1. Classification and Organisation of Two-component Systems

David E. Whitworth

Two-component systems (TCSs) are common signal transduction pathways found abundantly in most phyla except animals. The basic TCS pathway involves two multi-domain proteins. The first is a histidine protein kinase (HPK) whose autokinase activity is dependent upon an environmental stimulus. The second is a response regulator (RR), onto which a phosphoryl group is transferred from the phosphorylated HPK, and which mediates phosphorylation-dependent effects within the cell. TCS proteins can be readily identified from genomic sequences, however, approaches for identifying and classifying TCS proteins are non-trivial because TCSs are multi-domain, multi-gene pathways, which exhibit considerable heterogeneity in their gene and domain organisation. This chapter provides an overview of the domain architectures and genetic organisation of TCSs, including the common TCS variants known as phosphorelays and chemosensory systems. Strategies for the identification of TCS genes are described, alongside systems for categorising TCS proteins that exploit the evolutionary relationships between TCS proteins to provide biologically relevant insights. The development of such classification systems are reviewed in addition to the current state of the art, to help the reader navigate through the extensive literature on this topic.

2. Structural Basis of Signal Transduction and Specificity in Two-components Systems

Patricia Casino, Marisa López-Redondo and Alberto Marina

Two-component systems (TCSs) constitute a signal transduction mechanism, mainly found in prokaryotes, which is relatively simple as they are basically formed by two proteins: a histidine kinase (HK) and a response regulator (RR). These two proteins are able to transmit all kinds of signals productively and elegantly by the use of phosphorelays in order to ensure cell survival. For this purpose, the HK possesses many qualities; first it is able to sense a signal, second it binds ATP, and third it autophosphorylates on a catalytic His residue. Subsequently, the RR comes into play to promote the final response, but first it needs to be phosphorylated on a catalytic Asp residue via a phosphoryl group transfer from the phosphorylated His of the HK. The phosphorylated RR (RR~P) can thus elicit many diverse responses, which generally involve binding to DNA to activate specific genes. Finally, the system is shut down by the dephosphorylation of the RR~P, a mechanism which can be performed by the RR itself, or assisted by the HK. Undoubtedly, understanding the molecular basis of specificity in the interaction between HK and RR couples as well as the complete catalytic mechanism between these two molecules in the signaling process is especially important from many viewpoints, including the medical, and recent advances in the structural and biochemical field have contributed decidedly.

3. Redox Responding Sensor Kinases

Jiang Wu, Vladimira Dragnea and Carl Bauer

Sensor kinases that respond to changes in redox have been described in several bacterial systems. This review is centered on the molecular mechanism of redox sensing in a select group of sensor kinases that have been well-defined biochemically. We cover the role of oxidized ubiquinone and redox active cysteine in controlling the kinase activity of the sensor kinases RegB and ArcB of Rhodobacter capsulatus and Escherichia coli, respectively. We also review inhibition of kinase activity that occurs upon the binding of dioxygen to heme to a PAS domain in FixL of rhizobia and upon the binding of dioxygen to heme to a GAF domain in DosT of Mycobacterium tuberculosis. Finally, we discuss recent evidence that the photoreceptor LovK of Caulobacter crescentus contains a redox active flavin that when reduced inhibits the ability of LovK to undergo a photocycle that regulates kinase activity.

4. BvgS of Pathogenic Bordetellae: a Paradigm for Sensor-kinases with Venus Flytrap Perception Domains

Françoise Jacob-Dubuisson, René Wintjens, Julien Herrou, Elian Dupré and Rudy Antoine

The whooping cough agent Bordetella pertussis regulates the expression of its virulence regulon through the two-component system BvgAS. BvgA is a canonical response regulator serving as a transcriptional activator when phosphorylated. BvgS is a multidomain, hybrid sensor-kinase harbouring several cytoplasmic domains that mediate a complex phospho-transfer cascade. BvgS also contains two periplasmic Venus flytrap (VFT) domains in tandem. It is thus the prototype for a large family of bacterial VFT-containing sensor-kinases, whose molecular mechanisms for signal perception and transduction remain to be deciphered. Ubiquitous in nature, VFT domains usually function along a clamshell model, with two lobes that enclose specific ligands. Structure/function analyses of the second VFT domain of BvgS have indicated a non-canonical behaviour, whereby this domain adopts a closed conformation that gives a positive signal to the system in the absence of a bona fide ligand and can also bind negative signals that turn off the system. Sequence analyses of BvgS have shown a strong conservation of the region linking the periplasmic and cytoplasmic domains, indicating that it is essential for signal transduction. On the contrary, the linkers between VFT domains are not conserved, and thus the VFT most likely communicate with each other via their interfaces.

