Split histidine kinases enable ultrasensitivity and bistability in two-component signaling networks

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

Response dynamics of phosphorelays suggest their potential utility in cell signalling

Phosphorelays are extended two-component signalling systems found in diverse bacteria, lower eukaryotes and plants. Only few of these systems are characterized, and we still lack a full understanding of their signalling abilities. Here, we aim to achieve a global understanding of phosphorelay signalling and its dynamical properties. We develop a generic model, allowing us to systematically analyse response dynamics under different assumptions. Using this model, we find that the steady-state concentration of phosphorylated protein at the final layer of a phosphorelay is a linearly increasing, but eventually saturating function of the input. In contrast, the intermediate layers can display ultrasensitivity. We find that such ultrasensitivity is a direct result of the phosphorelay biochemistry; shuttling of a single phosphate group from the first to the last layer. The response dynamics of the phosphorelay results in tolerance of cross-talk, especially when it occurs as cross-deactivation. Further, it leads to a high signal-to-noise ratio for the final layer. We find that a relay length of four, which is most commonly observed, acts as a saturating point for these dynamic properties. These findings suggest that phosphorelays could act as a mechanism to reduce noise and effects of cross-talk on the final layer of the relay and enforce its input–response relation to be linear. In addition, our analysis suggests that middle layers of phosphorelays could embed thresholds. We discuss the consequence of these findings in relation to why cells might use phosphorelays along with enzymatic kinase cascades

Physical, functional and conditional interactions between ArcAB and phage shock proteins upon secretin-induced stress in Escherichia coli

The phage shock protein (Psp) system found in enterobacteria is induced in response to impaired inner membrane integrity (where the Psp response is thought to help maintain the proton motive force of the cell) and is implicated in the virulence of pathogens such as Yersinia and Salmonella. We provided evidence that the two-component ArcAB system was involved in induction of the Psp response in Escherichia coli and now report that role of ArcAB is conditional. ArcAB, predominantly through the action of ArcA regulated genes, but also via a direct ArcB–Psp interaction, is required to propagate the protein IV (pIV)-dependent psp-inducing signal(s) during microaerobiosis, but not during aerobiosis or anaerobiosis. We show that ArcB directly interacts with the PspB, possibly by means of the PspB leucine zipper motif, thereby allowing cross-communication between the two systems. In addition we demonstrate that the pIV-dependent induction of psp expression in anaerobiosis is independent of PspBC, establishing that PspA and PspF can function as a minimal Psp system responsive to inner membrane stress

Exploring and Understanding Signal-response Relationships and Response Dynamics of Microbial Two-Component Signaling Systems

Two-component signaling systems are found in bacteria, fungi and plants. They mediate many of the physiological responses of these organisms to their environment and display several conserved biochemical and structural features. This thesis identifies a potential functional role for two commonly found architectures in two-component signaling system, the split kinases and phosphate sink, which suggests that by enabling switch-like behaviors they could underlie physiological decision making. I report that split histidine kinases, where autophosphorylation and phosphotransfer activities are segregated onto distinct proteins capable of complex formation, enable ultrasensitivity and bistability. By employing computer simulations and analytical approaches, I show that the specific biochemical features of split kinases “by design” enable higher nonlinearity in the system response compared to conventional two-component systems and those using bifunctional (but not split) kinases. I experimentally show that one of these requirements, namely segregation of the phosphatase activity only to the free form of one of the proteins making up the split kinase, is met in proteins isolated from Rhodobacter sphaeroides. While the split kinase I study from R. sphaeroides is specifically involved in chemotaxis, other split kinases are involved in diverse responses. Genomics studies suggest 2.3% of all chemotaxis kinases, and 2.8% of all kinases could be functioning as split kinases. Combining theoretical and experimental approaches, I show that the phosphate sink motif found in microbial and plant TCSs allows threshold behaviors. This motif involves a single histidine kinase that can phosphotransfer reversibly to two separate response regulators and examples are found in bacteria, yeast and plants. My results show that one of the response regulators can act as a “sink” or “buffer” that needs to be saturated before the system can generate significant responses. This sink, thereby allows the generation of a signal threshold that needs to be exceeded for there to be significant phosphoryl group flow to the other response regulator. Thus, this system can enable cells to display switch-like behavior to external signals. Using an analytical approach, I identify mathematical conditions on the system parameters that are necessary for threshold dynamics. I find these conditions to be satisfied in both of the natural systems where the system parameters have been measured. Further, by in vitro reconstitution of a sample system, I experimentally demonstrate threshold dynamics for a phosphate-sink containing two-component system. This study provides a link between these architectures of TCSs and signal-response relationship, thereby enabling experimentally testable hypotheses in these diverse two-component systems. These findings indicate split kinases and phosphate as a microbial alternative for enabling ultrasensitivity and bistability - known to be crucial for cellular decision making. By demonstrating ultrasensitivity, threshold dynamics and their mechanistic basis in a common class of two-component system, this study allows a better understanding of cellular signaling in a diverse range of organisms and will open the way to the design of novel threshold systems in synthetic biology. Thus, I believe that this study will have broad implications not only for microbiologists but also systems biologists who aim to decipher conserved dynamical features of cellular networks.University of Exete

