Amino acid sensor conserved from bacteria to humans.

SignificanceAmino acids are the building blocks of life and important signaling molecules. Despite their common structure, no universal mechanism for amino acid recognition by cellular receptors is currently known. We discovered a simple motif, which binds amino acids in various receptor proteins from all major life-forms. In humans, this motif is found in subunits of calcium channels that are implicated in pain and neurodevelopmental disorders. Our findings suggest that γ-aminobutyric acid-derived drugs bind to the same motif in human proteins that binds natural ligands in bacterial receptors, thus enabling future improvement of important drugs.

Study of NIT domain-containing chemoreceptors from two global phytopathogens and identification of NIT domains in eukaryotes.

Bacterial signal transduction systems are typically activated by the binding of signal molecules to receptor ligand binding domains (LBDs), such as the NIT LBD. We report here the identification of the NIT domain in more than 15,000 receptors that were present in 30 bacterial phyla, but also in 19 eukaryotic phyla, expanding its known phylogenetic distribution. The NIT domain formed part of seven receptor families that either control transcription, mediate chemotaxis or regulate second messenger levels. We have produced the NIT domains from chemoreceptors of the bacterial phytopathogens Pectobacterium atrosepticum (PacN) and Pseudomonas savastanoi (PscN) as individual purified proteins. High-throughput ligand screening using compound libraries revealed a specificity for nitrate and nitrite binding. Isothermal titration calorimetry experiments showed that PacN-LBD bound preferentially nitrate ( K D = 1.9 µM), whereas the affinity of PscN-LBD for nitrite ( K D = 2.1 µM) was 22 times higher than that for nitrate. Analytical ultracentrifugation experiments indicated that PscN-LBD is monomeric in the presence and absence of ligands. The R182A mutant of PscN did not bind nitrate or nitrite. This residue is not conserved in the NIT domain of the Pseudomonas aeruginosa chemoreceptor PA4520, which may be related to its failure to bind nitrate/nitrite. The magnitude of P. atrosepticum chemotaxis towards nitrate was significantly greater than that of nitrite and pacN deletion almost abolished responses to both compounds. This study highlights the important role of nitrate and nitrite as signal molecules in life and advances our knowledge on the NIT domain as universal nitrate/nitrite sensor module.

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.

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.

A bacterial chemoreceptor that mediates chemotaxis to two different plant hormones.

Indole-3-acetic acid (IAA) is the main naturally occurring auxin and is produced by organisms of all kingdoms of life. In addition to the regulation of plant growth and development, IAA plays an important role in the interaction between plants and growth-promoting and phytopathogenic bacteria by regulating bacterial gene expression and physiology. We show here that an IAA metabolizing plant-associated Pseudomonas putida isolate exhibits chemotaxis to IAA that is independent of auxin metabolism. We found that IAA chemotaxis is based on the activity of the PcpI chemoreceptor and heterologous expression of pcpI conferred IAA taxis to different environmental and human pathogenic isolates of the Pseudomonas genus. Using ligand screening, microcalorimetry and quantitative chemotaxis assays, we found that PcpI failed to bind IAA directly, but recognized and mediated chemoattractions to various aromatic compounds, including the phytohormone salicylic acid. The expression of pcpI and its role in the interactions with plants was also investigated. PcpI extends the range of central signal molecules recognized by chemoreceptors. To our knowledge, this is the first report on a bacterial receptor that responds to two different phytohormones. Our study reinforces the multifunctional role of IAA and salicylic acid as intra- and inter-kingdom signal molecules.

Flagella, Chemotaxis and Surface Sensing.

Based on genome analyses, it has been estimated that more than half of the bacteria have made an important investment into motility since they possess genes encoding the flagellar motor, the flagellum, chemosensory pathways and chemoreceptors. The metabolic burden associated with gene maintenance, protein synthesis and operating these systems is very important. A central question is thus to establish the physiological benefits that compensate such an important investment. In this chapter, we illustrate that benefits are multiple and diverse, including access to nutrients and preferred niches, biofilm formation and bacterial dispersal. There is also evidence that the complete range of advantages still remains to be defined. In these research efforts, Pseudomonas aeruginosa (PA) has played a central role and is among the central model species. Research conducted on PA had a significant impact in the field and has motivated many experiments in the study of other model bacterial species.

