INSERTIONAL MUTAGENESIS IN THE EXTREME THERMOPHILIC EUBACTERIA THERMUS-THERMOPHILUS HB8

The transcription and translation signals of the S-layer gene (slpA) from Thermus thermophilus HB8 have been used to express a thermostable kanamycin adenyl transferase gene in this organism. The chimaeric resistance gene was inserted in vitro into slpA to produce different inactive forms of the gene, which were used to transform T. thermophilus HB8. After 48 hours of incubation at 70-degrees-C, only two constructions that contained the kat gene flanked by Thermus sequences from both sides of slpA were able to produce protein layer (P100)-defective mutants. The mutants obtained with both constructions showed identical protein patterns, in which a major 50 kDa protein and two other minor proteins were tentatively identified as P100 fragments, expressed from the extreme 5' end of slpA. They also exhibited important phenotypic defects, such as slow growth in liquid broth, a tendency to aggregate as 'rotund bodies', a twisted filamentous shape, and an extreme sensitivity to lysozyme, suggesting protective and shaping roles for the S-layer in T. thermophilus HB8. These results also demonstrate for the first time the feasibility of using selective antibiotic-resistance markers in extreme thermophiles.