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Charakterisierung und Nutzung der mikrobiellen Diversität extremer Habitate der Kamtschatka-Region

Characterization of the microbial diversity of extreme habitats in the Kamchatka-region

by Jörg Schuldes
Doctoral thesis
Date of Examination:2008-04-30
Date of issue:2009-01-06
Advisor:PD Dr. Rolf Daniel
Referee:Prof. Dr. Gerhard Gottschalk
crossref-logoPersistent Address: http://dx.doi.org/10.53846/goediss-1344

 

 

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Abstract

English

The Kamchatka Peninsula, located in the northeast of Russia is known for its variety of volcanic areas. Samples from these environments are excellent sources for extremophilic microorganisms and natural product discovery. Several samples of water and sediment derived from Kamchatka hot springs, geysers and volcanoes with temperatures ranging from 50 to 91°C and pH values from 2 to 3.5 and from 8.3 to 9.5 were used as starting materials for isolation of metagenomic DNA and phylogenetic surveys. To encounter novel biocatalysts metagenomic DNA was isolated directly from the Kamchatka samples by bead-beating and subsequently direct lysis of the microorganisms present. Large-insert and small-insert libraries were constructed and screened for genes encoding proteases, lipases, cellulases and amylases. In summary we identified and partially characterized 4 different lipases/esterases, 2 proteases and 1 amylase. The isolated DNA was also employed for 16S rRNA gene analysis by PCR using primer pairs directed against Bacteria or Archaea. The resulting PCR products were cloned and sequenced. A total of 508 16S rRNA genes were analyzed and subsequently phylogenetic trees were created. The majority of the recovered 16S rRNA gene sequences were affiliated to members of the phyla Proteobacteria, Bacteroidetes, Chlorobi, Acidobacteria, Firmicutes, Nitrospirae, Planctomyces, Actinobacteria, Chloroflexi, Gemmatimonadetes, Cyanobacteria, Aquificae, Thermotogae, Crenarchaeota and Euryarchaeota and to genera represented only by 16S rRNA gene sequences such as candidates divisions OP8, OP10, OP11, TM6 and BRC1.
Keywords: Metagenomic; metagenomic DNA libraries; screening strategies; 16SrRNA gene analyses; Kamchatka; hot springs; esterases; lipases; proteases

Other Languages

Die im äußersten Nordosten von Russland gelegene Halbinsel Kamtschatka ist überzogen von heißen Quellen, Geysiren und Vulkanen. Proben aus diesen extremen Habitaten sind für die Entdeckung neuartiger Biokatalysatoren interessant. Als Ausgangsmaterial für diese Arbeit dienten Sedimente, Wasser und Bodenproben im Temperaturbereich von 50 bis 91 °C und pH-Bereichen von 2 bis 3,5 und 8,3 bis 9,5. Durch die Methode des mechanischen Bead-beating wurde die metagenomische DNA isoliert. Diese diente als Grundlage zur Konstruktion von Metagenombanken und schließlich zur Identifizierung neuartiger Biokatalysatoren und Wirkstoffe. Im Rahmen der auf Aktivität basierenden Durchmusterung der Genbanken konnten vier verschiedene Lipasen/Esterasen, zwei Proteasen und eine Amylase identifiziert und teilweise charakterisiert werden. Die isolierte metagenomische DNA diente schließlich auch zur Untersuchung der mikrobiellen Diversität. Mittels 16S rRNA-Genanalysen wurden 508 16S rRNA-Gensequenzen generiert, analysiert und in phylogenetische Stammbäume integriert. Es konnten 16S rRNA-Gensequenzen der Phyla Proteobacteria, Bacteroidetes, Chlorobi, Acidobacteria, Firmicutes, Nitrospirae, Planctomyces, Actinobacteria, Chloroflexi, Gemmatimonadetes, Cyanobacteria, Aquificae, Thermotogae, Crenarchaeota und Euryarchaeota sowie der Candidates Divisions OP8, OP10, OP11, TM6 und BRC1 identifiziert werden.
Schlagwörter: Metagenomik; Umweltgenbanken; Screening-Strategien; 16SrRNA-Genanalysen; Kamtschatka; heiße Quellen; Esterasen; Lipasen; Proteasen;
 

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