Rates of spontaneous mutation in an archaeon from geothermal environments

Abstract

To estimate the efficacy of mechanisms which may prevent or repair thermal damage to DNA in thermophilic archaea, a quantitative assay of forward mutation at extremely high temperature was developed for Sulfolobus acidocaldarius, based on the selection of pyrimidine-requiring mutants resistant to 5-fluoro-orotic acid. Maximum-likelihood analysis of spontaneous mutant distributions in wild-type cultures yielded maximal estimates of (2.8 ؎ 0.7) ؋ 10 ؊7 and (1.5 ؎ 0.6) ؋ 10 ؊7 mutational events per cell per division cycle for the pyrE and pyrF loci, respectively. To our knowledge, these results provide the first accurate measurement of the genetic fidelity maintained by archaea that populate geothermal environments. The measured rates of forward mutation at the pyrE and pyrF loci in S. acidocaldarius are close to corresponding rates reported for protein-encoding genes of Escherichia coli. The normal rate of spontaneous mutation in E. coli at 37°C is known to require the functioning of several enzyme systems that repair spontaneous damage in DNA. Our results provide indirect evidence that S. acidocaldarius has cellular mechanisms, as yet unidentified, which effectively compensate for the higher chemical instability of DNA at the temperatures and pHs that prevail within growing Sulfolobus cells.In the past 25 years, diverse microorganisms have been isolated from geothermal environments and have been shown to require temperatures of 75 to 105°C (depending on the species) for optimal growth; these organisms have been variously termed extreme thermophiles or hyperthermophiles (30). The fact that the preferred growth temperatures of these organisms can effectively denature the RNA, DNA, and protein of mesophilic microorganisms raises basic questions regarding the potential stability of biological macromolecules and the means by which extreme thermophiles and hyperthermophiles realize this potential stability in vivo. Considerable effort has been invested in understanding, in molecular terms, how the tertiary and secondary structures of enzymes and nucleic acids are preserved in these microorganisms. The fact that growth temperatures of 75 to 105°C should also disrupt the primary structure of biological macromolecules has received much less attention, despite indications that spontaneous processes of this type affect the growth and survival of bacteria growing at 37°C (16,29).The chemical instability of DNA is a major source of spontaneous mutation in mesophiles and has been studied extensively. Hydrolytic depurination represents the most frequent spontaneous decomposition of duplex DNA, followed by hydrolytic deamination of cytosine and adenine residues (19)(20)(21)(22)29). In aerobes, additional oxidative reactions form a variety of damaged bases (21). Biochemical and genetic studies using bacteria have shown that these various spontaneous reactions (i) occur in vivo (indeed, some metabolic processes actually contribute to them [21,29]); (ii) are potentially mutagenic, i.e., they cause mutation if the lesions escape rep...

Genetic fidelity under harsh conditions: Analysis of spontaneous mutation in the thermoacidophilic archaeon Sulfolobus acidocaldarius

“…Data from the second f luctuation test were used to confirm the genic mutation rate realized under our conditions. The value (3.37 Ϯ 0.36) ϫ 10 Ϫ7 , agrees well with an average value of (3.29 Ϯ 1.35) ϫ 10 Ϫ7 determined previously for larger cultures under similar but not identical growth conditions (5). The 79 intragenic pyrE mutations from the first two tests were pooled and designated set I.…”

“…Three corresponding estimates of the mutation rate per cell generation, , were calculated by using the relationship ϭ (ln2)(m͞N av ). There was no apparent bias in the rate estimates as a function of the quartile used, reinforcing direct experimental evidence that possible distortions caused by phenotypic lag or differences in growth rate between mutant and parent strains in uracil-supplemented media are negligible (5).…”

“…Fluctuation tests were performed as described (5) except that independent cultures were smaller and grown without mechanical agitation in microdilution plates. For each mutant-isolation experiment, one isolated colony was suspended in 3 ml of nonselective liquid medium and incubated until the culture reached a density of about 5 ϫ 10 7 cells͞ml.…”

“…The use of 5-fluoroorotic acid (FOA) to select pyrimidine auxotrophs facilitated the measurement of an aggregate mutation rate for the pyrE and pyrF genes (about 3.3 ϫ 10 Ϫ7 mutations per cell division) in S. acidocaldarius (5), but no sequence information was available then for either gene. In the present study, we isolated numerous independent mutants resistant to FOA under similar conditions, identified their mutations by sequencing, and used this information to estimate the size of the mutational target and the fraction of base pair substitutions (BPSs) that were not detected.…”

DNA repair glycosylases with a [4Fe–4S] cluster: A redox cofactor for DNA-mediated charge transport?

“…This is especially noteworthy given that the primary process that leads to uracil in DNA, cytosine deamination, has an enhanced rate at high temperatures [61]. Yet these organisms do not display a higher mutation rate [62]. Might the presence of this cofactor help fulfill this greater requirement for repair?…”