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From 2011.igem.org

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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.

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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).