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Bacterial chemotaxis as a simple model for a sensory system
TIBS - January 19 76
I
try routinely erases artificial barriers of this sort. Chemotaxis in bacteria was discovered in the 1880s by Englemann [l] and Pfeffer [2]. Pfeffer showed that bacteria swam towards some chemicals which he called attractants and away from others which acted as repellents. These beautiful studies were performed with great experimental simplicity utilizing a capillary filled with the appropriate chemical and measuring the bacterial response by microscopic assay. Adler [3] using modern methods in a resourceful way put the capillary assay of Pfeffer on a quantitative basis and used it to screen a wide variety of chemical compounds by competition tests. In these studies he deduced that Escherichia coli have on the order of twenty receptors, some for attractants and some for repellents, which provide the sensory signals from the outer environment [4]. Furthermore he demonstrated that the compounds need not be metabolized and need not be transported to act as attractants, a situation analogous to saccharin and other stimulants in man.
REVIEWS Bacterial chemotaxis as a simple model for a sensory system D. E. Koshland, Jr Bacteria have a rudimentary memory and a system for interpreting responses to positive and negative stimuli. The biochemical pathways qf thk system may have close analogies to the behavioral mechanisms qf higher species.
In current biochemistry there is growing interest in the study of sensory and neural systems. The tools for understanding behavior are becoming available and a large number of investigators are seeking the fundamental bases on which sensory systems operate. The choice of the model varies enormously. Some individuals prefer direct work on mammals because they are most relevant to man. The complexity of a mammalian system and the difficulty of manipulating the species leads others to utilize less complex organisms such as Drosophila, nematodes and crustaceans. In our case, we chose bacteria for three reasons : (1) the simplicity of the system indicated that we might be able to understand it in its entirety; (2) the known methods of harvesting bacteria make it possible to isolate all the proteins in sizable amounts so that they can be studied independently in the test tube; and (3) the ease of genetic manipulation means that mutations can be used to define the system and also for subsequent modification to test the hypotheses developed. The study of simple systems always raises the question of its relevance to higher species. Only time will tell the extent of the similarity, but historical perspective has almost invariably shown the biochemical unity of all organisms. In the early studies of ATP, the genetic code and biochemical pathways in microorganisms, some critics repeatedly claimed that the extrapolation from simple microorganisms to D.E.K. Jr is Professor of Biochemistry at the Univetsity of California. Berkeley, California 94720. USA
higher species was invalid and almost monotonously proved to be wrong. There is no doubt that there are species differences and certainly bacteria cannot be expected to have the complex neural networks characteristic of higher species. In man alone a wide variety of neurotransmitters are used, indicating that there is no unique chemical for providing a sensory signal. But there are fundamental similarities in behavior between widely different species and these probably are based on common principles. One might next ask whether ‘bacterial behavior’ is not in itself pretentiousness, an anthropomorphic implication of a sophistication that does not exist. In the case of bacteria, the sensory apparatus is used to control the motion of bacteria towards more favorable environments. This means that the bacteria must have receptors for recognizing changes in their environment, a processing system to interpret the information they receive and a means of conveying these interpretations to their flagella to alter the direction of motion. Stated in these terms, the analogy to higher systems is obvious and in fact, chemotaxis, the migration of a species under the influence of a chemical gradient, is ubiquitous. Pheromones attract insects, perfumes attract man, and nutrients attract bacteria. In fact, the resistance of some to the concept that bacteria have behavioral responses is analogous to the resistance of others to the concept that mental behavior is controlled by chemistry. The line between ‘instinct’ and ‘reason’ is not easy to draw and modern biochemis-
Biased random walk
