Concentration Dependent Effect of Plant Root Exudates on the Chemosensory Systems of Pseudomonas putida KT2440

Plant root colonization by rhizobacteria can protect plants against pathogens and promote plant growth, and chemotaxis to root exudates was shown to be an essential prerequisite for efficient root colonization. Since many chemoattractants control the transcript levels of their cognate chemoreceptor genes, we have studied here the transcript levels of the 27 Pseudomonas putida KT2440 chemoreceptor genes in the presence of different maize root exudate (MRE) concentrations. Transcript levels were increased for 10 chemoreceptor genes at low MRE concentrations, whereas almost all receptor genes showed lower transcript levels at high MRE concentrations. The exposure of KT2440 to different MRE concentrations did not alter c-di-GMP levels, indicating that changes in chemoreceptor transcripts are not mediated by this second messenger. Data suggest that rhizosphere colonization unfolds in a temporal fashion. Whereas at a distance to the root, exudates enhance chemoreceptor gene transcript levels promoting in turn chemotaxis, this process is reversed in root vicinity, where the necessity of chemotaxis toward the root may be less important. Insight into KT2440 signaling processes were obtained by analyzing mutants defective in the three cheA paralogous genes. Whereas a mutant in cheA1 showed reduced c-di-GMP levels and impaired biofilm formation, a cheA2 mutant was entirely deficient in MRE chemotaxis, indicating the existence of homologs of the P. aeruginosawsp and che (chemotaxis) pathways. Signaling through both pathways was important for efficient maize root colonization. Future studies will show whether the MRE concentration dependent effect on chemoreceptor gene transcript levels is a feature shared by other species

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. [EN]FEDER funds and Fondo Social Europeo through grants from the Junta de Andalucía (grant CVI-7335) and the Spanish Ministry for Economy and Competitiveness (grant BIO2013-42297). MM was supported by the Spanish Ministry of Economy and Competitiveness Postdoctoral Research Program, Juan de la Cierva (JCI-2012-11815).Peer reviewe

Inhibition of HIV-1 Replication and Dimerization Interference by Dual Inhibitory RNAs

The 5’-untranslated region (5’UTR) of the HIV-1 RNA is an attractive target for engineered ribozymes due to its high sequence and structural conservation. This region encodes several conserved structural RNA domains essential in key processes of the viral replication and infection cycles. This paper reports the inhibitory effects of catalytic antisense RNAs composed of two inhibitory RNA domains: an engineered ribozyme targeting the 5’ UTR and a decoy or antisense domain of the dimerization initiation site (DIS). These chimeric molecules are able to cleave the HIV-1 5’UTR efficiently and prevent viral genome dimerization in vitro. Furthermore, catalytic antisense RNAs inhibited viral production up to 90% measured as p24 antigen levels in ex vivo assays. The use of chimeric RNA molecules targeting different domains represents an attractive antiviral strategy to be explored for the prevention of side effects from current drugs and of the rapid emergence of escape variants of HIV-1

by Dual Inhibitory RNAs

molecule

In Vitro and Ex Vivo Selection Procedures for Identifying Potentially Therapeutic DNA and RNA Molecules

molecule

Bacterial chemotaxis towards aromatic hydrocarbons in Pseudomonas

12 pages, 5 figures, 2 tables, 48 references.Bacterial chemotaxis is an adaptive behaviour, which requires sophisticated information-processing capabilities that cause motile bacteria to either move towards or flee from chemicals. Pseudomonas putida DOT-T1E exhibits the capability to move towards different aromatic hydrocarbons present at a wide range of concentrations. The chemotactic response is mediated by the McpT chemoreceptor encoded by the pGRT1 megaplasmid. Two alleles of mcpT are borne on this plasmid and inactivation of either one led to loss of this chemotactic phenotype. Cloning of mcpT into a plasmid complemented not only the mcpT mutants but also its transfer to other Pseudomonas conferred chemotactic response to high concentrations of toluene and other chemicals. Therefore, the phenomenon of chemotaxis towards toxic compounds at high concentrations is gene-dose dependent. In vitro experiments show that McpT is methylated by CheR and McpT net methylation was diminished in the presence of hydrocarbons, what influences chemotactic movement towards these chemicals.Peer Reviewe

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 (K = 301 μM, K = 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.We acknowledge financial support from FEDER funds and Fondo Social Europeo through grants from the Junta de Andalucía (grant CVI-7335) and the Spanish Ministry for Economy and Competitiveness (grant BIO2013-42297). MM was supported by the Spanish Ministry of Economy and Competitiveness Postdoctoral Research Program, Juan de la Cierva (JCI-2012-11815).Peer Reviewe

Evidence for chemoreceptors with bimodular ligand binding regions harboring two signal binding sites

Chemoreceptor-based signaling is a central mechanism in bacterial signal transduction. Receptors are classified according to the size of their ligand-binding region. The well-studied cluster I proteins have a 100- to 150-residue ligand-binding region that contains a single site for chemoattractant recognition. Cluster II receptors, which contain a 220- to 300-residue ligand-binding region and which are almost as abundant as cluster I receptors, remain largely uncharacterized. Here, we report high-resolution structures of the ligand-binding region of the cluster II McpS chemotaxis receptor (McpS-LBR) of Pseudomonas putida KT2440 in complex with different chemoattractants. The structure of McpS-LBR represents a small-molecule binding domain composed of two modules, each able to bind different signal molecules. Malate and succinate were found to bind to the membrane-proximal module, whereas acetate binds to the membrane-distal module. A structural alignment of the two modules revealed that the ligand-binding sites could be superimposed and that amino acids involved in ligand recognition are conserved in both binding sites. Ligand binding to both modules was shown to trigger chemotactic responses. Further analysis showed that McpS-like receptors were found in different classes of proteobacteria, indicating that this mode of response to different carbon sources may be universally distributed. The physiological relevance of the McpS architecture may lie in its capacity to respond with high sensitivity to the preferred carbon sources malate and succinate and, at the same time, mediate lower sensitivity responses to the less preferred but very abundant carbon source acetate

RecA protein plays a role in the chemotactic response and chemoreceptor clustering of Salmonella enterica

The RecA protein is the main bacterial recombinase and the activator of the SOS system. In Escherichia coli and Salmonella enterica sv. Typhimurium, RecA is also essential for swarming, a flagellar-driven surface translocation mechanism widespread among bacteria. In this work, the direct interaction between RecA and the CheW coupling protein was confirmed, and the motility and chemotactic phenotype of a S. Typhimurium DrecA mutant was characterized through microfluidics, optical trapping, and quantitative capillary assays. The results demonstrate the tight association of RecA with the chemotaxis pathway and also its involvement in polar chemoreceptor cluster formation. RecA is therefore necessary for standard flagellar rotation switching, implying its essential role not only in swarming motility but also in the normal chemotactic response of S. Typhimurium