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One or more of the terZ–terF genes have been identified in pathogenic bacteria. Although it is not clear how they function, their primary role might be to protect against mammalian host defenses, possibly by counteracting toxic substances produced by macrophages or other human cellular defenses. The maintenance of several unrelated sets of genes in many different microorganisms, all of which confer TeR, implies that they are important for survival. Acknowledgements This work was supported by a grant from the Medical Research Council of Canada (1977–1999). I am grateful to my present and former colleagues, Joan Hou, David Kelly, Mingfu Liu, Michelle Rooker, Raymond Turner, Emily Walter, Joel Weiner and Ken Whelan, who have contributed to the work in my laboratory on tellurite resistance, and Heather Mitchell, Moylum Lee and Richard Sherburne for assistance in preparing the manuscript and the figures. I also thank Val Burland, Nicole Perna and Cathy Trieber for helpful discussion. References 1 Cooper, W.C. (1971) Tellurium, Van Nostrand Reinhold 2 Fleming, A. (1932) J. Pathol. Bacteriol. 35, 831–842 3 Zadik, P.M., Chapman, P.A. and Siddons, C.A. (1993) J. Med. Microbiol. 39, 155–158
4 Summers, A.O. and Jacoby, G.A. (1977) J. Bacteriol. 129, 275–281 5 Walter, E.G. and Taylor, D.E. (1992) Plasmid 27, 52–64 6 Turner, R.J., Weiner, J.H. and Taylor, D.E. (1995) Microbiology 141, 3133–3140 7 Avazeri, C. et al. (1997) Microbiology 143, 1181–1189 8 Turner, R.J., Weiner, J.H. and Taylor, D.E. (1995) Can. J. Microbiol. 41, 92–98 9 Hou, Y. and Taylor, D.E. (1994) Plasmid 32, 306–311 10 Whelan, K.F., Sherburne, R.K. and Taylor, D.E. (1997) J. Bacteriol. 178, 63–71 11 Pansegrau, W. et al. (1994) J. Mol. Biol. 239, 623–663 12 Cournoyer, B., Watanabe, S. and Vivian, A. (1998) Biochim. Biophys. Acta 1397, 161–168 13 Taylor, D.E. et al. (1994) J. Bacteriol. 176, 2740–2742 14 Fu, Z. et al. (1996) Biochemistry 35, 11985–11993 15 Turner, R.J., Taylor, D.E. and Weiner, J.H. (1997) Antimicrob. Agents Chemother. 41, 440–444 16 O’Gara, J.P., Gomelsky, M. and Kaplan, S. (1997) Appl. Environ. Microbiol. 63, 4713–4720 17 Fleischmann, R.D. et al. (1995) Science 269, 496–512 18 Kumano, M., Tamakoshi, A. and Yamane, K. (1997) Microbiology 143, 2775–2782 19 Turner, R.J. et al. (1992) J. Bacteriol. 174, 3092–3094 20 Chen, C.M. et al. (1986) J. Biol. Chem. 261, 15030–15038 21 Chen, C.M., Mobley, H.L.T. and Rosen, B.P. (1985) J. Bacteriol. 161, 758–763
Signalling pathways in twocomponent phosphorelay systems Anne-Laure Perraud, Verena Weiss and Roy Gross
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wo-component signal- Two-component systems are characterized The phosphate is subsequently ling systems were previby phosphotransfer reactions involving transferred from the phosphoously thought to occur histidine and aspartate residues in highly histidine to an aspartate residue only in eubacteria, but are conserved signalling domains. Although located in an acidic pocket of now also known in Archaea the basic principles of signal transduction the receiver domain. Low-moland eukaryotes, including Sacby these systems have been elucidated, ecular-weight phosphodonors, charomyces cerevisiae, Canseveral important aspects, such as their including acetyl phosphate, dida albicans, Dictyostelium integration into more complex cellular might function in vitro, and in discoideum, Neurospora crasregulatory networks and the molecular some cases in vivo, as a subsa and Arabidopsis thaliana1–3. basis of the specificity of signal strate for phosphorylation inThe mechanism of signal transduction, remain unknown. stead of the phosphohistidine transduction by these systems presented by a transmitter or is based on phosphotransfer A-L. Perraud and R. Gross* are in the Lehrstuhl für HPt domain. Thus, the receiver Theodor-Boveri-Institut, Biozentrum reactions between histidine Mikrobiologie, domain harbours the enzymatic der Universität Würzburg, Am Hubland, D-97074 and aspartate residues. Three activity for the phosphotransfer Würzburg, Germany; V. Weiss is in the Fakultät für types of conserved signalling reaction, and the phosphorylBiologie, Universität Konstanz, D-78434 Konstanz, Germany. domains are known to be inated receiver should be envisaged *tel: 1931 888 4403, volved in the phosphorylation as an active phospho-enzyme fax: 1931 888 4402, reactions: the ‘transmitter’, the intermediate that is unstable e-mail: [email protected] ‘receiver’ and the histidineand decays with a half-life that containing phosphotransfer module, HPt. varies considerably depending on the receiver domain The transmitter acts as an autokinase and carries an involved4. In addition, transmitter domains might harATP-binding site and a short histidine-containing motif bour phosphatase activities that control the phosphoryl(H-motif) that is the target of autophosphorylation4. ation status of their cognate receiver domains4. 0966-842X/99/$ - see front matter © 1999 Elsevier Science. All rights reserved. TRENDS
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Fig. 1. (facing page) Modular architecture of two-component systems. (a) The osmoregulatory EnvZ/OmpR system of Escherichia coli is an example of ‘classical’ two-component systems. In such systems, phosphotransfer to the effector protein occurs in a single step from the transmitter domain of the histidine kinase to an aspartic acid in the receiver of the effector protein. (b) The sporulation control system of Bacillus subtilis is an example of an ‘unorthodox’ system, in which additional signalling domains – a second receiver and the HPt domain – are employed, leading to a His–Asp–His–Asp phosphorelay between the sensor and the effector proteins. In this case, all signalling domains form independent proteins. Membrane-spanning and soluble histidine kinases, KinA and KinB, respectively, feed into the signalling pathway. The phosphorelay can be further modulated by additional regulatory elements, e.g. by the action of specific asparttussis and the EvgAS system of E. coli, and (e) the TodST system of Pseudomonas putida show the high degree of structural variability among two-component systems. The two-component signalling domains are shown in green; receiver domains are marked with Asp, and histidine-containing transmitter and HPt domains are marked with His. The transmitter is depicted as a rectangle and the HPt domain is depicted as a circle. Sensory domains are shown in blue, and effector domains are shown in orange or yellow. bZIP indicates the presence of a leucine zipper motif. Membranes are indicated as grey bars, and membrane-spanning domains of sensor proteins are shown. The phosphate flow in the various systems, as far as it is known, is shown by black arrows.
