Team:Imperial College London/Project/Chemotaxis/Overview

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<h1>Chemotaxis Overview</h1>
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Movement performed by bacteria based on attraction or repulsion of chemicals in the environment is known as chemotaxis. In our project we are using this mechanism for location of plant root by modified bacteria, which will be attracted to the root and will actively swim towards it.<br><br>
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<h1>Overview</h1>
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Chemotaxis in Escherichia coli is well documented. E. Coli can perform two types of movement, tumbling or smooth swimming. The difference between the two is in flagellar movement. During tumbling movement,flagella move clockwise due to the formation of complex between CheY-P and FliM ,one of the flagellum associated  proteins. During smooth swimming, CheY is not phosphorylated and therefore cannot associate with flagellar proteins, which causes flagellum to move counterclockwise. Smooth swimming is the movement performed by bacteria, while the bacteria are being attracted towards localised chemical source. The control of smooth swimming is done by a number of chemotaxis proteins, which together concise a signalling pathway.<br><br>
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At the start of the signalling pathway there is a receptor which is  associated with CheW and CheA, when no attractant is bound. This complex leads to phosphorylation of CheY, which then carries on to be associated with flagellar proteins. When ligand (attractant or repellent) binds to receptor, CheW & CheA dissociates from the receptor and leads to inability to phosphorylate CheY that leads to flagellar FliM protein not being associated with CheY and that leads to counterclockwise flagellar movement. Also there is CheZ a phosphatase, which removes phosphate group from CheY. Another aspect of bacterial  chemotaxis is a simple memory that bacteria use to move up the concentration gradient. This is achieved by protein CheR a methyltransferase, which methylates MCP (methyl accepting chemotaxis protein) and this way affect the capability of receptor to form association with CheW & CheA. CheW & CheA dissociation from the chemoreceptor depends on the rising concentration of attractant, which in turn depends on the bacterium moving towards the source of attraction. Also there is CheB, which acts as a methylesterase and can remove methyl groups from MCP receptor (Chelsky & Dahlquist, 1980). <br><br>
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[[File:ICL-tinker cell mcpS.jpg]]
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<p>Movement performed by bacteria based on attraction or repulsion of chemicals is known as chemotaxis. In our project we are engineering this mechanism in order to enable our microbes to swim towards plant roots. Plant roots naturally secrete a variety of compounds that <i>Escherichia coli</i> are not attracted to naturally. Accordingly, we engineered a chemoreceptor that can sense the root exudates into our chassis. This receptor will enable the bacteria to swim towards roots.</p>
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<p>Following bacterial movement to the roots, the microbes will be taken up into the roots of the plants. A recent paper (1) described the uptake of <i>E. coli</i> into the roots of watercress and tomato plants. We have replicated these findings (Figure 1). </p>
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Malate and other root exudates have been identified as chemotactic attractants in a number of other bacteria. For our project we are therefore rewiring chemotaxis in E. coli, by adding a chemoreceptor mcpS from Pseudomonas putida KT2440 strain. McpS is a receptor, which responds to a number of TCA cycle intermediates such as malate, fumarate, oxaloacetate, succinate, citrate, isocitrate and butyrate (Lacal et al, 2010). P. putida uses chemotaxis proteins in a different way to control its chemotactic response, however structurally are very similar to Che proteins in E. coli. Therefore the idea is that mcpS domain that interacts with Che proteins is sufficiently similar to native chemoreceptors, so that upon malate binding to the mcpS receptor, E. coli with expressed mcpS will exhibit chemotactic response towards malate. In a similar way we are also utilising chemotaxis receptor PA2652 from Pseudomonas aeruginosa PA01 strain, which also uses malate as attractant ligand. Therefore we can also compare which of the two introduced chemoreceptors responds more efficiently in terms of concentration of malate attractant. <br><br>
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<p style="text-align:center;"><img src="https://static.igem.org/mediawiki/igem.org/b/bf/Gfpinroot10xnostack.jpg" width=450/></p>
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This way we can show that foreign chemoreceptors are compatible with e. coli chemotaxis system, provided the parts integrated share substantial structural similarity. Also we are capable of increasing a number of attractants to which e. coli is attracted, by addition of compatible chemoreceptors.
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<br><br>
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<p>Our primary chassis for wet lab experiments is <i>Escherichia coli</i>. Chemotaxis in <i>E. coli</i> is well documented. These bacteria can perform two types of movement, tumbling and smooth swimming. The difference between the two is determined by flagellar movement. During tumbling movement, the flagella move clockwise. This is caused by the formation of a complex between CheY-P and FliM, one of the flagella-associated proteins. During smooth swimming, the flagella move counter-clockwise. CheY is not phosphorylated and therefore cannot associate with flagellar proteins, causing the flagella to rotate in the opposite direction.</p>