If one observes the bacterium under the microscope, it appears to travel in a roughly straight line and then turn abruptly. Sometimes the bacteria appear to be tumbling head over tails for a brief period before swimming off in a new direction. This led early workers to deduce that some type of random walk was likely for bacteria [5]. These qualitative observations were placed on a quantitative basis by Berg and Brown [6] who utilized an automatic tracking apparatus. The quantitative measurement of bacterial motion showed (1) that the length of the runs, i.e. the distance between tumbles, was Poissonian, and (2) the angle of the turn averaged 62” with a Poissonian distribution about this average. When the bacteria were observed in a gradient, the pattern changed so that the length of an average run was increased when the bacteria were travelling up a gradient of attractant. The length of the fun on going down a gradient remained the same as in an isotropic solution. While these tracking studies were proceeding, Macnab and Koshland [7] were utilizing a temporal gradient apparatus to study tumbling frequency and concluded that the path’s length is increased on going up and decreased on going down, a gradient of attractant. The apparent discrepancy between these results and those of Berg and Brown was quickly reconciled on a quantitative basis. Bacteria do increase their tumbling frequency on going down a gradient of attractant, but the increase is small ; whereas the decrease in
3
tumbling frequency on going up a gradient is very great. Hence, the quantitatively important contribution to the migration is caused by the decrease in tumbling frequency (or increase in path length) in going up a gradient of attractant. Thus, these experiments established that the bacteria proceed through space by a biased random walk in which the sensory system essentially encourages the bacteria to proceed smoothly when it is going in the right direction and discourages it by frequent tumbling when it is going in the wrong direction. Temporal sensing mechanism
How could an organism as small as a bacterium sense a gradient? Two logical alternatives seemed obvious based on other sensory systems : an instantaneous spatial sensing or a temporal sensing mechanism. In the first, the bacteria would compare the concentration of attractant at its head with the concentration of attractant at its tail in a given instant of time. In the second, the bacteria, swimming through space, would compare the concentration over its whole body at time TVwith the concentration over its whole body at time tz. The choice between these for bacteria were resolved by Macnab and Koshland [7] who used a rapid mixing device to plunge the bacteria from one uniform concentration of attractant (C,) into a uniform second concentration (Cr). Control experiments had established that the absolute concentration of attractant did not affect the motility pattern. Therefore the experiment involved a change in sensory information over time while producing an isotropic chemical distribution eliminating any instantaneous difference in concentration between the heads and tails of the bacteria. The results showed clearly that the bacteria sense over time, i.e. they had some kind of ‘memory’ which allowed them to compare the environment of their past with that of their present and interpret this signal. Thus, the random motion of bacteria through space is used to sense gradients by incorporating into the bacteria a simple memory system. The bacterium compares the concentration observed at tt with the concentration at t2. If it is going up a favorable gradient (CZ > Cl) tumbling is suppressed. If it is going in an unfavorable direction (C? < Cl), for example down a gradient of attractant, the difference increases tumbling so the bacteria sets off in a new direction. This conclusion was later confirmed utilizing an enzymatically generated gradient [8]. The utilization of a ‘memory process’ for sensing means that the bacterium can use more than its own body length for
TIBS - Januagl 19 76 sensing a gradient, a considerable advantage in the analytical problem for such a small organism since it can in effect extend its body length for sensing [9]. It may also be worth mentioning how frequently man measures distance by means of time. In fact, in ordinary conversation if we are asked how far away a certain city is, we frequently answer ‘three hours’, an indication that distance measurement from the light years of the galaxies to the hours of the commuter is frequently expressed in units of time. The receptors
The receptors which generate the primary signal from the environment are located in the bacterial membrane. Hazelbauer and Adler [lo] showed that the periplasmic galactose binding protein was the receptor for galactose taxis in E. cob. Other periplasmic proteins are the ribose receptor of Salmonella typhimurium [l l] and the maltose receptor of E. co/i [12]. However, some receptors are not shockable (i.e. solubilized by osmotic shock) and a glucose receptor has been identified [ 131 as part of the phosphotransferase transport system of Kundig and Roseman [ 141. Hence the receptors are not identified with only one type of protein molecule, but they are located on the periphery of the bacterium just like the sensory receptors of higher species. By using a quantitative procedure for measuring tumbling frequency, Spudich and Koshland [ 151 were able to make a precise quantitative correlation of the properties of the purified ribose receptor with the behavior of the intact organism. The purified ribose receptor was found to have a dissociation constant (Kd) for ribose of 3 x lo-’ and the dissociation constant of the in vivo receptor for ribose chemotaxis was 3 x lo-‘. Moreover, allose which has a constant of 3 x lop4 M for the purified
10-a
10.’
