# Structural Biochemistry/Chemotaxis

Chemotaxis is a behavioral response to a chemical stimulus in which an organism moves either toward or away from the stimulus. A chemical that causes an organism to move closer to it is called an attractant, whereas a chemical that causes an organism to move further away is called a repellent.

## Overview

Cells use a flagella motor to move about, either toward or away from an attractant. When the cell moves away from an attractant, the receptors send a signal that allows one or more flagella to switch rotation from counterclockwise (CCW) to clockwise (CW). When the rotation switch is spinning counter-clockwise the cell goes into a run in a single direction, usually toward an attractant, which is favorable. When the flagella motor is rotating clockwise, the flagella disrupts the run, causing the cell to tumble. Each tumble causes the cell to swims off in a new direction. A counter clockwise rotation allows the cell to move faster than when the cell tumbles.
The pattern of movement resulting from alternating swimming and tumbling is a “biased random walk” in which the cell sometimes moves randomly but overall tends to migrate toward the attractant. When no attractant is present, the movement results in a "random walk", tumbling more often.

## Research of Bacterial Chemotaxis

Intact flagellum is isolated and purified from bacterium such as E. coli. The structure of the E. coli is then determined through the electron microscopy. It is with this data that the structure and function of the flagella can be understood. Further research into the motility of the bacterial flagella revealed that there are several genes required to conduct chemotaxis called the che genes. Mutants that failed to conduct chemotaxis to a singular attractant were discovered by Jerry Hazelbaur and Bob Mesibov and called "specifically nonchemotactic mutants" (Adler 2011). These mutants were later used in tests to determine what chemicals were attractants and which were repellents.
Motile bacteria respond to environmental cues to move to more favorable locations. The components of the chemotaxis signal transduction systems that mediate these responses are highly conserved among prokaryotes including both eubacterial and archaeal species. The best-studied system is that found in Escherichia coli. Attractant and repellant chemicals are sensed through their interactions with transmembrane chemoreceptor proteins that are localized in multimeric assemblies at one or both cell poles together with a histidine protein kinase, CheA, an SH3-like adaptor protein, CheW, and a phosphoprotein phosphatase, CheZ. These multimeric protein assemblies act to control the level of phosphorylation of a response regulator, CheY, which dictates flagellar motion. Bacterial chemotaxis is one of the most-understood signal transduction systems, and many biochemical and structural details of this system have been elucidated. This is an exciting field of study because the depth of knowledge now allows the detailed molecular mechanisms of transmembrane signaling and signal processing to be investigated.

## Che genes

John Armstrong worked with E. coli and discovered the first nonchemotactic mutants. The mutants were basically bacteria that did not exhibit any attracting or repelling response to a chemical stimulus. However, the bacteria were still motile. With further analysis, three che genes were found necessary for E. coli to undergo chemotaxis. Those che genes were cheA, cheB, and cheC. The che mutants were investigated further in another study by Jerry Hazelbauer and Bob Mesibov, which led to chemoattractants and chemorepellants.
Che genes are responsible for the Che proteins in the chemotaxis process, which comes right after the MCP (methyl-accepting chemotaxis protein) receptors that receive the chemical stimulus and before the flagella. The Che proteins perform “excitation” and “adaptation” processes. Four more che genes (cheW, cheY, cheZ, and cheR) were discovered that played an important role in bacteria chemotaxis. After the MCP receptors have received the attractant or repellant stimulus, cheA, cheW, cheY, and cheZ signals the flagella to perform excitation and cheB and cheR signals the flagella to perform adaptation. These che genes are found commonly among bacteria (ones that utilizes flagella for mobility and ones that utilizes pili for mobility) and in the archaea. (Adler 2011)
Hybrid Escherichia coli ColE1 plasmids carrying the genes for motility (mot) and chemotaxis (che) were transferred to a minicell-producing strain. The mot and che genes on the hybrid plasmid directed protein synthesis in minicells. Polypeptides synthesized in minicells were identical to the products of the motA, motB, cheA, cheW, cheM, cheX, cheB, cheY, and cheZ genes previously identified by using hybrid lambda and ultraviolet-irradiated host cells (Silverman and Simon, J. Bacteriol. 130:1317-1325, 1977), thus confirming these gene product assignments. The products of some che genes (cheA and cheM) appeared as more than one band on polyacrylamide gel electrophoresis, but analysis of partial peptide digests of these polypeptides suggested that the multiple forms were coded for by a single gene. Measurement of the physical length of the hybrid plasmids allowed an estimate of the amount of coding capacity of the cloned deoxyribonucleic acid, which was devoted to the synthesis of the mot and che gene products. These estimates were also consistent with the hypothesis that the multiple polypeptides corresponding to cheA and cheM were the products of single genes.
The products of three chemotaxis-specific genes in Escherichia coli, cheM, cheD, and cheZ, are methylated. The cheZ gene codes for the synthesis of a 24,000 molecular weight polypeptide that appears in the cytoplasm. cheM codes for the synthesis of a membrane-bound polypeptide with a molecular weight of 61,000. cheD codes for another membrane-bound polypeptide with an apparent molecular weight of 64,000. CheM- mutants show chemotaxis toward some attractants (Tar- phenotype), while CheD- mutants respond to other attractants (Tsr- phenotype). The double mutant (CheD-, CheM-) does not respond to any attractant or repellent tested. Therefore, these polypeptides play a central role in chemotaxis. They collect information from two subsets of chemoreceptors and act as the last step in the chemoreceptor pathway and the first step in the general processing of signals for transmission to the flagellar rotor. It is suggested that they may be involved in both an initial process that reflects the instantaneous state of the chemoreceptors and in an integrative, adaptive process. Two other genes, cheX and cheW, are required for the methylation of the cheD and cheM gene products.

