Jesús Lacal

Bacterial Sensor Kinases: Diversity in the Recognition of Environmental Signals

Bacteria sense and respond to a wide range of physical and chemical signals. Central to sensing and responding to these signals are two-component systems, which have a sensor histidine kinase (SK) and a response regulator (RR) as basic components. Here we review the different molecular mechanisms by which these signals are integrated and modulate the phosphorylation state of SKs. Apart from the basic mechanism, which consists of signal recognition by the SK that leads to an alteration of its autokinase activity and subsequently a change in the RR phosphorylation state, a variety of alternative modes have evolved. The biochemical data available on SKs, particularly their molecular interactions with signals, nucleotides, and their cognate RRs, are also reviewed.

Sensing of environmental signals: classification of chemoreceptors according to the size of their ligand binding regions.

Central to the different forms of taxis are methyl-accepting chemotaxis proteins (MCPs). The increasing number of genome sequences reveals that MCPs differ enormously in sequence, topology and genomic abundance. This work is a one-by-one bioinformatic analysis of the almost-totality of MCP genes available and a classification of motile bacteria according to their lifestyle. On average, motile archaea have 6.7 MCP genes per genome whereas motile bacteria have more than twice as much. We show that the number of MCPs per genome depends on bacterial lifestyle and metabolic diversity, but weakly on genome size. Signal perception at an MCP occurs at the N-terminal ligand binding region (LBR). Here we show that around 88% of MCPs possess an LBR that remains un-annotated in SMART. MCPs can be classified into two clusters according to the size of the LBR. Cluster I receptors have an LBR between 120 and 210 amino acids whereas cluster II receptors have larger LBRs of 220-299 amino acids. There is evidence that suggests that some cluster II LBRs are composed of two cluster I LBRs. Further evidence indicates that other cluster II LBRs might harbour novel sensor domains. Cluster II receptors are dominant in archaea whereas cluster I receptors are prevalent in bacteria. MCPs can be classified into six different receptor topologies and this work contains a first estimation of the relative abundance of different receptor topologies in bacteria and archaea. Topologies involving extracytoplasmic sensing are prevalent in bacteria whereas topologies with cytosolic signal recognition are abundant in archaea.

Diversity at its best: bacterial taxis

Bacterial taxis is one of the most investigated signal transduction mechanisms. Studies of taxis have primarily used Escherichia coli and Salmonella as model organism. However, more recent studies of other bacterial species revealed a significant diversity in the chemotaxis mechanisms which are reviewed here. Differences include the genomic abundance, size and topology of chemoreceptors, the mode of signal binding, the presence of additional cytoplasmic signal transduction proteins or the motor mechanism. This diversity of chemotactic mechanisms is partly due to the diverse nature of input signals. However, the physiological reasons for the majority of differences in the taxis systems are poorly understood and its elucidation represents a major research need.

Bacterial sensor kinase TodS interacts with agonistic and antagonistic signals

The TodS/TodT two-component system controls expression of the toluene dioxygenase (TOD) pathway for the metabolism of toluene in Pseudomonas putida DOT-T1E. TodS is a sensor kinase that ultimately controls tod gene expression through its cognate response regulator, TodT. We used isothermal titration calorimetry to study the binding of different compounds to TodS and related these findings to their capacity to induce gene expression in vivo. Agonistic compounds bound to TodS and induced gene expression in vivo. Toluene was a powerful agonist, but ortho-substitutions of toluene reduced or abolished in vivo responses, although TodS recognized o-xylene with high affinity. These compounds were called antagonists. We show that agonists and antagonists compete for binding to TodS both in vitro and in vivo. The failure of antagonists to induce gene expression in vivo correlated with their inability to stimulate TodS autophosphorylation in vitro. We propose intramolecular TodS signal transmission, not molecular recognition of compounds by TodS, to be the phenomenon that determines whether a given compound will lead to activation of expression of the tod genes. Molecular modeling identified residues F46, I74, F79, and I114 as being potentially involved in the binding of effector molecules. Alanine substitution mutants of these residues reduced affinities (2- to 345-fold) for both agonistic and antagonistic compounds. Our data indicate that determining the inhibitory activity of antagonists is a potentially fruitful alternative to design specific two-component system inhibitors for the development of new drugs to inhibit processes regulated by two-component systems.

