Antioxidant pathways are up-regulated during biological nitrogen fixation to prevent ROS-induced nitrogenase inhibition in Gluconacetobacter diazotrophicus

Abstract

Gluconacetobacter diazotrophicus, an endophyte isolated from sugarcane, is a strict aerobe that fixates N(2). This process is catalyzed by nitrogenase and requires copious amounts of ATP. Nitrogenase activity is extremely sensitive to inhibition by oxygen and reactive oxygen species (ROS). However, the elevated oxidative metabolic rates required to sustain biological nitrogen fixation (BNF) may favor an increased production of ROS. Here, we explored this paradox and observed that ROS levels are, in fact, decreased in nitrogen-fixing cells due to the up-regulation of transcript levels of six ROS-detoxifying genes. A cluster analyses based on common expression patterns revealed the existence of a stable cluster with 99.8% similarity made up of the genes encoding the α-subunit of nitrogenase Mo-Fe protein (nifD), superoxide dismutase (sodA) and catalase type E (katE). Finally, nitrogenase activity was inhibited in a dose-dependent manner by paraquat, a redox cycler that increases cellular ROS levels. Our data revealed that ROS can strongly inhibit nitrogenase activity, and G. diazotrophicus alters its redox metabolism during BNF by increasing antioxidant transcript levels resulting in a lower ROS generation. We suggest that careful controlled ROS production during this critical phase is an adaptive mechanism to allow nitrogen fixation.

Fig. 1

ROS levels are decreased in nitrogen-fixing cells. G. diazotrophicus grown under fixing (FIX) and non-fixing (NFIX) conditions were stained with CM-H₂DCFDA, sensitive to ROS. a, c, e and g represent bright field images. b, d, f, h are the fluorescent images, an indicative of the presence of ROS. All images were acquired with the same exposure time to allow comparison of signal intensities. Scale bar 10 μm

Fig. 2

Expression and activity of ROS detoxification in G. diazotrophicus during nitrogen fixation. a Catalase activity of cells grown in fixing and non-fixing conditions. b mRNA expression of the genes involved in ROS-detoxification from fixing and non-fixing cells. The data are expressed as the relative expression of the respective mRNAs normalized to the housekeeping gene 23S. The data are expressed as the average of three replicates ± error bars

Fig. 3

Cluster analysis of redox genes. Notice that there are two major clusters: one formed by gorB alone and the other formed by gorA, kat, katC, nifD, sodA, and katE, with similarities up to 98% inside this group. This last cluster can be divided in three minor clusters kat + gorA, katC and katE + sodA + nifD

Fig. 4

ROS effect on cell growth and BNF activity. a Effect of different concentrations of PQ on the growth of fixing (black) and non-fixing (gray) cells. After an initial growth period of 24 h, PQ was added at a concentration of 0.5 mM or 5 mM (dashed and dotted lines, respectively). b Acetylene reduction activity (ARA) of G. diazotrophicus after 72 h cultivation. The activity was measured after 48 h in the presence of 0.5 and 5 mM of PQ