In the past, it has also been used with several haloarchaeal species including H. volcanii and it was shown that it also induces oxidative stress at the high salt concentrations of the haloarchaeal cytoplasm (May & Dennis, 1989; May et al., 1989; Joshi & Dennis, 1993). Paraquat was added in concentrations from 1 μM to 4 mM to exponentially growing cultures of H. volcanii (Fig. 4). All cultures continued to grow for several hours and then exhibited a difference from the nonstressed
control. The growth curves after the addition of paraquat in concentrations from 1 to 100 μM were identical, the cultures entered the transition phase earlier than the wild type, but the final growth yields were the same as that of the wild type. The selleck inhibitor growth yields of cultures after the addition of 1 mM or higher concentrations of paraquat were lower than that of the wild type. The effect was relatively mild after the addition of 1 and 2 mM paraquat, in contrast to the addition of 4 mM, which led to a considerable reduction in growth yield (<50% after paraquat addition compared with the nonstressed control). The next application was the optimization of the supplementation of amino acid auxotrophic mutants. The tryptophan auxotrophic mutant H53 with
a deletion of the trpA gene was compared with the tryptophan prototrophic strain selleck H26 (Fig. 5a). Growth of the prototrophic strain is independent of the addition of tryptophan and the growth curves in the absence of tryptophan and in the presence of three different concentrations were absolutely identical. In contrast, mutant H53 was totally unable to grow without tryptophan addition. Growth after the addition of 2 and 10 μg mL−1 was strictly tryptophan limited, while the growth yield after the addition of 50 μg mL−1 was the same as that of the prototrophic strain and thus growth could fully be supplemented. Unexpectedly, the auxotrophic
mutant H53 grew faster than the prototrophic strain H26 after the addition of 10 and 50 μg mL−1 tryptophan. As already mentioned, the growth rate of the prototrophic strain was not influenced by tryptophan addition. PIK3C2G It seems that H. volcanii does not benefit from external tryptophan as long as the biosynthesis gene trpA is intact, in contrast to the expectation that saving of the energetically very costly tryptophan biosynthesis would be beneficial. Another unexpected result was obtained as the leucine auxotrophic mutant H66 with a deletion of the leuB gene was supplemented with leucine (Fig. 5b). Again, the growth curves of the prototrophic control strain H26 were independent of the addition of leucine. As expected, the auxotrophic mutant H66 was unable to grow in the absence of leucine.