The rate and character of spontaneous mutation in Thermus thermophilus

Selection of spontaneous, loss-of-function mutations at two chromosomal loci (pyrF and pyrE) enabled the first molecular-level analysis of replication fidelity in the extremely thermophilic bacterium Thermus thermophilus. Two different methods yielded similar mutation rates, and mutational spectra determined by sequencing of independent mutants revealed a variety of replication errors distributed throughout the target genes. The genomic mutation rate estimated from these targets, 0.00097 +/- 0.00052 per replication, was lower than corresponding estimates from mesophilic microorganisms, primarily because of a low rate of base substitution. However, both the rate and spectrum of spontaneous mutations in T. thermophilus resembled those of the thermoacidophilic archaeon Sulfolobus acidocaldarius, despite important molecular differences between these two thermophiles and their genomes.     

Loss of genetic accuracy in mutants of the thermoacidophile Sulfolobus acidocaldarius

To investigate how hyperthermophilic archaea can propagate their genomes accurately, we isolated Sulfolobus acidocaldarius mutants exhibiting abnormally high rates of spontaneous mutation. Our isolation strategy involved enrichment for mutator lineages via alternating selections, followed by screening for the production of spontaneous, 5-fluoro-orotate-resistant mutants in micro-colonies. Several candidates were evaluated and found to have high frequencies of pyrE and pyrF mutation and reversion. Neither an increased efficiency of plating of mutants on selective medium, nor the creation of a genetically unstable pyrE allele, could be implicated as the cause of these high frequencies. The strains had elevated frequencies of other mutations, and exhibited certain phenotypic differences among themselves. A large increase in sensitivity to DNA-damaging agents was not observed, however. These properties generally resemble those of bacterial mutator mutants and suggest loss of functions specific to genetic accuracy.     

Identification of Weissella species by the genus-specific amplified ribosomal DNA restriction analysis

To investigate the generality of efficient double-strand break repair and damage-induced mutagenesis in hyperthermophilic archaea, we systematically measured the effects of five DNA-damaging agents on Sulfolobus acidocaldarius and compared the results to those obtained for Escherichia coli under corresponding conditions. The observed lethality of gamma-radiation was very similar for S. acidocaldarius and E. coli, arguing against unusually efficient double-strand break repair in S. acidocaldarius. In addition, DNA-strand-breaking agents (gamma-radiation or bleomycin), as well as DNA-cross-linking agents (mechlorethamine, butadiene diepoxide or cisplatin) stimulated forward mutation, reverse mutation, and formation of recombinants via conjugation in Sulfolobus cells. Although two of the five DNA-damaging agents failed to revert the E. coli auxotrophs under these conditions, all five reverted S. acidocaldarius auxotrophs.     

Construction of a shuttle vector for,and spheroplast transformation of,the hyperthermophilic archaeon Pyrococcus abyssi

Our understanding of the genetics of species of the best-studied hyperthermophilic archaea, Pyrococcus spp., is presently limited by the lack of suitable genetic tools, such as a stable cloning vector and the ability to select individual transformants on plates. Here we describe the development of a reliable host-vector system for the hyperthermophilic archaeon Pyrococcus abyssi. Shuttle vectors were constructed based on the endogenous plasmid pGT5 from P. abyssi strain GE5 and the bacterial vector pLitmus38. As no antibiotic resistance marker is currently available for Pyrococcus spp., we generated a selectable auxotrophic marker. Uracil auxotrophs resistant to 5-fluoorotic acid were isolated from P. abyssi strain GE9 (devoid of pGT5). Genetic analysis of these mutants revealed mutations in the pyrE and/or pyrF genes, encoding key enzymes of the pyrimidine biosynthetic pathway. Two pyrE mutants exhibiting low reversion rates were retained for complementation experiments. For that purpose, the pyrE gene, encoding orotate phosphoribosyltransferase (OPRTase) of the thermoacidophilic crenarchaeote Sulfolobus acidocaldarius, was introduced into the pGT5-based vector, giving rise to pYS2. With a polyethylene glycol-spheroplast method, we could reproducibly transform P. abyssi GE9 pyrE mutants to prototrophy, though with low frequency (10(2) to 10(3) transformants per micro g of pYS2 plasmid DNA). Transformants did grow as well as the wild type on minimal medium without uracil and showed comparable OPRTase activity. Vector pYS2 proved to be very stable and was maintained at high copy number under selective conditions in both Escherichia coli and P. abyssi.     

