Amine-recognizing domain in diverse receptors from bacteria and archaea evolved from the universal amino acid sensor.

Bacteria possess various receptors that sense different signals and transmit information to enable an optimal adaptation to the environment. A major limitation in microbiology is the lack of information on the signal molecules that activate receptors. Signals recognized by sensor domains are poorly reflected in overall sequence identity, and therefore, the identification of signals from the amino acid sequence of the sensor alone presents a challenge. Biogenic amines are of great physiological importance for microorganisms and humans. They serve as substrates for aerobic and anaerobic growth and play a role of neurotransmitters and osmoprotectants. Here, we report the identification of a sequence motif that is specific for amine-sensing sensor domains that belong to the Cache superfamily of the most abundant extracellular sensors in prokaryotes. We identified approximately 13,000 sensor histidine kinases, chemoreceptors, receptors involved in second messenger homeostasis and Ser/Thr phosphatases from 8,000 bacterial and archaeal species that contain the amine-recognizing motif. The screening of compound libraries and microcalorimetric titrations of selected sensor domains confirmed their ability to specifically bind biogenic amines. Mutants in the amine-binding motif or domains that contain a single mismatch in the binding motif had either no or a largely reduced affinity for amines. We demonstrate that the amine-recognizing domain originated from the universal amino acid-sensing Cache domain, thus providing insight into receptor evolution. Our approach enables precise "wet"-lab experiments to define the function of regulatory systems and therefore holds a strong promise to enable the identification of signals stimulating numerous receptors.

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.

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.

A Nitrate-Sensing Domain-Containing Chemoreceptor Is Required for Successful Entry and Virulence of Dickeya dadantii 3937 in Potato Plants.

Nitrate metabolism plays an important role in bacterial physiology. During the interaction of plant-pathogenic bacteria with their hosts, bacteria face variable conditions with respect to nitrate availability. Perception mechanisms through the chemosensory pathway drive the entry and control the colonization of the plant host in phytopathogenic bacteria. In this work, the identification and characterization of the nitrate- and nitrite-sensing (NIT) domain-containing chemoreceptor of Dickeya dadantii 3937 (Dd3937) allowed us to unveil the key role of nitrate sensing not only for the entry into the plant apoplast through wounds but also for infection success. We determined the specificity of this chemoreceptor to bind nitrate and nitrite, with a slight ligand preference for nitrate. Gene expression analysis showed that nitrate perception controls not only the expression of nitrate reductase genes involved in respiratory and assimilatory metabolic processes but also the expression of gyrA, hrpN, and bgxA, three well-known virulence determinants in Dd3937.

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.

Low CyaA expression and anti-cooperative binding of cAMP to CRP frames the scope of the cognate regulon of Pseudomonas putida.

Although the soil bacterium Pseudomonas putida KT2440 bears a bona fide adenylate cyclase gene (cyaA), intracellular concentrations of 3',5'-cyclic adenosine monophosphate (cAMP) are barely detectable. By using reporter technology and direct quantification of cAMP under various conditions, we show that such low levels of the molecule stem from the stringent regulation of its synthesis, efflux and degradation. Poor production of cAMP was the result of inefficient translation of cyaA mRNA. Moreover, deletion of the cAMP-phosphodiesterase pde gene led to intracellular accumulation of the cyclic nucleotide, exposing an additional cause of cAMP drain in vivo. But even such low levels of the signal sustained activation of promoters dependent on the cAMP-receptor protein (CRP). Genetic and biochemical evidence indicated that the phenomenon ultimately rose from the unusual binding parameters of cAMP to CRP. This included an ultratight cAMP-CrpP. putida affinity (KD of 45.0 ± 3.4 nM) and an atypical 1:1 effector/dimer stoichiometry that obeyed an infrequent anti-cooperative binding mechanism. It thus seems that keeping the same regulatory parts and their relational logic but changing the interaction parameters enables genetic devices to take over entirely different domains of the functional landscape.

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.

Chemotaxis of Beneficial Rhizobacteria to Root Exudates: The First Step towards Root-Microbe Rhizosphere Interactions.

Chemotaxis, the ability of motile bacteria to direct their movement in gradients of attractants and repellents, plays an important role during the rhizosphere colonization by rhizobacteria. The rhizosphere is a unique niche for plant-microbe interactions. Root exudates are highly complex mixtures of chemoeffectors composed of hundreds of different compounds. Chemotaxis towards root exudates initiates rhizobacteria recruitment and the establishment of bacteria-root interactions. Over the last years, important progress has been made in the identification of root exudate components that play key roles in the colonization process, as well as in the identification of the cognate chemoreceptors. In the first part of this review, we summarized the roles of representative chemoeffectors that induce chemotaxis in typical rhizobacteria and discussed the structure and function of rhizobacterial chemoreceptors. In the second part we reviewed findings on how rhizobacterial chemotaxis and other root-microbe interactions promote the establishment of beneficial rhizobacteria-plant interactions leading to plant growth promotion and protection of plant health. In the last part we identified the existing gaps in the knowledge and discussed future research efforts that are necessary to close them.

