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Key Points Chemotaxis allows bacteria to swim towards environments that are better for growth. The process is involved in pathogenicity, biofilm formation and the establishment of symbiotic relationships. Changes in attractant and repellent concentrations are detected by clusters of chemoreceptors. Bacteria can sense very small changes in attractant concentration over a wide range of background concentrations. The chemoreceptor clusters control the activity of a two-component system comprising the histidine protein kinase CheA and the response regulators CheY and CheB. Phosphorylated CheY controls flagellar motor switching, whereas phosphorylated CheB mediates adaptation. The Escherichia coli chemotaxis signalling pathway is one of the simplest and best understood, but it is becoming increasingly apparent that most bacteria have more complex chemosensory pathways involving multiple homologues of the E. coli chemotaxis proteins. Rhodobacter sphaeroides has one of the best understood complex chemotaxis pathways; it has two distinct types of chemosensory cluster: one that is positioned at the cell pole and detects changes in the external attractant and repellent concentrations, and another that is cytoplasmic and is believed to monitor the metabolic state of the cell (a form of energy taxis). Structural studies have revealed the specificity determinants in the interaction of CheY proteins with CheA proteins and allowed rewiring of the signalling pathway. Mechanisms of signal integration and signal termination have been elucidated by mathematical modelling. Some bacteria have complex chemotaxis pathways that go beyond what is found in E. coli and R. sphaeroides . For example, in addition to the methylation-based adaptation system, Bacillus subtilis has two further adaptation pathways, one involving CheC and CheD and another using CheV. Some bacteria exploit the ability of the chemotaxis circuitry to sense small changes in ligand concentrations, and use the system to control behaviour other than chemotaxis. For example, Myxococcus xanthus has a chemotaxis-like pathway controlling development of the fruiting body, and Pseudomonas aeruginosa has one controlling biofilm formation. In this Review, Armitage and colleagues describe how some bacterial species, as typified by Rhodobacter sphaeroides , have evolved to contain complex chemotaxis signalling networks that integrate sensory information from the environment with metabolic information from within the cell to produce a balanced response at the flagellar motor. Bacteria use chemotaxis to migrate towards environments that are better for growth. Chemoreceptors detect changes in attractant levels and signal through two-component systems to control swimming direction. This basic pathway is conserved across all chemotactic bacteria and archaea; however, recent work combining systems biology and genome sequencing has started to elucidate the additional complexity of the process in many bacterial species. This article focuses on one of the best understood complex networks, which is found in Rhodobacter sphaeroides and integrates sensory data about the external environment and the metabolic state of the cell to produce a balanced response at the flagellar motor.

Bacteria and many non-metazoan Eukaryotes respond to stresses and threats using two-component systems (TCSs) comprising sensor kinases (SKs) and response regulators (RRs). Multikinase networks, where multiple SKs work together, detect and integrate different signals to control important lifestyle decisions such as sporulation and virulence. Here, we study interactions between two SKs from Pseudomonas aeruginosa , GacS and RetS, which control the switch between acute and chronic virulence. We demonstrate three mechanisms by which RetS attenuates GacS signalling: RetS takes phosphoryl groups from GacS-P; RetS has transmitter phosphatase activity against the receiver domain of GacS-P; and RetS inhibits GacS autophosphorylation. These mechanisms play important roles in vivo and during infection, and exemplify an unprecedented degree of signal processing by SKs that may be exploited in other multikinase networks. Bacteria respond to stresses using two-component systems consisting of sensor kinases (SKs) and response regulators. Here, Francis et al . reveal three specific interaction mechanisms between a pair of SKs that are important for regulation of virulence in the pathogen Pseudomonas aeruginosa .

Melioidosis is an emerging, potentially life-threatening infection caused by the bacterium Burkholderia pseudomallei , killing ~89,000 people per year globally. Antibiotic therapy fails in ~10%–40% of cases, and hence, an improved understanding of the molecular mechanisms that control B. pseudomallei virulence could reveal new approaches for improving melioidosis treatment. Biofilm formation and resistance to the antimicrobial radical NO are virulence traits that help bacteria establish infections. Here, we show that two proteins in B. pseudomallei , NosP and NosK, work together to detect NO and regulate a suite of virulence traits, including NO resistance, biofilm formation, growth, and swimming motility. This work, therefore, improves our understanding of the molecular mechanisms that control infection-related phenotypes in B. pseudomallei .

