INTERACTIVE REGULATION OF AZORHIZOBIUM NIFA TRANSCRIPTION VIA OVERLAPPING PROMOTERS

The Azorhizobium nifA promoter (PnifA) is positively regulated by two physiological signal transduction pathways, NtrBC, which signals anabo lic N status, and FixLJK, which signals prevailing O-2 status. Yet, Pn ifA response (gene product per unit time) to these two activating sign als together is more than twice that of the summed, individual signals . In the absence of NIFA, a negative PnifA autoregulator, the fully in duced PnifA response is more than 10-fold greater than that of summed, individual signals. Given this synergism, these two signal transducti on pathways must interactively regulate PnifA activity. PnifA carries three cia-acting elements, an anaerobox, which presumably binds FIXK, a NIFAbox, which presumably binds NIFA itself, and a sigma(54)box, whi ch presumably binds sigma(54) initiator, a subunit of RNA polymerase. For combinatorial analysis, single, double, and triple promoter mutati ons were constructed in these cia-acting elements, and PnifA activitie s were measured in six different tr ans-acting backgrounds, i.e., fixK , fixJ, nifA, ntrC, rpoF, and wild type. Under all physiological condi tions studied, high-level PnifA activity required both FIXK in trans a nd the anaerobox element in cis. Surprisingly, because PnifA was hyper active with a mutated sigma(54)box, this cia-acting element mediates b oth negative and positive control. Because PnifA hyperactivity also re quired a wild-type upstream NIFAbox element, even in the absence of NI FA, a second upstream nifA transcription start superimposed on the NIF Abox element was hypothesized. When nifA mRNA 5' start points were map ped by primer extension, both a minor upstream transcript(s) starting 45 bp distal to the anaerobox and a major downstream transcript starti ng 10 bp distal to the sigma(54)box were observed. In Azorhizobium, RN A polymerase sigma(54) initiator subunits are encoded by a multigene f amily, which includes rpoF and rpoN genes. Because rpoF mutants show a n Ntr(+) phenotype, whereas rpoN mutants are Ntr(-), multiple sigma(54 ) initiators are functionally distinct. Two independent rpoF mutants b oth show a tight Nif(-) phenotype. Moreover, rpoF product sigma(54)F i s absolutely required for high-level PnifA activity. In summary, the A zorhizobium nifA gene carries overlapping housekeeping-type and sigma( 54)-type promoters which interactively respond to different signals. E ffectively, the upstream, housekeeping-type promoter responds to FIXK and positively regulates the downstream, sigma(54)-type promoter. The downstream, sigma(54)-type promoter responds to NTRC and negatively re gulates the upstream, housekeeping-type promoter. In terms of transcri pt yield, the upstream, housekeeping-type promoter is therefore weak, and the downstream, sigma(54)-type promoter is strong.