Histamine: A Bacterial Signal Molecule.

Bacteria have evolved sophisticated signaling mechanisms to coordinate interactions with organisms of other domains, such as plants, animals and human hosts. Several important signal molecules have been identified that are synthesized by members of different domains and that play important roles in inter-domain communication. In this article, we review recent data supporting that histamine is a signal molecule that may play an important role in inter-domain and inter-species communication. Histamine is a key signal molecule in humans, with multiple functions, such as being a neurotransmitter or modulator of immune responses. More recent studies have shown that bacteria have evolved different mechanisms to sense histamine or histamine metabolites. Histamine sensing in the human pathogen Pseudomonas aeruginosa was found to trigger chemoattraction to histamine and to regulate the expression of many virulence-related genes. Further studies have shown that many bacteria are able to synthesize and secrete histamine. The release of histamine by bacteria in the human gut was found to modulate the host immune responses and, at higher doses, to result in host pathologies. The elucidation of the role of histamine as an inter-domain signaling molecule is an emerging field of research and future investigation is required to assess its potential general nature.

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.

An auxin controls bacterial antibiotics production.

The majority of clinically used antibiotics originate from bacteria. As the need for new antibiotics grows, large-scale genome sequencing and mining approaches are being used to identify novel antibiotics. However, this task is hampered by the fact that many antibiotic biosynthetic clusters are not expressed under laboratory conditions. One strategy to overcome this limitation is the identification of signals that activate the expression of silent biosynthetic pathways. Here, we report the use of high-throughput screening to identify signals that control the biosynthesis of the acetyl-CoA carboxylase inhibitor antibiotic andrimid in the broad-range antibiotic-producing rhizobacterium Serratia plymuthica A153. We reveal that the pathway-specific transcriptional activator AdmX recognizes the auxin indole-3-acetic acid (IAA). IAA binding causes conformational changes in AdmX that result in the inhibition of the expression of the andrimid cluster and the suppression of antibiotic production. We also show that IAA synthesis by pathogenic and beneficial plant-associated bacteria inhibits andrimid production in A153. Because IAA is a signalling molecule that is present across all domains of life, this study highlights the importance of intra- and inter-kingdom signalling in the regulation of antibiotic synthesis. Our discovery unravels, for the first time, an IAA-dependent molecular mechanism for the regulation of antibiotic synthesis.

Evidence for Pentapeptide-Dependent and Independent CheB Methylesterases.

Many bacteria possess multiple chemosensory pathways that are composed of homologous signaling proteins. These pathways appear to be functionally insulated from each other, but little information is available on the corresponding molecular basis. We report here a novel mechanism that contributes to pathway insulation. We show that, of the four CheB paralogs of Pseudomonas aeruginosa PAO1, only CheB2 recognizes a pentapeptide at the C-terminal extension of the McpB (Aer2) chemoreceptor (KD = 93 µM). McpB is the sole chemoreceptor that stimulates the Che2 pathway, and CheB2 is the methylesterase of this pathway. Pectobacterium atrosepticum SCRI1043 has a single CheB, CheB_Pec, and 19 of its 36 chemoreceptors contain a C-terminal pentapeptide. The deletion of cheB_Pec abolished chemotaxis, but, surprisingly, none of the pentapeptides bound to CheB_Pec. To determine the corresponding structural basis, we solved the 3D structure of CheB_Pec. Its structure aligned well with that of the pentapeptide-dependent enzyme from Salmonella enterica. However, no electron density was observed in the CheB_Pec region corresponding to the pentapeptide-binding site in the Escherichia coli CheB. We hypothesize that this structural disorder is associated with the failure to bind pentapeptides. Combined data show that CheB methylesterases can be divided into pentapeptide-dependent and independent enzymes.

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.

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.

Biosynthesis of the acetyl-CoA carboxylase-inhibiting antibiotic, andrimid in Serratia is regulated by Hfq and the LysR-type transcriptional regulator, AdmX.

