Determination of archaeal diversity of hot water sources inAydin

Archaea, which constitute one of the three major domains of living things, in terms of both biochemical properties and structural features, are prokaryotic cell types that are separated from eukaryotes and bacteria. The vast majority of members of Archea survive in extreme ambient conditions thanks to their metabolic and molecular adaptations. Typical environments in which pure cultures of archaea species isolated; are hot springs, hydrothermal vents, solfataras, salt lakes, soda lakes. In recent years, the use of molecular techniques involving PCR-based amplification of 16S rRNA genes in environmental samples has allowed a culture-independent microbial diversity assessment. In this study, it was aimed to investigate the archeal diversity of hot water sources in Aydın and its surroundings using culture independent methods. Total DNA isolation was performed from water, sludge and debris samples and then 16S rRNA gene region was amplified by PCR method using domain-specific primers. After the amplicons were ligated into the puc19 plasmid, E.coli DH10B cells was transformed by recombinant plasmid. By selecting white colonies, PCR reactions were established with M13 primers, and the sequences of the 16S rDNA amplicons obtained were determined to find the similarity rates with other prokaryotes in the database. In addition, three different primer sets (751F/1406R- 25F/1492R- 1F/1000R) and two different cloning methods (TA cloning and cloning with restriction enzyme) were compared. Among the 112 clones obtained in the study 45 of them were found to be belong to domain Bacteria, while 67 of the clones were belong to the domain Bacteria and a wide biodiversity were determined. The sequences of archeal clones were found to be mostly similar to Ignisphaera aggregans and Thermofilum spp., also some methanogenic members of Archea like Methanocaldococcus spp. and Methanospirillum spp. On the other hand, bacterial lineages were found to be mostly similar with some species of genus Thermus, members of Cyanobacteria and Proteobacteria phyla in the samples. In our country, researches on determining archeal diversity in hot water resources are very limited. Our thesis study will make an important contribution to the biodiversity of our country today, where extreme microorganisms are popular in many fields, especially in biotechnology. Comparison of different PCR and cloning methods will be a guide for the researchers working on this subject.