Bacillus subtilis biofilm formation under extreme terrestrial and simulated extraterrestrial conditions

Kurzfassung

Since the Apollo 16 mission in 1972, Bacillus subtilis served as bacterial model organism in space. Due to the ability to form highly resistant endospores and complex biofilms, it was used to investigate the effects of unshielded space radiation as well as the limits of life in space. Within the International Space Station (ISS), fungal and bacterial biofilms already emerged as a burden that could harm the spacecraft due to material corrosion as well as the crew by causing infections. The ISS is a sensitive environment in which biofilm associated clogging or contaminating of life-support systems (water, electricity, cooling or ventilation) could lead to a termination of the mission. So far, little is known about the effects of biofilm formation under the influence of space conditions, such as altered gravity or enhanced radiation levels. Unfortunately, space research is very expensive, time-consuming and experimentally limited. In order to investigate single space conditions without conducting a space experiment, single parameters can be simulated under laboratory conditions. In frame of this thesis, B. subtilis was used as model organism to intensively study biofilm formation and sporulation as well as germination properties of spores grown under simulated microgravity (sim-µg). The aim was to elucidate the structure and the biological response of biofilms to sim-µg and further spaceflight-relevant conditions to optimize the preparation of flight experiments and to make space travel safer. B. subtilis NCIB 3610 and biofilm-deficient mutants in the same background were exposed to sim-µg by using a fast-rotating 2-D clinostat and biofilm formation was compared to terrestrial gravity (1g). First, a method was developed to generate standardized colony biofilms on membrane filters to guarantee reproducible results. Surface structures (topography) of biofilms grown under both conditions did not exhibit structural differences by white-light profilometry, but exhibited changes in the surface hydrophobicity. Whereas REM and TEM images of biofilm cross sections showed differences in cell phenotypes and in the abundance of matrix components. Phenotypic appearance of biofilms as well as growth(rates) were not affected by sim-µg, neither in CFU or spore composition. A transcriptome analysis of young biofilms showed that approximately 7 % of the transcripts differed due to the influence of sim-µg. Based on proteome analyses 10 (72 h) - 20 % (24 h) differences in the proteome of young and mature B. subtilis biofilms were found. In addition, no differences in sporulation rates, but in the germination behavior of spores isolated from biofilms were observed. Spores isolated after sporulation in sim-µg, tended to germinate spontaneously in water, which is atypical compared to 1g-cultivated spores. The time-resolved heterogeneity in germination of individual spores was reduced in sim-µg spores, which exhibited a uniform germination behavior. In addition, various space parameters were investigated, such as ionizing and heavy ion radiation, which showed no difference in survivability between spores/biofilms formed under both gravitational conditions. In the frame of this thesis it was shown that the simulation of microgravity changes the B. subtilis biofilm formation with respect to its structure but not its resistance to space parameters. In addition, spores produced under sim-µg showed a more homogeneous germination behavior than 1g spores and tended to germinate spontaneously.