5. Phosphatase Activity of Two-component System Transmitter Proteins

Alexander J. Ninfa

Many of the two-component system transmitter proteins bring about both the phosphorylation and dephosphorylation of their cognate receiver proteins. While the mechanism of the transmitter protein kinase activity was relatively easy to discern biochemically, investigation of the phosphatase activity has proven more difficult owing to the involvement of widely dispersed portions of the transmitter protein in the activity and the corresponding requirement to study the activity using intact, full-length transmitter proteins. Here, I review the genetics, physiology, and biochemistry of the transmitter protein phosphatase activity, with special emphasis on possible mechanisms of receiver protein dephosphorylation, relationship of the transmitter phosphatase activity to the autophosphatase activity of the receiver protein, and physiological significance of the activity.

6. Deviations from the Rule: Orphan and Atypical Response Regulators

Dagmar Beier

In a classical two-component system a response regulator is activated via phosphoryl group transfer from a cognate histidine kinase in order to elicit a cellular output response to a specific stimulus. However, it is increasingly recognized that response regulator proteins do not necessarily rely on this standard activation mode. The so-called orphan response regulators lack a cognate histidine kinase as phosphorylation partner. In many cases these proteins retain their function upon substitution of the phosphate-accepting aspartic acid residue in the receiver domain suggesting phosphorylation-independent mechanisms for output response control. The concept of unorthodox signaling to and activation of a response regulator is supported by the occurrence of atypical response regulators in several bacteria. Atypical response regulators comprise degenerate receiver domains with conserved residues required for the phosphotransfer reaction and signal propagation within the response regulator protein being absent, which certainly precludes canonical receiver phosphorylation. Nevertheless, atypical response regulators are involved in various cellular functions including vegetative cell growth, differentiation, secondary metabolism and virulence.

7. Essential Two-component Systems of Gram-positive Bacteria

Hendrik Szurmant

The YycF/YycG two-component signal transduction system has become an enigma since its initial discovery as the only signal transduction system essential for viability in Bacillus subtilis, Staphylococcus aureus and Streptococcus pneumoniae. Conserved across the low G+C Gram-positives, over a decade worth of dedicated molecular investigations have identified some of the factors that control the YycG kinase activity and the genes that are regulated by the YycF transcription factor. A picture has emerged where the YycFG system is embedded in the physiology of the cell and serves to coordinate essential physiological processes such as cell wall homeostasis and cell division. Several regulatory connections with other signaling systems have been recognized. The YycFG system has surfaced as an important player in the acquisition of antibiotic resistances in clinical isolates of significant human pathogens and has become the target of dedicated antimicrobial drug screening efforts. This chapter summarizes the current state of knowledge on this and other essential two-component signaling systems in Gram-positive bacteria.

8. Molecular Mechanism of Bacterial Two-component Signal Transduction Networks via Connectors

Yoko Eguchi, Eiji Ishii and Ryutaro Utsumi

Two-component system (TCS) connectors are proteins that connect different TCSs. By connecting TCSs, they integrate the signals to which each TCS responds, and enable the cell to respond flexibly to the fluctuating environment. A number of auxiliary proteins of TCSs, which bind to the components of TCSs for modification, have been reported. When the expression of such TCS auxiliary proteins is controlled by a TCS, it connects the two TCSs and is termed a TCS connector. Several TCS connectors have been identified in Salmonella and Escherichia coli. In this chapter, we describe how these TCS connectors and the TCSs they connect form a signal transduction network. TCS connectors in Bacillus subtilis as well as TCS auxiliary proteins as candidates of TCS connectors are also introduced. We have clarified a small portion of the predicted two-component signal transduction network as an initial description of the complicated signal network that exists in bacteria.

9. Antikinases: their Structures and Roles in Two-component Signalling

David A. Jacques, J. Mitchell Guss and Jill Trewhella

Antikinases are protein inhibitors of the bacterial sensor histidine kinases found in two-component systems. Antikinases act by binding the highly-conserved autokinase domains and thereby inhibit autophosphorylation. Antikinases offer unique opportunities to understand histidine kinase function and inhibition, and as histidine kinases are not found in mammals, knowledge of their structures and binding can potentially be exploited for the development of novel antimicrobial compounds. This chapter describes our current understanding of the three known antikinases: Sda and KipI from Bacillus subtilis and FixT from Sinorhizobium meliloti, with a focus on understanding of their detailed structures and mechanisms of action.