Abstract Pseudomonas aeruginosa is a versatile opportunistic pathogen capable of infecting a broad range of hosts, in addition to thriving in a broad range of environmental conditions outside of hosts. With this versatility comes the need to tightly regulate its genome to optimise its gene expression and behaviour to the prevailing conditions. Two-component systems (TCSs) comprising sensor kinases and response regulators play a major role in this regulation. This minireview discusses the growing number of TCSs that have been implicated in the virulence of P. aeruginosa, with a special focus on the emerging theme of multikinase networks, which are networks comprising multiple sensor kinases working together, sensing and integrating multiple signals to decide upon the best response. The networks covered in depth regulate processes such as the switch between acute and chronic virulence (GacS network), the Cup fimbriae (Roc network and Rcs/Pvr network), the aminoarabinose modification of lipopolysaccharide (a network involving the PhoQP and PmrBA TCSs), twitching motility and virulence (a network formed from the Chp chemosensory pathway and the FimS/AlgR TCS), and biofilm formation (Wsp chemosensory pathway). In addition, we highlight the important interfaces between these systems and secondary messenger signals such as cAMP and c-di-GMP. Two-component systems controlling the virulence of the opportunistic pathogen, Pseudomonas aeruginosa.

Synthetic biology aims to design de novo biological systems and reengineer existing ones. These efforts have mostly focused on transcriptional circuits, with reengineering of signaling circuits hampered by limited understanding of their systems dynamics and experimental challenges. Bacterial two-component signaling systems offer a rich diversity of sensory systems that are built around a core phosphotransfer reaction between histidine kinases and their output response regulator proteins, and thus are a good target for reengineering through synthetic biology. Here, we explore the signal-response relationship arising from a specific motif found in two-component signaling. In this motif, a single histidine kinase (HK) phosphotransfers reversibly to two separate output response regulator (RR) proteins. We show that, under the experimentally observed parameters from bacteria and yeast, this motif not only allows rapid signal termination, whereby one of the RRs acts as a phosphate sink towards the other RR (i.e. the output RR), but also implements a sigmoidal signal-response relationship. We identify two mathematical conditions on system parameters that are necessary for sigmoidal signal-response relationships and define key parameters that control threshold levels and sensitivity of the signal-response curve. We confirm these findings experimentally, by in vitro reconstitution of the one HK-two RR motif found in the Sinorhizobium meliloti chemotaxis pathway and measuring the resulting signal-response curve. We find that the level of sigmoidality in this system can be experimentally controlled by the presence of the sink RR, and also through an auxiliary protein that is shown to bind to the HK (yielding Hill coefficients of above 7). These findings show that the one HK-two RR motif allows bacteria and yeast to implement tunable switch-like signal processing and provides an ideal basis for developing threshold devices for synthetic biology applications.

Two-component signal transduction pathways comprising histidine protein kinases (HPKs) and their response regulators (RRs) are widely used to control bacterial responses to environmental challenges. Some bacteria have over 150 different two-component pathways, and the specificity of the phosphotransfer reactions within these systems is tightly controlled to prevent unwanted crosstalk. One of the best understood two-component signalling pathways is the chemotaxis pathway. Here, we present the 1.40 A crystal structure of the histidine-containing phosphotransfer domain of the chemotaxis HPK, CheA(3), in complex with its cognate RR, CheY(6). A methionine finger on CheY(6) that nestles in a hydrophobic pocket in CheA(3) was shown to be important for the interaction and was found to only occur in the cognate RRs of CheA(3), CheY(6), and CheB(2). Site-directed mutagenesis of this methionine in combination with two adjacent residues abolished binding, as shown by surface plasmon resonance studies, and phosphotransfer from CheA(3)-P to CheY(6). Introduction of this methionine and an adjacent alanine residue into a range of noncognate CheYs, dramatically changed their specificity, allowing protein interaction and rapid phosphotransfer from CheA(3)-P. The structure presented here has allowed us to identify specificity determinants for the CheA-CheY interaction and subsequently to successfully reengineer phosphotransfer signalling. In summary, our results provide valuable insight into how cells mediate specificity in one of the most abundant signalling pathways in biology, two-component signal transduction.

