Auxin-mediated regulation of susceptibility to toxic metabolites, c-di-GMP levels, and phage infection in the rhizobacterium Serratia plymuthica.

The communication between plants and their microbiota is highly dynamic and involves a complex network of signal molecules. Among them, the auxin indole-3-acetic acid (IAA) is a critical phytohormone that not only regulates plant growth and development, but is emerging as an important inter- and intra-kingdom signal that modulates many bacterial processes that are important during interaction with their plant hosts. However, the corresponding signaling cascades remain largely unknown. Here, we advance our understanding of the largely unknown mechanisms by which IAA carries out its regulatory functions in plant-associated bacteria. We showed that IAA caused important changes in the global transcriptome of the rhizobacterium Serratia plymuthica and multidisciplinary approaches revealed that IAA sensing interferes with the signaling mediated by other pivotal plant-derived signals such as amino acids and 4-hydroxybenzoic acid. Exposure to IAA caused large alterations in the transcript levels of genes involved in amino acid metabolism, resulting in significant metabolic alterations. IAA treatment also increased resistance to toxic aromatic compounds through the induction of the AaeXAB pump, which also confers resistance to IAA. Furthermore, IAA promoted motility and severely inhibited biofilm formation; phenotypes that were associated with decreased c-di-GMP levels and capsule production. IAA increased capsule gene expression and enhanced bacterial sensitivity to a capsule-dependent phage. Additionally, IAA induced the expression of several genes involved in antibiotic resistance and led to changes in the susceptibility and responses to antibiotics with different mechanisms of action. Collectively, our study illustrates the complexity of IAA-mediated signaling in plant-associated bacteria. IMPORTANCE: Signal sensing plays an important role in bacterial adaptation to ecological niches and hosts. This communication appears to be particularly important in plant-associated bacteria since they possess a large number of signal transduction systems that respond to a wide diversity of chemical, physical, and biological stimuli. IAA is emerging as a key inter- and intra-kingdom signal molecule that regulates a variety of bacterial processes. However, despite the extensive knowledge of the IAA-mediated regulatory mechanisms in plants, IAA signaling in bacteria remains largely unknown. Here, we provide insight into the diversity of mechanisms by which IAA regulates primary and secondary metabolism, biofilm formation, motility, antibiotic susceptibility, and phage sensitivity in a biocontrol rhizobacterium. This work has important implications for our understanding of bacterial ecology in plant environments and for the biotechnological and clinical applications of IAA, as well as related molecules.

Regulation of indole-3-acetic acid biosynthesis and consequences of auxin production deficiency in Serratia plymuthica.

Indole-3-acetic acid (IAA) is emerging as a key intra- and inter-kingdom signal molecule that modulates a wide range of processes of importance during plant-microorganism interaction. However, the mechanisms by which IAA carries out its functions in bacteria as well as the regulatory processes by which bacteria modulate auxin production are largely unknown. Here, we found that IAA synthesis deficiency results in important global transcriptional changes in the broad-range antibiotic-producing rhizobacterium Serratia plymuthica A153. Most pronounced transcriptional changes were observed in various gene clusters for aromatic acid metabolism, including auxin catabolism. To delve into the corresponding molecular mechanisms, different regulatory proteins were biochemically characterized. Among them, a TyrR orthologue was essential for IAA production through the activation of the ipdc gene encoding a key enzyme for IAA biosynthesis. We showed that TyrR specifically recognizes different aromatic amino acids which, in turn, alters the interactions of TyrR with the ipdc promoter. Screening of mutants defective in various transcriptional and post-transcriptional regulators allowed the identification of additional regulators of IAA production, including PigP and quorum sensing-related genes. Advancing our knowledge on the mechanisms that control the IAA biosynthesis in beneficial phytobacteria is of biotechnological interest for improving agricultural productivity and sustainable agricultural development.

Role of the RNA-binding protein ZC3H41 in the regulation of ribosomal protein messenger RNAs in trypanosomes.

