Diversity of thermostable DNA enzymes from Icelandic hot springs

University dissertation from Biotechnology, Lund University

Abstract: The diversities of bacteria as well as thermostable DNA enzymes in Icelandic hot springs were studied using different techniques. First of all the bacterial diversity of different microbial mats as well as sediment samples was described by using both molecular methods and cultivation methods. The molecular methods showed a low level of diversity and it was apparently lower as the environment became more extreme, e.g. in terms of higher temperature or higher sulfide concentration. The cultivation methods used gave mainly Thermus isolates. It was suggested that Thermus was the dominant bacterial organotroph in the hot spring studied. There were four different media used in the cultivation approach and they gave a biased selection of isolated species compared to the direct molecular diversity analysis. Some media were also more selective for one Thermus species than another. One new species of Thermus, T. scotoductus was isolated and described. Secondly, activity screenings for three different thermostable DNA enzymes were performed on cultivated strains from a large collection of thermophilic bacteria. The strains tested were from the genera Thermus, Rhodothermus, Bacillus and Hydrogenobacter. The enzymes screened for were thermostable DNA ligases, DNA polymerases and type II restriction endonucleases. Several new enzymes were found. Two of them were purified characterized and commercialized. The third and last approach was a sequence based screening performed both on cultivated strains and on complex environmental DNA samples. A PCR based method using CODEHOP degenerate primers was used for obtaining DNA polymerase gene fragments for sequencing. The phylogenetic relationship of the polymerase gene fragments was analysed. The genetic variability obtained among Thermus species indicated that Thermus is a diverse and still not a fully exhausted resource for searching for potentially valuable DNA polymerases. The polymerase gene fragments obtained from the complex hot spring DNA samples also showed a high number of new thermophilic DNA polymerases. Uncultivated biomass is therefore clearly also a good source for new DNA polymerases.

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