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From 2011.igem.org

Chemotaxis Overview

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.

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.

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).

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.

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.

Root uptake

As part of the chemotaxis module, we will also be looking at the uptake of our bacteria into plant roots. We want the bacteria to get taken up into the plant roots to ensure that the concentration of indole-3-acetic acid in the plant is increased. If the bacteria remained outside the roots, this goal may also be reached but the risk that we would not increase the internal IAA concentration would be significantly higher. In addition, uptake of bacteria into the roots followed by secretion of chemicals presents a novel platform for modifying plants without engineering their genomes.

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.

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.

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.

References

Chelsky, D. & Dahlquist, F. W. (1980) Chemotaxis in Escherichia coli: Association of protein components. Biochemistry, 19, 4633 – 4639.
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.
Paungfoo-Lonhienne et al. (2010) Turning the table: plants consume microbes as a source of nutrients. PLoS ONE 5(7): e11915. http://www.nih.gov/science/models/arabidopsis/index.html
Paungfoo-Lonhienne et al. (2010) Turning the table: Plants consume microbes as a source of nutrients. PLoS ONE 5:1-11.