BACKGROUND: Trypanosomes are single-celled eukaryotes that rely heavily on post-transcriptional mechanisms to regulate gene expression. RNA-binding proteins play essential roles in regulating the fate, abundance and translation of messenger RNAs (mRNAs). Among these, zinc finger proteins of the cysteine3histidine (CCCH) class have been shown to be key players in cellular processes as diverse as differentiation, regulation of the cell cycle and translation. ZC3H41 is an essential zinc finger protein that has been described as a component of spliced leader RNA granules and nutritional stress granules, but its role in RNA metabolism is unknown. METHODS: Cell cycle analysis in ZC3H41- and Z41AP-depleted cells was carried out using 4',6-diamidino-2-phenylindole staining, microscopic examination and flow cytometry. The identification of ZC3H41 protein partners was done using tandem affinity purification and mass spectrometry. Next-generation sequencing was used to evaluate the effect of ZC3H41 depletion on the transcriptome of procyclic Trypanosoma brucei cells, and also to identify the cohort of mRNAs associated with the ZC3H41/Z41AP complex. Levels of 5S ribosomal RNA (rRNA) species in ZC3H41- and Z41AP-depleted cells were assessed by quantitative reverse transcription-polymerase chain reaction. Surface sensing of translation assays were used to monitor global translation. RESULTS: We showed that depletion of the zinc finger protein ZC3H41 resulted in marked cell cycle defects and abnormal cell morphologies. ZC3H41 was found associated with an essential protein, which we named Z41AP, forming a stable heterodimer, and also with proteins of the poly(A)-binding protein 1 complex. The identification of mRNAs associated with the ZC3H41/Z41AP complex revealed that it is primarily composed of ribosomal protein mRNAs, and that binding to target transcripts is diminished upon nutritional stress. In addition, we observed that mRNAs encoding several proteins involved in the maturation of 5S rRNA are also associated with the ZC3H41/Z41AP complex. Finally, we showed that depletion of either ZC3H41 or Z41AP led to the accumulation of 5S rRNA precursors and a decrease of protein translation. CONCLUSIONS: We propose that ZC3H41 and Z41AP play important roles in controlling the fate of ribosomal components in response to environmental cues.

Emergence of an Auxin Sensing Domain in Plant-Associated Bacteria.

Bacteria have evolved a sophisticated array of signal transduction systems that allow them to adapt their physiology and metabolism to changing environmental conditions. Typically, these systems recognize signals through dedicated ligand binding domains (LBDs) to ultimately trigger a diversity of physiological responses. Nonetheless, an increasing number of reports reveal that signal transduction receptors also bind antagonists to inhibit responses mediated by agonists. The mechanisms by which antagonists block the downstream signaling cascade remain largely unknown. To advance our knowledge in this field, we used the LysR-type transcriptional regulator AdmX as a model. AdmX activates the expression of an antibiotic biosynthetic cluster in the rhizobacterium Serratia plymuthica. AdmX specifically recognizes the auxin phytohormone indole-3-acetic acid (IAA) and its biosynthetic intermediate indole-3-pyruvic acid (IPA) as signals. However, only IAA, but not IPA, was shown to regulate antibiotic production in S. plymuthica. Here, we report the high-resolution structures of the LBD of AdmX in complex with IAA and IPA. We found that IAA and IPA compete for binding to AdmX. Although IAA and IPA binding does not alter the oligomeric state of AdmX, IPA binding causes a higher degree of compactness in the protein structure. Molecular dynamics simulations revealed significant differences in the binding modes of IAA and IPA by AdmX, and the inspection of the three-dimensional structures evidenced differential agonist- and antagonist-mediated structural changes. Key residues for auxin binding were identified and an auxin recognition motif defined. Phylogenetic clustering supports the recent evolutionary emergence of this motif specifically in plant-associated enterobacteria. IMPORTANCE Although antagonists were found to bind different bacterial signal transduction receptors, we are still at the early stages of understanding the molecular details by which these molecules exert their inhibitory effects. Here, we provide insight into the structural changes resulting from the binding of an agonist and an antagonist to a sensor protein. Our data indicate that agonist and antagonist recognition is characterized by small conformational differences in the LBDs that can be efficiently transmitted to the output domain to modulate the final response. LBDs are subject to strong selective pressures and are rapidly evolving domains. An increasing number of reports support the idea that environmental factors drive the evolution of sensor domains. Given the recent evolutionary history of AdmX homologs, as well as their narrow phyletic distribution within plant-associated bacteria, our results are in accordance with a plant-mediated evolutionary process that resulted in the emergence of receptor proteins that specifically sense auxin phytohormones.

Herbicolin A production and its modulation by quorum sensing in a Pantoea agglomerans rhizobacterium bioactive against a broad spectrum of plant-pathogenic fungi.

