A chromosome integration system for stable gene transfer into Thermus flavus.

We have developed a chromosomal integration system for gene transfer into the extreme thermophile Thermus flavus. The system relies on integration at the site of leuB (3-isopropylmalate dehydrogenase) which was cloned from T. flavus. The leuB gene was insertionally inactivated in vitro with a thermostable kanamycin-resistance gene and transformed in single-copy into the chromosome of T. flavus on a plasmid vector. Gene replacement strains required leucine for growth, were stably kanamycin-resistant and could grow in the presence of kanamycin at temperatures up to 55 degrees C.