Molecular and cellular factors control signal transduction via switchable allosteric modulator proteins (SAMPs)

Background: Rap proteins from Bacilli directly target response regulators of bacterial two-component systems and modulate their activity. Their effects are controlled by binding of signaling peptides to an allosteric site. Hence Raps exemplify a class of monomeric signaling receptors, which we call switchable allosteric modulator proteins (SAMPs). These proteins have potential applications in diverse biomedical and biotechnical settings, but a quantitative understanding of the impact of molecular and cellular factors on signal transduction is lacking. Here we introduce mathematical models that elucidate how signals are propagated though the network upon receptor stimulation and control the level of active response regulator. Results: Based on a systematic parameter analysis of the models, we show that key features of the dose-response behavior at steady state are controlled either by the molecular properties of the modulator or the signaling context. In particular, we find that the biochemical activity (i.e. non-enzymatic vs. enzymatic) and allosteric properties of the modulator control the response amplitude. The Hill coefficient and the EC50 are controlled in addition by the relative ligand affinities. By tuning receptor properties, either graded or more switch-like (memory-less) response functions can be fashioned. Furthermore, we show that other contextual factors (e.g. relative concentrations of network components and kinase activity) have a substantial impact on the response, and we predict that there exists a modulator concentration which is optimal for response amplitude. Conclusion: We discuss data on Rap-Phr systems in B. subtilis to show how our models can contribute to an integrated view of SAMP signaling by combining biochemical, structural and physiological insights. Our results also suggest that SAMPs could be evolved or engineered to implement diverse response behaviors. However—without additional regulatory controls—they can generate rather variable cellular outputs

STRUCTURAL AND MUTAGENESIS STUDIES OF THE YEAST PHOSPHORELAY SIGNALING PROTEINS YPD1 AND SSK1

His-Asp signaling systems are ubiquitous in bacteria, archaea, and certain plants and fungi. Little structural information is known about the protein-protein interactions within these signaling pathways, leaving an incomplete picture of how these essential systems operate. In this dissertation, the focus of my work in the West laboratory was the receiver domain of the fungal response regulator protein Ssk1, and its interaction with the histidine phosphotransfer protein Ypd1. In Saccharomyces cerevisiae (Sc), Ypd1 interacts with receiver domains from upstream Sln1 and downstream Ssk1 on a common hydrophobic docking site. The main portion of this thesis presents the co-crystal complex of Ypd1 and Ssk1-R2W638A and the accompanying analysis to explain key differences in the physiological functions of Ssk1-R2 and Sln1-R1. Protein-protein interactions were characterized using a newly developed fluorescence binding assay and in vitro 32P-phosphotransfer experiments. In addition, the co-crystallization of Ssk1-R2W638A and a point mutant of Ypd1 (Ypd1-G68Q) is described. Ssk1 protein constructs from the human pathogen Cryptococcus neoformans (Cn) were designed in order to biochemically characterize interactions with C. neoformans Ypd1, but these proteins were either insoluble or inactive. Lastly, my work in the Cichewicz laboratory describes the discovery of three new secondary metabolites from a previously uncharacterized microbial mat fungus (clearanols C, D and E)

Regulation and localisation of PSP proteins in E-Coli

The phage shock protein (Psp) response is found in many Gram-negative enterobacteria, where it helps to maintain the proton motive force (PMF) when the integrity of the inner membrane (IM) is impaired and promotes virulence of pathogens such as Yersinia or Salmonella. In Escherichia coli, Psp comprises seven genes (pspF pspABCDE and pspG) which are organised in a regulon under the control of two Sigma54-dependent promoters. Despite considerable advances, neither the mechanism of Psp induction nor the functioning of the Psp response is fully understood. Recent findings comparing the roles of ArcB in Yersinia enterocolitica and E. coli caused a dispute over the requirement of the twocomponent system ArcAB in Psp signal-transduction. The present study now establishes that ArcAB involvement is conditional and appears to be mediated via protein-protein interactions between ArcB and PspB. The study further suggests that the cellular ubiquinone pool, which acts upstream of ArcAB, may also play a role in Psp signalling whereas dissipation of proton motive force (PMF), generally inferred to be the inducing signal, is not sufficient to mount a Psp response. To gain further insight into its functioning, PspA (a negative regulator and effector of Psp) and PspG (an effector of Psp) were visualised in vivo using fusions to Green fluorescent protein (GFP). To maintain PMF, PspA was proposed to uniformly cover the cytoplasmic face of the IM. However, the present study demonstrates that PspA (and PspG) is highly organised into distinct complexes at the cell pole and the lateral cell membrane. Real-time observations revealed lateral PspA and PspG complexes are highly mobile, but absent in cells lacking MreB. Without the MreB cytoskeleton, induction of the Psp response is still observed, yet these cells fail to maintain PMF under stress conditions