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1.  Endogenous Mutagenesis in Recombinant Sulfolobus Plasmids 
Journal of Bacteriology  2013;195(12):2776-2785.
Low rates of replication errors in chromosomal genes of Sulfolobus spp. demonstrate that these extreme thermoacidophiles can maintain genome integrity in environments with high temperature and low pH. In contrast to this genetic stability, we observed unusually frequent mutation of the β-d-glycosidase gene (lacS) of a shuttle plasmid (pJlacS) propagated in Sulfolobus acidocaldarius. The resulting Lac− mutants also grew faster than the Lac+ parent, thereby amplifying the impact of the frequent lacS mutations on the population. We developed a mutant accumulation assay and corrections for the effects of copy number and differential growth for this system; the resulting measurements and calculations yielded a corrected rate of 5.1 × 10−4 mutational events at the lacS gene per plasmid replication. Analysis of independent lacS mutants revealed three types of mutations: (i) G·C-to-A·T transitions, (ii) slipped-strand events, and (iii) deletions. These mutations were frequent in plasmid-borne lacS expressed at a high level but not in single-copy lacS in the chromosome or at lower levels of expression in a plasmid. Substitution mutations arose at only two of 12 potential priming sites of the DNA primase of the pRN1 replicon, but nearly all these mutations created nonsense (chain termination) codons. The spontaneous mutation rate of plasmid-borne lacS was 175-fold higher under high-expression than under low-expression conditions. The results suggest that important DNA repair or replication fidelity functions are impaired or overwhelmed in pJlacS, with results analogous to those of the “transcription-associated mutagenesis” seen in bacteria and eukaryotes.
doi:10.1128/JB.00223-13
PMCID: PMC3697259  PMID: 23564176
2.  Roles of the Y-family DNA Polymerase Dbh in Accurate Replication of the Sulfolobus Genome at Extremely High Temperature 
DNA repair  2012;11(4):391-400.
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.
doi:10.1016/j.dnarep.2012.01.005
PMCID: PMC3591481  PMID: 22305938
Trans-lesion DNA synthesis; Y-family DNA polymerase; Sulfolobus DNA polymerase Dbh; DNA-damage sensitivity; Spontaneous mutation spectra
3.  Heteroduplex Formation, Mismatch Resolution, and Genetic Sectoring During Homologous Recombination in the Hyperthermophilic Archaeon Sulfolobus Acidocaldarius 
Hyperthermophilic archaea exhibit certain molecular-genetic features not seen in bacteria or eukaryotes, and their systems of homologous recombination (HR) remain largely unexplored in vivo. We transformed a Sulfolobus acidocaldarius pyrE mutant with short DNAs that contained multiple non-selected genetic markers within the pyrE gene. From 20 to 40% of the resulting colonies were found to contain two Pyr+ clones with distinct sets of the non-selected markers. The dual-genotype colonies could not be attributed to multiple DNAs entering the cells, or to conjugation between transformed and non-transformed cells. These colonies thus appear to represent genetic sectoring in which regions of heteroduplex DNA formed and then segregated after partial resolution of inter-strand differences. Surprisingly, sectoring was also frequent in cells transformed with single-stranded DNAs. Oligonucleotides produced more sectored transformants when electroporated as single strands than as a duplex, although all forms of donor DNA (positive-strand, negative-strand, and duplex) produced a diversity of genotypes, despite the limited number of markers. The marker patterns in the recombinants indicate that S. acidocaldarius resolves individual mismatches through un-coordinated short-patch excision followed by re-filling of the resulting gap. The conversion events that occur during transformation by single-stranded DNA do not show the strand bias necessary for a system that corrects replication errors effectively; similar events also occur in pre-formed heteroduplex electroporated into the cells. Although numerous mechanistic details remain obscure, the results demonstrate that the HR system of S. acidocaldarius can generate remarkable genetic diversity from short intervals of moderately diverged DNAs.
doi:10.3389/fmicb.2012.00192
PMCID: PMC3367456  PMID: 22679441
linear DNA; genetic transformation; mismatch repair; gene conversion
4.  Sulfolobus Mutants, Generated via PCR Products, Which Lack Putative Enzymes of UV Photoproduct Repair 
Archaea  2011;2011:864015.
In order to determine the biological relevance of two S. acidocaldarius proteins to the repair of UV photoproducts, the corresponding genes (Saci_1227 and Saci_1096) were disrupted, and the phenotypes of the resulting mutants were examined by various genetic assays. The disruption used integration by homologous recombination of a functional but heterologous pyrE gene, promoted by short sequences attached to both ends via PCR. The phenotypic analyses of the disruptants confirmed that ORF Saci_1227 encodes a DNA photolyase which functions in vivo, but they could not implicate ORF Saci_1096 in repair of UV- or other externally induced DNA damage despite its similarity to genes encoding UV damage endonucleases. The success of the gene-disruption strategy, which used 5′ extensions of PCR primers to target cassette integration, suggests potential advantages for routine construction of Sulfolobus strains.
doi:10.1155/2011/864015
PMCID: PMC3139894  PMID: 21785574
5.  Discontinuity and Limited Linkage in the Homologous Recombination System of a Hyperthermophilic Archaeon▿  
Journal of Bacteriology  2010;192(18):4660-4668.