The third signalling device, the HPt domain, contains a short consensus motif with an invariant histidine. Apart from the short histidine motif, which is unrelated to the H-motif of the transmitter, sequence conservation among HPt domains is far less pronounced than for other domains5. The histidine typically obtains a phosphoryl group from a receiver domain and transfers it to another receiver within the signalling cascade. The HPt domain itself does not exhibit any kinase or phosphatase activity5,6. The conserved signalling domains are connected to other non-conserved domains involved in signal transduction reactions that are specific for each system. The histidine kinases contain a sensory domain, which triggers the autophosphorylation reaction of the transmitter in response to the perceived stimulus. In most cases, the effector proteins contain output domains in addition to the receivers, which frequently mediate DNA binding and transcriptional control of target genes4. Modular architecture of two-component systems Two classes of two-component systems have been distinguished: the ‘classical’ and the ‘unorthodox’ type. The classical models of two-component systems predominate. They are composed of two proteins, the sensor kinase and the effector protein, that are characterized by the transmitter and receiver domains, respectively. These systems lack an HPt domain and, as exemplified by the EnvZ/OmpR system of Escherichia coli (Fig. 1a), a one-step phosphotransfer occurs between the sensor and the effector protein. Unorthodox systems are characterized by a multistep phosphorelay that alternates between several histidine and aspartate residues (His–Asp–His–Asp) and involves additional receiver and HPt domains. The signalling domains are either isolated proteins or are fused with each other in various combinations7 (Fig. 1). The intriguing variability in the modular architecture of unorthodox two-component systems highlights their adaptive potential. For example, in the sporulation control system of Bacillus subtilis, all four signalling domains are separated from each other and belong to independent proteins. The two histidine kinases, KinA and KinB, function as the phosphodonor for the intermediate receiver domain, Spo0F. Spo0F~P is the phosphodonor for the HPt domain Spo0B, and Spo0B~P is the phosphate
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source for the DNA-binding effector protein, Spo0A (Refs 8,9) (Fig. 1b). The osmoregulatory system of S. cerevisiae shows an interesting structural variation (Fig. 1c). The Sln1p histidine kinase is fused with an intermediate receiver domain, allowing an intramolecular phosphotransfer reaction within the sensor protein from the H-motif to the aspartate of its receiver. After phosphotransfer to the isolated HPt domain Ypd1p, the effector protein, Ssk1p, is phosphorylated and activates the mitogen-activated protein kinase (MAPK) cascade10. Moreover, as exemplified by the TodST system of Pseudomonas putida11, additional structural phosphorelay variants have been detected (Fig. 1e). In a growing family of phosphorelay systems, a further structural variation is observed in which three of the phosphotransfer domains – the transmitter, a receiver and HPt – are combined in a multi-domain histidine kinase. Such systems include the two virulence regulatory systems: BvgAS of Bordetella pertussis and GacA/GacS (formerly LemA) of Pseudomonas syringae, and the ArcAB, TorRS and EvgAS systems of E. coli6,12–21 (Fig. 1d). ArcAB controls the adaptation to the redox environment, and TorRS induces the trimethylamine N-oxide reductase respiratory system. The function of the EvgAS system remains elusive. Phosphorelays as targets for negative control elements What is the advantage of an unorthodox two-component system over a classical system? The extension of the signalling pathway between the sensor and the effector components might allow different regulatory circuits to communicate with the phosphorelay systems. Experimental evidence for this idea mainly comes from the B. subtilis sporulation control system. The level of phosphorylated effector protein should directly reflect the activation status of the respective kinase. However, there are aspartate-specific phosphatases that can dephosphorylate the effector proteins and counteract the activity of the kinases; for example, the Spo0E phosphatase specific for the Spo0A~P protein in B. subtilis22. Moreover, in a multi-step phosphorelay, the intermediate signalling domains can also be the targets of specific phosphatases. For example, in the B. subtilis system, the RapA and RapB proteins have recently been shown to dephosphorylate the intermediate receiver domain, Spo0F~P
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Fig. 2. Examples of (a) optional (ArcAB) and (b) obligate (BvgAS) phosphorelay systems. (a) Phosphorylation of the ArcA effector protein can be mediated by either the ArcB transmitter or the HPt domain. The His–Asp–His–Asp phosphorelay is therefore optional. In addition to the ArcA protein, other non-cognate effector proteins, such as CheY and OmpR, can be phosphorylated efficiently via the ArcB HPt domain in vitro. SixA is a phosphohistidine phosphatase that might interfere with the phosphorylation status of the ArcB HPt domain. (b) Phosphorylation of the BvgA protein can be mediated only by the HPt domain of the histidine kinase BvgS. As no direct phosphorylation via the transmitter is possible, the His–Asp–His–Asp phosphorelay pathway is obligate. No cross-reactivity with the highly homologous non-cognate effector protein EvgA has been detected. The two-component signalling domains are shown in green; receiver domains are marked with Asp, and histidine-containing transmitter and HPt domains are marked with His. The transmitter is depicted as a rectangle and the HPt domain is depicted as a circle. Sensory domains are shown in blue, and effector domains are shown in orange or yellow. Membranes are indicated as grey bars, and membranespanning domains of sensor proteins are shown. The phosphate flow in the various systems is shown by black arrows.
