Introduction
Pseudomonas putida F1 is a gram-negative rod-shaped soil bacterium. It is known to be chemotactic to and can degrade many different compounds, such as aromatic compounds, which are typically toxic environmental compounds. Specifically, the compound that I studied was phenylacetate, and F1 is known to degrade phenylacetate. F1 can demonstrate taxis towards phenylacetate.
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| Figure 1: Electron Micrograph of Pseudomonas putida |
Chemotaxis is the ability for bacteria to move towards an attractant. Chemotactic bacteria utilizes methyl-accepting chemotaxis proteins(MCPs). In the genome for Pseudomonas putida F1, there have been 27 MCPs identified. The significance to studying chemotaxis is to improve mechanism for biodegradation. Typically, bacteria that are chemotactic towards certain compounds can also typically degrade the compound.
As seen in Figure 2, the left region, the area shaded lighter, indicates an area of lower concentration of the chemoattractant. The right region, the area shaded darker red, indicates an area of higher concentration of the chemoattractant. Although the bacteria exhibits random walk, where it moves around, chemotaxis is demonstrated when the bacteria has a net movement towards the area of higher concentration of the chemoattractant.
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| [Low Concentration] [High Concentration] Figure 2: Directional "random" walk on a concentration gradient from low to high. |
Energy taxis is a type of chemotaxis with a slight difference. Energy taxis is when the bacteria detects the change in energy produced by metabolism of the chemoattractant. Aer2 is the MCP responsible for energy taxis in F1.
Chemotaxis Machinery
As seen in Figure 3, there are two receptors the normal standard 27 MCPs and the Aer2 energy taxis receptor. The standard MCPs are a transmembrane protein that senses the chemoattractant directly. Once the MCP senses the chemoattractant, it causes a chemical signaling cascade, which then in turn causes the flagella to rotate a certain direction, propelling the bacteria to move towards the area with the higher concentration of chemoattractant. The Aer2 receptor is a protein within the bacterial cell. It doesn't sense the chemoattractant directly, but it senses the energy generated through the metabolism of the chemoattractant. The key difference between the MCP receptor and the Aer2 receptor is that the MCP receptor directly senses the chemoattractant, whereas the Aer2 receptor sense the energy generated through the metabolism of the chemoattractant, which is a cellular process.
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| Figure 3: Cell Diagram of F1 |
Phenylacetate is a model compound for studying other toxic aromatic compounds because it has a similar structure as other aromatic compounds, but it itself is not toxic. It is found naturally as an active auxin in plants. Currently, it is used in perfumes to hold the aroma, and it can be used as a treatment for type II hyperammonemia, a disease where the body has trouble excreting ammonia.
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| Figure 4: Chemical Structure of Phenylacetate |
Figure 5 is a diagram of the degradation pathway of phenylacetate. This degradation pathway has been annotated based on similar gene clusters in other bacterial organisms that have been known to degrade phenylacetate. These gene clusters have been identified by function and sequence in other bacterial organisms, and it is presumed that because F1 has the same set of gene clusters that it can also degrade phenylacetate. My project seeks to confirm the function of these genes.
There are two important enzymatic proteins that must be identified. This includes the paaF and the paaI proteins. The paaF protein is the first protein used in the breakdown of phenylacetate, and it turns phenylacetate into phenylacetyl-coA. The second important protein is paaI, and this protein is used to breakdown phenylacetly-coA into 2'-OH phenylacetly-coA.
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| Figure 5: Degradation Pathway of Phenylacetate |
The first question I was interested in studying was which methyl-accepting chemotaxis protein is involved in the detection of phenylacetate. The second question I was interested in studying was is phenylacetate or a metabolite of phenylacetate the true attractant.
My hypothesis is that one of the 27 standard MCPs is responsible for detecting phenylacetate directly and initiating chemotaxis.
Experimental Methodology
First Test: Screening for MCPs in Deletion Strains of F1
Each of the 27 MCPs were deleted from wild type and tested for a chemotactic response to phenylacetate using swarm assays.
The way swarm assays work is that the bacterial strain grows on the agar that is filled with the chemoattractant. In this case, phenlyacetate acts as the chemoattractant as well as the carbon source for the bacterial strain to grow. Chemotaxis is demonstrated in this assay when the bacteria swarms out and forms a circular growth. The bacteria basically creates its own concentration gradient. By breaking down the carbon source, which is also the attractant, to grow on it, it depletes that area of the chemoattractant, lowering the concentration of that compound. At a lower concentration, the bacterial strain will want to swim out and swarm to the areas of higher concentration of the chemoattractant. Thus, in swarm assays, the size of the growth of the bacterial strain is crucial.
