Molecular Breeding of an Extreme Thermophile Thermus thermophilus HB27
Takayuki HOSHINO*
Institute of Applied Biochemistry, University of Tsukuba, 1-1-1 Ten-nodai, Tsukuba, Ibaraki 305, Japan
Thermus thermophilus is a Gram-negative, aerobic eubacterium which can grow at temperatures ranging from 50 to 82℃. The organism produces carotenoids. We have determined the carotenogenic pathway of T. thermophilus HB27 (1). The organism is capable of incorporating foreign DNA efficiently via natural transformation system. We have already elucidated the basic mechanism of natural transformation system. We have also shown that transformation can be used as a tool for genetic fine mapping in T thermophilus (2).
Transformation efficiency with plasmid DNAs were relatively lower than that with chromosomal DNA. We have shown that transformation efficiency with plasmid DNAs can be raised by about 30 folds using the marker rescue transformation method (3). We constructed a promoter probe vector and performed a systematic screening and analysis of promoters. The consensus sequence for strong T. thermophilus promoters has been clarified (4). Kanamycin resistance (KmR) gene, the only antibiotic resistance gene for T. thermophilus, was expressed in T. thermophilus cells at temperatures of up to 60℃. We have shown that fusion of the ribosomal protein L32 to the N-end of the KmR gene product increased the thermostability of the KmR gene product in T. thermophilus (5). The new T. thermophilus expression vectors carrying the heat-resistant KmR gene and cloning sites downstream of the strong promoter sequences have been constructed.
The physical maps of the HB27 chromosome (1.82 Mb) and large plasmid pTT27 (250 kb) have been constructed (6). A total of 49 cleavage sites with five restriction enzymes and the location of 61 genes are determined on the maps. Comparison of the genomic organization of the chromosomes of HB27 and T. thermophilus HB8 revealed that they were basically identical, but some genes were located in different regions. The IS1000 sequences were found at such regions in HB8 chromosome, suggesting that IS 1000 may play an important role in genomic rearrangements in Thermus strains.
Since T. thermophilus takes up double-stranded DNAs of any species at almost same efficiencies, even a non-cognate DNA fragment should be incorporated into the genome if homologous sequence(s) is added to the fragment. To confirm this possibility, we inserted a kanamycin resistance (KmR) gene cassette into various
