Poster Session
S1 Genetics and Molecular Biology
S2 Structure and Function of Proteins
S3 Physiology and Metabolisms
S4 Ecology and Diversity
S5 Genome
Exhibition
January 19, Monday 13:15
to
January 20, Tuesday 16:00
Obligation time
January 19, Monday 13:15〜14:00 (odd number)
January 19, Monday 14:00〜14:45 (even number)
Development of a Gene Manipulation System for an Extreme Thermohile, Thermus thermophilus, and Its Application to the Screening of Stabilized Proteins
Masatada TAMAKOSHI*, Akihiko YAMAGISHI, and Tairo OSHIMA
Department of Molecular Biology, Tokyo University of Pharmacy and Life Science, 1432 Horinouchi, Hachioji, Tokyo 192-03 Japan
Thermus thermophilus is a gram-negative aerobic eubacterium which can grow at temperatures up to 85℃. Genetic manipulation of the thermophile has many potential applications for basic as well as applied research. Homologous recombination occurs at high frequencies in the thermophile. We have developed integration vector systems for the thermophile by using leuB or pyrE genes as a marker. Integration occurred by single or double crossing over events at defined sites. The vector integrated through a single crossover could be recovered into Escherichia coli after recircularization in vitro (1). We have succeeded in selecting stabilized mutant enzymes with the system (2,3,4).
We have also developed a plasmid vector system in which the plasmid vector can be maintained stably without the integration into the chromosome. A host strain in which the leuB gene was totally deleted was constructed by means of sequential recombinations of the pyrE marker gene, which is an efficient method for gene targeting in microorganisms and lower eukaryoteS. The plasmid vector was constructed by ligating pTT8, a cryptic plasmid in T. thermophilus HB8, a synthetic promoter designed for the gene expression in the thermophile, T. thermophilus leuB or pyrE gene as a marker and an Escherichia coli plasmid vector, pUC119. No recombination between the plasmid vector and the chromosomal DNA has been observed for several months. The transformant with the plasmid vector expressing E. coli leuB gene, which encodes 3-isopropylmalate dehydrogenase (IPMDH), a member of the leucine biosynthetic pathway could grow at higher temperature than the transformant with an integration vector. A plasmid with a mutant E. coli leuB gene which encodes a stabilized mutant enzyme with decreased activity could complement the leuB deficiency of the thermophile. The results show that the complementation with the integration vector system requires the higher activity of the gene product than that with the plasmid vector system. It implies that various stabilized enzymes even with decreased activity can be selected with the plasmid vector system.
The leu2 gene encodes IPMDH of an eukaryote, Saccharomyces cerevisiae. Very recently we have succeeded in expressing the eukaryotic gene in T. thermophilus