lo-”
10-s
10-a
10-z
10-z
10-1
[ATTRACTANT]
Fig. 1. A correlation of an in vivo behavioral response qf a bacterium with the properties of a pure protein in vitro [ 151.Points represent recovery times of bacterial tumbling after being subjected to stimuli ?f an attractant whose concentration is shown on the abscissa. Measurements utilized the quantitative tumble frequency assay of Spudich and Koshland [ 151. Solid lines were calculated assuming previously determined binding constants[ I I]. For attractants to thepurified ribose receptor (3.3 x 10e7 M for ribose and 3.0 x 10m4 M for allose). Reproduced by permission from Proc. Nat. Acad. Sri. USA [IS].
receptor gave a behavioral response curve with a constant of 3x 10m4 M (Fig. 1). Sugars such as arabinose which did not interact with the receptor did not cause chemotaxis. Thus the receptor showed the same specificity in vivo and in vitro, a linding of interest to higher species where the isolation of receptors is not as simple. The quantitative studies further showed that the stimulus was proportional to the change in receptor occupancy, i.e. d(RC) where R is the receptor and C is the chemoeffector. Moreover, the stimuli were additive, i.e. two concentration changes of O-0.02 mM and 0.0220.5 mM gave the same overall response as one from O-0.5 mM. Repellents were shown to act as the inverse of attractants [ 161, e.g. increasing tumbling on going down a gradient, and specificity patterns indicated there are receptors for repellents as well as attractants [ 171. Moreover, the bacteria can add the signals from attractants and repellents in an algebraic way [ 16,181, not unlike inhibitory and excitatory signals in higher species. Transmission system
How is the information from the receptors processed ? Armstrong and Adler [ 191 initially studied some mutants in E. cofi which were incapable of transmitting signals from any receptors and identified three genes which they called the A, the B and the C [19]. Parkinson found a fourth gene, the D [20]. Recently in our laboratory we have developed new selection methods [21] which made it very easy to obtain large numbers of mutants, and applying these techniques to Salmonella have so far discovered nine genes identified with the transmission system [22]. We have labelled these the p, q, r, s, t, etc. to avoid confusion with the E. coli genes. From the methods employed, it is clear that we have not yet uncovered all of the genes, but it appears that we are approaching the maximum number. Even assuming that we have only located half of the genes this means that the system requires twenty gene products, a rather simple sensory system. The selection of these mutants illustrates one of the great advantages of bacteria. Not only are mutant selection methods easily performed but gene products can be isolated and the genes are readily mapped. Five of the genes are clustered together near the motility region of the bacteria. Two can be placed in gene loci which are also loci for flagella assembly, i.e. genes whose damage causes a lack of any flagella being formed. This has also been found for one of the E. coli genes [23]. Since the mutants which we have selected are motile, these results suggest that some protein involved in flagella
TIBS - January I9 76
3
assembly can be modified in two ways one which prevents assembly ofthe flagella entirely and another which allows assembly but does not allow the flagellum to receive signals of the sensory apparatus. Further studies of these genes and gene products will be extraordinarily revealing. For the interim, at least, they define the scope of the problem.
