. A number of other examples demonstrate that purified LBDs can contain bound ligand which prevents further binding. In such cases, the apo-form can be generated by denaturation and subsequent refolding. We are convinced that this approach will accelerate the functional annotation of sensor proteins which will help to understand regulatory circuits in bacteria.

Identification of a Chemoreceptor for C2 and C3 Carboxylic Acids.

Chemoreceptors are at the beginnings of chemosensory signaling cascades that mediate chemotaxis. Most bacterial chemoreceptors are functionally unannotated and are characterized by a diversity in the structure of their ligand binding domains (LBDs). The data available indicate that there are two major chemoreceptor families at the functional level, namely, those that respond to amino acids or to Krebs cycle intermediates. Since pseudomonads show chemotaxis to many different compounds and possess different types of chemoreceptors, they are model organisms to establish relationships between chemoreceptor structure and function. Here, we identify PP2861 (termed McpP) of Pseudomonas putida KT2440 as a chemoreceptor with a novel ligand profile. We show that the recombinant McpP LBD recognizes acetate, pyruvate, propionate, and l-lactate, with KD (equilibrium dissociation constant) values ranging from 34 to 107 µM. Deletion of the mcpP gene resulted in a dramatic reduction in chemotaxis toward these ligands, and complementation restored a native-like phenotype, indicating that McpP is the major chemoreceptor for these compounds. McpP has a CACHE-type LBD, and we present data indicating that CACHE-containing chemoreceptors of other species also mediate taxis to C2 and C3 carboxylic acids. In addition, the LBD of NbaY of Pseudomonas fluorescens, an McpP homologue mediating chemotaxis to 2-nitrobenzoate, bound neither nitrobenzoates nor the McpP ligands. This work provides further insight into receptor structure-function relationships and will be helpful to annotate chemoreceptors of other bacteria.