Optional and obligate phosphorelays Interesting differences in the phosphorylation of the effector components have been noted among several phosphorelay systems. For the multi-domain redox sensor ArcB, it has been shown in vitro that either the transmitter or the HPt domain can act as the phosphodonor for ArcA (Ref. 6) (Fig. 2a). Recent data suggest that both pathways might operate in vivo19: although the role of the ArcB receiver domain remains unclear, the HPt-mediated phosphorelay appears to be essential for adaptation to anaerobic conditions, whereas the short-cut phosphorylation pathway is relevant under aerobic conditions. Because of its ability to transduce two types of stimuli via different phosphorylation pathways, ArcB has been designated a dual-signalling sensor protein19. The possibility of switching between these two phosphorylation pathways classifies the ArcAB system as an optional phosphorelay system; i.e. a system that
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either uses the classical pathway, allowing direct phosphotransfer from the transmitter to the effector protein, or the phosphorelay pathway, employing the HPt domain. Similarly, the GacAS system might use both the short-cut and the phosphorelay pathway, as deletion of the GacS HPt domain does not affect the phenotype significantly20. In contrast, the phosphorelay employing the HPt domain is obligate for the BvgAS, EvgAS and TorRS systems. Only the phosphohistidine in the HPt domain can be used for transphosphorylation of their cognate effector proteins BvgA, EvgA and TorR (Refs 12,13,15,21). Deletion of the HPt domain, or mutation of its conserved histidine, results in inactivation of signal transduction (Fig. 2b). Cross-reactivity of heterologous transmitter and receiver domains The high level of sequence conservation among the transmitter and receiver domains exposes two-component
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systems to the danger of nonspecific cross-reactions. Cross-reactivity between different two-component systems has been observed under certain experimental conditions; for example, when using one of the noncognate partners in excess25–27. Inactivation or a lack of negative regulatory functions, such as those supplied by specific phosphatases, might contribute to the appearance of cross-reactivity. The rates of phosphotransfer between non-cognate partners have been estimated to be at least two orders of magnitude lower than those between cognate reaction partners27. Although the potential for cross-reactivity can vary considerably for different two-component systems, most experimental data available so far indicate that, under physiological conditions, transmitter and receiver domains interact with high specificity. Real cross-communication between two-component systems appears to be a rare event. The intriguing question is how undesirable cross-reactivity between these systems can be avoided, as the highly conserved signalling domains can occur in very high numbers per cell. For example, in E. coli, 32 two-component effector proteins and 28 histidine kinases are known5. Recently, selection for mutated PhoB effector proteins that are able to cross-react with a non-cognate histidine kinase has allowed the identification of amino acid residues in the PhoB receiver that contribute to specificity in the protein–protein interaction28. Most importantly, the three-dimensional structures of the signalling domains and of complexes of interacting signalling domains are currently being elucidated and will provide detailed insights into the molecular basis of signalling specificity29–33.
possess very significant sequence conservation, even outside the two-component signalling domains. We have analysed the interactions of the various domains of the two systems, either after their expression as isolated domains or by the construction of chimaeric sensor and effector proteins composed of domains from both systems. Surprisingly, and in contrast to the HPt domain of ArcB, we have not detected any significant cross-reactivity, either in vitro or in vivo, between the BvgS or EvgS HPt domains and the highly related effector proteins BvgA or EvgA (Refs 13,21). The interaction of the HPt domains of BvgS and EvgS with the respective receiver domains in the effector proteins occurs with high specificity. The apparent differences in the specificity of the HPt domains in their interactions with receiver domains in the Arc system and, alternatively, in the Bvg and Evg systems correlate with the intramolecular phosphorelay within the multi-domain histidine kinases being optional for ArcA phosphorylation but obligate for BvgA and EvgA phosphorylation (Fig. 2). Are there different subtypes of unorthodox phosphorelay systems emerging? One subtype might be represented by ArcB and, possibly, by GacS. These systems are characterized by an optional intramolecular phosphorelay that might employ both the multi-step phosphorelay and a short-cut phosphotransfer, thereby possibly facilitating interconnections with other twocomponent systems. The second subtype might be represented by BvgS, EvgS and TorS. In these systems, the intramolecular phosphorelay is obligatory for the activation of the effector protein, and the interaction between HPt and the effector is highly specific.
