New investigations into oxygen-tolerant (superoxide-dependent) nitrogen fixation by Streptomyces thermoautotrophicus
Streptomyces thermoautotrophicus, a thermophilic chemoautotrophic bacterium, was first isolated from the soil covering a burning charcoal pile in Germany in 1990. The organism was later reported to fix nitrogen by a unique biochemical pathway devoid of traditional nitrogenase enzymes, which are inactivated in the presence of oxygen. In contrast, the proposed pathway for nitrogen fixation by S. thermoautotrophicus features oxygen-tolerant enzymes and actually relies on oxygen as an electron carrier through the formation and subsequent oxidation of a superoxide molecule. Despite the large consequences for nitrogen cycling, nothing has been published on superoxide-dependent nitrogen fixation since 2000 and cultures of S. thermoautotrophicus are no longer available in strain depositories. Here we sought to re-isolate S. thermoautotrophicus and related organisms from similar high-temperature, high-CO environments, with the goal of retesting the nitrogen-fixing ability of this organism and eventually outlining the role of superoxide-dependent nitrogen fixation in natural systems.
To re-isolate S. thermoautotrophicus, we obtained soil from a charcoal pile in Germany similar to that used for the original isolation of this organism. We also obtained soil from the Kilauea volcanic crater in Hawaii, and soil covering a coal-seam fire beneath Centralia Pennsylvania with the hope that these high-temperature, high-CO environments would harbor similar organisms. We were able to isolate strains of S. thermoautotrophicus from both Germany (strain H1) and Pennsylvania (strain P1-2), though we did not obtain cultures from Hawaii. Interestingly, we were able to obtain a sample of the original strain (UBT1) directly from another researcher as well. We incubated all strains in the presence of isotopically-labeled nitrogen gas to test for diazotrophy, the ability to fix nitrogen, by tracking isotopic incorporation into biomass. After repeated testing, we find no evidence that any strain of S. thermoautotrophicus is a diazotroph. Since S. thermoautotrophicus is the only organism thought to harbor this novel mechanism for nitrogen fixation, we further conclude that there is no evidence for the existence of superoxide-dependent diazotrophy as an alternative pathway for nitrogen fixation in natural systems.