Dual Stress Planetary Protection: UV Sterilization During Rocket Launch Disrupts RecA Ortholog-mediated DNA Repair in Saccharomyces cerevisiae
Space Dynamics Laboratory Post Flight Analysis for the Hermes I Launch
Annaliese C.S. Meyer, Sarah N. Ebert, Sean Farley, Sean Waugh, Andy Martinez, Tessa Charlton, and Sachi Premathilaka
UVic Rocketry, University of Victoria, Victoria, BC, V8P 5C2
FLIGHT RESULTS
Due to a presumable static failure of the UV LED units used in this experiment, DNA damage from UV lights could not accurately be assessed. A lawn of growth was seen in all dishes incubated. As a contingency measure, to investigate late-stage survival of the S. cerevisiae specimens, sub-cultures were taken every millimeter across the diameter of each flight example and incubated to look for a trend in growth decreasing towards the UV source. No trend was visible after incubation, suggesting that the lights failed early in the launch.
DISCUSSION OF EXPECTED RESULTS
E. coli RecA from an expression plasmid effectively rescues UV sensitivity of S. cerevisiae rad51 knockout strains at both 245 nm and 275 nm in lab trials. We had expected flight trials to decrease in viability more so than the lab trials – this would indicate that the mechanism of action of Rad51 and RecA is similar, and is affected by launch conditions as demonstrated by epistasis with the knockout rad51 – i.e. the phenotype conferred by the launch environment gets masked by the existing phenotype of UV sensitivity in the rad51 knockout.
RecA analogs are conserved in most species observed and share similar ATP- and DNA-binding domains (1). The physical interaction between the RecA/Rad51 filament and the damaged strand of DNA that is impacted by the launch conditions may be based on these conserved domains. To that point, it is worth noting that several multi-amino acid sequence both within and at ends of Rad51 is conserved in E. coli RecA, but is not shared in yeast paralogs to Rad51, such as Rad52 (2). Brozmanova et al. (3) determined that E. coli RecA could rescue strains lacking Rad51, but not so those lacking Rad52, possibly to these amino acid conserved domains. A broad chemical inhibitor could be also be used to target this conserved interaction and domains involved and thus reduce viability of bioburden. Other effects of high vibration and acceleration stress of launch include sedimentation of cytoplasmic components, slight mechanical deformation, and artificially increased cell density (4).
Several potential sources of error exist in this experiment even when the UV LEDs function nominally. Reduced overall cellular viability due to the heat of launch environment may appear to be due to the test stress of launch acceleration and vibrational loading. To mitigate this, cultures were kept below room temperature using cold packs and reflective sheeting. Other sources of error, such as distortion of the growth limit due to condensation collecting in the petri dishes as temperature changes, can be corrected by taking the average of several different chords from edge of plate to edge of growth area.
FUTURE WORK
Other resistance mechanisms that may be impacted by launch conditions should be investigated with a genome-wide screen, and longer-exposure test conditions with lower acceleration can be used to accurately mimic deep space rocket launches.
Testing should be repeated with species known to be resistant to sterilization methods, such as Bacillus pumilus SAFR-032 and Deinococcus radiodurans, and species such Actineobacter sp. that have been shown to be prevalent in NASA clean rooms. (5)
ACKNOWLEDGEMENTS
UVic Rocketry would like to extend their profound thanks the MIT Rocket Team for their flexibility and enthusiams, and to the Space Dynamics Laboratory for their encouragement, support and rad Frisbee skills. We would like to thank Drs. Chris Nelson and Afzal Suleman for their belief in our abilities, Dr. Immo Hansen for the use of his laboratory space at NMSU, and Dr. Miroslav Chovanec for providing the PVT103-LrecA plasmid from the Slovak Academy of Sciences. Our project would not be possible without the support of our sponsors and we cannot thank them enough. We are very grateful for the camaraderie and enthusiasm of all the other teams. We would also like to thank Mr. Jonathan Sykes for encouraging this important step towards space beer.
REFERENCES
- Kim J-I, Sharma AK, Abbott SN, Wood EA, Dwyer DW, Jambura A, Minton KW, Inman RB, Daly MJ, Cox MM. 2002. RecA Protein from the extremely radioresistant bacterium Deinococcus radiodurans: expression, purification, and characterization. J Bacteriol 184:1649–60.
- Goujon M, McWilliam H, Li W, Valentin F, Squizzato S, Paern J, Lopez R. 2010. A new bioinformatics analysis tools framework at EMBL-EBI. Nucleic Acids Res 38:W695–W699.
- Brozmanová J, Vlčková V, Chovanec M. 2004. How heterologously expressed Escherichia coli genes contribute to understanding DNA repair processes in Saccharomyces cerevisiae. Curr Genet 46:317–330.
- Deguchi S, Shimoshige H, Tsudome M, Mukai S, Corkery RW, Ito S, Horikoshi K. 2011. Microbial growth at hyperaccelerations up to 403,627 x g. Proc Natl Acad Sci U S A 108:7997–8002.
- La Duc MT, Vaishampayan P, Nilsson HR, Torok T, Venkateswaran K. 2012. Pyrosequencing-derived bacterial, archaeal, and fungal diversity of spacecraft hardware destined for Mars. Appl Environ Microbiol 78:5912–22.
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