Characterization of intragenic recombination in a hyperthermophilic archaeon via conjugational DNA exchange

Sulfolobus acidocaldarius is so far the only hyperthermophilic archaeon in which genetic recombination can be assayed by conjugation and simple selections. Crosses among spontaneous pyr mutants were able to resolve closely spaced chromosomal mutations, identify deletions and rearrangements, and map mutations to a given deletion interval. Frameshift mutations in pyrE exerted polar effects that depressed orotidine-5'-monophosphate decarboxylase activity (encoded by pyrF), whereas base pair substitutions and an 18-bp deletion had no effect.     

Genetic responses of the thermophilic archaeon Sulfolobus acidocaldarius to short-wavelength UV light.

The archaea which populate geothermal environments are adapted to conditions that should greatly destabilize the primary structure of DNA, yet the basic biological aspects of DNA damage and repair remain unexplored for this group of prokaryotes. We used auxotrophic mutants of the extremely thermoacidophilic archaeon Sulfolobus acidocaldarius to assess genetic and physiological effects of a well-characterized DNA-damaging agent, short-wavelength UV light. Simple genetic assays enabled quantitative dose-response relationships to be determined and correlated for survival, phenotypic reversion, and the formation of genetic recombinants. Dose-response relationships were also determined for survival and phenotypic reversion of the corresponding Escherichia coli auxotrophs with the same equipment and procedures. The results showed S. acidocaldarius to be about twice as UV sensitive as E. coli and to be equally UV mutable on a surviving-cell basis. Furthermore, UV irradiation significantly increased the frequency of recombinants recovered from genetic-exchange assays of S. acidocaldarius. The observed UV effects were due to the short-wavelength (i.e., UV-C) portion of the spectrum and were effectively reversed by subsequent illumination of S. acidocaldarius cells with visible light (photoreactivation). Thus, the observed responses are probably initiated by the formation of pyrimidine dimers in the S. acidocaldarius chromosome. To our knowledge, these results provide the first evidence of error-prone DNA repair and genetic recombination induced by DNA damage in an archaeon from geothermal habitats.     

Molecular characteristics of spontaneous deletions in the hyperthermophilic archaeon Sulfolobus acidocaldarius

Prokaryotic genomes acquire and eliminate blocks of DNA sequence by lateral gene transfer and spontaneous deletion, respectively. The basic parameters of spontaneous deletion, which are expected to influence the course of genome evolution, have not been determined for any hyperthermophilic archaeon. We therefore screened a number of independent pyrimidine auxotrophs of Sulfolobus acidocaldarius for deletions and sequenced those detected. Deletions accounted for only 0.4% of spontaneous pyrE mutations, corresponding to a frequency of about 10(-8) per cell. Nucleotide sequence analysis of five independent deletions showed no significant association of the endpoints with short direct repeats, despite the fact that several such repeats occur within the pyrE gene and that duplication mutations in pyrE reverted at high frequencies. Endpoints of the spontaneous deletions did not coincide with short inverted repeats or potential stem-loop structures. No consensus sequence common to all the deletions could be identified, although two deletions showed the potential of being stabilized by octanucleotide sequences elsewhere in pyrE, and another pair of deletions shared an octanucleotide at their 3' ends. The unusually low frequency and low sequence dependence of spontaneous deletions in the S. acidocaldarius pyrE gene compared to other genetic systems could not be explained in terms of possible constraints imposed by the 5-fluoroorotate selection.     

