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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.

Figure 1: Escherichia coli cells expressing superfolder GFP (sfGFP) can be seen inside an Arabidopsis thaliana root using confocal microscopy after overnight incubation of the plants with bacteria. Roots were washed in PBS prior to imaging to avoid "false positives" of bacteria adhering to the outside of the root. For a cool 3D video of bacteria inside the roots, check out our Results page. (Data and imaging by Imperial iGEM 2011).

Overview

The Phyto Route module mainly consists of bacterial movement towards plant roots. Following bacterial movement to the roots, the microbes will be taken up into the roots of the plants. The fact that bacteria are taken up into plant roots, where they are used for nutrients by the plant itself, is a novel finding that was only described last year. Paungfoo-Lonhienne et al. (1) described the uptake of Escherichia coli into the roots of watercress and tomato plants. We have replicated these findings (Figure 1). This is especially interesting for our project as the plants will be exposed to bacterial auxin inside the root cells.

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 [2].

In E. coli chemotaxis there are a number of other proteins, which have functions associated with the two-component system and as a result they enable the 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 a memory reset [3].

References

[2]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.

[3]Chelsky, D. & Dahlquist, F. W. (1980) Chemotaxis in Escherichia coli: Association of protein components. Biochemistry, 19, 4633 – 4639.
Sourjik, V. & Armitage, J. (2010) Spatial organization in bacterial chemotaxis. The EMBO Journal, 29, 2724 - 2733.