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Bacterial Chemotaxis: The motor connection
BACTERIAL CHEMOTAXIS
JEFF STOCK AND MICHAEL SURETTE
The motor connection In bacterial chemotaxis, membrane receptors control the phosphorylation of the response regulator CheY; phospho-CheY has been found to control motility by binding to a switching apparatus attached to the flagellar motor. In the 1960s, Julius Adler began his pioneering studies of how Escherichia coli can sense and move towards nutrients in their environment, a process known as chemotaxis [1]. The underlying motivation of the undertaking was the idea that to understand the basic biochemistry of sensory-motor regulation, one should start with a very simple organism like E. coli, rather than the much more complex species normally considered within the province of neurobiology. The validity of Adler's idea has been established as bacterial chemotaxis has emerged as a paradigm for studies of membrane receptor function and intracellular signal transduction [2,3]. E. coli chemotaxis was the first behavioral system to be dissected genetically, and the first to be subjected to molecular genetic analysis. This analysis has progressed from molecular to atomic resolution with the determination of crystal structures of both membrane receptors and signal transduction components. We now know more about the molecular mechanism of chemotaxis in E. coli than we know about any other signal transduction system; but we are still a long way from knowing everything. A major gap in our understanding of the chemotaxis signal transduction pathway has recently been filled by Welch and colleagues [4], in a collaborative study involving groups in Israel and Japan. In the early genetic analysis of chemotaxis, three different classes of mutant, designated Fla, Mot and Che mutants, were defined on the basis of their phenotype [5]. Fla mutants lack flagella and Mot mutants have paralyzed flagella; Che mutants have fully functional flagella, but their activities are not properly regulated in response to sensory information. Studies of the chemotaxis system have diverged along the lines defined by these genetic results. Some groups have focused on Fla and Mot products, trying to understand how the flagellar motor works; others have identified and characterized Che products, attempting to determine how sensory information from membrane receptors controls motor activity. Studies of the flagellar motor have identified three proteins, FliG, FIiM and FIiN, that function as a switching complex to control the motor in response to signals from the Che system [6]. At the same time, studies of receptor-mediated signal transduction indicated that a small protein called CheY functions as a response regulator that controls the motor in response to environmental signals [7,81. Welch et al. [4] have now provided the first direct biochemical demonstration that CheY actually binds to the switch, thus connecting the Che system to the motor.
The switch proteins were initially identified genetically. The genes that encode FliG, FliM and FIiN are members of the Fla class because the deletion of any of them renders a cell unable to synthesize flagella. Nevertheless, some missense alleles at these loci produce Che phenotypes, in which normal-looking flagella are made but not properly regulated in response to environmental stimuli. More importantly, it was possible to select missense mutations at the fliG, fliM and fliN loci that suppress the Che defect caused by missense cheY mutations. Although these observations strongly supported the notion that the FIiG, FliM and FIiN proteins constitute a switching apparatus, there was one major caveat: purified preparations of the motor apparatus did not contain the putative switch. The localization of the switch to the motor required a more gentle method for preparing motors. Previous procedures used a detergent extraction method that stripped away peripheral components, leaving a core structure that lacked the switch. Recently, DeRosier and colleagues [9] have developed a procedure for isolating this core structure with the switch attached. Analysis of electron micrographs of this structure has revealed a large cone of proteins extending into the cytoplasm from the flagellar basal structure. This cone, which contains the FIiG, FliM and FIiN proteins, corresponds to the switch apparatus and is precisely where it should be - at the interface between the motor and the cytoplasm. One might imagine that the conical shape of the switch apparatus helps it to catch efficiently a molecule of CheY that diffuses into the neighborhood of the motor, much as the conical shape of the neuromuscular acetylcholine receptor [101 facilitates the efficient binding of neurotransmitter. The structure and function of CheY have also been intensively studied over the past few years [7,8]. The structure of CheY was solved by X-ray crystallography at about the same time as that of an analogous signaling protein from eukaryotic cells, the Ras protein [11]. CheY and Ras share many features (Fig. 1 and [2]): they are both doubly wound a/P proteins with essentially the same folding topology, and both proteins function as on/off switching components in signal transduction pathways. CheY is activated by addition of a phosphoryl group, which is joined by an anhydride bond to the carboxylate side of residue aspartate 57; Ras is also activated by a phosphoryl group, in this case linked by an anhydride bond to a bound guanine nucleotide.