Recombination of synthetic oligonucleotides with prokaryotic chromosomes: substrate requirements of the Escherichia coli/lambdaRed and Sulfolobus acidocaldarius recombination systems

In order to reveal functional properties of recombination involving short ssDNAs in hyperthermophilic archaea, we evaluated oligonucleotide-mediated transformation (OMT) in Sulfolobus acidocaldarius and Escherichia coli as a function of the molecular properties of the ssDNA substrates. Unmodified ssDNAs as short as 20–22 nt yielded recombinants in both organisms, as did longer DNAs forming as few as 2–5 base pairs on one side of the genomic mutation. The two OMT systems showed similar responses to certain end modifications of the oligonucleotides, but E. coli was found to require a 5' phosphate on 5'-limited ssDNA whereas this requirement was not evident in S. acidocaldarius . The ability of both E. coli and S. acidocaldarius to incorporate short, mismatched ssDNAs into their genomes raises questions about the biological significance of this capability, including its phylogenetic distribution among microorganisms and its impact on genome stability. These questions seem particularly relevant for S. acidocaldarius , as this archaeon has natural competence for OMT, encodes no MutSL homologues and thrives under environmental conditions that accelerate DNA decomposition.     

A new phosphotriesterase from Sulfolobus acidocaldarius and its comparison with the homologue from Sulfolobus solfataricus

The phosphotriesterase PTE, identified in the soil bacterium Pseudomonas diminuta, is thought to have evolved in the last several decades to degrade the pesticide paraoxon with proficiency approaching the limit of substrate diffusion (k(cat)/K(M) of 4 x 10(7)M(-1)s(-1)). It belongs to the amidohydrolase superfamily, but its evolutionary origin remains obscure. The enzyme has important potentiality in the field of the organophosphate decontamination. Recently we reported on the characterization of an archaeal member of the amidohydrolase superfamily, namely Sulfolobus solfataricus, showing low but significant and extremely thermostable paraoxonase activity (k(cat)/K(M) of 4 x 10(3)M(-1)s(-1)). Looking for other thermostable phosphotriesterases we assayed, among others, crude extracts of Sulfolobus acidocaldarius and detected activity. Since the genome of S. acidocaldarius has been recently reported, we identified there an open reading frame highly related to the S. solfataricus enzyme. The gene was cloned, the protein overexpressed in Escherichia coli, purified, and proven to have paraoxonase activity. A comparative analysis detected some significant differences between the two archaeal enzymes.     

Stability and repair of DNA in hyperthermophilic Archaea

Evolutionary and physiological considerations argue that study of hyperthermophilic archaea should reveal new molecular aspects of DNA stabilization and repair. So far, these unusual prokaryotes have yielded a number of genes and enzymatic activities consistent with known mechanisms of excision repair, photo-reversal, and trans-lesion synthesis. However, other DNA enzymes of hyperthermophilic archaea show novel biochemical properties which may be related to DNA stability or repair at extremely high temperature but which remain difficult to evaluate rigorously in vivo. Perhaps the most striking feature of the hyperthermophilic archaea is that all of them whose genomes have been sequenced lack key genes of both the nucleotide excision repair and DNA mismatch repair pathways, which are otherwise highly conserved in biology. Although the growth properties of these micro-organisms hinder experimentation, there is evidence that some systems of excision repair and mutation avoidance operate in Sulfolobus spp. It will therefore be of strategic significance in the next few years to formulate and test hypotheses in Sulfolobus spp. and other hyperthermophilic archaea regarding mechanisms and gene products involved in the repair of UV photoproducts and DNA mismatches.     

Roles of the Y-family DNA polymerase Dbh in accurate replication of the Sulfolobus genome at high temperature

The intrinsically thermostable Y-family DNA polymerases of Sulfolobus spp. have revealed detailed three-dimensional structure and catalytic mechanisms of trans-lesion DNA polymerases, yet their functions in maintaining their native genomes remain largely unexplored. To identify functions of the Y-family DNA polymerase Dbh in replicating the Sulfolobus genome under extreme conditions, we disrupted the dbh gene in Sulfolobus acidocaldarius and characterized the resulting mutant strains phenotypically. Disruption of dbh did not cause any obvious growth defect, sensitivity to any of several DNA-damaging agents, or change in overall rate of spontaneous mutation at a well-characterized target gene. Loss of dbh did, however, cause significant changes in the spectrum of spontaneous forward mutation in each of two orthologous target genes of different sequence. Relative to wild-type strains, dbh(-) constructs exhibited fewer frame-shift and other small insertion-deletion mutations, but exhibited more base-pair substitutions that converted G:C base pairs to T:A base pairs. These changes, which were confirmed to be statistically significant, indicate two distinct activities of the Dbh polymerase in Sulfolobus cells growing under nearly optimal culture conditions (78-80°C and pH 3). The first activity promotes slipped-strand events within simple repetitive motifs, such as mononucleotide runs or triplet repeats, and the second promotes insertion of C opposite a potentially miscoding form of G, thereby avoiding G:C to T:A transversions.     

