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 five 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, you can download our protocol which is based on the work 2011_Imperial_College_London had done before.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 compare with the work what 2011_Imperial_College_London had done, we chose McfR which also responds to malate. The mechanisms of prokaryote chemoreceptors are similar, so we designed a plasmid to express chemoreceptor. McfR in the E.coli makes the bacteria have the ability of chemotaxis towards TCA intermediates. As our experimental condition is limited, we just did experiments on chemotaxis towards succinate and malate. What’s more, in an attractant gradient, the chemoreceptors monitor chemoeffector concentration changes as they move around. And they 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 detect the response of E.coli to different attractants and different concentrations of each attractant. Because the agar assay is difficult to replicate and collect the data from, we just show the datas of capillary assay. We made a negative control using washing buffer and five concentration gradients (100mM/10mM/1mM/0.01mM/0.0001mM) of attractants. These E.coli were 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 was designed by us. BBa_K608003 (5A) only has a strong promoter and medium RBS, so it doesn’t have specific chemotaxis towards TCA intermediates. BBa_K515102 (8F) is a biobrick from 2011_Imperial_College_London, which responds to L(-)malic acid (HO2CCH2CH(OH)CO2H).

Fig .3 E. coli’s ability of chemotaxis towards different concentrations of succinate or malate.
The cells were diluted 20000 times. 5A is a control which doesn’t have chemotaxis towards malate or succinate.
Malate: 8F showed the strongest respond. The tendency of curve of 8F was biphasic with maximums at attractant concentration of about 10^-2 M and 10^-5 M, and reached minimum at attractant concentration of about 10^-1 M, which is similar to others’ work. With attractant concentration decreasing for 8F, the number of cells decreased slowly. McfR stayed stable at range of 10^-7 M to 10^-3 M, and had only one phase which reached its maximum at attractant concentration of 10^-2 M, then it went down sharply at 10^-1 M. There was a significant difference among 8F and 5A (p < 0.05), and quantity of cells of 8F was much more than that of 5A. So 8F has chemotaxis towards malate. On the other hand, McfR and 5A were not significantly different and quantity of cells of McfR was little less than it of 5A. So McfR has no chemotaxis towards malate.
Succinate: The tendencies of curves of 8F and McfR towards succinate are same. As the attractant concentration increased, the number of cells arose and reached the maximum at attractant concentration of about 10^-2 M and fell sharply with the minimum at attractant concentration of 10^-1 M. The quantities of cells of 8F and McfR were almost equivalent and did not have significant difference. But there were significant differences among 8F & 5A, and McfR & 5A: the number of cells of 5A are far less than 8F or McfR (p < 0.05), which demonstrated that 8F and McfR have chemotaxis towards succinate and the capacity of chemotaxis of 8F and McfR towards succinate are almost equal.

*You can download the protocol of this part here: Protocol on Delivery System.pdf

The Story of E.coli Prometheus

BNU-China