©Current Biology 1994, Vol 4 No 2
143
144
Current Biology 1994, Vol 4 No 2 no binding to a mutant form of CheY that cannot be phosphorylated. These experiments provide elegant support for the model of sensory-motor regulation in E. coli that has emerged from previous, less direct studies.
Fig. 1. Ribbon diagrams of CheY (left) and Ras (right). Red, a helices; green, [~sheets. Graphics generated using UCSF Midas Plus (Computer graphics laboratory, School of Pharmacy, UCSF).
It will now be possible to characterize the CheY-FliM interaction in detail, and to analyze directly the effects of cbeY and fliM mutations on the interaction. Chemotaxis research is poised to enter a new phase, where one can begin to design specific mutations to introduce biochemically defined perturbations, and use genetic methods to work back from in vitro behavior to in vivo function. As envisaged by Adler over two decades ago, we shall, for the first time, understand the molecular mechanisms that underlie the behavior of an individual organism as it goes about its business of searching for a favorable environment in which to grow.
In the case of CheY, the activating phosphoryl group is transferred from a phosphohistidine residue of a kinase, the activity of which is controlled by membrane chemoreceptors. In the case of Ras, the activating phosphoryl group is introduced by exchange of GTP for GDP, a process that is catalyzed by exchange proteins that are also regulated by membrane receptors (for growth factors). The additional phosphoryl group of GTP causes Ras to undergo a conformational change that allows it to bind to an effector target and generate a growth response. It has been suggested that CheY might function similarly. The idea that the interaction between CheY and the flagellar motor is controlled by a conformational change is supported by the identification of CheY mutants that can cause motor responses in the absence of phosphorylation and of others that can be phosphorylated but cannot cause a response [2,3].
References
The recent work of Welch et al. [4] directly demonstrates both the binding of CheY to the switch apparatus and the role of phosphorylation in the control of this interaction. The experimental approach involved attaching the CheY protein to a solid support and looking for binding of radioactively labeled switch proteins to the immobilized CheY. The results were straightforward and dramatic: the FliM protein binds to CheY, but only under conditions in which CheY is phosphorylated. CheY that is not phosphorylated has a relatively low affinity for the switch protein, and under conditions that do allow phosphorylation there is
1.
2.
3.
4.
5.
6.
J: The sensing of chemicals by bacteria. Sci Am 1976, 243:40-47. STOCK Jl3, LUKAT GS, STOCK AM: Bacterial chemotaxis and the molecular logic of intracellular signal transduction networks. Annu Rev Biopbys Bophys Cbem 1991, 20:109-136. BOURRET RB, BORKOVICII KA, SIMON MI: Signal transduction pathways involving protein phosphorylation in prokaryotes. Annu Rev Biochem 1991, 60:401-442. ADLER
WELCH
M,
OOSAWA
K,
AIZAWA
S-I,
EISENBACII
M:
Phosphorylation-dependent binding of a signal molecule to the flagellar switch of bacteria. Proc Natl Acad Sci USA 1993, 90:8787-8791. PARKINSON JS: Behavioral genetics in bacteria. Annu Rev Genet 1977, 11:397-414. SOCKErI' H, YAMAGUCIII S, KItIARA M, IRIKURA VM, MACNAB RM:
Molecular analysis of the flagellar switch protein FliM of Salmonella typhlmurlum. JBact 1992, 174:793-806. 7.
STOCK JB, STOCK AM, MOIONEN JM: Signal transduction in
8.
bacteria. Nature 1990, 344:395-400. VoLrz K: Structural conservation in the CheY superfamily. Biochem 1993, 32:11741-11753.
9.
FRANCIS NR, SOSINSKY GE, TIIOMAS D, DEROSIER DJ: Isolation,
characterization and structure of bacterial flagellar motors containing the switching complex. JMol Biol 1994, in press. 10.
TOYOSHIMA C, UNWIN N: Ion channel of acetylcholine receptor
reconstructed from images of postsynaptic membranes. Nature 1988, 336:247-250. 11.
BOURNE HR, SANDERS DA, MCCORMICK F: The GTPase superfam-
ily: conserved structure and molecular mechanism. Nature 1991, 349:117-127.
Jeff Stock and Michael Surette, Department of Molecular Biology, Princeton University, Princeton, New Jersey 08544, USA.