Tumbling Mutant
Che+ I smooth motility
threshold
Motor response
The way bacteria tumble is now known. They simply reverse the direction of rotation of their flagella which act as propellors. This was shown by the tethering experiments of Simon and co-workers [24], and analyzed theoretically by Berg [25]. Adler and his co-workers showed that the reversals of rotation correlated with tumbling [26]. Hence a very simple all-or-none switching is sufficient to cause tumbling. The analogy to the firing ofa neuron seems obvious. Working hypothesis
A tentative model can be devised in which the tumbling frequency is controlled by variations in the concentration of a tumble regulator (Fig. 2). Such a model can rationalize the role of mutants, the effect of methionine, etc. but it is too early to define the tumble regulator itself. It may be a small molecule, a membrane potential, or a protein complex. A more extensive discussion of the tumble regulator and its mechanistic implications has been presented elsewhere [7,27]. The future
The bacterial behavior is designed to allow the organism to survive, to swim towards nutrients and favorable conditions and away from toxic or unfavorable conditions. The system designed to elicit this behavior seems related to the brain of man in the way that a hand calculator is related to a giant computer. The unit processes would appear to be similar; there are just a lot more of them in the large computer. The analogies in the biochemistry of the unit processes between the simple bacterium and higher species (which have been elaborated on more completely elsewhere [27]) are extensive. Receptors are proteins located on the periphery of the organism. A rudimentary memory is present. A system for integrating responses to positive and negative stimuli exists. s-Adenosylmethionine, implicated in higher systems, is important in the bacterial behavior [28301.Hence it seems likely that the biochemical pathways of the bacterial system will have close analogies to higher species. More important than the detailed biochemistry, however, are the principles
attractant
attractant
Time Fig. 2. A model for the regulation of rumbling frequency by gradients. When the rumble regulator concenrralion (wavy solid or dotted line) rises above rhe threshold vale (horizontal solid line), rumbling is suppressed (smooth motility); when it falls below, tumbling (rnolilirv) ir increaed. In rhe absence of a gradient the regulator level flucruates randomly near rhe threshold level, but a rapid temporal increase in attractant makes rhe twnble regulator concenrration rise far above the threshold level, suppressing rumbling complerely until it returns to normal. The regulator is maintained aI a steady slate level by a delicate balance between its synthesis and degradation. S-Adenosyl melhionine may be necessary for rhe degradation reaction. Thus, during merhionine (met) starvation of the wild type Salmonella (Che+). the level of regulator is higher than normal so tumbling is suppressed. When a consranrly tumbling mutant [IS) is perturbed by a temporal gradienr ir rakes longer than normal lo return to its steady state level when methionine is absent. By assuming that the consrantly rumbling mutant has an abnormally high threshold. one can easily explain its behavior in both the presence and absence of methionine. Reproduced by permission from J. Mol. Bid. 1301.
which we hope are revealed by the chemotactic system. Bacteria can detect extremely small changes in concentration. Even using their memory the analytical problem involves the detection of about 1 part in 103.This small difference is then amplified by the sensing system to lead ultimately to the motor response. The ability to detect and the capacity to amplify is at the heart of all sensing systems. It is also at the heart of many other problems in biology, e.g. differentiation. Hence, the little bacteria moving through space in a random but purposeful manner may be cleverly leading us to new principles in biology. Acknowledgement
This work was supported by USPHS Grant AM9765 and NSF Grant BMS 710133A03.
References Engelmann, T.W. (1881) P@gers Arch. gesammle Physiol. 25,285-292
Pfeffer, W. (1883) Ber. deur. boran. Ges. 1, 524 c’1’1
Adler, J. (1969) Science 166, 1588-I 597 Adler, J. (1975) Ann. Rev. Biochem. 44, 341-356 Weibull, C. (1960) The Bacteria (Guns&q I.C. and Stanier; R.Y., eds), Vol. 1, pp. 153-205, Academic Press, New York Berg, H.C. and Brown, D.A. (1972) Nature 239,
Sci. USA 71,2895-2899
14 Kundig, W. and Roseman, S. (1971)
Macnab, R.M. and Koshland, D.E., Jr (1972) Proc. Nat. Acad. Sci. USA 69,2509-2512 Brown, D.A. and Berg, H.C. (1974) Proc. Nat. Acad. Sci. USA 71,1388-l 392 Macnab, R.M. and Koshland, D.E., Jr (1973) Cell Motility
