Research Project in the Parales Lab at UC Davis Department of Microbiology

Characterizing Phenylacetate Degradation and Taxis in Pseudomonas putida F1: Identification of the Receptor Used in Phenylacetate Taxis

Christie C. Ho

Abstract

            Pseudomonas putida F1 is a gram-negative rod-shaped soil bacterium capable of growth on and taxis to phenylacetate. Phenylacetate, a common aromatic compound used in perfumes, provides a model system for the study of the degradation of toxic aromatic compounds because it is a non-toxic alternative to various other toxic compounds. P. putida F1 has 27 identified methyl-accepting chemotaxis proteins (MCPs) encoded in its genome ,including one functional energy taxis protein that detects the energy obtained from metabolism of compounds that serve as carbon and energy sources.

We aim to characterize the taxis response to phenylacetate in P. putida F1 by identifying the MCP(s) responsible for detecting phenylacetate, the specific chemoattractant that P. putida F1 recognizes, and the regulation of this pathway via enzymatic proteins. This includes determining the role of metabolism towards phenylacetate taxis, which involves finding the relationship between degradation of phenylacetate to the taxis of phenylacetate. This is determined by indentifying the proteins involved in the regulation and degradation of phenylacetate. 

By studying different mutants of P. putida F1 with deletions of genes predicted to encode enzymes required for phenylacetate degradation based on studies of other phenylacetate degrading organisms, we can test for similar functionality in phenylacetate degradation in this strain. Results of this project demonstrated that paaF and paaI, which were predicted to encode the first and second steps of phenylacetate degradation are necessary for growth on phenylacetate in P. putida F1, and the ability to metabolize phenylacetate is necessary for the chemotactic response. P. putida F1 responds to phenylacetate through energy taxis, and Aer2 is the receptor responsible for this response.

Introduction

Phenylacetate is a chemical compound made from phenylacetic acid, which involves combining phenol and acetic acid. Found naturally as an active auxin, a plant hormone, phenylacetate is commonly produced in many plants. With an aromatic benzene ring in its structure, these chemical properties make phenylacetate useful in perfumes because they have the capability to retain the infused aroma. In addition, phenylacetate is commonly used to treat type II hyperammonemia, a disease in which the human body has difficulty excreting the excess ammonia in the blood stream  (Honda et. al, 2002).

By studying phenylacetate degradation, we can increase the basic knowledge about bacterial assimilation of aromatic compounds within other similar species of bacteria. It opens possibilities of new biotechnological and environmental applications for bioremediation and biodegradation. In addition, steps in phenylacetate degradation compared with the degradation of other related toxic compounds, such as styrene, are extremely similar. Thus, studying phenylacetate can reveal common degradation pathways and mechanisms in other toxic aromatic compounds. The use of phenylacetate provides a non-toxic alternative to the study of toxic aromatic compounds. It highlights potential pathways for how other complex aromatic compounds are broken down into phenylacetate (Olivera et. al, 1998).

Phenylacetate can be broken down and degraded into simple energy sources by some bacteria, specifically the experimental strain in this project, which is Pseudomonas putida F1. The phenylacetate degradation pathway in other bacteria has been well characterized. Annotation of the P. putida F1 genome has revealed similar phenylacetate degradation genes, and it is presumed that F1 shares a similar pathway (Figure 1). The image shows the hypothesized phenylacetate degradation pathway in P. putida F1 with the sequenced genes encoding enzymes in various other bacterial strains. (Olivera et. al, 1998).

Figure 1: Phenylacetate Degradation Process in Pseudomonas putida sp. F1

As shown in Figure 1, PaaF is responsible for the first step in the degradation of phenylacetate, converting phenylacetate into phenylacetyl-CoA. The product of paaI – another gene involved in the degradation pathway – converts phenylacetyl-CoA into 2’-OH phenylacetyl-CoA. Without this gene, the chain of degradation is broken, and thus, phenylacetate cannot be degraded.

