The basis of ammonium release in nifL mutants of Azotobacter vinelandii

In Azotobacter vinelandii, nitrogen fixation is regulated at the transcript ional level by an unusual two-component system encoded by nifLA. Certain mu tations in nifL result in the bacterium releasing large quantities of ammon ium into the medium, and earlier work suggested that this occurs by a mecha nism that does not involve NifA, the activator of nif gene transcription, W e have investigated a number of possible alternative mechanisms and find no evidence for their involvement in ammonium release. Enhancement of NifA-me diated transcription, on the other hand, by either elimination of nifL or o verexpression of nifA, resulted in ammonium release, correlating with enhan ced levels of nifH mRNA, raised levels of nitrogenase and acetylene-reducin g activity, and increased concentrations of intracellular ammonium, Up to 3 5 mM ammonium can accumulate in the medium. Where measured, intracellular l evels exceeded extracellular levels, indicating that rather than being acti vely transported, ammonium is lost from the cell passively, possibly by rev ersal of an NH4+ uptake system. The data also indicate that in the wild typ e the bulk of NifA is inactivated by NifL during steady-state growth on din itrogen.