Team:BNU-China/Chemotaxis.html

Overview

The plants root exudates contain TCA intermediates that can attract bacteria having the ability of chemotaxis. Click here to learn more about root exudates. E.coli has four kinds of chemoreceptors, which interact with factors of the flagella that leads to chemotaxis. But E.coli doesn’t have specific chemotaxis towards some TCA intermediates while Pseudomonas putida has. Pseudomonas putida has some McpS, like McfQ and McfR. We made a part BBa- containing the sequence of McfR, which detects succinate, malate and fumarate. Then we detected its chemotaxis towards malate and fumarate, click here to see the results. At last, we designed a model to mimic the movement pattern and predict the efficiency of the Prometheus E.coli.

Root exudates

Plants secrete both high- and low-molecular weight compounds from their roots, and these root exudates play an important role not only as nutrients for soil microbes but also as signal molecules in plant–microbe interactions. In the wild, legume plants establish symbiotic interactions with rhizobia and arbuscular mycorrhizal fungi to obtain several nutrients such as nitrogen and phosphate. The root exudates of legume plants contain organic acids, including some TCA intermediates, such as malate, succinate, citrate and fumarate. Especially, in response to phosphorus starvation, malonie, succinic, fumaric, malic, citric, and t-aconitic acids were detected in the root exudates. (cite 2 papers)Here, we design the Prometheus E.coli that responses to malate and succinate and will swim to the roots to improve the interaction between the roots and fungi .

Chemoreceptor and Chemotaxis

In isotropic chemical environments, E. coli swims in a random - walking pattern produced by alternating episodes of counter-clockwise (CCW) and clockwise (CW) flagellar rotation (Fig. 1, left panel). While an attractant or repellent appear somewhere in the environment, the cells’ locomotor responses specific runs that take the cells in favorable directions (toward attractants and away from repellents), resulting in net movement towards preferred environments. (Fig. 1, right panel).

Fig. 1 - Random and biased walks. Left: A random walk in isotropic environments. When the cell's motors rotate CCW, the flagellar filaments push the cell forward. When one or more of the flagellar motors reverses to CW rotation, that filament undergoes a shape change (owing to the torque reversal) that disrupts the bundle. Until all motors once again turn in the CCW direction, the filaments act independently to push and pull the cell in a chaotic tumbling motion. Tumbling episodes enable the cell to try new, randomly-determined swimming directions. Right: A biased walk in a chemoeffector gradient. Sensory information suppresses tumbling to help the cell head in a favorable direction.

E. coli has five chemoreceptors, four are methyl-accepting chemotaxis proteins (MCPs) which have periplasmic ligand binding sites and conserved cytoplasmic signaling domains (Fig. 2). The MCPs initiate a motor control response and a feedback circuit that updates the methylation record to achieve sensory adaptation and cessation of the motor response. A fifth MCP-like protein, Aer, mediates methylation-independent aeroTCAtic responses by monitoring redox changes in the electron transport chain. The five MCP-family receptors in E. coli utilize a common set of cytoplasmic signaling proteins to control flagellar rotation and sensory adaptation (Fig. 2). CheW and CheA generate receptor signals; CheY and CheZ modulate rotation of the flagellar motor and thus change cell behavior and movement; CheR and CheB regulate MCP methylation state to make feedback to CheW and CheA.

Fig. 2 Signaling components and circuit logic. E. coli receptors employ a common set of cytoplasmic signaling proteins: CheW and CheA interact with receptor molecules to form stable ternary complexes that generate stimulus signals; CheY transmits those signals to the flagellar motors, CheZ controls their lifetime; CheR (methyltransferase) and CheB (methylesterase) regulate MCP methylation state. Abbreviations: OM (outer membrane); PG (peptidoglycan layer of the cell wall); CM (cytoplasmic membrane).

But the original chemoreceptors of E.coli do not respond to the root exudates, while the MCPs of the Pseudomonas putida , McfQ and McfR do. McfQ responds to citrate and fumarate, and McfR detects succinate, malate and fumarate. To make a control with the work 2011_Imperial_College_London done, we chose McfR which also responds to malate. The mechanism of prokaryote chemoreceptors is similar, so we design a plasmid to express chemoreceptor, McfR in the E.coli to make it have chemotaxis towards TCA intermediates. As our experimental condition is limited, we just made chemotaxis towards succinate and malate. What’s more, in an attractant gradient, the chemoreceptors monitor chemoeffector concentration changes as they move about and use that information to modulate the probability of the next tumbling event. And we searched the pattern of their chemotaxis towards a concentration gradient of attractant using capillary assay. The results are demonstrated below.

Results

We did both agar assay and capillary assay to compare the response of E.coli to different attractants and different concentration of each attractants. As our agar assay can not replicate very well or gain data, so we just show the results of capillary assay. We made a negative control using wash buffer and five concentration gradients (100mM/10mM/1mM/0.01mM/0.0001mM) of attractants. These E.coli are divided into three groups based on the plasmid they have been transformed into. The plasmids are biobricks, BBa_K608003 and BBa_K515102 (they are from 5A and 8F wells in plate1), and the McfR plasmid designed by us. BBa_K608003 only has a strong promoter and medium RBS, so it doesn’t have specific chemotaxis towards TCA intermediates. BBa_K515102 is a biobrick from 2011_Imperial_College_London, which responds to L(-)malic acid (HO2CCH2CH(OH)CO2H).

The Story of E.coli Prometheus

BNU-China