Specificity of HPt domains One of the most promiscuous phosphodonors for various prokaryotic and eukaryotic receiver domains appears to be the HPt domain of ArcB (Refs 34–36). In vitro, this domain has been found to be an efficient phosphodonor, not only for its cognate effector protein, ArcA, but also for the chemotaxis protein, CheY, and the osmoregulator, OmpR. In vivo, a deletion in arcB changes the chemotactic behaviour of E. coli under microaerobic conditions32. Accordingly, it has been suggested that ArcB might be a multisignal transducer involved in cross-communication6. In fact, HPt domains might be more suitable than the classical signalling modules for connecting subsets of different two-component systems in a specific fashion. The employment of HPt domains for specific crosscommunication might reduce the risk of non-specific cross-reactivity, as these domains are generally present in low numbers (in E. coli, only four HPt-containing histidine kinases have been found5), and sequence conservation seems to be limited to the short histidine consensus motif5. To investigate the specificity of interactions of HPt domains with receiver domains, we have started to characterize the two highly related, unorthodox, BvgAS and EvgAS systems13,21. These systems are particularly suitable for such analysis because they not only show the same modular architecture but also
Evolutionary considerations The discovery of unorthodox phosphorelay systems has interesting implications for the evolution of two-component systems. The employment of additional signalling domains intercalated between the sensor and effector proteins might have permitted the connection of effector proteins to sensor kinases, which were not originally their primary reaction partners. In fact, two-component systems represent a protein family in which domainshuffling has occurred frequently during evolution37. However, only in rare cases have such mutations resulted in hybrid multi-domain proteins retaining a functional primary signalling pathway sufficient to avoid impairment of cellular fitness. Accordingly, optional phosphorelay systems might have been relevant evolutionary intermediates, because they allow the further development of either the primary signalling pathway, the multi-step phosphorelay or the maintenance of both. Apparently, there is a correlation between the use of multistep phosphorelay systems and the complexity of the organism possessing them. The classical type of twocomponent system predominates in bacteria, whereas the unorthodox systems are prevalent in eukaryotes. Whether this reflects a better adaptability by unorthodox systems to more complex organisms is currently unknown. The results of genome projects involving various higher eukaryotic organisms, including humans, will help to reveal to what extent these organisms use two-
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component systems and how they are organized. The comparative characterization of the prokaryotic and eukaryotic systems has important implications for the development of novel antimicrobials that target twocomponent signal transduction systems38. Acknowledgements We thank Dagmar Beier, Justin Daniels, Johannes Gross and Michael Kuhn for many discussions and careful reading of the manuscript, and Simone Sauze for assistance with graphics. We apologize that, owing to space limitations, the selected references are only a subset of recent publications. Work in both of our laboratories is supported by the Schwerpunktprogramm ‘Analysis of Regulatory Networks in Bacteria’ of the Deutsche Forschungsgemeinschaft and by the Fonds der Chemischen Industrie. References 1 Kennelly, P.J. and Potts, M. (1996) J. Bacteriol. 178, 4759–4764 2 Loomis, W.F. et al. (1997) J. Cell Sci. 110, 1141–1145 3 Rudolph, J. and Oesterhelt, D. (1995) EMBO J. 14, 667–673 4 Stock, J.B. et al. (1995) in Two-component Signal Transduction (Hoch, J.A. and Silhavy, T.J., eds), pp. 25–51, ASM Press 5 Mizuno, T. (1997) DNA Res. 4, 161–168 6 Tsuzuki, M. et al. (1995) Mol. Microbiol. 18, 953–962 7 Appleby, J.L. et al. (1996) Cell 20, 845–848 8 Hoch, J.A. (1995) in Two-component Signal Transduction (Hoch, J.A. and Silhavy, T.J., eds), pp. 129–144, ASM Press 9 Perego, M. (1998) Trends Microbiol. 6, 366–370 10 Wurgler-Murphy, S.M. and Saito, H. (1997) Trends Biochem. Sci. 22, 172–176 11 Lau, P.C. et al. (1997) Proc. Natl. Acad. Sci. U. S. A. 94, 1453–1458 12 Uhl, M.A. and Miller, J.F. (1996) EMBO J. 15, 1028–1036
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Beier, D. et al. (1995) J. Mol. Biol. 248, 596–610 Utsumi, R. et al. (1994) Gene 140, 73–77 Jourlin, C. et al. (1997) J. Mol. Biol. 267, 770–777 Rich, J.J. et al. (1994) J. Bacteriol. 176, 7468–7475 Georgellis, D. et al. (1997) J. Bacteriol. 179, 5429–5435 Kitten, T. et al. (1998) Mol. Microbiol. 28, 917–929 Matsushika, A. and Mizuno, T. (1998) J. Bacteriol. 180, 3973–3977 Hrabak, E.M. and Willis, D.K. (1992) J. Bacteriol. 174, 3011–3020 Perraud, A.L. et al. (1998) Mol. Microbiol. 27, 875–888 Ohlsen, K.L. et al. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 1756–1760 Perego, M. et al. (1996) Mol. Microbiol. 19, 1151–1157 Ogino, T. et al. (1998) Mol. Microbiol. 27, 573–585 Ninfa, A.J. et al. (1988) Proc. Natl. Acad. Sci. U. S. A. 85, 5492–5496 Wanner, B.L. (1992) J. Bacteriol. 174, 2053–2058 Fisher, S.L. et al. (1996) Biochemistry 35, 4732–4740 Haldimann, A. et al. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 14361–14366 McEvoy, M.M. et al. (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 7333–7338 Welch, M. et al. (1998) Nat. Struct. Biol. 5, 25–29 Martinez-Hackert, E. and Stock, A.M. (1997) Structure 5, 109–124 Kato, M. et al. (1997) Cell 88, 717–723 Baikalov, I. et al. (1998) Biochemistry 37, 3665–3676 Ishige, K. et al. (1994) EMBO J. 13, 5195–5202 Yaku, H. et al. (1997) FEBS Lett. 408, 337–340 Imamura, A. et al. (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 2691–2696 Reizer, J. and Saier, M.H., Jr (1997) Curr. Opin. Struct. Biol. 7, 407–415 Barrett, J.F. and Hoch, J.A. (1998) Antimicrob. Agents Chemother. 42, 1529–1536
Selection of drug-resistant HIV P. Richard Harrigan and Christopher S. Alexander
