Characterization Of Spontaneous Mutations In A Moderate Thermophile Deinococcus Geothermalis

The genus Deinococcus is well known for its extreme resistance to ionizing radiation. Deinococcus geothermalis is distinguished from other Deinococcus species by its ability to grow at temperatures as high as 55℃. The radioresistant and thermophilic bacterium D. geothermalis can potentially be used as a genetic-engineering bacterium for the bioremediation of radioactive wastes in situ, in environments where temperatures are elevated by the decay of long-lived radionuclides, e.g.,137Cs and 90Sr. However, the mechanism underlying its growth in detrimental environments is poorly understood. Comparative genome analysis failed to identify a unique DNA repair systems in D. radiodurans or D. geothermalis, but highly efficient DNA repair proteins and cell-cleaning systems have been suggested to contribute to their resistant phenotypes.The investigation of spontaneous mutations patterns is not only helpful in understanding the relationships between the nature of mutagenesis and thermophilic growth behaviour, but may also reveal the cellular resistance mechanism used by this bacterium The mutation rate is also an important parameter in studying bacterial adaptation and evolutionary mechanisms. An rpoB/Rif system was developed to analyze mutations in Escherichia coli. The resistance of E. coli to rifampin is attributed almost exclusively to amino acid exchanges in a limited region of the P subunit of the RNA polymerase which is involved in rifampin binding, and the related sequence is highly conserved among prokaryotes.The rpoB/Rif mutation analysis system has been widely used, including E. coili,Bacillus subtilis, Helicobacter pylori and D. radiodurans, Mycobacterium tuberculosis, Pseudomonas stutzeri, Neisseria species.The rpoB/Rifr mutational analysis system was extended to analyze mutations in mesophilic D. radiodurans, based on the conservation of the rpoB gene. We have previously used this system to determine the mutation rates of the mutant recQ in D.radiodurans and the four-mutation strain uvsE uvrAl uvrA2 recQ. The results indicated that recQ is involved in the ultraviolet damage repair pathway of D.radiodurans. Moreover, reactive oxygen species (ROS), generated as by-products of aerobic metabolism, are thought to be the inducers of spontaneous mutations of bacteria. The antioxidant enzymes including superoxide dismutase (SOD) and catalase (CAT) plays an important role in the resistance of D. radiodurans to oxidative stress by their ROS scavenging activity. Hence, antioxidant enzyme may have effects on the mutation rates and spectra of D. geothermalis.In this study, we determined the mutation rates and spectra of D. geothermalis using the rpoB/Rifr mutational analysis system. The results provided the first detailed description of mutagenesis in D. geothermalis. More base-pair substitutions (BPSs) were detected from D. geothermalis grown at 30℃than that at the optimal growth temperature (50℃). The activities and gene expression levels of SOD and CAT of D. geothermalis grown at 50℃are significantly higher than those grown at 30℃. These results indicated that the elevation of mutation rate when D. geothermalis grown at 30℃was associated with a decline of antioxidant enzymes in this extreme bacterium.