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<h2>Root uptake</h2>
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<p>Smooth swimming is the movement performed by bacteria towards an attractant or away from a repellent. Smooth swimming is controlled by a number of chemotaxis proteins that make up a signalling pathway, with basic functioning having same as typical prokaryotic two component system.
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As part of the chemotaxis module, we will also be looking at the uptake of our bacteria into plant roots. In a paper published last year, Paungfoo-Lonhienne et al. showed that Arabidopsis and tomato plants are able to actively break down their cell wall to take up GFP-tagged E. coli and S. cerevisiae and use them as a source of nutrients. For simplicity, we will be working with Arabidopsis.<br><br>
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First part of the mechanism is sensory kinase, which consists of input domain and autokinase domain. Second part of the mechanism is the response regulator, with reciever and output domains. In the case of chemotactic system, sensory kinase is chemoreceptor associated with CheA and CheW proteins. This association remains present only in the absence of a ligand. During that period CheA autophosphorylates and is capable of phosphorylating CheY, protein which acts as a response regulator in this mechanism. Phosphorylated CheY has the capability of associating itself with flagellar proteins, thereby controlling the direction which flagellum rotates. However, in the presence of ligand, sensory kinase domain is not functional due to dissociation of CheA from chemoreceptor. This way CheY does nto associate with flagellar proteins and result is counterclockwise flagellar movement (Sourjik & Armitage, 2010).</p>
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Arabidopsis thaliana is a common plant model organism. It belongs to the mustard family and fulfils many important requirements for model organisms. As such, its genome has been almost completely sequenced and replicates quickly, producing a large number of seeds. It is easily transformed and many different mutant strains have been constructed to study different aspects (National Institute of Health, no date). While Arabidopsis may not represent plant populations naturally occurring in arid areas threatened by desertification, it is a handy model organism we will be using to study the effect of auxin on roots, observe chemotaxis towards them and look at uptake of bacteria into the roots. <br><br>
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<p> In E. coli chemotaxis there is a number of other proteins, which have functions associated with the two component system and as a result it enables bacterium to move up or down a concentration gradient. This is mediated by CheR, a methyltransferase that methylates MCP (methyl accepting chemotaxis protein). This affects the receptor’s ability to associate with CheW and CheA. Dissociation of CheW and CheA from the chemoreceptor depends on the rising concentration of attractant, which in turn depends on the bacterium moving towards the source of attraction. This is driven by CheZ, a phosphatase that removes phosphate groups from CheY, while sensory kinase is dissociated. In addition, CheB  acts as a methylesterase and can remove methyl groups from the MCP receptor, to act as some kind of memory reset (Chelsky & Dahlquist, 1980).</p>
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We will be using Arabidopsis to look at the uptake of our engineered bacteria into the plants. For this, we will be using wild type Arabidopsis and E. coli that constitutively express green fluorescent protein. The natural fluorescence produced by plant roots and green fluorescence produced by the bacteria can be used to image the uptake of bacteria using confocal microscopy.<br><br>
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<p><embed src="https://static.igem.org/mediawiki/igem.org/5/54/ICL_Chemotaxis.swf" width="935px" height="500px" /></p>
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<h2>References</h2>
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Chelsky, D. & Dahlquist, F. W. (1980) Chemotaxis in Escherichia coli: Association of protein components. Biochemistry, 19, 4633 – 4639.<br>
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Lacal, J., Alfonso, C.,  Liu, X. & et al. (2010) Identification of a chemoreceptor for tricarboxylic acid cycle intermediates: differential chemotactic response towards receptor ligands. Journal of Biological Chemistry, 285 (30), 23126 – 23136.<br>
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<a href="http://www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone.0011915">Paungfoo-Lonhienne et al. (2010) Turning the table: plants consume microbes as a source of nutrients. PLoS ONE 5(7): e11915.</a>
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<a href="http://www.nih.gov/science/models/arabidopsis/index.html">http://www.nih.gov/science/models/arabidopsis/index.html</a><br>
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<a href="http://www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone.0011915">Paungfoo-Lonhienne et al. (2010) Turning the table: Plants consume microbes as a source of nutrients. PLoS ONE 5:1-11.</a>
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Latest revision as of 16:07, 14 September 2011