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ombinations of anti- Resistance to antiretroviral drugs by HIV pletely prevented over treatretroviral drugs inhibit- develops from competition among several ment periods of 10–20 years. ing the HIV reverse different virus variants within an Indeed, variants resistant to transcriptase (RT) and/or proindividual. Recent studies have measured commonly prescribed triple tease can suppress plasma levthe changing proportions of HIV therapies are already emergels of the virus and delay populations, which differ by single nucleic ing (and have even been disease progression in HIV- acids, under the selective pressures exerted reported to be transmitted infected individuals. This repby the addition or removal of from person to person), just resents an important breakantiretroviral drugs. two years after the widethrough in the treatment of spread application of protease P.R. Harrigan* and C.S. Alexander are in the BC AIDS. Unfortunately, current inhibitors2. Centre for Excellence in HIV/AIDS, 603–1081 HIV therapy requires longResistance to HIV infection Burrard Street, Vancouver, British Columbia, term medication – perhaps lifecan be modeled as an evoCanada V6Z 1Y6. long. As one year of therapy lutionary competition among *tel: 11 604 631 5281, fax: 11 604 631 5464, corresponds to hundreds of viral variants3–6. In a constant e-mail: [email protected] HIV generations, the evolution environment, relative viral of drug resistance is a critical ‘fitness’ is a measure of the issue1. Resistance can be delayed to a certain extent ability of an HIV variant to produce viable progeny. by initiating therapies with combinations of drugs, In the simplest case, the accumulation of mutants in a but it is not clear whether resistance can be com- viral population can be modeled as a function of the 0966-842X/99/$ - see front matter © 1999 Elsevier Science. All rights reserved. TRENDS
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One or more of the terZ–terF genes have been identified in pathogenic bacteria. Although it is not clear how they function, their primary role might be to protect against mammalian host defenses, possibly by counteracting toxic substances produced by macrophages or other human cellular defenses. The maintenance of several unrelated sets of genes in many different microorganisms, all of which confer TeR, implies that they are important for survival. Acknowledgements This work was supported by a grant from the Medical Research Council of Canada (1977–1999). I am grateful to my present and former colleagues, Joan Hou, David Kelly, Mingfu Liu, Michelle Rooker, Raymond Turner, Emily Walter, Joel Weiner and Ken Whelan, who have contributed to the work in my laboratory on tellurite resistance, and Heather Mitchell, Moylum Lee and Richard Sherburne for assistance in preparing the manuscript and the figures. I also thank Val Burland, Nicole Perna and Cathy Trieber for helpful discussion. References 1 Cooper, W.C. (1971) Tellurium, Van Nostrand Reinhold 2 Fleming, A. (1932) J. Pathol. Bacteriol. 35, 831–842 3 Zadik, P.M., Chapman, P.A. and Siddons, C.A. (1993) J. Med. Microbiol. 39, 155–158
4 Summers, A.O. and Jacoby, G.A. (1977) J. Bacteriol. 129, 275–281 5 Walter, E.G. and Taylor, D.E. (1992) Plasmid 27, 52–64 6 Turner, R.J., Weiner, J.H. and Taylor, D.E. (1995) Microbiology 141, 3133–3140 7 Avazeri, C. et al. (1997) Microbiology 143, 1181–1189 8 Turner, R.J., Weiner, J.H. and Taylor, D.E. (1995) Can. J. Microbiol. 41, 92–98 9 Hou, Y. and Taylor, D.E. (1994) Plasmid 32, 306–311 10 Whelan, K.F., Sherburne, R.K. and Taylor, D.E. (1997) J. Bacteriol. 178, 63–71 11 Pansegrau, W. et al. (1994) J. Mol. Biol. 239, 623–663 12 Cournoyer, B., Watanabe, S. and Vivian, A. (1998) Biochim. Biophys. Acta 1397, 161–168 13 Taylor, D.E. et al. (1994) J. Bacteriol. 176, 2740–2742 14 Fu, Z. et al. (1996) Biochemistry 35, 11985–11993 15 Turner, R.J., Taylor, D.E. and Weiner, J.H. (1997) Antimicrob. Agents Chemother. 41, 440–444 16 O’Gara, J.P., Gomelsky, M. and Kaplan, S. (1997) Appl. Environ. Microbiol. 63, 4713–4720 17 Fleischmann, R.D. et al. (1995) Science 269, 496–512 18 Kumano, M., Tamakoshi, A. and Yamane, K. (1997) Microbiology 143, 2775–2782 19 Turner, R.J. et al. (1992) J. Bacteriol. 174, 3092–3094 20 Chen, C.M. et al. (1986) J. Biol. Chem. 261, 15030–15038 21 Chen, C.M., Mobley, H.L.T. and Rosen, B.P. (1985) J. Bacteriol. 161, 758–763
Signalling pathways in twocomponent phosphorelay systems Anne-Laure Perraud, Verena Weiss and Roy Gross
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wo-component signal- Two-component systems are characterized The phosphate is subsequently ling systems were previby phosphotransfer reactions involving transferred from the phosphoously thought to occur histidine and aspartate residues in highly histidine to an aspartate residue only in eubacteria, but are conserved signalling domains. Although located in an acidic pocket of now also known in Archaea the basic principles of signal transduction the receiver domain. Low-moland eukaryotes, including Sacby these systems have been elucidated, ecular-weight phosphodonors, charomyces cerevisiae, Canseveral important aspects, such as their including acetyl phosphate, dida albicans, Dictyostelium integration into more complex cellular might function in vitro, and in discoideum, Neurospora crasregulatory networks and the molecular some cases in vivo, as a subsa and Arabidopsis thaliana1–3. basis of the specificity of signal strate for phosphorylation inThe mechanism of signal transduction, remain unknown. stead of the phosphohistidine transduction by these systems presented by a transmitter or is based on phosphotransfer A-L. Perraud and R. Gross* are in the Lehrstuhl für HPt domain. Thus, the receiver Theodor-Boveri-Institut, Biozentrum reactions between histidine Mikrobiologie, domain harbours the enzymatic der Universität Würzburg, Am Hubland, D-97074 and aspartate residues. Three activity for the phosphotransfer Würzburg, Germany; V. Weiss is in the Fakultät für types of conserved signalling reaction, and the phosphorylBiologie, Universität Konstanz, D-78434 Konstanz, Germany. domains are known to be inated receiver should be envisaged *tel: 1931 888 4403, volved in the phosphorylation as an active phospho-enzyme fax: 1931 888 4402, reactions: the ‘transmitter’, the intermediate that is unstable e-mail: [email protected] ‘receiver’ and the histidineand decays with a half-life that containing phosphotransfer module, HPt. varies considerably depending on the receiver domain The transmitter acts as an autokinase and carries an involved4. In addition, transmitter domains might harATP-binding site and a short histidine-containing motif bour phosphatase activities that control the phosphoryl(H-motif) that is the target of autophosphorylation4. ation status of their cognate receiver domains4. 0966-842X/99/$ - see front matter © 1999 Elsevier Science. All rights reserved. TRENDS