Chemotaxis is the ability bacteria to move towards attractant substances or away from repellant substances. In order for chemotaxis to occur, bacteria utilize chemoreceptors called methyl-accepting chemotaxis proteins, also known as MCPs. MCPs are found in the cell membrane of bacteria and can sense specific chemical compounds. By binding to certain chemicals, the MCP activates a signaling cascade to trigger a change in the rotation of the flagella to propel the cell either towards attractants or away from repellants. Preliminary studies have suggested that Pseudomonas putida F1 is chemotactic to phenylacetate. However, the MCP(s) involved in phenylacetate chemotaxis in P. putida F1 is unknown. Genome sequencing of Pseudomonas putida F1 has revealed the presence of 27 MCPs, but the specific function of each MCP has not been characterized.

Chemotaxis can also be initiated indirectly through energy taxis. This form of taxis occurs when bacteria are attracted to a substance that it can degrade and capture energy from its degradation.  Energy taxis is the orientation of bacteria towards conditions of optimal metabolic activity by sensing the energetic conditions of the cell. There is one major difference between chemotaxis and energy taxis. In energy taxis the cell responds on an intracellular stimulus, whereas chemotaxis is response towards a specific extracellular chemical compound. P. putida F1 has a specific MCP involved in energy taxis, called Aer2. Aer2 has the ability to sense the change in energy generated from the metabolism of certain compounds and initiate chemotaxis.

Chemotaxis has the potential to enhance the biodegradation of toxic compounds. By discovering how bacteria move towards certain pollutants, the field of bioremediation and biodegradation can be greatly improved. 

The goal of this project is to characterize phenylacetate chemotaxis in P. putida F1 and make generalizations to other organisms as well. Furthermore, we seek to identify one or multiple MCPs that are responsible for phenylacetate chemotaxis. Also, the true attractant and the inducer of phenylacetate chemotaxis has not yet been identified. Different compounds may trigger the complete activation of phenylacetate degradation genes in P. putida F1. Finally, another goal of this project is to confirm the functions of each protein in the phenylacetate degradation process in P. putida F1 as characterized in similar strains of bacteria. Moreover, the regulation of gene expression and the regulation of phenylacetate degradation and chemotaxis need to be clarified.

Materials and Methods

Bacterial Strains Used:

Two different experimental tests were used. In the first experiment, bacterial strains used included wild-type P. putida F1, F1∆aer2, F1∆paaX, F1∆paaF, and F1 ∆paaI. In the second experiment, bacterial strains used included F1(pRK 415), F1∆aer2(pRK 415), and F1∆aer2 + aer2. F1∆aer2 + aer2 is wild-type F1 with the deletion of the aer2 gene with a complementing aer2 gene that is reinserted into the bacteria on a plasmid.

Growth Conditions:

P. putida F1 cultures were grown overnight at 30°C in minimal medium  containing 10 mM succinate and 20 μg/mL tetracycline when appropriate. On the following day, overnight cultures were harvested and normalized to an OD₆₆₀ of approximately 0.3 to 0.4.

Growth Study:

Fresh cultures were inoculated to an initial OD₆₆₀ of approximately 0.05 from overnight cultures into minimal medium and a concentration of 2.5 mM phenylacetate from a 0.5 M stock solution. Cultures were incubated at 30°C. The OD₆₆₀ was recorded every hour for a total of 10 hours to track the growth of each strain.

Chemotaxis Assays:

Soft Agar Swarm Plate Assay:

Soft agar plates were used to quantitatively measure chemotaxis in MSB soft agar. These plates consist of minimal medium (MSB) containing 0.3% Noble agar and 1 mM glycerol. Phenylacetate was added into the plate as the carbon source for the growth of the bacterial strains to a concentration of 1 mM phenylacetate from a 0.5 M stock solution . It was then autoclaved, poured into petri dishes, and cooled for at least 12 hours before used.

Bacterial strains were inoculated using toothpicks or from normalized cell suspensions (see below). Swarm plates were incubated at 30°C for at least 20 hours.

