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Chemotaxis in Bacteria

Marty Player

Introduction

"A Taste of Smell" was a course of many topics that centered around the sense of smell. Projects included looking at possible pheromone production by humans, scent markings of wolves, and smell myths that pervade our society. Most of the topics dealt with the use of smell of higher organisms with a large bias toward humans. Unfortunately, many aspects of the effects of smell on humans and other organisms are not clearly defined. For my project, I wanted to look at the possibility of the existence of the ability to "smell" in other, less complex organisms in the hopes that there it would be better understood. One of the simplest organisms to look at is motile bacteria, many of which respond to certain chemicals as attractants or repellants in a process equivalent to smell known as chemotaxis.

Structures and Mechanisms of Bacterial Motility

Motile bacteria move themselves using a prokaryotic structure known as the flagellum, which acts as a motor of sorts to propel a bacterium through a fluid medium. Three basic parts compose the flagellum all of which are highly conserved throughout prokaryotic organisms: the filament, the hook, and the basal body. The filament is a long, helical structure composed of approximately 20,000 monomer proteins known as flagellin (1). The flagellin monomers self-assemble into a long, helical structure that is about 20nm in width and 10um in length. The filament is the actual moving, rotating structure of the flagellum, which occurs at its base. The next part of the prokaryotic flagellum is the hook, a bent, cylindrical structure also composed of many units of a single protein. It is believed that the function of the hook is to act to connect the filament to the anchor/motor structure known as the basal body. The hook may also facilitate a change in the rotation of the filament, but that is inconclusive. Finally, the basal body acts both to anchor the entire flagellular structure to the cell, as well as contains the various protein complexes that control the rotational direction of the filament (discussed later). Gram negative bacteria such as Escherichia coli have four protein rings associated with the basal body of their flagella. These rings include the L-ring found in the lipopolysaccharide (LPS) layer of the outer membrane, the P-ring in the peptidoglycan layer (the LPS and peptidoglycan layers compose the cell wall), and the S- and M-rings associated with the cytoplasmic membrane (1). Together, these proteins compose the final structure of the prokaryotic flagellum.

Studies done on the movement of bacteria have often been performed using E. coli as a model. The motion of flagellated bacteria has often been described as a type of random running and tumbling. When rotating their flagella in one direction, bacteria run or swim. Conversely, if rotated in the opposite direction, the bacteria randomly tumble without forward motion. To study which direction of flagellular rotation causes which type of movement, anti-flagellin antibodies were bound to the filament of the flagella, which in turn stuck to a glass surface immobilizing the bacteria (1). In this experimental set up, the flagella were unable to rotate which caused the actual bacteria to spin instead, allowing the scientist to observe more easily. The results showed that for bacteria with peritrichous flagella (multiple flagella around the perimeter of the cell) a counter-clockwise (CCW) rotation results in the drawing together of the structures and swimming of the bacteria in one direction (2). When rotated clockwise (CW), however, the flagella fly apart, and the bacteria tumble chaotically (2). This alternation between running and tumbling is believed to be random; studies have shown that some bacteria rotate their flagella CCW 50% of the time and CW 50% of the time, while others have shown that enteric bacteria rotate flagella CCW 95% of the time and CW 5% of the time (3).

Chemotaxis

Having explained the structures and mechanisms many bacteria use to move, we can now turn our attention to the focus of this paper, the chemotactic behavior of some bacteria. As mentioned above, motile bacteria move in a random running and tumbling pattern when in an isotonic solution. While this type of movement may be totally random in some situations, in others motile bacteria bias this random walk. Chemotactic bacteria are able to detect changes in concentrations of certain chemicals, such as sugars and amino acids (4). This detection is both through space and over time (a few seconds) (5), and allows the bacteria to change movement patterns based on the nature of the chemical and the gradient present. Essentially, they must continually test the chemical environment as they move and compare each sample with the one before.

To the advantage of these bacteria, they are able to detect both attractant and repellant chemicals (6). When an increasing concentration of an attractant (sugar) or decreasing concentration of a repellant (fatty acid) is detected, the bacteria will decrease the frequency at which it tumbles and will thus swim for longer periods (5). This gives the bacteria more of an opportunity to swim into an area where the concentration of a needed chemical is high and away from areas which contain toxic chemicals. Conversely, detection of decreasing concentrations of attractants or increasing concentrations of repellants causes bacteria to increase their tumbling frequencies (5). While the tumbling remains random, increasing the amount of tumbling allows bacteria to change direction more often, hopefully oriented away from a repellant and toward an attractant gradient. What must be understood here is that the movement of these bacteria is still random just as it would be in a solution where there was no detection of chemical gradients; however, the bacteria modify their movement patterns by increasing and decreasing the frequency at which they change from swimming to tumbling.