Global population growth makes it necessary to increase agricultural production yields. However, climate change impacts and diseases caused by plant pathogens are challenging modern agriculture. Therefore, it is necessary to look for alternatives to the excessive use of chemical fertilizers and pesticides. The plant microbiota plays an essential role in plant nutrition and health, and offers enormous potential to meet future challenges of agriculture. In this context, here we characterized the antifungal properties of the rhizosphere bacterium Pantoea agglomerans 9Rz4, which is active against a broad spectrum of plant pathogenic fungi. Chemical analyses revealed that strain 9Rz4 produces the antifungal herbicolin A and its biosynthetic gene cluster was identified and characterized. We found that the only acyl-homoserine lactone-based quorum sensing system of 9Rz4 modulates herbicolin A gene cluster expression. No role of plasmid carriage in the production of herbicolin A was observed. Plant assays revealed that herbicolin A biosynthesis does not affect the root colonization ability of P. agglomerans 9Rz4. Current legislative restrictions are aimed at reducing the use of chemical pesticides in agriculture, and the results derived from this study may lay the foundations for the development of novel biopesticides from rhizosphere microorganisms.

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.

An RNA-binding protein complex regulates the purine-dependent expression of a nucleobase transporter in trypanosomes.

Post-transcriptional regulation of gene expression is particularly important in trypanosomatid protozoa. RNA-binding proteins (RBPs) regulate mRNA stability and translation, yet information about how RBPs are able to link environmental cues to post-transcriptional control is scarce. In Trypanosoma brucei, we have previously characterized a short RNA stem-loop cis-element (PuRE, Purine Responsive Element) within the 3'-UTR of the NT8 nucleobase transporter mRNA that is necessary and sufficient to confer a strong repression of gene expression in response to purines. In this study, we have identified a protein complex composed of two RNA-binding proteins (PuREBP1 and PuREBP2) that binds to the PuRE in vitro and to NT8 mRNA in vivo. Depletion of PuREBP1 by RNA interference results in the upregulation of just NT8 and the mRNAs encoding the amino acid transporter AATP6 paralogues. Moreover, we found that the PuREBP1/2 complex is associated with only a handful of mRNAs, and that it is responsible for the observed purine-dependent regulation of NT8 expression.

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.

How Bacterial Chemoreceptors Evolve Novel Ligand Specificities.

Chemoreceptor-based signaling pathways are among the major modes of bacterial signal transduction, and Pseudomonas aeruginosa PAO1 is an important model to study their function. Of the 26 chemoreceptors of this strain, PctA has a broad ligand range and responds to most of the proteinogenic amino acids, whereas PctB and PctC have a much narrower range and show strong ligand preference for l-glutamine and γ-aminobutyrate, respectively. Using several comparative genomics approaches, we show that these receptors are paralogs: pctA gene duplication in the common ancestor of the genus Pseudomonas led to pctC, whereas pctB originated through another, independent pctA duplication in the common ancestor of P. aeruginosa Thus, the broad-range amino acid chemoreceptor was evolutionarily older, and chemoreceptors that complemented "missing" amino acid sensing abilities arose later in specific Pseudomonas lineages. Using comparative sequence analysis, newly solved crystal structures of PctA, PctB, and PctC ligand-binding domains, and their molecular dynamics simulations, we identified a conserved amino acid recognition motif and changes in the ligand-binding pocket that led to novel ligand specificities. In addition, we determined major forces driving the evolution of this group of chemoreceptors.IMPORTANCE Many bacteria possess a large number of chemoreceptors that recognize a variety of different compounds. More than 60% of the genomes analyzed in this study contain paralogous chemoreceptors, suggesting that they emerge with high frequency. We provide first insight on how paralogous receptors have evolved and show that two chemoreceptors with a narrow ligand range have evolved from an ancestral protein with a broad chemoeffector spectrum. Protein structures show that multiple changes in the ligand-binding site account for the differences in the ligand spectrum. This work lays the ground for further studies aimed at establishing whether the principles of ligand-binding evolution reported here can be generalized for a wider spectrum of sensory proteins in bacteria.

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.

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.

Two different mechanisms mediate chemotaxis to inorganic phosphate in Pseudomonas aeruginosa.

Inorganic phosphate (Pi) is a central signaling molecule that modulates virulence in various pathogens. In Pseudomonas aeruginosa, low Pi concentrations induce transcriptional alterations that increase virulence. Also, under low Pi levels, P. aeruginosa exhibits Pi chemotaxis-a process mediated by the two non-paralogous receptors CtpH and CtpL. Here we show that the two receptors operate via different mechanisms. We demonstrate that the ligand binding domain (LBD) of CtpH but not CtpL binds Pi directly. We identify the periplasmic ligand binding protein PstS as the protein that binds in its Pi loaded state to CtpL, resulting in receptor stimulation. PstS forms part of the Pi transporter and has thus a double function in Pi transport and chemotaxis. The affinity of Pi for CtpH was modest whereas that for PstS very high, which may explain why CtpH and CtpL mediate chemotaxis to high and low Pi concentrations, respectively. The pstS/ctpH double mutant was almost devoid of Pi taxis, indicating that PstS is the only CtpL Pi-shuttle. Chemotaxis mechanisms based on indirect ligand recognition were unambiguously identified in enterobacteria. The discovery of a similar mechanism in a different bacterial order, involving a different chemoreceptor type and chemoeffector suggests that such systems are widespread.

