Horizontal gene transfer in space - preparation of the ISS experiment Bacterial Conjugation -

Kurzfassung

Horizontal gene transfer (HGT) enables microorganisms to transmit genetic information to neighboring cells. On Earth, different mechanisms of HGT are known, namely conjugation, transduction and transformation. Most importantly, the mechanism of conjugation permits bacteria to rapidly adapt to environmental conditions, for example by conveying antibiotic resistances. This advantageous ability poses a huge threat to humans on Earth as well as in space as multi¬ resistant strains are on the rise and only few new antibiotics are being discovered. In regard to long-term space missions, the issue of microorganisms developing antibiotic resistances compromises the health of astronauts as they can only rely on limited medical support. Even though the driving force of emerging antibiotic resistances by bacterial conjugation has been studied on Earth, it has not been studied extensively in space. Live Cell Imaging facility FLUMIAS on board the International Space Station which was developed by the German Space Agency. The experiment aims to shed light on bacterial conjugation in microgravity compared to Earth gravity by microscopic fluorescence observation. The results of these experiments are of high importance to ensure the safety and health of astronauts during future long-term missions. However, the challenging technical and experiment constrains imposed by space conditions require a meticulous planning and procedure development to guarantee a campaign with significant results. The presented study was designed to determine the optimal experiment parameters to track the conjugation. The model organism E. coli will carry different plasmids, each encoding a different fluorescent protein. First it was shown that within the set constrains it is possible to observe bacterial growth and fluorescence, as well as that within the defined field of view (350pm x 400pm) and available objectives of FLUMIAS it is possible to observe a sufficient cell number to reach conclusive results. With an average of 7500 cells per field of view and an expected conjugation ratio of 1.8 - 4.1 x 10⁻² it will be possible tovisualize 200 transconjugants in an adequate resolution. Along with the tests and evaluation of different fluorophores encoded by different plasmids, it was shown that experiment will work within the borders of the set constrains. Furthermore, it can be shown that an ibidi® glass microscopy channel slide coated with Poly-DLysin and lysogeny broth (LB) as growth media is advantageous for biofilm formation compared to other growth conditions. It was also tested how different circumstances such as donor or recipient in biofilm and simulated p-gravity can influence the conjugation rate, and how storage conditions influence the stability of the plasmid. Moreover, the presented study provides a preliminary sample preparation procedure to enable the space experiment and delivers a proof of concept for the setup within the set of experimental and technical constrains.