In vitro activity of the nitrogen fixation regulatory protein NIFA

Abstract

We have detected activity of the nitrogen fixation regulatory protein NIFA of Klebsiella pneumoniae in vitro. To do so we directed synthesis of NIFA in a coupled transcription-translation system and detected its ability to activate expression of a translational fusion between the nifH and lacZ genes. We infer that NIFA stimulates initiation of transcription by sigma 54 holoenzyme from the nifHDK promoter. The activity of NIFA was lost rapidly under both aerobic and anaerobic conditions at 30 degrees C and was lost somewhat less rapidly at 0 degrees C. Loss of activity was not accompanied by degradation of NIFA polypeptide. Loss of activity was approximately exponential and was not affected by NIFA concentration over a 5-fold range. Therefore, NIFA inactivation does not appear to be due to self-association. We found that the factor in crude extracts previously demonstrated to bind to the nifHDK promoter-regulatory region [Beynon, J., Cannon, M., Buchanan-Wollaston, V., and Cannon, F. (1983) Cell 34, 665-671] is the integration host factor, which is known to bend DNA. Since the binding site for integration host factor lies between the upstream binding site for NIFA and the nifHDK promoter, integration host factor may bend the DNA between these two sites to facilitate productive interactions between NIFA and sigma 54 holoenzyme.