Dynamic properties of the Sulfolobus CRISPR/Cas and CRISPR/Cmr systems when challenged with vector-borne viral and plasmid genes and protospacers

The adaptive immune CRISPR/Cas and CRISPR/Cmr systems of the crenarchaeal thermoacidophile Sulfolobus were challenged by a variety of viral and plasmid genes, and protospacers preceded by different dinucleotide motifs. The genes and protospacers were constructed to carry sequences matching individual spacers of CRISPR loci, and a range of mismatches were introduced. Constructs were cloned into vectors carrying pyrE/pyrF genes and transformed into uracil auxotrophic hosts derived from Sulfolobus solfataricus P2 or Sulfolobus islandicus REY15A. Most constructs, including those carrying different protospacer mismatches, yielded few viable transformants. These were shown to carry either partial deletions of CRISPR loci, covering a broad spectrum of sizes and including the matching spacer, or deletions of whole CRISPR/Cas modules. The deletions occurred independently of whether genes or protospacers were transcribed. For family I CRISPR loci, the presence of the protospacer CC motif was shown to be important for the occurrence of deletions. The results are consistent with a low level of random dynamic recombination occurring spontaneously, either inter-genomically or intra-genomically, at the repeat regions of Sulfolobus CRISPR loci. Moreover, the relatively high incidence of single-spacer deletions observed for S. islandicus suggests that an additional more directed mechanism operates in this organism.     

Spontaneous mutagenesis: the roles of DNA repair, replication, and recombination

There appears to be no dearth of mechanisms to explain spontaneous mutagenesis. In the case of base substitutions, data for bacteriophage T4 and especially for E. coli and S. cerevisiae suggest important roles in spontaneous mutagenesis for the error-prone repair of DNA damage (to produce mutations) and for error-free repair of DNA damage (to avoid mutagenesis). Data from the very limited number of studies on the subject suggest that about 50% of the spontaneous base substitutions in E. coli, and perhaps 90% in S. cerevisiae are due to error-prone DNA repair. On the other hand, spontaneous frameshifts and deletions seem to result from mechanisms involving recombination and replication. Spontaneous insertions have been shown to be important in the strongly polar inactivation of certain loci, but it is less important at other loci. Perhaps with continued study, the term "spontaneous mutagenesis" will be replaced by more specific terms such as 5-methylcytosine deamination mutagenesis, fatty acid oxidation mutagenesis, phenylalanine mutagenesis, and imprecise-recombination mutagenesis. While most studies have concentrated on mutator mutations, the most conclusive data for the actual source of spontaneous mutations have come from the study of antimutator mutations. Further study in this area, perhaps along with an understanding of chemical antimutagens, should be invaluable in clarifying the bases of spontaneous mutagenesis.     

The sub-cellular localization of Sulfolobus DNA replication

Analyses of the DNA replication-associated proteins of hyperthermophilic archaea have yielded considerable insight into the structure and biochemical function of these evolutionarily conserved factors. However, little is known about the regulation and progression of DNA replication in the context of archaeal cells. In the current work, we describe the generation of strains of Sulfolobus solfataricus and Sulfolobus acidocaldarius that allow the incorporation of nucleoside analogues during DNA replication. We employ this technology, in conjunction with immunolocalization analyses of replisomes, to investigate the sub-cellular localization of nascent DNA and replisomes. Our data reveal a peripheral localization of replisomes in the cell. Furthermore, while the two replication forks emerging from any one of the three replication origins in the Sulfolobus chromosome remain in close proximity, the three origin loci are separated.     