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.

Specific gamma-aminobutyrate chemotaxis in pseudomonads with different lifestyle.

The PctC chemoreceptor of Pseudomonas aeruginosa mediates chemotaxis with high specificity to gamma-aminobutyric acid (GABA). This compound is present everywhere in nature and has multiple functions, including being a human neurotransmitter or plant signaling compound. Because P. aeruginosa is ubiquitously distributed in nature and able to infect and colonize different hosts, the physiological relevance of GABA taxis is unclear, but it has been suggested that bacterial attraction to neurotransmitters may enhance virulence. We report the identification of McpG as a specific GABA chemoreceptor in non-pathogenic Pseudomonas putida KT2440. As with PctC, GABA was found to bind McpG tightly. The analysis of chimeras comprising the PctC and McpG ligand-binding domains fused to the Tar signaling domain showed very high GABA sensitivities. We also show that PctC inactivation does not alter virulence in Caenorhabditis elegans. Significant amounts of GABA were detected in tomato root exudates, and deletion of mcpG reduced root colonization that requires chemotaxis through agar. The C. elegans data and the detection of a GABA receptor in non-pathogenic species indicate that GABA taxis may not be related to virulence in animal systems but may be of importance in the context of colonization and infection of plant roots by soil-dwelling pseudomonads.

Qualitative and quantitative assays for flagellum-mediated chemotaxis.

A primary driving force during bacterial evolution was the capacity to access compounds necessary for growth and survival. Since the species of the genus Pseudomonas are characterized by metabolic versatility, these bacteria have developed chemotactic behaviors towards a wide range of different compounds. The specificity of a chemotactic response is determined by the chemoreceptor, which is at the beginning of the signaling cascade and to which chemoattractants and chemorepellents bind. The number of chemoreceptor genes of Pseudomonas species is significantly higher than the average number in motile bacteria. Although some of the receptors have been annotated with a function, the cognate signal molecules for the majority of them still need to be identified. Different qualitative and quantitative methods are presented that can be used to study flagellum-mediated taxis.

Characterization of molecular interactions using isothermal titration calorimetry.

Isothermal titration calorimetry (ITC) is based on a simple titration of one ligand with another and the small heat changes caused by the molecular interaction are detected. From one ITC experiment the complete set of thermodynamic parameters of binding including association and dissociation constants as well as changes in enthalpy, entropy, and free energy can be derived. Using this technique almost any type of molecular interaction can be analyzed. Both ligands are in solution, and there is no need for their chemical derivatization. There are no limits as to the choice of the analysis buffer, and the analysis temperature can be set between 4 and 80 °C. This technique has been primarily applied to study the interaction between various proteins of Pseudomonas with small molecule ligands. In addition, ITC has been used to study the binding of Pseudomonas proteins to target DNA fragments.

Purification, crystallization and preliminary crystallographic analysis of the ligand-binding regions of the PctA and PctB chemoreceptors from Pseudomonas aeruginosa in complex with amino acids.

Pseudomonas aeruginosa is an opportunistic pathogen and one of the major model organisms for the study of chemotaxis. The bacterium harbours 26 genes encoding chemoreceptors, most of which have not been annotated with a function. The paralogous chemoreceptors PctA and PctB (Pseudomonas chemotactic transducer A and B) were found to mediate chemotaxis towards L-amino acids. However, the ligand spectrum of the receptors is quite different since the recombinant ligand-binding region (LBR) of PctA binds 17 different L-amino acids whereas that of PctB recognizes only five. To determine the molecular basis underlying this ligand specificity, PctA-LBR and PctB-LBR have been purified and crystals have been produced after pre-incubation with L-Ile and L-Arg, respectively. Initial crystallization conditions have been identified by the counter-diffusion method and X-ray data have been collected at 2.5 Å (PctA-LBR bound to L-Ile) and 3.14 Å (PctB-LBR bound to L-Arg) resolution. Crystals belonged to space groups P2(1)2(1)2(1) and P3(1)2(1), with unit-cell parameters a = 72.2, b = 78.5, c = 116.6 Å and a = b = 111.6, c = 117.4, respectively, for PctA-LBR and PctB-LBR. Molecular-replacement methods will be pursued for structural determination.

Paralogous chemoreceptors mediate chemotaxis towards protein amino acids and the non-protein amino acid gamma-aminobutyrate (GABA).

