Isolation of auxotrophic and antimetabolite-resistant mutants of the hyperthermophilic bacterium Thermotoga neapolitana

Antimetabolite-resistant and auxotrophic mutants of the hyperthermophilic bacterium Thermotoga neapolitana were isolated to provide strains with genetic backgrounds amenable to genetic analyses. Norleucine, azaleucine, 4-nitropyridine-N-oxide, and 3-amino-1, 2, 4-triazole did not affect growth, while 5-fluorouracil (5 g/ml), 5-methyltryptophan (250g/ml), 6-azauracil (100 g/ml), and 4-fluorophenylalanine (30 g/ml) inhibited growth at the indicated minimum inhibitory concentrations. The effect of 5-fluorouracil was analyzed and found to be bacteriostatic. These inhibitors were used to select spontaneously arising resistant mutants. In addition, auxotrophic mutants requiring leucine, tryptophan, adenine, and histidine were isolated following mutagenesis with ethyl methanesulfonate. Six other auxotrophs with undefined growth requirements were also isolated. These strains will be useful for the development of genetic methods for T. neapolitana.