DOE Human Genome Program Contractor-Grantee Workshop VIII
February 27-March 2, 2000  Santa Fe, NM

118. The Haloferax volcanii Genome Project

Rajendra J. Redkar, Joe J. Shaw, Gary G. Bolus, Mary Lee Ferguson, Troy A. Horn, and Vito G. Delvecchio

Institute of Molecular Biology and Medicine, University of Scranton, Corner of Monroe and Ridge Row, Scranton, PA 18510

Vimbm@aol.com

Haloferax volcanii is a salt-loving archaeon belonging to the family Halobacteriaceae. Halophilic archaea exhibit obligate halophilism and require 2-5 M salt concentration for viability. At lower concentrations of salt (about 1 M), cells become distorted, leading to cell lysis and death. The cytoplasm of halophiles contains very high internal concentrations of K+ and Na+, and is iso-osmotic with the environment. These bacteria have evolved metabolic and synthetic machinery that functions at high concentrations of salts, concentrations that are typically lethal for other organisms. Future research on halophilic archaea will provide better insight into early evolution of microorganisms and fundamental knowledge of biochemical and genetic events in organisms living in extreme environments.

H. volcanii cells are disk shaped and show involuted forms in the presence of NaCl. H. volcanii is a chemoorganotroph requiring complex nutrient medium and 1.5-2.5 M NaCl for growth. Cultures may be grown in the laboratory at 37°C with gentle shaking, however better growth is achieved at 42°C. In the laboratory, H. volcanii produces a characteristic pink pigment. Overall, the organism can be easily cultivated and maintained in the laboratory. Moreover, auxotrophic mutants are available, the cells are easily transformable, and genetic manipulation systems such as shuttle vectors and expression vectors are available for functional studies.

The genome size of the H. volcanii is estimated to be ~4.2 Mb. About 90% of the genome has 65% (G + C) content while remaining genome has 55% (G + C) content. The genome is composed of a chromosome (2,920 kb) and 4 plasmids, viz. pHV1 (86 kb), pHV2 (6.4 kb), pHV3 (442 kb) and pHV4 (690 kb). Plasmid pHV3 was selected for the first phase of sequencing operation. Sixteen overlapping cosmids were supplied by our collaborator Dr. Robert Charlebois, University of Ottawa, Ottawa, Canada, and used to make individual shotgun libraries. The average size of inserts in these randomly fragmented libraries is 2-3 kb. The sequencing reactions were performed on both ends of the clones using Big Dye Terminator chemistry to achieve 6-8X coverage. The data has been collated into several large assemblies using different software packages and the gaps in the assemblies are being located for closure. The annotation of data has started simultaneously to identify genes on pHV3. A progress report and the future plans on H. volcanii sequencing project will be presented.