Inhibition of Biosynthesis and Activity of Nitrogenase in Azospirillum brasilense Sp7 Under Salinity Stress

Azospirillum brasilenseis a microaerophilic, plant growth-promoting bacterium, whose nitro- genase activity has been shown to be sensitive to salinity stress. Growth of A. brasilense in semi-solid medium showed that diazotrophic growth in N-free medium was relatively less sensitive to high NaCl concentrations (200 - 400 mM) than that in presence of NH4 . Increase in salinity stress to diazotrophic A. brasilense in the semi-solid medium led to the migration of the pellicle to deeper anaerobic zones. Assays of acetylene reduction and nifH-lacZ and nifA-lacZ fusions indicated that salinity stress inhibited nitrogenase biosynthesis more strongly than nitrogenase activity. Under salt stress, the amount of dinitrogenase reductase inactivated by ADP-ribosylation was strongly reduced, indicating that the dinitrogenase reductase ADP ribosyl transferase (DRAT) activity was also inhibited by increased NaCl concentrations. Movement of the pellicle to the anaerobic zone and inhibition of DRAT might be adaptive responses of A. brasilense to salinity stress under diazotrophic conditions. Supplementation of glycine betaine, which alleviates salt stress, partially reversed both responses. Bacteria of the genus Azospirillum colonize the rhizo- sphere of several important cereals and grasses and are regarded as plant growth-promoting rhizobacteria (13). Azospirillum brasilense is a microaerophilic, nitrogen- fixing bacterium. Since microaerobic conditions are typ- ical of the plant rhizosphere, aerotaxis plays an important role in attracting azospirilla to plant roots and establish- ing interrelationships between bacteria and plants (12). It has been shown that Azospirillum brasilense generates maximum energy (the proton motive force) at a specific low oxygen concentration that appears to be optimal for both chemotaxis to oxygen and nitrogen fixation (26). The preferred oxygen concentration for aerotaxis (3-5 M )i nA. brasilense coincides with a preferred oxygen concentration for nitrogen fixation (2- 8 M). This sup- ports the hypothesis that aerotaxis, in A. brasilense ,i s an adaptive response to optimize the efficiency of nitrogen fixation. The cells of A. brasilense accumulate in a form of pellicle at the oxygen concentration that supports the maximum proton motive force in the cells (26). This