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Fig. 1. (facing page) Modular architecture of two-component systems. (a) The osmoregulatory EnvZ/OmpR system of Escherichia coli is an example of ‘classical’ two-component systems. In such systems, phosphotransfer to the effector protein occurs in a single step from the transmitter domain of the histidine kinase to an aspartic acid in the receiver of the effector protein. (b) The sporulation control system of Bacillus subtilis is an example of an ‘unorthodox’ system, in which additional signalling domains – a second receiver and the HPt domain – are employed, leading to a His–Asp–His–Asp phosphorelay between the sensor and the effector proteins. In this case, all signalling domains form independent proteins. Membrane-spanning and soluble histidine kinases, KinA and KinB, respectively, feed into the signalling pathway. The phosphorelay can be further modulated by additional regulatory elements, e.g. by the action of specific asparttussis and the EvgAS system of E. coli, and (e) the TodST system of Pseudomonas putida show the high degree of structural variability among two-component systems. The two-component signalling domains are shown in green; receiver domains are marked with Asp, and histidine-containing transmitter and HPt domains are marked with His. The transmitter is depicted as a rectangle and the HPt domain is depicted as a circle. Sensory domains are shown in blue, and effector domains are shown in orange or yellow. bZIP indicates the presence of a leucine zipper motif. Membranes are indicated as grey bars, and membrane-spanning domains of sensor proteins are shown. The phosphate flow in the various systems, as far as it is known, is shown by black arrows.
The third signalling device, the HPt domain, contains a short consensus motif with an invariant histidine. Apart from the short histidine motif, which is unrelated to the H-motif of the transmitter, sequence conservation among HPt domains is far less pronounced than for other domains5. The histidine typically obtains a phosphoryl group from a receiver domain and transfers it to another receiver within the signalling cascade. The HPt domain itself does not exhibit any kinase or phosphatase activity5,6. The conserved signalling domains are connected to other non-conserved domains involved in signal transduction reactions that are specific for each system. The histidine kinases contain a sensory domain, which triggers the autophosphorylation reaction of the transmitter in response to the perceived stimulus. In most cases, the effector proteins contain output domains in addition to the receivers, which frequently mediate DNA binding and transcriptional control of target genes4. Modular architecture of two-component systems Two classes of two-component systems have been distinguished: the ‘classical’ and the ‘unorthodox’ type. The classical models of two-component systems predominate. They are composed of two proteins, the sensor kinase and the effector protein, that are characterized by the transmitter and receiver domains, respectively. These systems lack an HPt domain and, as exemplified by the EnvZ/OmpR system of Escherichia coli (Fig. 1a), a one-step phosphotransfer occurs between the sensor and the effector protein. Unorthodox systems are characterized by a multistep phosphorelay that alternates between several histidine and aspartate residues (His–Asp–His–Asp) and involves additional receiver and HPt domains. The signalling domains are either isolated proteins or are fused with each other in various combinations7 (Fig. 1). The intriguing variability in the modular architecture of unorthodox two-component systems highlights their adaptive potential. For example, in the sporulation control system of Bacillus subtilis, all four signalling domains are separated from each other and belong to independent proteins. The two histidine kinases, KinA and KinB, function as the phosphodonor for the intermediate receiver domain, Spo0F. Spo0F~P is the phosphodonor for the HPt domain Spo0B, and Spo0B~P is the phosphate
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source for the DNA-binding effector protein, Spo0A (Refs 8,9) (Fig. 1b). The osmoregulatory system of S. cerevisiae shows an interesting structural variation (Fig. 1c). The Sln1p histidine kinase is fused with an intermediate receiver domain, allowing an intramolecular phosphotransfer reaction within the sensor protein from the H-motif to the aspartate of its receiver. After phosphotransfer to the isolated HPt domain Ypd1p, the effector protein, Ssk1p, is phosphorylated and activates the mitogen-activated protein kinase (MAPK) cascade10. Moreover, as exemplified by the TodST system of Pseudomonas putida11, additional structural phosphorelay variants have been detected (Fig. 1e). In a growing family of phosphorelay systems, a further structural variation is observed in which three of the phosphotransfer domains – the transmitter, a receiver and HPt – are combined in a multi-domain histidine kinase. Such systems include the two virulence regulatory systems: BvgAS of Bordetella pertussis and GacA/GacS (formerly LemA) of Pseudomonas syringae, and the ArcAB, TorRS and EvgAS systems of E. coli6,12–21 (Fig. 1d). ArcAB controls the adaptation to the redox environment, and TorRS induces the trimethylamine N-oxide reductase respiratory system. The function of the EvgAS system remains elusive. Phosphorelays as targets for negative control elements What is the advantage of an unorthodox two-component system over a classical system? The extension of the signalling pathway between the sensor and the effector components might allow different regulatory circuits to communicate with the phosphorelay systems. Experimental evidence for this idea mainly comes from the B. subtilis sporulation control system. The level of phosphorylated effector protein should directly reflect the activation status of the respective kinase. However, there are aspartate-specific phosphatases that can dephosphorylate the effector proteins and counteract the activity of the kinases; for example, the Spo0E phosphatase specific for the Spo0A~P protein in B. subtilis22. Moreover, in a multi-step phosphorelay, the intermediate signalling domains can also be the targets of specific phosphatases. For example, in the B. subtilis system, the RapA and RapB proteins have recently been shown to dephosphorylate the intermediate receiver domain, Spo0F~P
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could be the target for additional control. A phosphohistidine phosphatase, SixA, has recently been identified in E. coli. SixA acts on the HPt domain of ArcB and dephosphorylates it in vitro, but its physiological role remains to be established24.
