Overview

As already explained in the description, the Quagga mussel (Dreissena rostriformis bugensis) is an invasive species coming from the eastern Europe and thriving in Swiss lakes. Current solutions are to clean the pipes by hand using active charcoal, chlorine, and other chemicals (Molloy et al.,2013). As these methods are either expensive or environmentally unacceptable as long-term solutions, we defined two projects to control the invasive species populations. The first sub-project aims to overexpress the FitD toxin in the strain Pseudomonas protegens CHA0, which will attack the digestive tract of the mussels and kill them (Molloy et al.,2013). The second sub-project is to produce zosteric acid in E. coli BL21 (DE3) to detach and prevent the attachment of the mussels. Information about the latter can be found in the design of the subproject zosteric acid.

The FitD toxin (or “P. fluorescens insecticidal toxin”) is produced by Pseudomonas protegens CHA0 to protect crop plants from root fungal diseases and can be found in the fitABCDEFGH operon of this strain (Péchy-Tarr et al., 2013; Ramette et al., 2011). This toxin is an outer membrane protein (OMP) of 327.3 kDa (Péchy-Tarr et al.,2008). It has insecticidal activity and can provoke the death of cells. It has been shown to kill Dreissenidae mussels and selectively destroy their digestive tract, with little or no impact on other aquatic organisms (Molloy et al., 2013). For these reasons, we created a way to use this toxin against the Quagga mussels.

Overview

On our journey to preserve the aquatic life of lake Geneva and the other Swiss lakes, we founded our project Quagg’out on two main pillars. The first pillar is control – we designed and built a plasmid which increases the native capability of the bacterium Pseudomonas protegens to express a toxin called FitD, deadly to most mollusks and therefore to quagga mussels. The second pillar is prevention and mainly tries to deal with the fact that quagga mussels, besides being a major threat to aquatic biodiversity, also clog up drinking water pipes which each year cost billions of dollars worldwide clean up. The goal is to reduce the number of mussels able to recolonize the newly cleaned drinking water pipes, thus increasing the time between each cleaning session and reducing the costs necessary for maintenance. The idea for this second pillar came from discussions we had with numerous experts in the field (click here for more info !). They told us that finding a way to remove quagga mussels from pipes would probably not be enough, considering that they would recolonize them almost immediately after. That is why we decided to implement the second level in our project by exploring the possibilities offered by antifouling compounds. To achieve this goal, we designed and built a plasmid containing all the necessary elements to produce zosteric acid (ZA), a bioactive molecule found in Zostera marina eelgrass (Taokaew et al. 2012) that possesses anti-adhesive properties. Combining those two pillars will create a new product with the potential to solve part of the global economic and ecological problem caused by quagga mussels.

What Is Zosteric Acid ?

In Zostera marina, a north American macrophyte, the production and release of ZA occur in the root and leaf of the plant. After its release into the environment, it binds to the leaf surface, which deters the attachment of marine microorganisms and bryozoans. This compound has already been studied and classified as an effective ecologically sound anti-biofilm against quagga mussels (Taokaew et al. 2012), making it a desirable candidate to be considered a major part of our project. For this reason, we decided to express ZA in E.coli and test its efficacy as an antifoulant directly on mussels, which has not been tried before.

Cells Factory Pathway

Based on the literature, we found a way to produce ZA in E. coli BL21 DE3 strain by adding a sulfate group to coumaric acid. This is possible in cells thanks to two sets of genes: one essential for the transport of sulfate inside the cell and the other necessary for the catalysis of the phosphorylation. The first genes responsible for sulfate intake are cysP (from Bacillus subtillis) or cysPUWA (from E.coli). cysPUWA encodes an ATP-dependent sulfate transporter that generates a flow of sulfate ions towards the inside of the cell, while cysP encodes a proton-sulfate cotransporter that imports sulfate into the cell. TAL gene encodes for a tyrosine amino lyase (from Flavobacterium johnsoniae) , which transforms L-tyrosine in p-coumaric acid. The p-coumaric acid will then be transformed in ZA thanks to the sulfotransferase encoded by SULT1A1. The metabolic pathway is visualized in figure 1, and the genes needed are explained in table 1. Therefore, the metabolic pathway for ZA production has already been explored and cloned in E.coli, but its production has only been assessed in 950µL of the medium. We will therefore clone the genes in plasmids different from the ones used in literature and try to swap the position of the genes relative to the inducible promoter to see if some of our constructs will produce more ZA than others. Bacteria will be grown in 200ml flasks instead of 96 well plates as found in the literature (Ram et al. 2012).

Our Project

For our project, we wished to produce ZA in large quantities using engineered bacterial cells as “cell factories” to assess its efficacy as an anti-foulant agent against quagga mussels. First, we had to engineer two plasmids, one containing cysPUWA and cysDNCQ or cysP and cysDNCQ and the other one containing SULT1A1 and TAL. For the second plasmid we tried two different versions to see which one is the most effective: the one with SULT1A1 upstream or the one with TAL upstream. We added all cys genes in a pCOLA plasmid backbone while SULT1A1 and TAL were placed in a pET17b plasmid. We used these plasmids because of their optimization for E. coli, their IPTG inducible promoters and because they have two different antibiotic resistances. We will then add to those two plasmids different antibiotic resistances to allow us to select them once transformed and put all the genes in the plasmids under the control of a Lac operon. The T7 promoter is activated once we add IPTG to the growth media, which switches on the expression of the genes.

All cys genes have been extracted from the genome of E. coli, with the exception of cysP which comes from B. subtilis, while SULT1A1 and TAL have been synthesized. We cloned the two plasmids individually in E. coli DH5α strain, then extracted them using a plasmid purification kit. We co-transformed E. coli BL21 DE3 cells with both extracted plasmids to allow the production of ZA. Successful co-transformants were isolated by plating cells on selective media supplemented with both the antibiotics kanamycin and ampicillin. The ZA produced was then released in the extracellular space and diluted in the media. We decided to use M9 as growing media for our transformed cells as it is a minimal media and contains low quantities of sugars and salts, making it easier to detect ZA. Detection of ZA was performed with high performance liquid chromatography (HPLC), as the sulfate group in the ZA made it undetectable using the GC-MS available in our department. Upon confirmation of the production of ZA, we have planned to concentrate and purify it using distillation. This would allow us to create either a liquid solution containing a high concentration of ZA or even a powder containing ZA and salts.

The testing on mussels was planned with known concentrations of ZA: 500ppm, 1000ppm, and 2000ppm. 100ml of lake water with the right concentration of ZA will be placed into a box with four quagga mussels, as we noticed along the way that having multiple mussels per box increases the probability of attachment. The attachment of each mussel will be tested at regular intervals of 3 hours: 9 am, 12 am, 3 pm, and 6 pm. Data will then be tabulated and subjected to an appropriate statistical test to understand if ZA plays a role in attachment probability. We analyzed the production of ZA via HPLC and the results can be seen in our results page.