2, 141-148
J. Biol.
Chem. 246,1393-1406
15 Spudich, J.L. and Koshland, D.E., Jr (1975) Proc. Nat. Acad. Sci. USA 72,710-713
16 Tsang, N., Macnab, R.M. and Koshland, D.E., Jr (1973) Science 181,6&63 17 Tso, W.-W. and Adler, J. (1974) J. Bacterial. 118, 560-576 18 Tso, W.-W. and Adler, J. (1974) Science 184, 1292-1294 19 Armstrong, J.B. and Adler, J. (1969) Genetics 61, 61-66 20 Parkinson, J.F. (1975) Nature 252, 317-319 21 Aswad, D. and Koshland, D.E., Jr (1975) J. Mol. Biol. 97, 225-236
22 Koshland, D.E., Jr, Warrick, H., Taylor, B. and Spudich, J. Cold Spring Harbor Symposium (in the press) 23 Silverman, M. and Simon, M. (1974) J. Bact. 117, 73-79 24 Silverman, M.R. and Simon, M.I. (1974) Nature 249,73-74 25 Berg, H.C. and Anderson,
R.A. (1973) Nature 239.500-504 26 Larsen,S.H. Reader, R.W..Kort, E.N., Tso, W.W. and Adler, J. (1974) Nature 249, 74-77 27 Koshland, D.E., Jr, Advan. Neurochem. (in the press) 28 Adler, J. and Dahl, M. (1967) J. Gen. Microbial. 46, 161-173 29
500-504
J. Mechanochem.
Hazelbauer, G.L. and Adler, J. (1971) Nature New Biol. 30, 101-104 Aksamit, R. and Koshland, D.E., Jr (1974) Biochemistry 13,4473-4478 Kellerman, O., Szmeloman, S. and Schwartz, M. (in preparation) Adler, J. and Epstein, W. (1974) Proc. Nat. Acad.
Armstrong, J. (1972) Can. J. Microbial. 18, 1695-
1701 30 Aswad, D. and Koshland. D.E., Jr, (1975) J. Mol. Biol. 97. 207-224
I
try routinely erases artificial barriers of this sort. Chemotaxis in bacteria was discovered in the 1880s by Englemann [l] and Pfeffer [2]. Pfeffer showed that bacteria swam towards some chemicals which he called attractants and away from others which acted as repellents. These beautiful studies were performed with great experimental simplicity utilizing a capillary filled with the appropriate chemical and measuring the bacterial response by microscopic assay. Adler [3] using modern methods in a resourceful way put the capillary assay of Pfeffer on a quantitative basis and used it to screen a wide variety of chemical compounds by competition tests. In these studies he deduced that Escherichia coli have on the order of twenty receptors, some for attractants and some for repellents, which provide the sensory signals from the outer environment [4]. Furthermore he demonstrated that the compounds need not be metabolized and need not be transported to act as attractants, a situation analogous to saccharin and other stimulants in man.
REVIEWS Bacterial chemotaxis as a simple model for a sensory system D. E. Koshland, Jr Bacteria have a rudimentary memory and a system for interpreting responses to positive and negative stimuli. The biochemical pathways qf thk system may have close analogies to the behavioral mechanisms qf higher species.
In current biochemistry there is growing interest in the study of sensory and neural systems. The tools for understanding behavior are becoming available and a large number of investigators are seeking the fundamental bases on which sensory systems operate. The choice of the model varies enormously. Some individuals prefer direct work on mammals because they are most relevant to man. The complexity of a mammalian system and the difficulty of manipulating the species leads others to utilize less complex organisms such as Drosophila, nematodes and crustaceans. In our case, we chose bacteria for three reasons : (1) the simplicity of the system indicated that we might be able to understand it in its entirety; (2) the known methods of harvesting bacteria make it possible to isolate all the proteins in sizable amounts so that they can be studied independently in the test tube; and (3) the ease of genetic manipulation means that mutations can be used to define the system and also for subsequent modification to test the hypotheses developed. The study of simple systems always raises the question of its relevance to higher species. Only time will tell the extent of the similarity, but historical perspective has almost invariably shown the biochemical unity of all organisms. In the early studies of ATP, the genetic code and biochemical pathways in microorganisms, some critics repeatedly claimed that the extrapolation from simple microorganisms to D.E.K. Jr is Professor of Biochemistry at the Univetsity of California. Berkeley, California 94720. USA
higher species was invalid and almost monotonously proved to be wrong. There is no doubt that there are species differences and certainly bacteria cannot be expected to have the complex neural networks characteristic of higher species. In man alone a wide variety of neurotransmitters are used, indicating that there is no unique chemical for providing a sensory signal. But there are fundamental similarities in behavior between widely different species and these probably are based on common principles. One might next ask whether ‘bacterial behavior’ is not in itself pretentiousness, an anthropomorphic implication of a sophistication that does not exist. In the case of bacteria, the sensory apparatus is used to control the motion of bacteria towards more favorable environments. This means that the bacteria must have receptors for recognizing changes in their environment, a processing system to interpret the information they receive and a means of conveying these interpretations to their flagella to alter the direction of motion. Stated in these terms, the analogy to higher systems is obvious and in fact, chemotaxis, the migration of a species under the influence of a chemical gradient, is ubiquitous. Pheromones attract insects, perfumes attract man, and nutrients attract bacteria. In fact, the resistance of some to the concept that bacteria have behavioral responses is analogous to the resistance of others to the concept that mental behavior is controlled by chemistry. The line between ‘instinct’ and ‘reason’ is not easy to draw and modern biochemis-