Genetic transformation of Sulfolobus acidocaldarius by a multiply marked pyrE gene provided a high-resolution assay of homologous recombination in a hyperthermophilic archaeon. Analysis of 100 Pyr+ transformants revealed that this recombination system could transfer each of 23 nonselected base pair substitutions to the recipient chromosome along with the selected marker. In 30% of the recombinants, donor markers were transferred as multiple blocks. In at least 40% of the recombinants, donor markers separated by 5 or 6 bp segregated from each other, whereas similar markers separated by 2 bp did not segregate. Among intermarker intervals, the frequency of recombination tract endpoints varied 40-fold, but in contrast to other recombination systems, it did not correlate with the length of the interval. The average length of donor tracts (161 bp) and the frequent generation of multiple tracts seemed generally consistent with the genetic properties observed previously in S. acidocaldarius conjugation. The efficiency with which short intervals of diverged pyrE sequence were incorporated into the genome raises questions about the threat of ectopic recombination in Sulfolobus spp. mediated by this apparently efficient yet permissive system.
doi:10.1128/JB.00447-10
PMCID: PMC2937427  PMID: 20644140
6.  Conjugational Genetic Exchange in the Hyperthermophilic Archaeon Sulfolobus acidocaldarius: Intragenic Recombination with Minimal Dependence on Marker Separation 
Journal of Bacteriology  2005;187(2):805-809.
In Sulfolobus acidocaldarius conjugation assays, recombinant frequency was relatively constant for marker separations from 1,154 bp down to about 50 bp and readily detectable at 10 bp. Three-factor crosses revealed little, if any, genetic linkage over distances of 500 to 600 bp, and large deletion mutants were good donors but poor recipients in matings. The results indicate that most intragenic recombination events occur at one of the mutations, not in the interval between them.
doi:10.1128/JB.187.2.805-809.2005
PMCID: PMC543538  PMID: 15629955
7.  Cytosine Methylation by the SuaI Restriction-Modification System: Implications for Genetic Fidelity in a Hyperthermophilic Archaeon 
Journal of Bacteriology  2003;185(15):4657-4661.
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 N4 methylation to block cleavage of its recognition site, thereby avoiding the creation of G · T mismatches by spontaneous deamination at extremely high temperature.
doi:10.1128/JB.185.15.4657-4661.2003
PMCID: PMC165766  PMID: 12867480
8.  Molecular Characteristics of Spontaneous Deletions in the Hyperthermophilic Archaeon Sulfolobus acidocaldarius 
Journal of Bacteriology  2003;185(4):1266-1272.
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.
doi:10.1128/JB.185.4.1266-1272.2003
PMCID: PMC142876  PMID: 12562797
9.  Loss of genetic accuracy in mutants of the thermoacidophile Sulfolobus acidocaldarius  
Archaea  2001;1(1):45-52.
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.
PMCID: PMC2685545  PMID: 15803658
5-fluoro-orotic acid; hyperthermophilic archaea; mismatch repair; mutator mutants; spontaneous mutation
10.  Characterization of Intragenic Recombination in a Hyperthermophilic Archaeon via Conjugational DNA Exchange 
Journal of Bacteriology  2001;183(9):2943-2946.
Sulfolobus acidocaldarius is so far the only hyperthermophilic archaeon in which genetic recombination can be assayed by conjugation and simple selections. Crosses among spontanteous 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.
doi:10.1128/JB.183.9.2943-2946.2001
PMCID: PMC99513  PMID: 11292816
11.  Evidence that β-Galactosidase of Sulfolobus solfataricus Is Only One of Several Activities of a Thermostable β-d-Glycosidase 
A survey of Sulfolobus isolates showed all to contain thermostable enzyme activities hydrolyzing various glycosidic compounds. Of those not previously reported, the β-glucosidase activity of Sulfolobus solfataricus isolate P2 was chosen for further study and found to have the same kinetics of inactivation, apparent molecular weight, and many (though not all) other biochemical properties of the β-galactosidase also present in this strain. The two activities copurified approximately 850-fold to apparent homogeneity. The enzyme, whose subunit Mr was estimated to be 60,000 to 65,000 by gel permeation chromatography of the active enzyme and 70,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the denatured form, hydrolyzed a variety of low-molecular-weight, β-linked glycosides and could account for most of the corresponding activities found in crude extract. Kinetic analyses indicated that chromogenic β-d-galactosides and β-d-glucosides are hydrolyzed at a common active site and that β-glucosides and β-fucosides represent the preferred substrates. The liberation of aglycone from aryl β-d-glucosides was stimulated by alcohols in a manner suggesting specific interaction between alcohol and enzyme.
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PMCID: PMC183446  PMID: 16348503

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