Identification of a Chemoreceptor for Tricarboxylic Acid Cycle Intermediates DIFFERENTIAL CHEMOTACTIC RESPONSE TOWARDS RECEPTOR LIGANDS

We report the identification of McpS as the specific chemoreceptor for 6 tricarboxylic acid (TCA) cycle intermediates and butyrate in Pseudomonas putida. The analysis of the bacterial mutant deficient in mcpS and complementation assays demonstrate that McpS is the only chemoreceptor of TCA cycle intermediates in the strain under study. TCA cycle intermediates are abundantly present in root exudates, and taxis toward these compounds is proposed to facilitate the access to carbon sources. McpS has an unusually large ligand-binding domain (LBD) that is un-annotated in InterPro and is predicted to contain 6 helices. The ligand profile of McpS was determined by isothermal titration calorimetry of purified recombinant LBD (McpS-LBD). McpS recognizes TCA cycle intermediates but does not bind very close structural homologues and derivatives like maleate, aspartate, or tricarballylate. This implies that functional similarity of ligands, such as being part of the same pathway, and not structural similarity is the primary element, which has driven the evolution of receptor specificity. The magnitude of chemotactic responses toward these 7 chemoattractants, as determined by qualitative and quantitative chemotaxis assays, differed largely. Ligands that cause a strong chemotactic response (malate, succinate, and fumarate) were found by differential scanning calorimetry to increase significantly the midpoint of protein unfolding (Tm) and unfolding enthalpy (ΔH) of McpS-LBD. Equilibrium sedimentation studies show that malate, the chemoattractant that causes the strongest chemotactic response, stabilizes the dimeric state of McpS-LBD. In this respect clear parallels exist to the Tar receptor and other eukaryotic receptors, which are discussed.

Bacterial chemotaxis towards aromatic hydrocarbons in Pseudomonas

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.

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.

The pGRT1 plasmid of Pseudomonas putida DOT‐T1E encodes functions relevant for survival under harsh conditions in the environment

Pseudomonas putida DOT-T1E has the capacity to grow in the presence of high concentrations of toluene. This ability is mainly conferred by an efflux pump encoded in a self-transmissible 133 kb plasmid named pGRT1. Sequence analysis of the pGRT1 plasmid revealed several key features. Most of the genes related to the plasmid maintenance functions show similarity with those encoded on pBVIE04 from Burkholderia vietnamensis G4, and knock-out mutants in several of these genes confirmed their roles. Two additional plasmid DNA fragments were incorporated into the plasmid backbone by recombination and/or transposition; in these DNA regions, apart from multiple recombinases and transposases, several stress-related and environmentally relevant functions are encoded. We report that plasmid pGRT1 not only confers the cells with tolerance to toluene but also resistance to ultraviolet light. We show here the implication of a new protein in solvent tolerance which controls the level of expression of the TtgGHI efflux pump, as well as the implication of a protein with homology to the universal stress protein in solvent tolerance and ultraviolet light resistance. Furthermore, this plasmid encodes functions that allow the cells to chemotactically respond to toluene and participate in iron scavenging.

Bioavailability of pollutants and chemotaxis

The exposure of bacteria to pollutants induces frequently chemoattraction or chemorepellent reactions. Recent research suggests that the capacity to degrade a toxic compound has co-evolved in some bacteria with the capacity to chemotactically react to it. There is an increasing amount of data which show that chemoattraction to biodegradable pollutants increases their bioavailability which translates into an enhancement of the biodegradation rate. Pollutant chemoreceptors so far identified are encoded on degradation or resistance plasmids. Genetic engineering of bacteria, such as the transfer of chemoreceptor genes, offers thus the possibility to optimize biodegradation processes.

Hierarchical binding of the TodT response regulator to its multiple recognition sites at the tod pathway operon promoter.

The TodS and TodT proteins form a highly specific two-component regulatory system that controls the expression of genes involved in the degradation of toluene, benzene, and ethylbenzene via the toluene dioxygenase pathway. The catabolic genes of the toluene dioxygenase pathway are transcribed from a single promoter called P(todX) once the response regulator TodT is phosphorylated by the TodS sensor kinase in response to pathway substrates. We show here that TodT is a monomer in solution and that it binds to three specific sites in the P(todX) promoter, centered at -57, -85, and -106 with respect to the transcription start site. The -85 and -106 sites are pseudopalindromic, whereas the -57 site is half a palindrome. TodT binding to its target sites is sequential, as shown by electrophoresis mobility gel shift assays and footprinting. The binding affinity values of TodT, as determined by isothermal titration calorimetry, are 1.8+/-0.2, 5+/-0.4, and 6.3+/-0.8 microM for the -106, -85, and -57 sites, respectively, and the binding stoichiometry is one monomer per half-palindromic element. Mutational analysis revealed that all three sites contribute to P(todX) strength, although the most relevant site is the distal one with respect to the -10 extended element of the downstream promoter element. The C-TodT [C-terminal TodT fragment (amino acids 154-206)], a truncated variant of TodT that contains the C-terminal half of the protein bearing the DNA binding domain, binds in vitro to all three sites with affinity similar to that of the full-length protein. However, C-TodT, in contrast to the full-length regulator, does not activate in vitro transcription from P(todX). We discuss the consequences of the organization of the binding sites on transcriptional control and propose that the N-terminal domain of TodT is necessary for appropriate interactions with other transcriptional elements.