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.

Assigning chemoreceptors to chemosensory pathways in Pseudomonas aeruginosa.

In contrast to Escherichia coli, a model organism for chemotaxis that has 5 chemoreceptors and a single chemosensory pathway, Pseudomonas aeruginosa PAO1 has a much more complex chemosensory network, which consists of 26 chemoreceptors feeding into four chemosensory pathways. While several chemoreceptors were rigorously linked to specific pathways in a series of experimental studies, for most of them this information is not available. Thus, we addressed the problem computationally. Protein-protein interaction network prediction, coexpression data mining, and phylogenetic profiling all produced incomplete and uncertain assignments of chemoreceptors to pathways. However, comparative sequence analysis specifically targeting chemoreceptor regions involved in pathway interactions revealed conserved sequence patterns that enabled us to unambiguously link all 26 chemoreceptors to four pathways. Placing computational evidence in the context of experimental data allowed us to conclude that three chemosensory pathways in P. aeruginosa utilize one chemoreceptor per pathway, whereas the fourth pathway, which is the main system controlling chemotaxis, utilizes the other 23 chemoreceptors. Our results show that while only a very few amino acid positions in receptors, kinases, and adaptors determine their pathway specificity, assigning receptors to pathways computationally is possible. This requires substantial knowledge about interacting partners on a molecular level and focusing comparative sequence analysis on the pathway-specific regions. This general principle should be applicable to resolving many other receptor-pathway interactions.

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.

Recognition of dominant attractants by key chemoreceptors mediates recruitment of plant growth-promoting rhizobacteria.

Chemotaxis to plant root exudates is supposed to be a prerequisite for efficient root colonization by rhizobacteria. This is a highly multifactorial process since root exudates are complex compound mixtures of which components are recognized by different chemoreceptors. Little information is available as to the key components in root exudates and their receptors that drive colonization related chemotaxis. We present here the first global assessment of this issue using the plant growth-promoting rhizobacterium (PGPR) Bacillus velezensis SQR9 (formerly B. amyloliquefaciens). This strain efficiently colonizes cucumber roots, and here, we show that chemotaxis to cucumber root exudates was essential in this process. We conducted chemotaxis assays using cucumber root exudates at different concentrations, individual exudate components as well as recomposed exudates, taking into account their concentrations detected in root exudates. Results indicated that two key chemoreceptors, McpA and McpC, were essential for root exudate chemotaxis and root colonization. Both receptors possess a broad ligand range and recognize most of the exudate key components identified (malic, fumaric, gluconic and glyceric acids, Lys, Ser, Ala and mannose). The remaining six chemoreceptors did not contribute to exudate chemotaxis. This study provides novel insight into the evolution of the chemotaxis system in rhizobacteria.

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.

Exploring the (Almost) Unknown: Archaeal Two-Component Systems.

Two-component systems (TCS) exist in bacteria and archaea. In contrast to the knowledge of bacterial TCSs, little information is available on their archaeal counterparts. In the current issue of Journal of Bacteriology, Galperin and coworkers present a bioinformatics analysis of TCS genes from archaeal genome sequences (M. Y. Galperin, K. S. Makarova, Y. I. Wolf, and E. V. Koonin, J Bacteriol 200:e00681-17, 2018, https://doi.org/10.1128/JB.00681-17). This study identifies different aspects in which TCS-mediated signaling differs in bacteria and archaea and forms a sound basis for the experimental design of studies to increase our knowledge of this poorly investigated protein family.

The plant compound rosmarinic acid induces a broad quorum sensing response in Pseudomonas aeruginosa PAO1.

The interference of plant compounds with bacterial quorum sensing (QS) is a major mechanism through which plants and bacteria communicate. However, little is known about the modes of action and effects on signal integrity during this type of communication. We have recently shown that the plant compound rosmarinic acid (RA) specifically binds to the Pseudomonas aeruginosa RhlR QS receptor. To determine the effect of RA on expression patterns, we carried out global RNA-seq analysis. The results show that RA induces the expression of 128 genes, amongst which many virulence factor genes. RA triggers a broad QS response because 88% of the induced genes are known to be controlled by QS, and because RA stimulated genes were found to be involved in all four QS signalling systems within P. aeruginosa. This finding was confirmed through the analysis of transcriptional fusions transferred to wt and a rhlI/lasI double mutant. RA did not induce gene expression in the rhlI/lasI/rhlR triple mutant indicating that the effects observed are due to the RA-RhlR interaction. Furthermore, RA induced seven sRNAs that were all encoded in regions close to QS and/or RA induced genes. This work significantly enhances our understanding of plant bacteria interaction.