A CHROMOSOME INTEGRATION SYSTEM FOR STABLE GENE-TRANSFER INTO THERMUS-FLAVUS

We have developed a chromosomal integration system for gene transfer i nto the extreme thermophile Thermus flavus. The system relies on integ ration 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 i n single-copy into the chromosome of T. flavus on a plasmid vector. Ge ne replacement strains required leucine for growth, were stably kanamy cin-resistant and could grow in the presence of kanamycin at temperatu res up to 55 degrees C.