Isolation of rpoB Mutations Causing Rifampicin Resistance in Bacillus subtilis Spores Exposed to Simulated Martian Surface Conditions

Bacterial spores are considered prime candidates for Earth-to-Mars transport by natural processes and human spaceflight activities. Previous studies have shown that exposure of Bacillus subtilis spores to ultrahigh vacuum (UHV) characteristic of space both increased the spontaneous mutation rate and altered the spectrum of mutation in various marker genes; but, to date, mutagenesis studies have not been performed on spores exposed to milder low pressures encountered in the martian environment. Mutations to rifampicin-resistance (Rif(R)) were isolated in B. subtilis spores exposed to simulated martian atmosphere (99.9% CO2, 710 Pa) for 21 days in a Mars Simulation Chamber (MSC) and compared to parallel Earth controls. Exposure in the MSC reduced spore viability by similar to 67% compared to Earth controls, but this decrease was not statistically significant (P = 0.3321). The frequency of mutation to Rif(R) was also not significantly increased in the MSC compared to Earth-exposed spores (P = 0.479). Forty-two and 51 Rif(R) mutant spores were isolated from the MSC- and Earth-exposed controls, respectively. Nucleotide sequencing located the Rif(R) mutations in the rpoB gene encoding the beta subunit of RNA polymerase at residue V135F of the N-cluster and at residues Q469K/L, H482D/P/R/Y, and S487L in Cluster I. No mutations were found in rpoB Clusters II or III. Two new alleles, Q469L and H482D, previously unreported in B. subtilis rpoB, were isolated from spores exposed in the MSC; otherwise, only slight differences were observed in the spectra of spontaneous Rif(R) mutations from spores exposed to Earth vs. the MSC. However, both spectra are distinctly different from Rif(R) mutations previously reported arising from B. subtilis spores exposed to simulated space vacuum.