Homologous recombination of exogenous DNA with the Sulfolobus acidocaldarius genome: properties and uses

In order to quantify recombination between exogenous DNA and the Sulfolobus acidocaldarius chromosome, we electroporated pyrE (uracil-auxtotrophic) recipient strains with functional pyrE sequences and counted Pyr+ transformants by direct plating. Certain culture and post-electroporation conditions increased the yield of Pyr+ recombinants from non-replicating pyrE plasmid, whereas cognate methylation of SuaI restriction sites in the plasmid decreased it. Recombination of linear DNAs with the S. acidocaldarius genome was proportional to the length of a limiting overlap, but even synthetic oligonucleotides produced reasonable numbers of recombinants with appropriate recipient strains. To investigate uses of this latter property, we electroporated an 18-bp pyrE deletion mutant with mixtures of synthetic oligonucleotides altering glycine-55 of the orotate phosphoribosyl transferase encoded by pyrE. Pyr+ transformants were recovered in which this codon was converted to each of the alternatives encoded by the oligonucleotide mixtures, thereby identifying five amino acid substitutions tolerated at this position of the thermostable enzyme.     

Cytosine methylation by the SuaI restriction-modification system: implications for genetic fidelity in a hyperthermophilic archaeon

5-methylcytosine in chromosomal DNA represents a potential source of frequent spontaneous mutation for hyperthermophiles. To determine the relevance of this threat for the archaeon Sulfolobus acidocaldarius, the mode of GGCC methylation by its restriction-modification system, SuaI, was investigated. Distinct isoschizomers of the SuaI endonuclease were used to probe the methylation state of GGCC in native S. acidocaldarius DNA. In addition, the methylation sensitivity of the SuaI endonuclease was determined with synthetic oligonucleotide substrates and modified natural DNAs. The results show that the SuaI system uses N(4) methylation to block cleavage of its recognition site, thereby avoiding the creation of G. T mismatches by spontaneous deamination at extremely high temperature.     

Multiple origins of replication in archaea

Until recently, the only archaeon for which a bona fide origin of replication was reported was Pyrococcus abyssi, where a single origin was identified. Although several in silico analyses have suggested that some archaeal species might contain more than one origin, this has only been demonstrated recently. Two studies have shown that multiple origins of replication function in two archaeal species. One study identified two origins of replication in the archaeon Sulfolobus solfataricus, whereas a second study used a different technique to show that both S. solfataricus and Sulfolobus acidocaldarius have three functional origins. These are the first reports of archaea having multiple origins. This finding has implications for research on the mechanisms of DNA replication and evolution.     

A restriction endonuclease SuaI from the thermoacidophilic archaebacterium Sulfolobus acidocaldarius

A type II restriction endonuclease (SuaI) has been isolated from the thermoacidophilic archaebacterium Sulfolobus acidocaldarius. The enzyme is an isoschizomer of BspRI. It does not cut S. acidocaldarius DNA, as the recognition sequence GGCC in this DNA contains modified nucleotide(s). The enzyme is most active at 60-70 degrees C and is highly thermostable.     

UV-inducible DNA exchange in hyperthermophilic archaea mediated by type IV pili

Archaea, like bacteria and eukaryotes, contain proteins involved in various mechanisms of DNA repair, highlighting the importance of these processes for all forms of life. Species of the order Sulfolobales of hyperthermophilic crenarchaeota are equipped with a strongly UV-inducible type IV pilus system that promotes cellular aggregation. Here we demonstrate by fluorescence in situ hybridization that cellular aggregates are formed based on a species-specific recognition process and that UV-induced cellular aggregation mediates chromosomal marker exchange with high frequency. Recombination rates exceeded those of uninduced cultures by up to three orders of magnitude. Knockout strains of Sulfolobus acidocaldarius incapable of pilus production could not self-aggregate, but were partners in mating experiments with wild-type strains indicating that one cellular partner can mediate the DNA transfer. Since pilus knockout strains showed decreased survival upon UV treatment, we conclude that the UV-inducible DNA transfer process and subsequent homologous recombination represents an important mechanism to maintain chromosome integrity in Sulfolobus. It might also contribute substantially to the frequent chromosomal DNA exchange and horizontal gene transfer in these archaea in their natural habitat.