by Bell, Greg David

Abstract (Summary)
Surveys of geothermal communities are necessary for understanding the genetic diversity, natural habitats and properties of hyperthermophiles. Past isolations of Sulfolobus have turned up new species and a variety of genetic elements, including plasmids, viruses, and insertion sequences. This project is the largest known survey of Sulfolobus species from four geographically isolated regions. To date, most previous surveys have used liquid culturing methods to isolate Sulfolobus species. To avoid the limitations of liquid culturing, a direct plating protocol was used in an attempt to capture greater diversity. Large degrees of diversity were observed amongst isolates, at the phenotypic and molecular level, particularly between those from different locations. Analysis of diversity at the molecular level using restriction profiles also revealed many isolates with multi-copy DNA fragments presumed to be viral and plasmid DNA. In the culturing process, an expansive collection of presumably diverse isolates was preserved for future detailed study. Preliminary DNA sequencing efforts suggest that the isolates analyzed thus far belong to two phylogenetically distinct groupings and that most of the observed diversity occurs below the species level. Hyperthermophilic archaea are the only organisms that lack widely conserved mismatch repair genes. In other organisms, including bacteria and eukaryotes, these mismatch repair genes are responsible for keeping spontaneous mutation rates at a low level. Previous work has shown that Sulfolobus acidocaldarius has a similar rate of spontaneous mutation to that of Eschericia coli. These observations suggest that some functionally equivalent DNA repair mechanism should operate in Sulfolobus that differs from other well-studied mismatch repair strategies. In order to study mismatch repair in Sulfolobus, mutants with elevated rates of spontaneous were isolated. The isolation strategy began with cells of strain DG64 being chemically mutagenized and then grown for several generations. Mutagenized cells were then subjected to an enrichment cycle, in an attempt to enrich for clones that mutate most frequently. The enrichment cycle was designed to generate alternating selections for the loss and then restoration of either of two pyrimidine biosynthetic enzymes, encoded by pyrE and pyrF. Following the enrichment scheme, cells were screened for high frequency mutators using a strategy that was patterned after the papillation assay of lacZ reversion in E. coli. Candidate clones were then purified and further screened for elevated rates of various types of forward and reverse mutation. Clones were also tested for elevated sensitivities to several chemical mutagens and to ultraviolet irradiation. No significant differences in sensitivity to DNA-damaging agents were found, but several strains displayed a 10-fold to greater than a 100-fold enhancement of mutant frequencies at independent genes, suggesting a general elevation in spontaneous mutation rates.
Bibliographical Information:


School:University of Cincinnati

School Location:USA - Ohio

Source Type:Master's Thesis

Keywords:archaea sulfolobus mutator mutant diversity


Date of Publication:01/01/2001

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