The paralogous receptors PctA, PctB and PctC of Pseudomonas aeruginosa were reported to mediate chemotaxis to amino acids, intermediates of amino acid metabolism and chlorinated hydrocarbons. We show that the recombinant ligand binding regions (LBRs) of PctA, PctB and PctC bind 17, 5 and 2 l-amino acids respectively. In addition, PctC-LBR recognized GABA but not any other structurally related compound. l-Gln, one of the three amino acids that is not recognized by PctA-LBR, was the most tightly binding ligand to PctB suggesting that PctB has evolved to mediate chemotaxis primarily towards l-Gln. Bacteria were efficiently attracted to l-Gln and GABA, but mutation of pctB and pctC, respectively, abolished chemoattraction. The physiological relevance of taxis towards GABA is proposed to reside in an interaction with plants. LBRs were predicted to adopt double PDC (PhoQ/DcuS/CitA) like structures and site-directed mutagenesis studies showed that ligands bind to the membrane-distal module. Analytical ultracentrifugation studies have shown that PctA-LBR and PctB-LBR are monomeric in the absence and presence of ligands, which is in contrast to the enterobacterial receptors that require sensor domain dimers for ligand recognition.

Genes encoding Cher-TPR fusion proteins are predominantly found in gene clusters encoding chemosensory pathways with alternative cellular functions.

Chemosensory pathways correspond to major signal transduction mechanisms and can be classified into the functional families flagellum-mediated taxis, type four pili-mediated taxis or pathways with alternative cellular functions (ACF). CheR methyltransferases are core enzymes in all of these families. CheR proteins fused to tetratricopeptide repeat (TPR) domains have been reported and we present an analysis of this uncharacterized family. We show that CheR-TPRs are widely distributed in GRAM-negative but almost absent from GRAM-positive bacteria. Most strains contain a single CheR-TPR and its abundance does not correlate with the number of chemoreceptors. The TPR domain fused to CheR is comparatively short and frequently composed of 2 repeats. The majority of CheR-TPR genes were found in gene clusters that harbor multidomain response regulators in which the REC domain is fused to different output domains like HK, GGDEF, EAL, HPT, AAA, PAS, GAF, additional REC, HTH, phosphatase or combinations thereof. The response regulator architectures coincide with those reported for the ACF family of pathways. Since the presence of multidomain response regulators is a distinctive feature of this pathway family, we conclude that CheR-TPR proteins form part of ACF type pathways. The diversity of response regulator output domains suggests that the ACF pathways form a superfamily which regroups many different regulatory mechanisms, in which all CheR-TPR proteins appear to participate. In the second part we characterize WspC of Pseudomonas putida, a representative example of CheR-TPR. The affinities of WspC-Pp for S-adenosylmethionine and S-adenosylhomocysteine were comparable to those of prototypal CheR, indicating that WspC-Pp activity is in analogy to prototypal CheRs controlled by product feed-back inhibition. The removal of the TPR domain did not impact significantly on the binding constants and consequently not on the product feed-back inhibition. WspC-Pp was found to be monomeric, which rules out a role of the TPR domain in self-association.

Responses of Pseudomonas putida to toxic aromatic carbon sources.

A number of bacteria can use toxic compounds as carbon sources and have developed complex regulatory networks to protect themselves from the toxic effects of these compounds as well as to benefit from their nutritious properties. As a model system we have studied the responses of Pseudomonas putida strains to toluene. Although this compound is highly toxic, several strains are able to use it for growth. Particular emphasis was given to the responses in the context of taxis, resistance and toluene catabolism. P. putida strains analysed showed chemotactic movements towards toluene. Strain DOT-T1E was characterised by an extreme form of chemotaxis, termed hyperchemotaxis, which is mediated by the McpT chemoreceptor encoded by plasmid pGRT1. Close McpT homologs are found in a number of other plasmids encoding degradation pathways of toxic compounds. The pGRT1 plasmid harbours also the genes for the TtgGHI efflux pump which was identified as the primary determinant for the resistance of strain DOT-T1E towards toluene. Pump expression is controlled by the TtgV repressor in response to a wide range of different mono- and biaromatic compounds. Strain DOT-T1E is able to degrade toluene, benzene and ethylbenzene via the toluene dioxygenase (TOD) pathway. The expression of the pathway operon is controlled by the TodS/T two component system. The sensor kinase TodS recognizes toluene with nanomolar affinity, which in turn triggers an increase in its autophosphorylation and consequently transcriptional activation. Data suggest that transcriptional activation of the TOD pathway occurs at very low toluene concentrations whereas TtgV mediated induction of pump expression sets in as the toluene concentration further increases.