Determination of complete nucleotide sequence of the genome of hyperthermophilic microorganism

Abstract

Decoding of microorganism genomes is a basis for research into the microbiology, molecular biology, and evolution of living organisms and is of practical importance for medicine and biotechnology. For instance, the sequencing of genomes of microorganisms living under extremal environmental conditions (e.g., high temperature) makes it possible to identify new practically important enzymes, many of which are already actively used today in medicine (molecular diagnostics, etc.) and biotechnology (manufacturing of detergents, foodstuff and fodders, as well as in pulp-and-paper industry. Since 1997, the complete sequences of genomes of approximately. 640 bacteria and 50 archaebacteria have been determined. In addition, a large number of microbial genomes, information about which was not deposited in GenBank, were decoded by companies specializing in search for biocatalysts. In the Russian Federation, not one complete structure of microorganism genomes has been decoded until 2007. In this study, we described the procedure of sequencing the complete microorganism genome, which is based on the combination of capillary electrophoresis and parallel pyrosequencing, and its application for decoding the complete nucleotide sequence of the genome of the hyperthermophilic archaea Desulfurococcus kamchatkensis. The D. kamchatkensis strain used in this study, stored in the collection of the Vinogradskii Institute of Microbiology, Russian Academy of Sciences (no. 1221n), was isolated from precipitates of the Treshchina hot spring of the Uzon Volcano caldera on the Kamchatka Peninsula. Cells of this microorganism are representative of cocci approximately 1 µ m in diameter, which have no flagella or pili. Strain 1221n grows under strictly anaerobic conditions at temperatures from 65 to 89 ° C (optimum temperature, 85 ° C) in the pH range from 5.5 to 7.5. An important characteristic of this strain is its ability to utilize various protein substrates—tryptone, peptone, yeast extract, meat extract, albumin, and even barely hydrolysable proteins, such as α -keratins—as sources of carbon and energy. In addition, this microorganism is able to utilize arabinose, agarose, and chitin as growth substrates.