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

Abstract

Microbes whose genomes are encoded by DNA and for which adequate information is available display similar genomic mutation rates (average 0.0034 mutations per chromosome replication, range 0.0025 to 0.0046). However, this value currently is based on only a few well characterized microbes reproducing within a narrow range of environmental conditions. In particular, no genomic mutation rate has been determined either for a microbe whose natural growth conditions may extensively damage DNA or for any member of the archaea, a prokaryotic lineage deeply diverged from both bacteria and eukaryotes. Both of these conditions are met by the extreme thermoacidophile Sulfolobus acidocaldarius. We determined the genomic mutation rate for this species when growing at pH 3.5 and 75°C based on the rate of forward mutation at the pyrE gene and the nucleotide changes identified in 101 independent mutants. The observed value of about 0.0018 extends the range of DNA-based microbes with rates close to the standard rate simultaneously to an archaeon and to an extremophile whose cytoplasmic pH and normal growth temperature greatly accelerate the spontaneous decomposition of DNA. The mutations include base pair substitutions (BPSs) and additions and deletions of various sizes, but the S. acidocaldarius spectrum differs from those of other DNA-based organisms in being relatively poor in BPSs. The paucity of BPSs cannot yet be explained by known properties of DNA replication or repair enzymes of Sulfolobus spp. It suggests, however, that molecular evolution per genome replication may proceed more slowly in S. acidocaldarius than in other DNA-based organisms examined to date.A rchaea isolated from geothermal environments grow optimally at temperatures that are lethal to all genetically well characterized microorganisms and damaging to DNA. The enzymes of these hyperthermophilic archaea are intrinsically thermostable due to a variety of structural features that discourage protein unfolding, which explains how metabolism can be maintained at extremely high temperatures (1). The strategy of intrinsic stabilization does not seem to apply to the chromosomes of these archaea, however, and does not address the problem of spontaneous DNA decomposition at physiological temperatures (2). Although information about the gene content, genome organization, and evolutionary divergence of hyperthermophilic archaea is expanding rapidly, their basic genetic processes remain largely unexplored. As a result, it is unclear how these organisms compare with well studied microbes with respect to genetic exchange, DNA repair, mutation, genetic exchange, and other fundamental processes important to their survival and evolution.Rates of spontaneous mutation measured in microbial systems provide evidence of the biological importance of genetic fidelity. Accurate rates of spontaneous mutation per genome are available for only six DNA-based microbes: phage M13, phage , phage T2͞T4, the bacterium Escherichia coli, the yeast Saccharomyces cerevisiae, and the filamentous...

Mutations and Rearrangements in the Genome ofSulfolobus solfataricusP2

“…For example, the 44-bp deletion had no recognizable direct repeats, inverted repeats, or secondary DNA structure at the endpoints, consistent with the deletions observed in S. acidocaldarius (12). Moreover, we observed two Ϯ1-bp mutations in runs of single bases (T 3 and A 6 in strains P2A-045 and -139, respectively) ( Table 2), although there was no strong preference for Ϫ1 bp mutations in long runs of the same base, as occurs in S. acidocaldarius, which is richer in homopolynucleotide sequences (11). However, in contrast to the pyrE bias observed for mutations in the pyrEF region of S. acidocaldarius (11), mutations were distributed fairly evenly throughout the region (Table 2).…”

“…Moreover, we observed two Ϯ1-bp mutations in runs of single bases (T 3 and A 6 in strains P2A-045 and -139, respectively) ( Table 2), although there was no strong preference for Ϫ1 bp mutations in long runs of the same base, as occurs in S. acidocaldarius, which is richer in homopolynucleotide sequences (11). However, in contrast to the pyrE bias observed for mutations in the pyrEF region of S. acidocaldarius (11), mutations were distributed fairly evenly throughout the region (Table 2). , and the lower part shows the changes found in strain P2A-003, where SSO0620 is partitioned and the arrow on the boxed ISC1359 indicates the direction of the transpose gene.…”

“…Moreover, since it carries substantially more IS elements and MITEs than Sulfolobus tokodaii, or than Sulfolobus acidocaldarius, which probably lacks active mobile elements (4,9,11), it was proposed that the genomic rearrangements are driven primarily by mobile-element-dependent mechanisms (5). This proposal complements the earlier hypothesis that the megaplasmid pNRC100 of the euryarchaeon Halobacterium halobium arose via a multistep IS element-mediated process (10,20).…”

“…Mutants were generated which were resistant to 5-fluoroorotic acid (FOA), which is converted into the toxic compound 5-fluoro-UMP by the gene products of pyrE and pyrF (16). Earlier studies, using a similar approach, detected only a few IS element insertions, mainly in the pyrE gene or the promoter region for the related strain Sulfolobus solfataricus P1 (18), while for the more distantly related S. acidocaldarius, only point mutations, small insertions, and deletions were observed at this locus (11).…”

Biogeography of theSulfolobus islandicuspan-genome

“…S2, ranging from 910,000 (Ϯ90,000) years ago for the North American and Mutnovsky populations to 140,000 (Ϯ25,000) years ago for the M.16.27 and M.14.25 divergence. Based on these dates, the average rate of nucleotide substitution was estimated to be 4.66 ϫ 10 Ϫ9 (Ϯ6.76 ϫ 10 Ϫ10 ) substitutions per site per year (Table S4), on the order of the universal rates estimated from comparisons of other species (46,47,63) and predicted from laboratory determinations of mutation rates in other Sulfolobus species (64). This unique method for dating microbial populations based on constraining colonization dates with geological events allows estimation of absolute rates of genome dynamics on more recent time scales than have previously been examined.…”