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.

Summary Central to the different forms of taxis are methylaccepting chemotaxis proteins (MCPs). The increasing number of genome sequences reveals that

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.histidine kinases ͉ isothermal titration calorimetry ͉ Pseudomonas ͉ two-component systems ͉ aromatic hydrocarbons

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 butdoes 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 (T m ) and unfolding enthalpy (⌬H) 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.

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.sensor domain | four-helix bundle T he ability to sense and respond to extracellular signals is of crucial importance for microorganisms. Bacteria have several types of signal-transduction systems that sense environmental signals and trigger a corresponding response. Genome analyses indicate a dominant role for one-component systems, two-component systems, and chemoreceptor-based mechanisms in bacterial signal transduction (1, 2).Chemoreceptors have been initially described in the context of chemotactic signaling. However, more recent studies reveal that chemoreceptors are also involved in the regulation of different cellular processes, such as the synthesis of second messengers (3) or the control of gene expression during development (4). Typically, chemoreceptors are composed of a ligand-binding region (LBR) and a signaling domain. Signal recognition by the LBR creates a molecular stimulus that is conveyed to the signaling domain, which forms a complex with CheA and CheW. This molecular stimulus modulates CheA autophosphorylation and, subsequently, transphosphorylation toward the response regulator (5).The enterobacterial chemoreceptors, and in particular Tar and Tsr, have been studied extensively (5, 6). The Tar-LBR forms a four-helix bundle structure (7), and there are two mechanisms by which Tar-LBR recognizes chemoattractants. One mode consists o...

The TodS and TodT proteins form a previously unrecognized and highly specific two-component regulatory system in which the TodS sensor protein contains two input domains, each of which are coupled to a histidine kinase domain. This system regulates the expression of the genes involved in the degradation of toluene, benzene, and ethylbenzene through the toluene dioxygenase pathway. In contrast to the narrow substrate range of this catabolic pathway, the TodS effector profile is broad. TodS has basal autophosphorylation activity in vitro, which is enhanced by the presence of effectors. Toluene binds to TodS with high affinity (K d ‫؍‬ 684 ؎ 13 nM) and 1:1 stoichiometry. The analysis of the truncated variants of TodS reveals that toluene binds to the N-terminal input domain (K d ‫؍‬ 2.3 ؎ 0.1 M) but not to the C-terminal half. TodS transphosphorylates TodT, which binds to two highly similar DNA binding sites at base pairs ؊107 and ؊85 of the promoter. Integration host factor (IHF) plays a crucial role in the activation process and binds between the upstream TodT boxes and the ؊10 hexamer region. In an IHF-deficient background, expression from the tod promoter drops 8-fold. In vitro transcription assays confirmed the role determined in vivo for TodS, TodT, and IHF. A functional model is presented in which IHF favors the contact between the TodT activator, bound further upstream, and the ␣-subunit of RNA polymerase bound to the downstream promoter element. Once these contacts are established, the tod operon is efficiently transcribed.Pseudomonas ͉ sensor kinase ͉ toluene dioxygenase ͉ transcriptional regulator M any Pseudomonas putida strains are able to use benzene, toluene, and ethylbenzene as the sole carbon and energy source through the toluene dioxygenase (TOD) pathway (1). In this pathway, the aromatic hydrocarbons are oxidized to their corresponding substituted catechols, which are further metabolized to Krebs cycle intermediates (1, 2). The catabolic genes of the TOD pathway form the operon todXFC1C2BADEGIH, which is transcribed from a single promoter called P todX , located upstream from the todX gene (1-3). The todST genes are found downstream and form an independent operon that is expressed constitutively (2, 3).TodS and TodT have been proposed to form a two-component regulatory system (TCS) that regulates the tod catabolic operon in P. putida F1 (3). TodT shows all of the characteristics of a response regulator, whereas sequence-based domain predictions indicate that the 108-kDa TodS belongs to a family of sensor histidine kinases that have not been studied at the biochemical level. TodS is predicted to comprise two supradomains, each containing a PAS͞PAC sensory domain and a histidine kinase domain. The supradomains are separated by the receiver domain of a response regulator. In contrast to other histidine kinases, TodS apparently lacks transmembrane regions (3). The mode of action of this previously unrecognized type of histidine kinase has yet to be established. On the basis of moderate sequence simil...

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 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.