Infections due to multidrug-resistant bacteria represent a major global health challenge. To combat this problem, new antibiotics are urgently needed and some plant-associated bacteria are a promising source. The rhizobacterium Serratia plymuthica A153 produces several bioactive secondary metabolites, including the anti-oomycete and antifungal haterumalide, oocydin A and the broad spectrum polyamine antibiotic, zeamine. In this study, we show that A153 produces a second broad spectrum antibiotic, andrimid. Using genome sequencing, comparative genomics and mutagenesis, we defined new genes involved in andrimid (adm) biosynthesis. Both the expression of the adm gene cluster and regulation of andrimid synthesis were investigated. The biosynthetic cluster is operonic and its expression is modulated by various environmental cues, including temperature and carbon source. Analysis of the genome context of the adm operon revealed a gene encoding a predicted LysR-type regulator, AdmX, apparently unique to Serratia strains. Mutagenesis and gene expression assays demonstrated that AdmX is a transcriptional activator of the adm gene cluster. At the post-transcriptional level, the expression of the adm cluster is positively regulated by the RNA chaperone, Hfq, in an RpoS-independent manner. Our results highlight the complexity of andrimid biosynthesis - an antibiotic with potential clinical and agricultural utility.

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.

Assessment of the contribution of chemoreceptor-based signalling to biofilm formation.

Although it is well established that one- and two-component regulatory systems participate in regulating biofilm formation, there also exists evidence suggesting that chemosensory pathways are also involved. However, little information exists about which chemoreceptors and signals modulate this process. Here we report the generation of the complete set of chemoreceptor mutants of Pseudomonas putida KT2440 and the identification of four mutants with significantly altered biofilm phenotypes. These receptors are a WspA homologue of Pseudomonas aeruginosa, previously identified to control biofilm formation by regulating c-di-GMP levels, and three uncharacterized chemoreceptors. One of these receptors, named McpU, was found to mediate chemotaxis towards different polyamines. The functional annotation of McpU was initiated by high-throughput thermal shift assays of the receptor ligand binding domain (LBD). Isothermal titration calorimetry showed that McpU-LBD specifically binds putrescine, cadaverine and spermidine, indicating that McpU represents a novel chemoreceptor type. Another uncharacterized receptor, named McpA, specifically binds 12 different proteinogenic amino acids and mediates chemotaxis towards these compounds. We also show that mutants in McpU and WspA-Pp have a significantly reduced ability to colonize plant roots. Data agree with other reports showing that polyamines are signal molecules involved in the regulation of bacteria-plant communication and biofilm formation.

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.

Biosynthesis of the antifungal haterumalide, oocydin A, in Serratia, and its regulation by quorum sensing, RpoS and Hfq.

Polyketides represent an important class of bioactive natural products with a broad range of biological activities. We identified recently a large trans-acyltransferase (AT) polyketide synthase gene cluster responsible for the biosynthesis of the antifungal, anti-oomycete and antitumor haterumalide, oocydin A (ooc). Using genome sequencing and comparative genomics, we show that the ooc gene cluster is widespread within biocontrol and phytopathogenic strains of the enterobacteria, Serratia and Dickeya. The analysis of in frame deletion mutants confirmed the role of a hydroxymethylglutaryl-coenzyme A synthase cassette, three flavin-dependent tailoring enzymes, a free-standing acyl carrier protein and two hypothetical proteins in oocydin A biosynthesis. The requirement of the three trans-acting AT domains for the biosynthesis of the macrolide was also demonstrated. Expression of the ooc gene cluster was shown to be positively regulated by an N-acyl-L-homoserine lactone-based quorum sensing system, but operating in a strain-dependent manner. At a post-transcriptional level, the RNA chaperone, Hfq, plays a key role in oocydin A biosynthesis. The Hfq-dependent regulation is partially mediated by the stationary phase sigma factor, RpoS, which was also shown to positively regulate the synthesis of the macrolide. Our results reveal differential regulation of the divergently transcribed ooc transcriptional units, highlighting the complexity of oocydin A production.