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Fig. 2. Examples of (a) optional (ArcAB) and (b) obligate (BvgAS) phosphorelay systems. (a) Phosphorylation of the ArcA effector protein can be mediated by either the ArcB transmitter or the HPt domain. The His–Asp–His–Asp phosphorelay is therefore optional. In addition to the ArcA protein, other non-cognate effector proteins, such as CheY and OmpR, can be phosphorylated efficiently via the ArcB HPt domain in vitro. SixA is a phosphohistidine phosphatase that might interfere with the phosphorylation status of the ArcB HPt domain. (b) Phosphorylation of the BvgA protein can be mediated only by the HPt domain of the histidine kinase BvgS. As no direct phosphorylation via the transmitter is possible, the His–Asp–His–Asp phosphorelay pathway is obligate. No cross-reactivity with the highly homologous non-cognate effector protein EvgA has been detected. The two-component signalling domains are shown in green; receiver domains are marked with Asp, and histidine-containing transmitter and HPt domains are marked with His. The transmitter is depicted as a rectangle and the HPt domain is depicted as a circle. Sensory domains are shown in blue, and effector domains are shown in orange or yellow. Membranes are indicated as grey bars, and membranespanning domains of sensor proteins are shown. The phosphate flow in the various systems is shown by black arrows.
Optional and obligate phosphorelays Interesting differences in the phosphorylation of the effector components have been noted among several phosphorelay systems. For the multi-domain redox sensor ArcB, it has been shown in vitro that either the transmitter or the HPt domain can act as the phosphodonor for ArcA (Ref. 6) (Fig. 2a). Recent data suggest that both pathways might operate in vivo19: although the role of the ArcB receiver domain remains unclear, the HPt-mediated phosphorelay appears to be essential for adaptation to anaerobic conditions, whereas the short-cut phosphorylation pathway is relevant under aerobic conditions. Because of its ability to transduce two types of stimuli via different phosphorylation pathways, ArcB has been designated a dual-signalling sensor protein19. The possibility of switching between these two phosphorylation pathways classifies the ArcAB system as an optional phosphorelay system; i.e. a system that
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either uses the classical pathway, allowing direct phosphotransfer from the transmitter to the effector protein, or the phosphorelay pathway, employing the HPt domain. Similarly, the GacAS system might use both the short-cut and the phosphorelay pathway, as deletion of the GacS HPt domain does not affect the phenotype significantly20. In contrast, the phosphorelay employing the HPt domain is obligate for the BvgAS, EvgAS and TorRS systems. Only the phosphohistidine in the HPt domain can be used for transphosphorylation of their cognate effector proteins BvgA, EvgA and TorR (Refs 12,13,15,21). Deletion of the HPt domain, or mutation of its conserved histidine, results in inactivation of signal transduction (Fig. 2b). Cross-reactivity of heterologous transmitter and receiver domains The high level of sequence conservation among the transmitter and receiver domains exposes two-component
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systems to the danger of nonspecific cross-reactions. Cross-reactivity between different two-component systems has been observed under certain experimental conditions; for example, when using one of the noncognate partners in excess25–27. Inactivation or a lack of negative regulatory functions, such as those supplied by specific phosphatases, might contribute to the appearance of cross-reactivity. The rates of phosphotransfer between non-cognate partners have been estimated to be at least two orders of magnitude lower than those between cognate reaction partners27. Although the potential for cross-reactivity can vary considerably for different two-component systems, most experimental data available so far indicate that, under physiological conditions, transmitter and receiver domains interact with high specificity. Real cross-communication between two-component systems appears to be a rare event. The intriguing question is how undesirable cross-reactivity between these systems can be avoided, as the highly conserved signalling domains can occur in very high numbers per cell. For example, in E. coli, 32 two-component effector proteins and 28 histidine kinases are known5. Recently, selection for mutated PhoB effector proteins that are able to cross-react with a non-cognate histidine kinase has allowed the identification of amino acid residues in the PhoB receiver that contribute to specificity in the protein–protein interaction28. Most importantly, the three-dimensional structures of the signalling domains and of complexes of interacting signalling domains are currently being elucidated and will provide detailed insights into the molecular basis of signalling specificity29–33.
possess very significant sequence conservation, even outside the two-component signalling domains. We have analysed the interactions of the various domains of the two systems, either after their expression as isolated domains or by the construction of chimaeric sensor and effector proteins composed of domains from both systems. Surprisingly, and in contrast to the HPt domain of ArcB, we have not detected any significant cross-reactivity, either in vitro or in vivo, between the BvgS or EvgS HPt domains and the highly related effector proteins BvgA or EvgA (Refs 13,21). The interaction of the HPt domains of BvgS and EvgS with the respective receiver domains in the effector proteins occurs with high specificity. The apparent differences in the specificity of the HPt domains in their interactions with receiver domains in the Arc system and, alternatively, in the Bvg and Evg systems correlate with the intramolecular phosphorelay within the multi-domain histidine kinases being optional for ArcA phosphorylation but obligate for BvgA and EvgA phosphorylation (Fig. 2). Are there different subtypes of unorthodox phosphorelay systems emerging? One subtype might be represented by ArcB and, possibly, by GacS. These systems are characterized by an optional intramolecular phosphorelay that might employ both the multi-step phosphorelay and a short-cut phosphotransfer, thereby possibly facilitating interconnections with other twocomponent systems. The second subtype might be represented by BvgS, EvgS and TorS. In these systems, the intramolecular phosphorelay is obligatory for the activation of the effector protein, and the interaction between HPt and the effector is highly specific.