In order to analyze the data, the grown plates were placed over the “bucket of light” to see the growth of the cells in the swarm plate. The chemotactic response was measured by diameter of the colony. The farther the growth extended from the point of inoculation, the stronger the chemotactic response. The chemotactic responses of the bacteria were recorded at 20, 22, and 24 hours (Rabinovitch-Deere et. al., 2012).

Swarm Plug Assay:

            Minimal semisolid agar contained 0.3% Noble agar and 1 mM glycerol. It was autoclaved, poured into petri dishes, and cooled for at least 12 hours before being used.

Phenylacetate-containing agar plugs were prepared by mixing molten 1% agar with a stock solution of 0.5 M phenylacetate to form a solution of 5 mM phenylacetate. The solution was poured into a petri dish to a depth of 5 mm and was allowed to cool and solidify for at least 2 hours.

Using the large end of a plastic pipette tip, an 8 mm diameter plug from the hardened phenylacetate agar was excised. The plug was pushed onto the top of the surface of the middle of the swarm plate.

Cells were harvested/inoculated either via the toothpick stab method or the liquid culture method. The cells were inoculated 2 cm from the center of the phenylacetate plug. Plates were incubated at 30°C for at least 20 hours.

In order to analyze the data, the grown plates were placed over the “bucket of light” to see the growth of the cells in the swarm plate. The chemotactic response was measured by the density of growth via the radial distance from the point of inoculation to the outside growth of the cells. The father the growth extended from the point of inoculation toward the phenylacetate plug, the stronger the chemotactic response. The chemotactic responses of the cultures were recorded at 20, 22, and 24 hours (Pham et. al., 2011).

Preparing Plate Inoculations:

Toothpick Stab:

Bacterial strains were grown on either LB plates or MSB with succinate plates. After sufficient growth had developed on the plates from incubation, such that single colonies had developed (approximately 24 hours), the plates were removed from the incubator. Toothpicks picked up single colony bacteria from the growth plates and stabbed onto the plates in their designated areas (Pham et. al., 2011).

Liquid Cultures:

The overnight cultures of the bacterial strains were washed down from the MSB with succinate media. Then it was resuspended in chemotaxis to an OD₆₆₀ of approximately 0.4. Then the plates were inoculated by pipetting 2μL of resuspended bacteria cells onto the agar(Rabinovitch-Deere et. al., 2012).

Results

PaaF and PaaI are Necessary for Growth on Phenylacetate

Growth studies were performed on Wild-type P. putida F1, and ΔpaaF and ΔpaaI, mutants, which have deletions of key genes predicted to encode and the first and second steps in phenylacetate degradation. Wild-type F1 was able to grow on phenylacetate as its sole carbon and energy source with a doubling time of 67.2 minutes (Figure 2). While ∆paaF and ∆paaI mutants displayed no significant growth, as the OD₆₆₀ was approximately the same with little fluctuation over the course of the 12 hours (Figure 2). This indicates that F1∆paaF and F1∆paaI cannot grow solely on phenylacetate as a carbon source and must rely on an alternate carbon source for growth. These genes, paaF and paaI, are therefore necessary for degradation of phenylacetate and for growth on phenylacetate.

Figure 2: Growth Curves of P. putida F1 Metabolic Knock-outs Over 12 Hours

Metabolism of Phenylacetate is Required for Chemotaxis

Chemotaxis to phenylacetate was tested in wild-type and in the mutants lacking the necessary enzymes – for phenylacetate degradation. Wild-type cells demonstrated a chemotactic response to phenylacetate as shown by the oblong shaped swarm towards the phenylacetate plug. The increased cell density towards the plug indicates the chemotactic response to phenylacetate for F1 (Figure 5 and Figure 6).

F1 ∆ paaF and F1 ∆ paaI, however, showed no response. The even distribution of cells with equal cell density in the swarms on the plate indicates a lack of a chemotactic response to phenylacetate in the deletion strains. These results suggested that metabolism is required for chemotaxis to phenylacetate, and that chemotaxis is metabolism dependent. In addition, it indicates that genes paaF and paaI are indeed necessary for phenylacetate degradation in P. putida F1.