10. K⁺ Supply, Osmotic Stress, and the KdpD/KdpE Two-component System

Ralf Heermann and Kirsten Jung

The KdpD/KdpE system is one of the most distributed histidine kinase/response regulator systems in bacteria. This two-component system controls expression of the kdpFABC operon encoding the high affinity K⁺ uptake system KdpFABC. KdpD/KdpE is activated whenever the constitutively expressed K⁺ uptake systems are unable to keep up with the cellular need for K⁺. Typical KdpD/KdpE activating conditions comprise K⁺ limitation or osmotic stress. The histidine kinase KdpD is composed of several subdomains that are important for stimulus perception, modulation of the kinase to phosphatase ratio, and signaling. The response regulator KdpE receives the phosphoryl group from KdpD and induces kdpFABC transcription. The three-dimensional structure of the KdpE receiver domain gives insights into the activation mechanism of this transcriptional regulator. Two accessory proteins, the universal stress protein UspC and the phosphotransferase system component IIA^Ntr, interact with KdpD, whereby kdpFABC expression is modulated under certain physiological conditions. Here we will review the nature of the stimulus of KdpD/KdpE in correlation with structural features as well as the interconnectivity of KdpD/KdpE with K⁺ supply, osmotic stress response and virulence.

11. Two-component Signaling in the Gram-positive Envelope Stress Response: Intramembrane-sensing Histidine Kinases and Accessory Membrane Proteins

Karen Schrecke, Anna Staroń and Thorsten Mascher

The cell envelope stress response (CESR) network monitors and maintains envelope integrity to counteract the damaging effects of cell wall antibiotics and membrane perturbating agents. Two-component systems (2CSs) involved in orchestrating CESR in Firmicutes bacteria (low G+C Gram-positive) are characterized by so-called intramembrane-sensing histidine kinases (IM-HKs). The N-terminal input domain of these proteins consists of two transmembrane helices with a very short extracellular linker of less than 20 amino acids, which is insufficient for stimulus perception. It was originally thought that these HKs sense their stimuli within the membrane interface. But subsequent studies identified accessory membrane proteins for all IM-HKs described so far. This chapter will specifically summarize the current state of knowledge on BceRS- and LiaRS-like 2CSs, which are ubiquitously distributed in Firmicutes bacteria. While BceRSAB-like systems represent antibiotic-specific detoxification modules, LiaFSR-like three-component systems mount more general CESR. These two types of systems are genetically and functionally linked to BceAB-like ABC transporters and LiaF-like membrane-anchored regulatory proteins, respectively, which play a crucial role in sensing envelope stress and transferring the information to the cognate HKs. Accordingly, BceS- and LiaS-like IM-HKs do not function as sensor proteins, but rather as signal transfer relays between the sensor and the cognate response regulators.

12. The CpxAR Two-component System Regulates a Complex Envelope Stress Response in Gram Negative Bacteria

Stefanie Vogt, Nicole Acosta, Julia Wong, Junshu Wang and Tracy Raivio

The CpxA membrane bound sensor kinase utilizes a periplasmic sensing domain to detect a wide variety of stresses to the bacterial envelope. This information is communicated via typical two-component phosphotransfer mediated reactions to the response regulator CpxR. Phosphorylated CpxR binds upstream of numerous promoters to mediate adaptation. Initial studies of CpxA inducing signals and CpxR regulated genes demonstrated a role for this two-component system in responding to protein misfolding in the envelope. In this chapter, we discuss recent progress regarding the mechanisms of signal detection, transduction, and gene regulation employed by CpxA and CpxR. The data indicate that the majority of inducing cues are sensed through the periplasmic domain of CpxA and lead to the relief of one or more inhibitory controls that function to maintain Cpx pathway activity at low basal levels in the absence of envelope stress. Some activating signals also enter the pathway downstream of CpxA, in the cytoplasm. Analysis of the Cpx regulon in multiple organisms indicates that, in addition to regulating the production of well studied envelope protein folding and degrading factors, CpxA and CpxR also control the expression of cellular functions linked to cell wall modification, transport, translation, and regulation, indicating that adaptation to envelope stress involves broad changes in cellular physiology. The Cpx two-component system has been shown to impact virulence in a number of pathogens, and our current knowledge of this field is discussed.

13. Cell Cycle and Developmental Regulation By Two-component Signaling Proteins in Caulobacter crescentus

Stephen C. Smith, Juan-Jesus Vicente and Kathleen R. Ryan

The intricate cell division and developmental cycle of the α-Proteobacterium Caulobacter crescentus has been studied for four decades. During that time, elegant genetic screens and comprehensive post-genomic methods have uncovered a branched network of two-component signaling proteins that orchestrates Caulobacter cell cycle progression and morphological development. In addition to yielding the first and most detailed picture of bacterial cell cycle control, Caulobacter studies have revealed novel ways in which two-component proteins generate cellular outputs, interact with each other, and are themselves regulated.

