ACCESS TO AVAILABLE NITROGEN COMMONLY LIMITS PLANT GROWTH, AND THE AGRICULTURAL DEMAND FOR NITROGEN IS LARGELY SUPPLEMENTED WITH SYNTHETIC NITROGEN FIRTILIZERS THAT CAUSE DELETARIOUS ENVIORNMENTAL EFFECTS INCLUDING SOIL ACIDIIFICATION AND GREENHOUSE GAS EMISSIONS.DIAZOTROPHS ARE BACTERIA AND ARCHAEA THAT CATALYZE BIOLOGICAL NITROGEN FIXATION (BNF), USING THE ENZYME NITROGENASE TOREDUCEATMOSPHERIC NITROGEN INTO AMMONIA THAT PLANTS READILY INCORPORATE INTO BIOMOLECULES. CURRENTLY, THE IMPRINT OF BNF IN AGRICULTURE IS LIMITED TO LEGUMINOUS CROPS THAT FORM ROOT NODULES TOFACILITATE AN ENVIRONMENT SUITABLE FOR NITROGEN FIXATION. HOWEVER, FREE-LIVING DIAZOTROPHS DO NOT REQUIRE NODULES TO FIX NITROGEN. WHILE SOME FREE-LIVING DIAZOTROPHS HAVE BEEN DEMONSTRATED TO SUPPLY NITROGEN TO PLANTS, THERE IS A STRONG SELECTIVE PRESSURE FOR THESE ORGANISMS TO KEEP NITROGEN FIXATION TIGHTLY REGULATED. THEREFORE, UNDERSTANDING HOW THESE FREE-LIVING DIAZOTORPHS REGULATE BNF OFFERS THE POTENTIAL TO EXPAND THE IMPRINT OF BNF IN AGRICULTURE,REDUCING OUR DEPENDENCE ON SYNTHETIC FERTILIZERS AND CONSEQUENTLY IMPROVING LAND STEWARDSHIP AND AGRICULTURAL SUSTAINABILITY.IN THE FREE-LIVINGDIAZOTROPHAZOTOBACTER VINELANDII, NITROGENASE EXPRESSION ISREGULATED BY THE TWO-COMPONENT NIFL-NIFA SYSTEM. IN THIS SYSTEM,NIFA IS THE TRANSCRIPTIONAL ACTIVATOR OF NITROGENASE, AND NIFL INHIBITS NITROGENAES EXPRESSION BY BINDINGNIFAIN CONDITIONS UNSUITABLE FOR NITROGEN FIXATION. WHILE WE KNOW THE PLAYERS IN THIS SYSTEM, NIFL-NIFA PROTEIN INTERACTIONS AND THE THE MECAHNISMS BY WHICH NIFL TRANSDUCES AND INTEGRATES SIGNALS TO MODULATE NITROGENASE EXPRESSION REMAIN UNCLEAR. THEREFORE, THIS PROJECT SEEKS TO IDENTIFY THE GENETIC REQUIREMENTS FOR NIFL SIGNAL TRANSDUCTION IN LIVING CELLS AND THE PROTEIN BIOCHEMISTRY OF SIGNAL PERCEPTION, TRANSDUCTION, AND INTEGRATION. SUCCESSFUL EXECUTION OF THE GOALS OF THIS PROJECT WILL INFORM THE MECHANISMS OF NITROGENASE REGULATION A FREE-LIVING DIAZOTROPH,KNOWLEDGE THAT IS REQUIRED TO ENGINEER HIGH-PERFORMANCE BIOFERTILIZERS FROM DIAZOTROPHS COMPATIBLE WITH A WIDE VARIETY OF CROPS.