The nifA gene product (NIFA protein) is required to activate transcription of the nitrogen fixation (nif) operons in a wide variety of gram-negative bacteria belonging to the phylum "Purple bacteria and their relatives". To activate, NIFA binds to sites 100 bp upstream of nif promoters and allows RNA polymerase sigma (54 holoenzyme form) to denature the DNA strands around the transcription startsite- that is, to isomerize from closed to open complexes. We succeeded in demonstrating activity of the NIFA protein from Klebsiella pneumonia in vitro by developing an assay that allowed us to detect activity in crude preparations. We found that NIFA is assisted in activating transcription by the integration host factor (IHF), a small protein that bends the DNA between upstream NIFA sites and nif promoters to increase contracts between the activator and polymerase. During the next grant period, we will attempt to purify active NIFA and study its properties-its DNA-binding and its presumed ATPase activity, which is rare for transcriptional activators. In addition, we will use electron microscopy to characterize specialized nucleoprotein structures (SNUPS) that form at nif promoters in the presence of activator, polymerase, and IHF. In the presence of the NIFL protein, Klebsiella NIFA is inactivated by molecular oxygen or combined nitrogen. We will attempt to demonstrate this inactivation convincingly in crude preparations and, using it as an assay, to purify and characterize NIFL.