14. Two-component Systems Involved in Regulation of Motility and Development in Myxococcus xanthus

Daniela Keilberg, Stuart Huntley and Lotte Søgaard-Andersen

The Myxococcus xanthus lifecycle is characterized by many social interactions. In particular, M. xanthus forms cooperatively spreading colonies in the presence of nutrients and multicellular, spore-filled fruiting bodies in the absence of nutrients. Formation of both cellular patterns depends on two intact motility systems. Moreover, fruiting body formation depends on intercellular communication and temporally regulated gene expression. The M. xanthus genome encodes a staggering 272 putative proteins of two-component system and most aspects of the M. xanthus lifecycle are regulated by one or more of these proteins. Interestingly, many of the corresponding genes encoding two-component system proteins possess an unusual organization in complex genes clusters and as orphan genes. However, major strides have been made in our understanding of a large number of these proteins. Here, we focus on the function of well-studied proteins of two-component systems in motility and development in M. xanthus.

15. Two-component Systems in Streptomyces

Juan-Francisco Martín, Alberto Sola-Landa and Antonio Rodríguez-García

Two-component systems (TCS) play a very important role in the regulation of metabolism in Streptomyces species in response to different nutritional or environmental signals. Streptomyces are Gram-positive soil-dwelling filamentous bacteria with large genomes that have the ability to produce thousands of different secondary metabolites. Streptomyces genomes contain a large number of paired two-component systems (usually more than 70) and some additional orphan sensor kinases and response regulators. Several of these systems have been studied in detail in the model species Streptomyces coelicolor. Particular attention has been paid to the PhoR/PhoP and the orphan GlnR systems due to their relevance in the control of primary metabolism and secondary metabolite biosynthesis. The PhoP binding sequence in many phosphate regulated promoters is formed by 11 nucleotide direct-repeats. A cross-talk between PhoP and other global regulators such as AfsR or GlnR has been found. Other two-component systems, particularly AbsA1/AbsA2, also control antibiotic biosynthesis in S. coelicolor, while others control chitinase synthesis, stress responses or cellular differentiation. Finally, some orphan response regulators named atypical response regulators (e.g. RedZ in S. coelicolor and JadR1 in Streptomyces venezuelae) bind as ligands the final product of the antibiotic biosynthetic pathway and act as feedback regulators of the biosynthesis of these secondary metabolites.

16. The Rcs Phosphorelay: Biofilm Formation and Virulence in the Enterobacteriaceae

David J. Clarke

The Rcs phosphorelay is a complex signaling network that is restricted in distribution to the Enterobacteriaceae. The core Rcs phosphorelay is composed of 3 proteins: the sensor kinase RcsC, the HPt domain protein RcsD and the response regulator RcsB. In addition to these core components the Rcs phosphorelay also contains some auxiliary proteins that are involved in mediating the inputs to and outputs from this signaling pathway. Therefore RcsF is a lipoprotein involved in signal perception, IgaA inhibits the activity of the phosphorelay and RcsA is required for the expression of some of the genes that are regulated by the phosphorelay. Whilst initially identified as a positive regulator of capsule production in Escherichia coli the Rcs phosphorelay is now recognized as a key regulator of motility, biofilm formation and virulence in many members of the Enterobacteriaceae.

17. Chemotactic Signal Transduction in Helicobacter pylori

Paphavee Lertsethtakarn, Jenny Draper and Karen M. Ottemann

The gastric pathogenic bacterium Helicobacter pylori has a relatively simple chemotaxis system, with four chemoreceptors plus a set of signal transduction proteins consisting of the CheA kinase, CheW and CheV coupling proteins, a CheY response regulator, the CheZ phosphatase and an unusual protein termed ChePep. H. pylori chemotaxis response has proved challenging to study, but studies have established that H. pylori uses its chemotaxis system to move away from low pH and autoinducer-2, and to respond to cellular energy status along with several chemicals . During stomach infection, H. pylori chemotaxis plays multiple roles including promoting early infection and critical epithelial interactions that drive a wild-type immune response.

18. Two-component Regulators in the Vibrio fischeri-Euprymna scolopes Symbiosis

Valerie A. Ray and Karen L. Visick

The symbiotic relationship between the marine bioluminescent bacterium Vibrio fischeri and its host, the Hawaiian bobtail squid Euprymna scolopes, depends upon the ability of the two partners to sense and respond to each other. V. fischeri colonizes a specialized squid organ called the light organ in three general stages: initiation, accommodation, and persistence. To respond to the different environments encountered during these stages of colonization, V. fischeri utilizes specialized two-component signal transduction systems to regulate processes such as biofilm formation, motility and chemotaxis, and luminescence. In this chapter, we discuss in detail the two component systems that regulate these processes and how they impact successful colonization of the squid host.