## Chemoattractants and Chemorepellents
### Chemoattractants

Bob Mesibov conducted attraction tests on E. coli against 53 amino acids and related chemicals. This was done by using mutants that reacted indifferently in L-serine and L-aspartate. Jerry Hazelbauer and Marge Dahl also tested the attraction of E. coli against the 85 common sugars and related chemicals. Among the highest attraction that were tested in this category were: D-galactose, D-glucose, D-mannitol, D-ribose, D-sorbitol, maltose and trehalose. A study later conducted by Wolfgang Epstein and Julius Adler revealed that phosphorotransferase sugars were also attractants. Some inorganic salts were also found to be attractants. (Adler 2011)
The study of leukocyte migration continues to provide new insights into the regulation of lymphocyte priming in secondary lymphoid organs and effector responses in inflamed tissues. Chemoattractant receptors have always been viewed as facilitators of cell movement into a tissue. This whole concept must now be revised with the discovery of sphingosine 1 phosphate receptors, which control cell exit from lymphoid tissues. The chemoattractants that regulate lymphoid tissue homing are usually different to those that regulate leukocyte recruitment to inflamed tissues. There is evidence, however, of inflammatory pathways of leukocyte recruitment in lymph nodes and, conversely of constitutive pathways in peripheral tissues. Finally, antagonists (or agonists) of chemoattractant receptors and their signalling pathways represent the most attractive strategy for the treatment of a wide range of inflammatory diseases, including allergy.

### Chemorepellents

Testing for chemorepellents was done in a similar manner as the testing for chemoattractants. For E. coli, 164 different chemicals were tested by Wung-Wai Tao. The results of these tests showed that the most effective repellents were: short-chain fatty acids, hydrophobic amino acids, benzoate, indole, skatol, salicylate, and the ions Co$^{2+}$, Ni$^{2+}$, H$^+$, and OH$^-$. (Adler 2011)
Although Paramecium has been widely used as a model sensory cell to study the cellular responses to thermal, mechanical and chemoattractant stimuli, little is known about their responses to chemorepellents. A convenient capillary tube repellent bioassay to describe 4 different compounds that are chemorepellents for Paramecium and compared their response with those of Tetrahymena. The classical Paramecium t-maze chemokinesis test was also used to verify that this is a reliable chemorepellent assay. The first two compounds, GTP and the oxidant NBT, are known to be depolarizing chemorepellents in Paramecium, but this is the first report of them as repellents in Tetrahymena. The second two compounds, the secretagogue alcian blue and the dye cibacron blue, have not previously been described as chemorepellents in either of these ciliates. Two other compounds, the secretagogue AED and the oxidant cytochrome c, were found to be repellents to Paramecium but not to Tetrahymena. The repellent nature of each of these compounds is not related to toxicity. The reason is because cells are completely viable in all of them. More importantly, all of these repellents are effective at micromolar to nanomolar concentrations, providing an opportunity to use them as excitatory ligands in future works concerning their membrane receptors and possible receptor operated ion channels.

### Two Component System

The two component system is constituted of che proteins that are not involved in chemotaxis. Rather these che genes are more related to the transcriptional regulator in the cell. An important phenomenon occurs in the two component system that affects the rotation and movement of the flagella of bacteria. As the histidine residue of the first component is phosphorylated with ATP, the phosphate is transferred to the aspartate residue of the second component, which affects the rotation and movement of the flagella. Phosphorylation in the two component system also controls the genes that is needed for protein synthesis. The two component system is mainly found in bacteria, archaea, and single-celled eukaryotes. Repellants bring about the phosphorylation of cheA and cheY. Consequentially, this leads to the tumbling of bacteria's flagella. This enables the bacteria to move away from the repellant. Attractants block the phosphorlyation of cheA and cheY, therefore, tumbling is stopped. On the other hand, this enables the bacteria to go running towards the attractant. The bacteria's interactions with the repellant and attractant are called excitations. Excitations determine the movement of the bacteria. Adaption, the process following excitation, determines the bacteria's rotation. Repellants cause demethylation of the methylated MCP (methyl-accepting-chemotaxis-protein), while the attractant causes its methylation. Generally, repellants cause bacteria to rotate in a clockwise fashion and attractants cause bacteria to rotate in a counter-clockwise fashion. http://www.pnas.org/content/94/14/7263/F1.expansion.html (Adler 2011)