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| Figure 6: Swarm Assay with F1 Deletion Mutants |
Second Test: Screening for MCPs in P.aeruginosa PAO1
Each of the 27 MCPs were introduced into P. aeruginosa and tested for a chemotactice response using swarm plug assays.
The swarm plug assays work in a different background carbon source for the bacteria, which is different from the chemoattractant. The chemoattractant is infused in a agar plug that is placed in the middle of the plate, surrounded by a plate with a different carbon source. Overtime, the chemoattractant will diffuse through the agar plate, creating a concentration gradient. Usually, the bacterial strain can demonstrate chemotaxis towards chemoattractant, but it cannot degrade the chemoattractant. The chemotaxis response is observed when we see an oblong shaped growth towards the plug - a denser and more oblong shaped growth. Circular growth demonstrates no chemotactic response.
In my project, I inserted each of the MCPs individually into the bacterial strain to detect whether there was a chemotactic response that developed. By inserting an MCP, we wanted to see which of the bacterial strains would gain the chemotactic response. However, none of the tested MCPs gained a chemotactic response phenylacetate, so we couldn't draw any conclusions to which MCP was responsible for phenylacetate chemotaxis.
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| Figure 7: Swarm Plug Assay with P. aeruginosa |
In order to test for energy taxis, first I had to use growth studies with the Wild Type, ∆PaaF, and ∆PaaI, which will help confirm the role of PaaF and PaaI as enzymes because the genes have only been annotated. The second test I used is swarm plug assays using Wild Type, ∆PaaF, and ∆PaaI, which will help to test to see if the response to phenylacetate is metabolism dependent. The third test I conducted is swarm plug assays using Wild Type, ∆Aer2, and ∆Aer2 + aer2, which will test to see if the response is mediated through the energy taxis receptor Aer2.
Growth Studies using Wild Type, ∆PaaF, and ∆PaaI
As seen in the figure, the wild type F1 grew on the phenylacetate. It had a doubling time of 67.2 minutes. This is significant because it indicates that F1 can metabolize phenylacetate and can grow on phenylacetate. ∆PaaF and ∆PaaI displayed no significant change in growth in the course of the 12 hours. This is significant because PaaF and PaaI are necessary for phenylacetate metabolism in F1, which means knocking out the genes for those proteins inhibits growth. This reconfirms the role of the annotated genes for the protein of PaaF and PaaI as being necessary steps in the phenylacetate degradation process.
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| Figure 8: Growth Curve of F1, ∆paaF, and ∆paaI |
Swarm Plug Assays using Wild Type, ∆PaaF, and ∆PaaI
Looking at the results from this assay, I can tell that metabolism is required for phenylacetate chemotaxis. In the swarm plug assay, the control group F1 displays the chemotaxis response towards the plug as seen by the oblong shaped growth on the plate. However, the ∆paaF and ∆paaI both indicate a regular, even, equal, circular growth, which means there is no chemotactic response to the phenylacetate. This indicates that the protein is necessary for phenylacetate chemotaxis. Thus, phenylacetate chemotaxis is metabolism dependent.
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| Figure 9: Swarm Plug Assays with F1, ∆paaF, and ∆paaI |
Swarm Plug Assays using Wild Type, ∆Aer2, and ∆Aer2 + aer2
In this assay, I concluded that the MCP Aer2 is responsible for phenylacetate energy taxis. In the control group, F1 demonstrates the oblong shaped growth towards the phenylacetate plug. This indicates that the control group demonstrates the phenylacetate chemotaxis. In the ∆aer2, a circular, evenly distributed growth indicates that the bacteria loses its chemotactic ability to phenylacetate without aer2. To strengthen the point that aer2 is necessary for phenylacetate energy taxis, the F1 deletion strain ∆aer2, once complemented back with aer2, regains the ability for phenylacetate chemotaxis, guaranteeing that aer2 is the receptor necessary for phenylacetate chemotaxis.
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| Figure 10: Swarm Plug Assays with F1, ∆aer2, and ∆aer2 + aer2 |
Taxis to phenylacetate is metabolism dependent. It is mediated through energy taxis. Aer2 is the MCP responsible for the response.
Future directions include testing for energy taxis towards phenylacetate in other bacteria.
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