Biased random walk
If one observes the bacterium under the microscope, it appears to travel in a roughly straight line and then turn abruptly. Sometimes the bacteria appear to be tumbling head over tails for a brief period before swimming off in a new direction. This led early workers to deduce that some type of random walk was likely for bacteria [5]. These qualitative observations were placed on a quantitative basis by Berg and Brown [6] who utilized an automatic tracking apparatus. The quantitative measurement of bacterial motion showed (1) that the length of the runs, i.e. the distance between tumbles, was Poissonian, and (2) the angle of the turn averaged 62” with a Poissonian distribution about this average. When the bacteria were observed in a gradient, the pattern changed so that the length of an average run was increased when the bacteria were travelling up a gradient of attractant. The length of the fun on going down a gradient remained the same as in an isotropic solution. While these tracking studies were proceeding, Macnab and Koshland [7] were utilizing a temporal gradient apparatus to study tumbling frequency and concluded that the path’s length is increased on going up and decreased on going down, a gradient of attractant. The apparent discrepancy between these results and those of Berg and Brown was quickly reconciled on a quantitative basis. Bacteria do increase their tumbling frequency on going down a gradient of attractant, but the increase is small ; whereas the decrease in
3
tumbling frequency on going up a gradient is very great. Hence, the quantitatively important contribution to the migration is caused by the decrease in tumbling frequency (or increase in path length) in going up a gradient of attractant. Thus, these experiments established that the bacteria proceed through space by a biased random walk in which the sensory system essentially encourages the bacteria to proceed smoothly when it is going in the right direction and discourages it by frequent tumbling when it is going in the wrong direction. Temporal sensing mechanism
How could an organism as small as a bacterium sense a gradient? Two logical alternatives seemed obvious based on other sensory systems : an instantaneous spatial sensing or a temporal sensing mechanism. In the first, the bacteria would compare the concentration of attractant at its head with the concentration of attractant at its tail in a given instant of time. In the second, the bacteria, swimming through space, would compare the concentration over its whole body at time TVwith the concentration over its whole body at time tz. The choice between these for bacteria were resolved by Macnab and Koshland [7] who used a rapid mixing device to plunge the bacteria from one uniform concentration of attractant (C,) into a uniform second concentration (Cr). Control experiments had established that the absolute concentration of attractant did not affect the motility pattern. Therefore the experiment involved a change in sensory information over time while producing an isotropic chemical distribution eliminating any instantaneous difference in concentration between the heads and tails of the bacteria. The results showed clearly that the bacteria sense over time, i.e. they had some kind of ‘memory’ which allowed them to compare the environment of their past with that of their present and interpret this signal. Thus, the random motion of bacteria through space is used to sense gradients by incorporating into the bacteria a simple memory system. The bacterium compares the concentration observed at tt with the concentration at t2. If it is going up a favorable gradient (CZ > Cl) tumbling is suppressed. If it is going in an unfavorable direction (C? < Cl), for example down a gradient of attractant, the difference increases tumbling so the bacteria sets off in a new direction. This conclusion was later confirmed utilizing an enzymatically generated gradient [8]. The utilization of a ‘memory process’ for sensing means that the bacterium can use more than its own body length for
TIBS - Januagl 19 76 sensing a gradient, a considerable advantage in the analytical problem for such a small organism since it can in effect extend its body length for sensing [9]. It may also be worth mentioning how frequently man measures distance by means of time. In fact, in ordinary conversation if we are asked how far away a certain city is, we frequently answer ‘three hours’, an indication that distance measurement from the light years of the galaxies to the hours of the commuter is frequently expressed in units of time. The receptors
