Team:Toulouse/Project/Spreading

Our engineered bacterium is designed to be inoculated in a tree and to cure fungal diseases. To target the possible environmental issues resulting of using a modified organism directly on trees bordering the Canal du Midi, our team worked on different aspects 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 imagined three modules.

Survival in the environment: proline auxotroph B. subtilis

SubtiTree will live in sap tree, thus we will use an endophyte B. subtilis strain. In order to contain our bacteria in this area during a short period of time, we thought of modifying some of its survival characteristics. To make the bacterium growth dependant on the presence of the tree (and therefore avoid spreading in the environment), we planned to use a B.subtilis strain auxotroph for proline. The bacterium should then be unable to synthesize this essential amino acid. Proline is the most abundant amino acid in the phloem sap. If the bacterium is in the sap, it should grow normally without any deficiency, but if it escapes from the tree and a fortiori from the sap, it will not be able to survive for a long time (proline is present only in very low quantities in the ground).
Auxotroph B.sutbilis strains already exist and are indexed in databases as BGSC (Bacillus Genetic Stock Center), therefore they should be easy to find.

Preventing sporulation of B. subtilis

It is known that endophyte bacteria must sporulate to survive to winter. In order to limit the spreading of our bacterium, we planned to limit its lifespan to only one season. The bacteria should be injected in spring, grow during the summer and finally should die in the fall. B. subtilis is a sporing bacterium: sporulation enables the microorganism to resist very harsh conditions and to spread from tree to tree.
To control any unwanted long-term development of SubtiTree, our strain should therefore be deficient for sporulating. We USED or COULD USE a B.subtilis strain deficient in the late genes for sporulation. Thus, during fall, when the sap become less nutritious and the temperature is low, the engineered bacterium will die and not pass through the following winter.
In addition, deleting all the engineered bacterial community every year puts a brake on the evolution due to random mutation, thus allowing a better faith on the genetic constructions.

These two first characteristics of SubtiTree demonstrate that it should be an annual bacterium, growing only in sap tree. By combining them, they should prevent any long term colonization of any other ecological niche than the plane trees

Gene transfer: toxin-antitoxin system

We also wondered about horizontal genes transfer. The goal of this module is to prevent horizontal transfers between bacteria and any exchange of synthetic genetic material that could be dangerous between wild type organisms and optimized organisms.
We thought about a system limiting 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 optimized 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 anther host bacterium receives the gene encoding for the toxin, it will be unable to survive since it will not possess the antitoxin. If it receives the antitoxin only, it will not be useful for the bacterium, and will not affect it.
In summary, since a simultaneous transfer is dimly probable, the bacterium will either die because of the toxin or live while expressing the antitoxin.

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.
While we have not constructed yet these modules, we definitely think that the measures that we designed should render the use of SubtiTree acceptable in the environment.

Using integrative plasmids

All our constructions are carried by integrative plasmids. Consequently, our different genetic modules should 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 dependent on a selective pressure, allowing a high level of transcription in planta.

References

• S. Dinant, J.L. Bonnemain, C. Girousse, J. Kehr. Phloem sap intricacy and interplay with aphid feeding.C R Biol. 2010 Jun-Jul;333(6-7):504-15. doi: 10.1016/j.crvi.2010.03.008. Epub 2010 May 14.

• Z.N. Senwo and M.A. Tabatabai. Amino acid composition of soil organic matter. Biology and Fertility of SoilsFebruary 1998, Volume 26, Issue 3, pp 235-242

• A.M. Guérout-Fleury, N. Frandsen and P. Stragier Plasmids for ectopic integration in Bacillus subtilis. Gene. 1996 Nov 21;180(1-2):57-61.

• G. Shang, X. Liu, D. Lu, J. Zhang, N. Li, C. Zhu, S. Liu, Q. Yu, Y. Zhao and L. Gu. Structural insight into how Pseudomonas aeruginosa peptidoglycanhydrolase Tse1 and its immunity protein Tsi1 function. Biochem J. 2012 Dec 1;448(2):201-11. doi: 10.1042/BJ20120668.

• W.Z. Hua, C. S. Yong and X.T. RenBiology and chemistry of endophytes. Nat. Prod. Rep., 2006, 23, 753–771, 753

Fungicides

Modeling