Team:Toulouse/Project/Spreading

Our engineered bacterium has been designed to be inoculated in a tree and to cure fungal diseases. Understanding the environmental issues resulting from the use of a modified organism in the nature, our team worked on different aspects in order to ensure a safe use of SubtiTree. The first objective is to avoid the spreading of our smart bacterium outside the tree. In other words, the purpose is to ensure that once SubtiTree is in the tree, it is unable to live anywhere else. Another issue concerns the horizontal transfers of the genetic material between different bacteria. Taking into account these issues, we thought about three modules.

Using a glutamin auxotroph B. subtilis strain

To make the bacterium dependant on the tree and to avoid its spreading in the environment, it should be preferable to use a strain of B.subtilis which is auxotroph to a particular amino acid. The bacterium should be unable to synthesize one essential amino acid, and should find it in its environment. The glutamine could be a good example since it is wide-spread in the phloem sap. It is the amino acid which is present in highest concentration in the phloem sap. If our bacterium is unable to synthesize the glutamine, it will be obliged to take it in its close environment, that is to say the phloem sap. Thus, if the bacterium is in the sap, it can grow normally without any deficiency since it uses the glutamine present in the sap ; but if it escapes from the tree and a fortiori from the sap, it will not be able to survive for a long time. Indeed, glutamine is found in low quantities in the ground. This system should guarantee that the bacterium develops only in the tree and not elsewhere in the surroundings of the tree.
Auxotroph B.sutbilis strains already exist and are indexed in databases as BGSC (Bacillus Genetic Stock Center), therefore it is easy to find.

Preventing sporulation of B. subtilis

In order to limit the spreading of our bacterium, we decided to limit its lifespan to only one season. The bacteria should be injected in spring, grow during the summer and finally should be inactivated in fall. Bacillus subtilis is a sporing bacterium : sporulation enable the microorganism to handle very harsh conditions and to spread tree to tree. Indeed, a spore is a very resistant form that is adapted for unfavorable conditions and for dispersal.
To keep the control on the development of SubtiTree, our strain should therefore be non-sporing. Thus, after a season of treatment, the sape become less nutritious, the temperature is low and the engineered bacterium cannot survive the following winter.
In addition, deleting all the engineered bacterial community every year puts a brake on the evolution due to random mutation, thus it allows to keep control on the genetic constructions.

Using a toxin-antitoxin system

The goal of this module is to prevent horizontal transfer between bacteria. Indeed, it is necessary to avoid any exchange of genetic material between wild type organisms and optimized organisms : it could be dangerous because of mutations, and considering ethics, it seems to be essential to avoid the spreading of synthetic genes.
Considering this issue, we thought about a system to avoid such transfers : a toxin-antitoxin module. It involves the addition of two genes to the bacterium : a gene encoding for a toxin (for example tse2) and a gene encoding for the antitoxin (tsi1), placing them in an opposite way on the genome. The large space between them prevents simultaneous transfers : if the optimised bacterium transfers the gene encoding for the toxin, the probability that the gene encoding for the antitoxin may be transferred simultaneously is really low since they are located far away from each other.
Therefore, if the host bacterium receives the gene encoding for the toxin, it will be unable to survive since it will not have the antitoxin. If it receives the antitoxin only, it will not be useful for the bacterium, and will not affect it.
To sum up, since a simultaneous transfer is dimly probable, the bacterium will either die because of the toxin or live while expressing the antitoxin (useless).

Using integrative plasmids

All our constructions are carried by integrative plasmids. Consequently, our different genetic modules would be integrated into the bacterium genome. The integration in the genome is more stable as the constructions are less likely to be transferred to other microorganisms. In addition to that, the expression of our genetic modules would not be dependant on a selective pressure based on an antibiotic resistance (as we can not inject antibiotics in the tree), allowing a high level of transcription in planta.

SubtiTree will live in sap tree, thus we use one endophyte Bacillus subtilis strain. In order to contain our bacteria in this area during a short period of time, we modified some of its survival characteristics. First of all, the control of synthetic bacterium location concerned us,that is why we border it inside the plane tree. Sap tree is known to contain a certain quantity of proline which could allow bacterial growth whereas the soil is composed by only trace of this amino acid. Thus, we decided to delete the proline production pathway to make an auxotrophic bacterium for this amino acid.

Secondly, SubtiTree is conceived as an annual bacterium to avoid any kind of mutations. It is known that endophyte bacteria must sporulate to survive to winter. Thus, we chose a B.subtilis strain without late genes of sporulation. So SubtiTree is a non-sporing bacterium, which means that under stress conditions (cold temperature, food lack …) or outside the sap, the bacterium will not resist and die.

These two characteristics of SubtiTree show that it is an annual bacterium, which can only grow in sap tree. By combinind them, they prevent any long term colonization of an ecological niche by SubtiTree against wild type bacteria. These prevent a long term effect.

While we were trying to respond to bacterial spreading problem, at the same time we wondered about horizontal gene transfer. The transmission of genes from our synthetic bacterium to wild type bacteria could be blocked with two modifications. First, as used in genetic engineering, plasmids are a tremendous way to transfer some genic information. In this way, it is not the best way to restrict the collecting transgenic DNA. That is why we inserted our genes in the bacterium chromosome.

Secondly, some bacteria are able to accept chromosomal DNA, like Bacillus subtilis. Thus, the previous strategy is not efficient enough to prevent transfer. We could include the toxin/anti-toxin system. The toxin gene is near our synthetic gene, contrary to the anti-toxin gene. So if there is a transfer of our synthetic gene, the toxin gene will be included in the transfer. Receiver bacteria will die by the production of the toxic protein.

Our synthetic genes are not the only problem in the design of SubtiTree. One of the side effects of our cloning method is the persistence of antibiotic resistance genes. This is incompatible with the introduction of SubtiTree in the environment. It is possible to delete this resistance in chromosome. To conclude, the spreading limitation shown previously makes the use of SubtiTree acceptable in the environment.

Fungicides

Modelling