The receptors which generate the primary signal from the environment are located in the bacterial membrane. Hazelbauer and Adler [lo] showed that the periplasmic galactose binding protein was the receptor for galactose taxis in E. cob. Other periplasmic proteins are the ribose receptor of Salmonella typhimurium [l l] and the maltose receptor of E. co/i [12]. However, some receptors are not shockable (i.e. solubilized by osmotic shock) and a glucose receptor has been identified [ 131 as part of the phosphotransferase transport system of Kundig and Roseman [ 141. Hence the receptors are not identified with only one type of protein molecule, but they are located on the periphery of the bacterium just like the sensory receptors of higher species. By using a quantitative procedure for measuring tumbling frequency, Spudich and Koshland [ 151 were able to make a precise quantitative correlation of the properties of the purified ribose receptor with the behavior of the intact organism. The purified ribose receptor was found to have a dissociation constant (Kd) for ribose of 3 x lo-’ and the dissociation constant of the in vivo receptor for ribose chemotaxis was 3 x lo-‘. Moreover, allose which has a constant of 3 x lop4 M for the purified
10-a
10.’
lo-”
10-s
10-a
10-z
10-z
10-1
[ATTRACTANT]
Fig. 1. A correlation of an in vivo behavioral response qf a bacterium with the properties of a pure protein in vitro [ 151.Points represent recovery times of bacterial tumbling after being subjected to stimuli ?f an attractant whose concentration is shown on the abscissa. Measurements utilized the quantitative tumble frequency assay of Spudich and Koshland [ 151. Solid lines were calculated assuming previously determined binding constants[ I I]. For attractants to thepurified ribose receptor (3.3 x 10e7 M for ribose and 3.0 x 10m4 M for allose). Reproduced by permission from Proc. Nat. Acad. Sri. USA [IS].
receptor gave a behavioral response curve with a constant of 3x 10m4 M (Fig. 1). Sugars such as arabinose which did not interact with the receptor did not cause chemotaxis. Thus the receptor showed the same specificity in vivo and in vitro, a linding of interest to higher species where the isolation of receptors is not as simple. The quantitative studies further showed that the stimulus was proportional to the change in receptor occupancy, i.e. d(RC) where R is the receptor and C is the chemoeffector. Moreover, the stimuli were additive, i.e. two concentration changes of O-0.02 mM and 0.0220.5 mM gave the same overall response as one from O-0.5 mM. Repellents were shown to act as the inverse of attractants [ 161, e.g. increasing tumbling on going down a gradient, and specificity patterns indicated there are receptors for repellents as well as attractants [ 171. Moreover, the bacteria can add the signals from attractants and repellents in an algebraic way [ 16,181, not unlike inhibitory and excitatory signals in higher species. Transmission system
How is the information from the receptors processed ? Armstrong and Adler [ 191 initially studied some mutants in E. cofi which were incapable of transmitting signals from any receptors and identified three genes which they called the A, the B and the C [19]. Parkinson found a fourth gene, the D [20]. Recently in our laboratory we have developed new selection methods [21] which made it very easy to obtain large numbers of mutants, and applying these techniques to Salmonella have so far discovered nine genes identified with the transmission system [22]. We have labelled these the p, q, r, s, t, etc. to avoid confusion with the E. coli genes. From the methods employed, it is clear that we have not yet uncovered all of the genes, but it appears that we are approaching the maximum number. Even assuming that we have only located half of the genes this means that the system requires twenty gene products, a rather simple sensory system. The selection of these mutants illustrates one of the great advantages of bacteria. Not only are mutant selection methods easily performed but gene products can be isolated and the genes are readily mapped. Five of the genes are clustered together near the motility region of the bacteria. Two can be placed in gene loci which are also loci for flagella assembly, i.e. genes whose damage causes a lack of any flagella being formed. This has also been found for one of the E. coli genes [23]. Since the mutants which we have selected are motile, these results suggest that some protein involved in flagella
TIBS - January I9 76
3
assembly can be modified in two ways one which prevents assembly ofthe flagella entirely and another which allows assembly but does not allow the flagellum to receive signals of the sensory apparatus. Further studies of these genes and gene products will be extraordinarily revealing. For the interim, at least, they define the scope of the problem.