Module 1: Phyto-Route

Chemotaxis is the movement of bacteria based on attraction or repulsion of chemicals. Roots secrete a variety of compounds that E. coli are not attracted to naturally. Accordingly, we engineered a chemoreceptor into our chassis that can sense malate, a common root exudate, so that it can swim towards the root. Additionally, E. coli are actively taken up by plant roots, which will allow targeted IAA delivery into roots by our system.





Overview

Movement performed by bacteria based on attraction or repulsion of chemicals is known as chemotaxis. In our project we are engineering this mechanism in order to enable our microbes to swim towards plant roots. Plant roots naturally secrete a variety of compounds that Escherichia coli are not attracted to naturally. Accordingly, we engineered a chemoreceptor that can sense the root exudates into our chassis. This receptor will enable the bacteria to swim towards roots.

Following bacterial movement to the roots, the microbes will be taken up into the roots of the plants. A recent paper (1) described the uptake of E. coli into the roots of watercress and tomato plants. We have replicated these findings (Figure 1).

Our primary chassis for wet lab experiments is Escherichia coli. Chemotaxis in E. coli is well documented. These bacteria can perform two types of movement, tumbling and smooth swimming. The difference between the two is determined by flagellar movement. During tumbling movement, the flagella move clockwise. This is caused by the formation of a complex between CheY-P and FliM, one of the flagella-associated proteins. During smooth swimming, the flagella move counter-clockwise. CheY is not phosphorylated and therefore cannot associate with flagellar proteins, causing the flagella to rotate in the opposite direction.

Smooth swimming is the movement performed by bacteria towards an attractant or away from a repellent. Smooth swimming is controlled by a number of chemotaxis proteins that make up a signalling pathway, with basic functioning having same as typical prokaryotic two component system. First part of the mechanism is sensory kinase, which consists of input domain and autokinase domain. Second part of the mechanism is the response regulator, with reciever and output domains. In the case of chemotactic system, sensory kinase is chemoreceptor associated with CheA and CheW proteins. This association remains present only in the absence of a ligand. During that period CheA autophosphorylates and is capable of phosphorylating CheY, protein which acts as a response regulator in this mechanism. Phosphorylated CheY has the capability of associating itself with flagellar proteins, thereby controlling the direction which flagellum rotates. However, in the presence of ligand, sensory kinase domain is not functional due to dissociation of CheA from chemoreceptor. This way CheY does nto associate with flagellar proteins and result is counterclockwise flagellar movement (Sourjik & Armitage, 2010).

In E. coli chemotaxis there is a number of other proteins, which have functions associated with the two component system and as a result it enables bacterium to move up or down a concentration gradient. This is mediated by CheR, a methyltransferase that methylates MCP (methyl accepting chemotaxis protein). This affects the receptor’s ability to associate with CheW and CheA. Dissociation of CheW and CheA from the chemoreceptor depends on the rising concentration of attractant, which in turn depends on the bacterium moving towards the source of attraction. This is driven by CheZ, a phosphatase that removes phosphate groups from CheY, while sensory kinase is dissociated. In addition, CheB acts as a methylesterase and can remove methyl groups from the MCP receptor, to act as some kind of memory reset (Chelsky & Dahlquist, 1980).

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