Disruption of Thermus thermophilus genes by homologous recombination usinga thermostable kanamycin-resistant marker

Genes of an extremely thermophilic bacterium, Thermus thermophilus, were di srupted by homologous recombination using a recently developed, thermostabl e kanamycin-resistant marker. First, the trpE gene was disrupted with vario us constructions of DNA. The transformation efficiency was exponentially in creased as the length of the homologous regions flanking the marker gene in creased above the minimum length (200-300 bp). We then disrupted five genes of the nucleotide excision repair system and examined their phenotypes. Th e convenience and high reliability of this method should prompt its applica tion to the high-throughput systematic disruption of the genes of this ther mophilic bacterium. (C) 2001 Federation of European Biochemical Societies. Published by Elsevier Science B.V. All rights reserved.