Tumbling Mutant
Che+ I smooth motility
threshold
Motor response
The way bacteria tumble is now known. They simply reverse the direction of rotation of their flagella which act as propellors. This was shown by the tethering experiments of Simon and co-workers [24], and analyzed theoretically by Berg [25]. Adler and his co-workers showed that the reversals of rotation correlated with tumbling [26]. Hence a very simple all-or-none switching is sufficient to cause tumbling. The analogy to the firing ofa neuron seems obvious. Working hypothesis
A tentative model can be devised in which the tumbling frequency is controlled by variations in the concentration of a tumble regulator (Fig. 2). Such a model can rationalize the role of mutants, the effect of methionine, etc. but it is too early to define the tumble regulator itself. It may be a small molecule, a membrane potential, or a protein complex. A more extensive discussion of the tumble regulator and its mechanistic implications has been presented elsewhere [7,27]. The future
The bacterial behavior is designed to allow the organism to survive, to swim towards nutrients and favorable conditions and away from toxic or unfavorable conditions. The system designed to elicit this behavior seems related to the brain of man in the way that a hand calculator is related to a giant computer. The unit processes would appear to be similar; there are just a lot more of them in the large computer. The analogies in the biochemistry of the unit processes between the simple bacterium and higher species (which have been elaborated on more completely elsewhere [27]) are extensive. Receptors are proteins located on the periphery of the organism. A rudimentary memory is present. A system for integrating responses to positive and negative stimuli exists. s-Adenosylmethionine, implicated in higher systems, is important in the bacterial behavior [28301.Hence it seems likely that the biochemical pathways of the bacterial system will have close analogies to higher species. More important than the detailed biochemistry, however, are the principles
attractant
attractant
Time Fig. 2. A model for the regulation of rumbling frequency by gradients. When the rumble regulator concenrralion (wavy solid or dotted line) rises above rhe threshold vale (horizontal solid line), rumbling is suppressed (smooth motility); when it falls below, tumbling (rnolilirv) ir increaed. In rhe absence of a gradient the regulator level flucruates randomly near rhe threshold level, but a rapid temporal increase in attractant makes rhe twnble regulator concenrration rise far above the threshold level, suppressing rumbling complerely until it returns to normal. The regulator is maintained aI a steady slate level by a delicate balance between its synthesis and degradation. S-Adenosyl melhionine may be necessary for rhe degradation reaction. Thus, during merhionine (met) starvation of the wild type Salmonella (Che+). the level of regulator is higher than normal so tumbling is suppressed. When a consranrly tumbling mutant [IS) is perturbed by a temporal gradienr ir rakes longer than normal lo return to its steady state level when methionine is absent. By assuming that the consrantly rumbling mutant has an abnormally high threshold. one can easily explain its behavior in both the presence and absence of methionine. Reproduced by permission from J. Mol. Bid. 1301.
which we hope are revealed by the chemotactic system. Bacteria can detect extremely small changes in concentration. Even using their memory the analytical problem involves the detection of about 1 part in 103.This small difference is then amplified by the sensing system to lead ultimately to the motor response. The ability to detect and the capacity to amplify is at the heart of all sensing systems. It is also at the heart of many other problems in biology, e.g. differentiation. Hence, the little bacteria moving through space in a random but purposeful manner may be cleverly leading us to new principles in biology. Acknowledgement
This work was supported by USPHS Grant AM9765 and NSF Grant BMS 710133A03.
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