Figure 5: Comparison of Phenylacetate Chemotaxis Response in F1 and F1∆paaF

Figure 6: Comparison of Phenylacetate Chemotaxis Response in F1 and F1∆paaI

Energy Taxis is Responsible for the Chemotactic Response to Phenylacetate

P. putida F1 shows a chemotactic response to phenylacetate as seen by the oblong swarm towards the phenylacetate plug. In contrast, F1∆aer2 swarms in a more even distribution, indicating a negative response. Although it seems as though there is higher cell density and more cell growth closer to the phenylacetate plug in the inoculation of the F1∆aer2, the circular shape and overall even distribution of bacterial cells is still present in the inoculation of the F1∆aer2 mutant.

As seen in the F1∆aer2 swarm, a light white back ring behind the F1∆aer2 swarm is present.  The results reveal that the deletion of to aer2 abolishes taxis towards phenylacetate, suggesting that aer2 may provide necessary function for phenylacetate detection. 

Figure 7: Comparison of Phenylacetate Chemotaxis Response in F1 and F1∆aer2 in MSB Swarm Plate

Aer2 is Responsible for Energy Taxis to Phenylacetate in P. putida F1

As demonstrated above, deletion of the gene encoding the Aer2 receptor eliminated the response to phenylacetate.  When the aer2 deletion was complemented, the taxis response was restored and was comparable to wild-type (Figure 8). This positive response is demonstrated by the oblong shape of the bacterial growth towards the phenylacetate plug. These results strongly suggest that Aer2 plays a central role in the taxis response to phenylacetate because the deletion of the Aer2 receptor causes the bacteria to lose the ability to respond to phenylacetate.

Figure 8: Comparison of Phenylacetate Chemotaxis Response in F1 pRK 415, F1∆aer2 pRK 415  and F1∆aer2+∆aer2 in MSB Swarm Plate

Discussion

Wild-type P. putida F1 was able to grow with phenylacetate as the sole carbon and energy source. The inability of F1∆paaF and F1∆paaI to grow on phenylacetate confirms the role of PaaF and PaaI in the degradation of this compound. To test whether metabolism of the chemoattractant is necessary for chemotaxis, swarm plug assays were performed on the PaaF and PaaI mutants. The PaaF and PaaI mutants were unable to respond to phenylacetate, demonstrating that metabolism of this compound is needed for chemotaxis. Because of this requirement, it is possible that P. putida F1 responds to phenylacetate through energy taxis. This was tested using a mutant strain with a deletion to of aer2, a gene that encodes for the MCP responsible for energy taxis. Results showed that the Aer2 mutant no longer responded to phenylacetate; however, a response was restored when the aer2 was used to complemented the mutant. These results suggest that P. putida F1 responds to phenylacetate through energy taxis, and this response is mediated through the MCP aer2.

These studies have increased the basic understanding of bacterial assimilation of aromatic compounds and the role chemotaxis plays in degradation. These results open possibilities for new biotechnological and biodegradation applications.

Acknowledgements

            Special thanks to Dr. Rebecca Parales for her guidance during the six weeks that I have worked at the UC Davis lab in the Department of Microbiology. Thanks and appreciation to the lab facility, the aid from many of the undergraduate and graduate students also working in the lab. They have helped me significantly by teaching me how to use the equipment. They have provided such a welcoming environment for me to conduct my research.

Special thanks to my mentor, graduate student Rita Luu for helping me throughout these six weeks by working closely with me, explaining abstract concepts to me, giving me clear instructions for the different experimental assays, and guiding me through my project every step of the way. Her guidance and aid must not be forgotten during my research opportunity here at UC Davis. In addition, I have learned so much in my stay here about chemotaxis and potential for bioremediation and biodegradation efforts that I had no idea before.

Thanks to UC Davis for providing the opportunity for me to conduct research here and learn more about the different fields of biological sciences in my stay here.

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