Motor Function and Rotational Switch

As discussed earlier, motile bacteria move via rotation of one or more flagella, turning counter-clockwise when swimming and clockwise when tumbling. Apparently, there must be some type of mechanisms and driving force for this flagellular movement and rotational switch. Eukaryotic organisms with flagella use ATP as the energy source for the movement of their flagella. Studies done with motile bacteria in which no ATP was present showed that flagellular movement did not change (1). When the environment was deficient in an electrochemical proton gradient, however, flagellular movement stopped. This evidence showed that rather than ATP, it is a proton motive force (PMF), more specifically a proton gradient across the cytoplasmic membrane of the bacteria, that provides the energy for movement (3).

The actual rotation of the flagellum is controlled by protein complexes located in the basal body of the flagellum. Rotation is actually controlled by the MotA/MotB complexes, which act as proton pores (1). Optimal rotation is achieved when all eight complexes of the flagellum are functioning. Changes in the direction of rotation are controlled by a switch complex in the cytoplasm of the bacteria and associated with the S/M ring of the basal body (1). This complex, composed of the Fli G, M, and N proteins, undergoes certain conformational changes based on the direction of flagellular rotation (7).

Mechanisms of Chemotaxis

Now that it has been established that some bacteria are chemotactic toward and away from certain chemicals and the mechanisms by which actual movement occurs, we must address the question of what the bacteria detect and how. Before this question was answered, it was unknown if chemotactic bacteria detect the actual chemical or if the detection is of some metabolic by-product of the attractant, such as ATP. Work done by Julius Adler in the late 1960s showed that the bacteria detect the actual chemical attractant or repellant (8). He found that bacteria that were attracted to certain chemicals showed no chemotactic attraction to the metabolized products of those attractants. By working with certain mutant strains of E. coli, he found that the detection of these attractants and repellants was due to certain sensory proteins he termed chemoreceptors.

Later work identified the genes for these chemoreceptors, as well as the receptors themselves. There are four types of receptors which are now known to be transmembrane proteins that send certain chemotactic signals across the membrane (2). Type I binds serine as well as certain repellants and is encoded for by the tsr gene. Type II encoded by the tar gene binds aspartate, maltose/maltose binding protein complex and some repellants. Type III binds galactose and ribose in conjunction with their specific binding proteins, while Type IV responds to certain dipeptides. The genes for Type III and Type IV are the trg and tap genes respectively (6). It has been estimated that there are approximately 2000 of each receptor type on the surface of a bacterium, which allows for extensive sampling of the environment for chemical gradients (3).

Signaling Pathway

Once these chemoreceptors have detected an attractant or a repellant and bind it, they undergo a conformational change that starts an intracellular signal the resembles a cascade of sorts (2). Four cytoplasmic proteins are involved in this pathway that send a message from the chemoreceptor to the flagellum, signaling it to rotate one way or the other; those proteins include CheA, CheW, CheY and CheZ (9). This cascade is actually responsible for the tumbling of bacteria, i.e. the clockwise rotation of the flagella. When not stimulated, the flagella rotate CCW and swimming occurs. Increasing concentrations of attractant (more binding of attractants to chemoreceptors) and decreasing concentrations of repellant (less binding) causes the chemoreceptors to be less active. The protein cascade is not stimulated as frequently and the bacteria swim longer. If, for example, there is an increasing concentration of repellant and thus more binding to the chemoreceptors, this signaling pathway will be stimulated. When the repellant binds, the CheA (a histidine kinase) (1) and CheW proteins bind to the cytoplasmic domain of the receptor protein causing CheA to be phosphorylated (2). The high energy phosphate on CheA is transferred to the CheY protein which then binds to the motor of the flagellum causing it to switch rotation from CCW to CW (7), and thus tumbling results. If the binding of attracts decrease, CheA will autophosphorylate to stimulate the cascade (9).

Work done by Welch et al. (7) showed that the CheY protein is active only when phosphorylated. Their work showed that CheY-phosphate binds to the FliM protein of the motor switch complex leading to a conformational change in the FliG protein and thus a switch to CW rotation. Lowry et al. (10) demonstrated that when phosphorylated, the binding face of the CheY protein that corresponds to the FliM protein of the motor switch complex undergoes a conformational change. This change facilitates binding to the switch complex. The dephosphorylation and thus inactivation of CheY is believed to be controlled by the CheZ protein (2). Mutants that lack CheZ tumble more often than those with the protein (9). The stimulation and regulation mechanisms for CheZ is unknown, however. Also, the phosphate bond on active CheY is unstable. Work done by Louis Tisa and Adler suggests that calcium ions (Ca2+) are involved in stabilizing this phosphate bond and maintaining the active CheY-phosphate (11).

