Response of the Endophytic Diazotroph Gluconacetobacter diazotrophicus on Solid Media to Changes in Atmospheric Partial O2 Pressure

摘要

Gluconacetobacter diazotrophicus is an N2-fixing endophyte isolated from sugarcane. G. diazotrophicus was grown on solid medium at atmospheric partial O2 pressures (pO2) of 10, 20, and 30 kPa for 5 to 6 days. Using a flowthrough gas exchange system, nitrogenase activity and respiration rate were then measured at a range of atmospheric pO2 (5 to 60 kPa). Nitrogenase activity was measured by H2 evolution in N2-O2 and in Ar-O2, and respiration rate was measured by CO2 evolution in N2-O2. To validate the use of H2 production as an assay for nitrogenase activity, a non-N2-fixing (Nif⁻) mutant of G. diazotrophicus was tested and found to have a low rate of uptake hydrogenase (Hup⁺) activity (0.016 ±  0.009 μmol of H2 10¹⁰ cells⁻¹ h⁻¹) when incubated in an atmosphere enriched in H2. However, Hup⁺ activity was not detectable under the normal assay conditions used in our experiments. G. diazotrophicus fixed nitrogen at all atmospheric pO2 tested. However, when the assay atmospheric pO₂ was below the level at which the colonies had been grown, nitrogenase activity was decreased. Optimal atmospheric pO₂ for nitrogenase activity was 0 to 20 kPa above the pO₂ at which the bacteria had been grown. As atmospheric pO₂ was increased in 10-kPa steps to the highest levels (40 to 60 kPa), nitrogenase activity decreased in a stepwise manner. Despite the decrease in nitrogenase activity as atmospheric pO₂ was increased, respiration rate increased marginally. A large single-step increase in atmospheric pO₂ from 20 to 60 kPa caused a rapid 84% decrease in nitrogenase activity. However, upon returning to 20 kPa of O₂, 80% of nitrogenase activity was recovered within 10 min, indicating a “switch-off/switch-on” O₂ protection mechanism of nitrogenase activity. Our study demonstrates that colonies of G. diazotrophicus can fix N₂ at a wide range of atmospheric pO₂ and can adapt to maintain nitrogenase activity in response to both long-term and short-term changes in atmospheric pO₂.