5:15 PM - 6:30 PM
[BAO01-P04] Analysis of mutations of rpoB gene in Deinococcus radiodurans R1 induced by simulated space conditions
Keywords:ISS, Tanpopo mission, mutatuion
To investigate the microbial viability and their DNA damage, the radioresistant bacteria Deinococcus spp. have been exposed at Exposure Facility of International Space Station (ISS) in Tanpopo mission since May 2015 [1]. The Exposure Panels (EPs) harboring dried-deinococcal cells will return to the ground after about one-, two- and three-year exposure. We are going to analyze the survival rate and DNA damage of dried deinococcal cells using pulsed-field gel electrophoresis, quantitative-PCR and mutation assay. The antibiotics rifampicin binds the RNA polymerase β-subunit, which is encoded by rpoB gene, and inhibits the initial step of the transcription activity. Certain mutations in the rpoB gene confer rifampicin resistance [2]. Based on the above understanding, we will determine mutant frequency and the mutation spectrum for the D. radiodurans rpoB gene. From these mutation data, we will estimate major DNA damage induced by space environment. For this purpose, the mutatagenic specificity of the D. radiodurans rpoB gene in simulated space conditions was investigated in this study.
The D. radiodurans R1 cell-suspension was dropped in the wells of aluminum plates (φ20 mm) and was dried under vacuum (vacuum-dried). The dried cells were exposed to vacuum (< 10−5 torr) or UVC254nm under the vacuum conditions. As a control, we analyzed the vacuum-dried cells without additional vacuum incubation. After exposure experiment, the cells were recovered from each well. inoculated to 10 ml of mTGE medium and cultured to show the OD590 nm to be about 4. The cell suspension was plated on mTGE agar containing 50μg/ml of rifampicin to determine the number of rifampicin resistant colonies (RifR), and on mTGE agar without rifampicin to determine the total number of viable colonies.
The rifampicin-resistant mutant frequency of vacuum-dried cells was 1.3 (±0.5) × 10−8. The rifampicin-resistant mutant frequency of the D. radiodurans R1 wet cells has been shown to be about 1.5 × 10−8 [3]. The result suggests that the rifampicin-resistant mutant frequencies of vacuum-dried cells and wet cells are comparable for D. radiodurans R1. Further, we will report and discuss the rifampicin-resistant mutant frequency and mutation spectra in the rpoB gene of rifampicin-resistant cells following exposure to UVC254nm and vacuum (< 10−5 torr).
[1] Yamagishi, A. et al., (2007) Bio. Sci. Space 21: 67−75.
[2] Campbell, E. A. et al., (2001) Cell 104: 901−912
[3] Kim, M. et al., (2004) Genetics 166: 661−668.
The D. radiodurans R1 cell-suspension was dropped in the wells of aluminum plates (φ20 mm) and was dried under vacuum (vacuum-dried). The dried cells were exposed to vacuum (< 10−5 torr) or UVC254nm under the vacuum conditions. As a control, we analyzed the vacuum-dried cells without additional vacuum incubation. After exposure experiment, the cells were recovered from each well. inoculated to 10 ml of mTGE medium and cultured to show the OD590 nm to be about 4. The cell suspension was plated on mTGE agar containing 50μg/ml of rifampicin to determine the number of rifampicin resistant colonies (RifR), and on mTGE agar without rifampicin to determine the total number of viable colonies.
The rifampicin-resistant mutant frequency of vacuum-dried cells was 1.3 (±0.5) × 10−8. The rifampicin-resistant mutant frequency of the D. radiodurans R1 wet cells has been shown to be about 1.5 × 10−8 [3]. The result suggests that the rifampicin-resistant mutant frequencies of vacuum-dried cells and wet cells are comparable for D. radiodurans R1. Further, we will report and discuss the rifampicin-resistant mutant frequency and mutation spectra in the rpoB gene of rifampicin-resistant cells following exposure to UVC254nm and vacuum (< 10−5 torr).
[1] Yamagishi, A. et al., (2007) Bio. Sci. Space 21: 67−75.
[2] Campbell, E. A. et al., (2001) Cell 104: 901−912
[3] Kim, M. et al., (2004) Genetics 166: 661−668.