Bacteria sense and respond to their environment through signaling cascades generally referred to as two-component signaling networks. These networks comprise histidine kinases and their cognate response regulators. Histidine kinases have a number of biochemical activities: ATP binding, autophosphorylation, the ability to act as a phosphodonor for their response regulators, and in many cases the ability to catalyze the hydrolytic dephosphorylation of their response regulator. Here, we explore the functional role of \"split kinases\" where the ATP binding and phosphotransfer activities of a conventional histidine kinase are split onto two distinct proteins that form a complex. We find that this unusual configuration can enable ultrasensitivity and bistability in the signal-response relationship of the resulting system. These dynamics are displayed under a wide parameter range but only when specific biochemical requirements are met. We experimentally show that one of these requirements, namely segregation of the phosphatase activity predominantly onto the free form of one of the proteins making up the split kinase, is met in Rhodobacter sphaeroides. These findings indicate split kinases as a bacterial alternative for enabling ultrasensitivity and bistability in signaling networks. Genomic analyses reveal that up 1.7% of all identified histidine kinases have the potential to be split and bifunctional.

Understanding how multiple signals are integrated in living cells to produce a balanced response is a major challenge in biology. Two-component signal transduction pathways, such as bacterial chemotaxis, comprise histidine protein kinases (HPKs) and response regulators (RRs). These are used to sense and respond to changes in the environment. Rhodobacter sphaeroides has a complex chemosensory network with two signaling clusters, each containing a HPK, CheA. Here we demonstrate, using a mathematical model, how the outputs of the two signaling clusters may be integrated. We use our mathematical model supported by experimental data to predict that: (1) the main RR controlling flagellar rotation, CheY(6), aided by its specific phosphatase, the bifunctional kinase CheA(3), acts as a phosphate sink for the other RRs; and (2) a phosphorelay pathway involving CheB(2) connects the cytoplasmic cluster kinase CheA(3) with the polar localised kinase CheA(2), and allows CheA(3)-P to phosphorylate non-cognate chemotaxis RRs. These two mechanisms enable the bifunctional kinase/phosphatase activity of CheA(3) to integrate and tune the sensory output of each signaling cluster to produce a balanced response. The signal integration mechanisms identified here may be widely used by other bacteria, since like R. sphaeroides, over 50% of chemotactic bacteria have multiple cheA homologues and need to integrate signals from different sources.

Phosphorylation-based signaling pathways employ dephosphorylation mechanisms for signal termination. Histidine to aspartate phosphosignaling in the two-component system that controls bacterial chemotaxis has been studied extensively. Rhodobacter sphaeroides has a complex chemosensory pathway with multiple homologues of the Escherichia coli chemosensory proteins, although it lacks homologues of known signal-terminating CheY-P phosphatases, such as CheZ, CheC, FliY or CheX. Here, we demonstrate that an unusual CheA homologue, CheA₃, is not only a phosphodonor for the principal CheY protein, CheY₆, but is also is a specific phosphatase for CheY₆-P. This phosphatase activity accelerates CheY₆-P dephosphorylation to a rate that is comparable with the measured stimulus response time of approximately 1 s. CheA₃ possesses only two of the five domains found in classical CheAs, the Hpt (P1) and regulatory (P5) domains, which are joined by a 794-amino acid sequence that is required for phosphatase activity. The P1 domain of CheA₃ is phosphorylated by CheA₄, and it subsequently acts as a phosphodonor for the response regulators. A CheA₃ mutant protein without the 794-amino acid region lacked phosphatase activity, retained phosphotransfer function, but did not support chemotaxis, suggesting that the phosphatase activity may be required for chemotaxis. Using a nested deletion approach, we showed that a 200-amino acid segment of CheA₃ is required for phosphatase activity. The phosphatase activity of previously identified nonhybrid histidine protein kinases depends on the dimerization and histidine phosphorylation (DHp) domains. However, CheA₃ lacks a DHp domain, suggesting that its phosphatase mechanism is different from that of other histidine protein kinases.

The truth of one's religious beliefs can be questioned by appeal to hypocrisy or blatant moral failure amongst the adherents of one's religion. Such an appeal implies that the absence of spiritual maturity within a religious individual or group can serve in some way as evidence against the truth of that religion and (presumably), conversely, that spiritual maturity within a religious individual or group can be thought of as providing some sort of evidence for the truth of that religion. The first part of this article attempts to get clear on what sort of evidential force the presence or absence of spiritual maturity has for the rational assessment of religious belief in general. This part of the article concludes that the evidential force of spiritual maturity must ultimately be assessed within the contours of a particular religion with a firm grasp on the sort of moral formational process envisaged by that religion. So, in the second part of the article, the evidential force of spiritual maturity is considered from a Christian perspective and an interpersonal model of sanctification is appealed to as an explanation of the lack of spiritual maturity amongst Christian believers.