Response of the Endophytic Diazotroph Gluconacetobacter diazotrophicus on Solid Media to Changes in Atmospheric Partial O 2 Pressure

Abstract

ABSTRACT Gluconacetobacter diazotrophicus is an N 2 -fixing endophyte isolated from sugarcane. G. diazotrophicus was grown on solid medium at atmospheric partial O 2 pressures (pO 2 ) 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 pO 2 (5 to 60 kPa). Nitrogenase activity was measured by H 2 evolution in N 2 -O 2 and in Ar-O 2 , and respiration rate was measured by CO 2 evolution in N 2 -O 2 . To validate the use of H 2 production as an assay for nitrogenase activity, a non-N 2 -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 H 2 10 10 cells −1 h −1 ) when incubated in an atmosphere enriched in H 2 . However, Hup + activity was not detectable under the normal assay conditions used in our experiments. G. diazotrophicus fixed nitrogen at all atmospheric pO 2 tested. However, when the assay atmospheric pO 2 was below the level at which the colonies had been grown, nitrogenase activity was decreased. Optimal atmospheric pO 2 for nitrogenase activity was 0 to 20 kPa above the pO 2 at which the bacteria had been grown. As atmospheric pO 2 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 pO 2 was increased, respiration rate increased marginally. A large single-step increase in atmospheric pO 2 from 20 to 60 kPa caused a rapid 84% decrease in nitrogenase activity. However, upon returning to 20 kPa of O 2 , 80% of nitrogenase activity was recovered within 10 min, indicating a “switch-off/switch-on” O 2 protection mechanism of nitrogenase activity. Our study demonstrates that colonies of G. diazotrophicus can fix N 2 at a wide range of atmospheric pO 2 and can adapt to maintain nitrogenase activity in response to both long-term and short-term changes in atmospheric pO 2 .