Specificity of HPt domains One of the most promiscuous phosphodonors for various prokaryotic and eukaryotic receiver domains appears to be the HPt domain of ArcB (Refs 34–36). In vitro, this domain has been found to be an efficient phosphodonor, not only for its cognate effector protein, ArcA, but also for the chemotaxis protein, CheY, and the osmoregulator, OmpR. In vivo, a deletion in arcB changes the chemotactic behaviour of E. coli under microaerobic conditions32. Accordingly, it has been suggested that ArcB might be a multisignal transducer involved in cross-communication6. In fact, HPt domains might be more suitable than the classical signalling modules for connecting subsets of different two-component systems in a specific fashion. The employment of HPt domains for specific crosscommunication might reduce the risk of non-specific cross-reactivity, as these domains are generally present in low numbers (in E. coli, only four HPt-containing histidine kinases have been found5), and sequence conservation seems to be limited to the short histidine consensus motif5. To investigate the specificity of interactions of HPt domains with receiver domains, we have started to characterize the two highly related, unorthodox, BvgAS and EvgAS systems13,21. These systems are particularly suitable for such analysis because they not only show the same modular architecture but also
Evolutionary considerations The discovery of unorthodox phosphorelay systems has interesting implications for the evolution of two-component systems. The employment of additional signalling domains intercalated between the sensor and effector proteins might have permitted the connection of effector proteins to sensor kinases, which were not originally their primary reaction partners. In fact, two-component systems represent a protein family in which domainshuffling has occurred frequently during evolution37. However, only in rare cases have such mutations resulted in hybrid multi-domain proteins retaining a functional primary signalling pathway sufficient to avoid impairment of cellular fitness. Accordingly, optional phosphorelay systems might have been relevant evolutionary intermediates, because they allow the further development of either the primary signalling pathway, the multi-step phosphorelay or the maintenance of both. Apparently, there is a correlation between the use of multistep phosphorelay systems and the complexity of the organism possessing them. The classical type of twocomponent system predominates in bacteria, whereas the unorthodox systems are prevalent in eukaryotes. Whether this reflects a better adaptability by unorthodox systems to more complex organisms is currently unknown. The results of genome projects involving various higher eukaryotic organisms, including humans, will help to reveal to what extent these organisms use two-
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component systems and how they are organized. The comparative characterization of the prokaryotic and eukaryotic systems has important implications for the development of novel antimicrobials that target twocomponent signal transduction systems38. Acknowledgements We thank Dagmar Beier, Justin Daniels, Johannes Gross and Michael Kuhn for many discussions and careful reading of the manuscript, and Simone Sauze for assistance with graphics. We apologize that, owing to space limitations, the selected references are only a subset of recent publications. Work in both of our laboratories is supported by the Schwerpunktprogramm ‘Analysis of Regulatory Networks in Bacteria’ of the Deutsche Forschungsgemeinschaft and by the Fonds der Chemischen Industrie. References 1 Kennelly, P.J. and Potts, M. (1996) J. Bacteriol. 178, 4759–4764 2 Loomis, W.F. et al. (1997) J. Cell Sci. 110, 1141–1145 3 Rudolph, J. and Oesterhelt, D. (1995) EMBO J. 14, 667–673 4 Stock, J.B. et al. (1995) in Two-component Signal Transduction (Hoch, J.A. and Silhavy, T.J., eds), pp. 25–51, ASM Press 5 Mizuno, T. (1997) DNA Res. 4, 161–168 6 Tsuzuki, M. et al. (1995) Mol. Microbiol. 18, 953–962 7 Appleby, J.L. et al. (1996) Cell 20, 845–848 8 Hoch, J.A. (1995) in Two-component Signal Transduction (Hoch, J.A. and Silhavy, T.J., eds), pp. 129–144, ASM Press 9 Perego, M. (1998) Trends Microbiol. 6, 366–370 10 Wurgler-Murphy, S.M. and Saito, H. (1997) Trends Biochem. Sci. 22, 172–176 11 Lau, P.C. et al. (1997) Proc. Natl. Acad. Sci. U. S. A. 94, 1453–1458 12 Uhl, M.A. and Miller, J.F. (1996) EMBO J. 15, 1028–1036
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Selection of drug-resistant HIV P. Richard Harrigan and Christopher S. Alexander
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ombinations of anti- Resistance to antiretroviral drugs by HIV pletely prevented over treatretroviral drugs inhibit- develops from competition among several ment periods of 10–20 years. ing the HIV reverse different virus variants within an Indeed, variants resistant to transcriptase (RT) and/or proindividual. Recent studies have measured commonly prescribed triple tease can suppress plasma levthe changing proportions of HIV therapies are already emergels of the virus and delay populations, which differ by single nucleic ing (and have even been disease progression in HIV- acids, under the selective pressures exerted reported to be transmitted infected individuals. This repby the addition or removal of from person to person), just resents an important breakantiretroviral drugs. two years after the widethrough in the treatment of spread application of protease P.R. Harrigan* and C.S. Alexander are in the BC AIDS. Unfortunately, current inhibitors2. Centre for Excellence in HIV/AIDS, 603–1081 HIV therapy requires longResistance to HIV infection Burrard Street, Vancouver, British Columbia, term medication – perhaps lifecan be modeled as an evoCanada V6Z 1Y6. long. As one year of therapy lutionary competition among *tel: 11 604 631 5281, fax: 11 604 631 5464, corresponds to hundreds of viral variants3–6. In a constant e-mail: [email protected] HIV generations, the evolution environment, relative viral of drug resistance is a critical ‘fitness’ is a measure of the issue1. Resistance can be delayed to a certain extent ability of an HIV variant to produce viable progeny. by initiating therapies with combinations of drugs, In the simplest case, the accumulation of mutants in a but it is not clear whether resistance can be com- viral population can be modeled as a function of the 0966-842X/99/$ - see front matter © 1999 Elsevier Science. All rights reserved. TRENDS
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