JEFF STOCK AND MICHAEL SURETTE
The motor connection In bacterial chemotaxis, membrane receptors control the phosphorylation of the response regulator CheY; phospho-CheY has been found to control motility by binding to a switching apparatus attached to the flagellar motor. In the 1960s, Julius Adler began his pioneering studies of how Escherichia coli can sense and move towards nutrients in their environment, a process known as chemotaxis [1]. The underlying motivation of the undertaking was the idea that to understand the basic biochemistry of sensory-motor regulation, one should start with a very simple organism like E. coli, rather than the much more complex species normally considered within the province of neurobiology. The validity of Adler's idea has been established as bacterial chemotaxis has emerged as a paradigm for studies of membrane receptor function and intracellular signal transduction [2,3]. E. coli chemotaxis was the first behavioral system to be dissected genetically, and the first to be subjected to molecular genetic analysis. This analysis has progressed from molecular to atomic resolution with the determination of crystal structures of both membrane receptors and signal transduction components. We now know more about the molecular mechanism of chemotaxis in E. coli than we know about any other signal transduction system; but we are still a long way from knowing everything. A major gap in our understanding of the chemotaxis signal transduction pathway has recently been filled by Welch and colleagues [4], in a collaborative study involving groups in Israel and Japan. In the early genetic analysis of chemotaxis, three different classes of mutant, designated Fla, Mot and Che mutants, were defined on the basis of their phenotype [5]. Fla mutants lack flagella and Mot mutants have paralyzed flagella; Che mutants have fully functional flagella, but their activities are not properly regulated in response to sensory information. Studies of the chemotaxis system have diverged along the lines defined by these genetic results. Some groups have focused on Fla and Mot products, trying to understand how the flagellar motor works; others have identified and characterized Che products, attempting to determine how sensory information from membrane receptors controls motor activity. Studies of the flagellar motor have identified three proteins, FliG, FIiM and FIiN, that function as a switching complex to control the motor in response to signals from the Che system [6]. At the same time, studies of receptor-mediated signal transduction indicated that a small protein called CheY functions as a response regulator that controls the motor in response to environmental signals [7,81. Welch et al. [4] have now provided the first direct biochemical demonstration that CheY actually binds to the switch, thus connecting the Che system to the motor.
The switch proteins were initially identified genetically. The genes that encode FliG, FliM and FIiN are members of the Fla class because the deletion of any of them renders a cell unable to synthesize flagella. Nevertheless, some missense alleles at these loci produce Che phenotypes, in which normal-looking flagella are made but not properly regulated in response to environmental stimuli. More importantly, it was possible to select missense mutations at the fliG, fliM and fliN loci that suppress the Che defect caused by missense cheY mutations. Although these observations strongly supported the notion that the FIiG, FliM and FIiN proteins constitute a switching apparatus, there was one major caveat: purified preparations of the motor apparatus did not contain the putative switch. The localization of the switch to the motor required a more gentle method for preparing motors. Previous procedures used a detergent extraction method that stripped away peripheral components, leaving a core structure that lacked the switch. Recently, DeRosier and colleagues [9] have developed a procedure for isolating this core structure with the switch attached. Analysis of electron micrographs of this structure has revealed a large cone of proteins extending into the cytoplasm from the flagellar basal structure. This cone, which contains the FIiG, FliM and FIiN proteins, corresponds to the switch apparatus and is precisely where it should be - at the interface between the motor and the cytoplasm. One might imagine that the conical shape of the switch apparatus helps it to catch efficiently a molecule of CheY that diffuses into the neighborhood of the motor, much as the conical shape of the neuromuscular acetylcholine receptor [101 facilitates the efficient binding of neurotransmitter. The structure and function of CheY have also been intensively studied over the past few years [7,8]. The structure of CheY was solved by X-ray crystallography at about the same time as that of an analogous signaling protein from eukaryotic cells, the Ras protein [11]. CheY and Ras share many features (Fig. 1 and [2]): they are both doubly wound a/P proteins with essentially the same folding topology, and both proteins function as on/off switching components in signal transduction pathways. CheY is activated by addition of a phosphoryl group, which is joined by an anhydride bond to the carboxylate side of residue aspartate 57; Ras is also activated by a phosphoryl group, in this case linked by an anhydride bond to a bound guanine nucleotide.