## Responses of other Stimuli

Understanding that the environment is a major factor in deciding what a cell will do, there are other sensory stimuli besides chemicals. E. Coli were discovered to be repelled by blue light (phototaxis) which was determined by the fact that the chemoreceptors in E. Coli respond to light because most species of bacteria were former photosynthetic. Using light to fix carbon, they generated behavioral signals which now allowed E. Coli to respond to light. (Adler 2011) (Berg 2011)
E. Coli had demonstrated some responses to heat. More tests were conducted to see the effects of chemotactic system for thermotaxis. If E. Coli was missing 3 of the chemo proteins (cheY|W|A) then they would not react to the change in temperature, but rather the chemoreceptors (Tsr,Tar,Trg,Tap) all demonstrated some hand in bacterial thermotaxis. Falling in two categories, the cold receptor is Tap, while Tsr, Tar, Trg showed an increase of tumbling when there was a decrease of temperature vice versa the cold receptor would produce an inverted response. Receptors, if near their attracts, will demonstrate a different behavior after being methylated. If there is Serin, there will be no response due to Tsr. Some explanation that maybe offered to understand why the E. Coli “swim” is due to the hot temperatures that will cause denaturation making it more fluid. (Adler 2011) (Jeffory,Salman,Libchaber 2011)
Osmolarity plays a major role in where E. Coli are repelled by both high & low osmolarity but seek optimum osmolarity. This osmotaxis is highly dependent on the environment of E. Coli and the changes have different effects. If there is a rapid flow of water from the cell, it will be turgor, causing a process to try to restore the original conditions. The amount and concentration of the cytoplasmic water are controllers of the growth and the lower/higher the osmolarity, the less it will move versus optimum states of osmolarity. (Adler 2011)

## Electrophysiology

If there are different responses to the environment, electrical properties must also play a role in E. Coli. Although experiments have played with the action potential of E. Coli there has yet to be any evidence. There proves to be electrical changes within E. Coli, but none that will come close to action potentials. The size of E. Coli also proves to be a difficulty because of its extremely small scale. If we were to measure the volts, it would degrade the cell wall and disrupt the cells function. To measure them, as of now, scientists monitor the blinking behaviors of E. Coli strains and determine their functions. (Adler 2011) (McDonald 2011)
Electrophysiology is the fastest growing of all the cardiovascular disciplines. Electrophysiologists are cardiologists who have additional education and training in the diagnosis and treatment of abnormal heart rhythms. Close collaboration between electrophysiologists and other doctors who treat patients with heart disease is very important.
An electrophysiology study (EPS) is a test that can help predict if an individual is at high risk for sudden cardiac arrest. Signals are administered to the heart muscle in patterns to see if they will stimulate ventricular tachycardia (VT). The test is performed in a safe and controlled electrophysiology laboratory at a hospital or clinic and the patient is in no danger. In an EP study, local anesthetics are used to numb areas in the groin or near the neck, and small catheters are passed into the heart to record its electrical signals. During the study, the physician studies the speed and flow of electrical signals through the heart, identifies rhythm problems and pinpoints areas in the heart's muscle that give rise to abnormal electrical signals. Anyway, electrophysiology (EP) is an emerging healthcare science and therapy. EP is a subspecialty of cardiology that focuses on diagnosing and treating cardiac arrhythmias. The cardiac electrophysiology technologist may come from a variety of allied health professionals (RT, RN, CVT, EMT, RRT and PA), assists an EP cardiologist during diagnostic and invasive procedures including programmed electrical stimulation, sterile scrub technique, electro-anatomical 3D mapping, catheter ablation for cardiac arrhythmias and device implantation for cardiac rhythm management such as pacemakers and other advanced implantable devices.
An electrophysiology study can: 1) Identify which patients who have had a prior heart attack, or MI, are at risk for serious ventricular arrhythmias and, perhaps, SCA. 2)Assist in determining which patients may require aggressive treatment to prevent sudden cardiac arrest. 3) Identify individuals whose hearts cannot be induced into dangerous arrhythmias. They appear at lower risk for developing spontaneous, sustained VT that can lead to ventricular fibrillation and sudden cardiac arrest.
We may need an EP study: 1)To determine the cause of an abnormal heart rhythm. 2)To locate the site of origin of an abnormal heart rhythm. 3)To decide the best treatment for an abnormal heart rhythm.

## References

Adler, Julius (2011). "My Life with Nature". The Annual Review of Biochemistry. Retrieved 2011-10-27.
Berg, Howard (2004). "E Coli in Motion". Biological and Medical Physics Biomedical Engineering. Retrieved 2011-10-29.
Jeffory,Salman,Libchaber, Marie,Hanna,Albert (2004/05). "Thermotaxis in E. Coli" (PDF). The Rockefeller University. Retrieved 2011-10-29. {{cite web}}: Check date values in: |date= (help)CS1 maint: multiple names: authors list (link)
McDonald, Casey (2011). "E. coli Voltmeters". Biotechniques. Retrieved 2011-10-29.
Foster, John W., Slonczewski, Joan L.. Microbiology. 1st ed. New York. 2009.