Adaptation

Finally, we must address one of the most interesting aspects of chemotactic bacteria-- their ability to adapt to their chemical environment. As discussed earlier, chemotactic bacteria bias their random walks toward an attractant gradient or away from a repellant gradient by decreasing or increasing their tumbling frequencies. Binding of attractants to chemoreceptors decreases the activity of the receptors and thus of the intracellular signal transduction proteins (less CheA and CheY activity). This overall decrease in activity leads to less tumbling and longer swimming. The binding of the attractant also induces adaptation back to normal running and tumbling frequencies (2) -- once in a high concentration of attractant, the bacteria need to stay there. They also must adapt so that they can respond to any changes that may occur.

The chemoreceptor proteins discussed earlier are also known as methyl-accepting chemotaxis proteins (MCPs) (2). When an attractant binds an MCP, a conformational change takes place that decreases the activity of the receptor. If the bacteria remain in the attract for a few minutes, specific glutamate residues on the cytoplasmic side of the MCPs will be methylated by the catalytic methyl transferase enzyme CheR (3). Methylation of the MCPs returns the proteins to their original conformation (1). All MCPs have eight methyl-binding sites; the more that are methylated, the more the MCPs are adapted (12). Higher concentrations of attractants would have a greater effect on the conformational change of the receptors and would therefore be more extensively methylated during adaptation (3). Once restored to original conformation and adapted, the methyl groups are removed by the methyl esterase CheB. CheB is part of a feedback mechanism because it is active only when phosphorylated (9). The means of CheB phosphorylation is that of an active CheA protein (1). CheA, therefore, controls both the signaling cascade (by phosphorylating CheY) and the level of chemoreceptor methylation, i.e. the level of adaptation.

Conclusion

As can be seen, many motile bacteria are chemotactic clearly for reasons of survival. While their movement is inherently random, they are able to bias this alternation of running and tumbling when moving in a concentration gradient of attractant or repellant. Spacial and temporal detection of these chemical gradients occur through the stimulation of specific chemoreceptor proteins on the surface of these bacteria. The activation of these receptors start an intracellular cascade that controls the direction of flagella rotation (the propulsive structure of motile bacteria). Once the bacteria move into an area of high attractant or low repellant concentration, they must then adapt to the new environment so that future stimulants can be detected. This adaptation process occurs through the methylation of the chemoreceptor proteins, which restores them to their original comformation. From this project, we have seen that chemotaxis is an important aspect of motile bacteria. And while bacteria may not be very complex organisms comparatively, even the process and mechanisms of chemotaxis illustrate that indeed at all levels life is intricate and facinating.

Works Cited

1. Armitage, J. P. (1992) Bacterial Motility and Chemotaxis. Science Progres. 76:451-477.

2. Alberts et al. (1992) Cell Signaling Cell Biology Chapter 15:773-778.

3. Armitage, J. P. and Sockett, R. E. (1987) Sensory Transduction in Flagellate Bacteria. Annals of the New York Academy of Sciences Olfaction and Taste IX 510: 9-15. ISBN 0-89766-416-7.

4. Adler, J. P. (1966) Chemotaxis in Bacteria. Science 153: 708-716.

5. Adler, J. P. (1975) Chemotaxis in Bacteria [Review]. Annual Review of Biochemistry 6. Adler, J. P. <1987) How Motile Bacteria Sense and Respond to Chemicals. Olfaction and Taste IX . 510: 95-97.

7. Welch, M. et al.(1993) Phosphorylation Dependent Binding of a Signal Molecule to the Flagellar Switch of Bacteria. Proc. Natl. Acad. Sci. 90: 8787-8791.

8. Adler, J. P. (1969) Chemoreceptors in Bacteria. Science. 166: 1588-1597.

9. Parkinson J. S.(1993) Signal transduction Schemes of Bacteria [Review]. Cell . 73(5): 857-871.

10. Lowry, D. F. et al. (1994) Signal Transduction in Chemotaxis. Journal of Biological Chemistry. 269(42): 26358-26362.

11. Tisa, L. S. and Adler, J. P. (1992) Calcium Ions Are Involved in E. coli Chemotaxis. Proc. Natl. Acad. Sci. 89: 11804-11808.

12. Shapiro, M. J. et al. (1995) Contributions Made by Individual Methylation Sites of the E. coli Aspartate Receptor to Chemotactic Behavior. Proc. Natl. Acad. Sci. USA 92: 1053-1056.