©Current Biology 1994, Vol 4 No 2
143
144
Current Biology 1994, Vol 4 No 2 no binding to a mutant form of CheY that cannot be phosphorylated. These experiments provide elegant support for the model of sensory-motor regulation in E. coli that has emerged from previous, less direct studies.
Fig. 1. Ribbon diagrams of CheY (left) and Ras (right). Red, a helices; green, [~sheets. Graphics generated using UCSF Midas Plus (Computer graphics laboratory, School of Pharmacy, UCSF).
It will now be possible to characterize the CheY-FliM interaction in detail, and to analyze directly the effects of cbeY and fliM mutations on the interaction. Chemotaxis research is poised to enter a new phase, where one can begin to design specific mutations to introduce biochemically defined perturbations, and use genetic methods to work back from in vitro behavior to in vivo function. As envisaged by Adler over two decades ago, we shall, for the first time, understand the molecular mechanisms that underlie the behavior of an individual organism as it goes about its business of searching for a favorable environment in which to grow.
In the case of CheY, the activating phosphoryl group is transferred from a phosphohistidine residue of a kinase, the activity of which is controlled by membrane chemoreceptors. In the case of Ras, the activating phosphoryl group is introduced by exchange of GTP for GDP, a process that is catalyzed by exchange proteins that are also regulated by membrane receptors (for growth factors). The additional phosphoryl group of GTP causes Ras to undergo a conformational change that allows it to bind to an effector target and generate a growth response. It has been suggested that CheY might function similarly. The idea that the interaction between CheY and the flagellar motor is controlled by a conformational change is supported by the identification of CheY mutants that can cause motor responses in the absence of phosphorylation and of others that can be phosphorylated but cannot cause a response [2,3].
References
The recent work of Welch et al. [4] directly demonstrates both the binding of CheY to the switch apparatus and the role of phosphorylation in the control of this interaction. The experimental approach involved attaching the CheY protein to a solid support and looking for binding of radioactively labeled switch proteins to the immobilized CheY. The results were straightforward and dramatic: the FliM protein binds to CheY, but only under conditions in which CheY is phosphorylated. CheY that is not phosphorylated has a relatively low affinity for the switch protein, and under conditions that do allow phosphorylation there is
1.
2.
3.
4.
5.
6.
J: The sensing of chemicals by bacteria. Sci Am 1976, 243:40-47. STOCK Jl3, LUKAT GS, STOCK AM: Bacterial chemotaxis and the molecular logic of intracellular signal transduction networks. Annu Rev Biopbys Bophys Cbem 1991, 20:109-136. BOURRET RB, BORKOVICII KA, SIMON MI: Signal transduction pathways involving protein phosphorylation in prokaryotes. Annu Rev Biochem 1991, 60:401-442. ADLER
WELCH
M,
OOSAWA
K,
AIZAWA
S-I,
EISENBACII
M:
Phosphorylation-dependent binding of a signal molecule to the flagellar switch of bacteria. Proc Natl Acad Sci USA 1993, 90:8787-8791. PARKINSON JS: Behavioral genetics in bacteria. Annu Rev Genet 1977, 11:397-414. SOCKErI' H, YAMAGUCIII S, KItIARA M, IRIKURA VM, MACNAB RM:
Molecular analysis of the flagellar switch protein FliM of Salmonella typhlmurlum. JBact 1992, 174:793-806. 7.
STOCK JB, STOCK AM, MOIONEN JM: Signal transduction in
8.
bacteria. Nature 1990, 344:395-400. VoLrz K: Structural conservation in the CheY superfamily. Biochem 1993, 32:11741-11753.
9.
FRANCIS NR, SOSINSKY GE, TIIOMAS D, DEROSIER DJ: Isolation,
characterization and structure of bacterial flagellar motors containing the switching complex. JMol Biol 1994, in press. 10.
TOYOSHIMA C, UNWIN N: Ion channel of acetylcholine receptor
reconstructed from images of postsynaptic membranes. Nature 1988, 336:247-250. 11.
BOURNE HR, SANDERS DA, MCCORMICK F: The GTPase superfam-
ily: conserved structure and molecular mechanism. Nature 1991, 349:117-127.
Jeff Stock and Michael Surette, Department of Molecular Biology, Princeton University, Princeton, New Jersey 08544, USA.