Team:Toulouse/Project/Spreading

Our engineered bacterium is designed to be inoculated in a tree and to cure fungal diseases. To avoid the environmental issues resulting from the use of a modified organism on the trees lining 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 of 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 key points, we elaborate different strategies.

Survival in the environment: Bacillus subtilis that is proline auxotrophic

SubtiTree will live in sap tree, thus we will use an endophyte Bacillus subtilis strain. In order to contain our bacteria in this area during a short period of time, we think about modifying some of its survival characteristics. To turn the bacterium growth dependant on the presence inside the tree (and therefore avoid spreading in the environment), we planned to use a B. subtilis strain that is proline auxotrophic. 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 as proline is present only in very low quantities in the soil.
Auxotrophic B. subtilis strains already exist and are indexed in the databases BGSC (Bacillus Genetic Stock Center).

Preventing sporulation of Bacillus subtilis

It is known that endophyte bacteria must sporulate to survive during the winter. We planned to limit Subtitree's 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 spore-forming bacterium: sporulation enables the microorganism to resist to very harsh conditions and to spread from tree to tree.
To control any unwanted long-term development of SubtiTree, our strain should therefore be unable to sporulate. Thus, during fall, when the sap become less nutritious and the temperature is lower, the engineered bacterium will die and not pass through the following winter. This question has been studied in our Modeling part .
In addition, deleting all the engineered bacterial community every year puts a brake on the evolution due to random mutations, thus allowing a better faith on the genetic constructions.

These two strategies aim to make B. subtilis an annual bacterium, growing only in the sap tree. By combining them, they should prevent any long term colonization of any other ecological niche than plane trees.

Gene transfer: toxin-antitoxin system

We also wondered about horizontal gene transfers. 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, placed next to the engineered genetic modules) and a gene encoding for the antitoxin (tsi1), placing them far away from each other in the genome. The large intergenic region 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 very low.
Therefore, if another 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.

Using integrative plasmids

One of the side effects of our cloning method is the persistence of antibiotic resistance genes. This is incompatible with the introduction in the tree, and with the stability of our constructs. To avoid this, 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.

While we have not constructed yet these modules, we definitely think that the elaborated strategies we designed should render the use of SubtiTree acceptable in real conditions.

References

• S. Dinant, J.L. Bonnemain, C. Girousse, and 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. Ren. Biology and chemistry of endophytes. Nat. Prod. Rep., 2006, 23, 753–771, 753

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