Determination of C/N ratios required for de-repression of nitrogenase in Rhodobacter capsulatus

Phototrophic continuous and batch cultures of Rhodobacter capsulatus were e mployed to identify the C/N ratio above which nitrogenase is de-repressed. The cultures were grown with limiting amounts of ammonium as source of boun d nitrogen and with L-lactate or L-malate as sources of carbon and reducing equivalents. De-repression of nitrogenase was determined on the basis of t he occurrence of dinitrogen fixation, acetylene reduction and nifH promoter activities as well as on the basis of hydrogen evolution and nitrogenase p olypeptides. In continuous culture, cells started to fix dinitrogen, to red uce acetylene, to activate the nifH promoter and to form nitrogenase polype ptides, when consuming lactate per ammonium at a C/N ratio of about 6 (this ratio represents the number of C and N atoms consumed). With malate as car bon source all of the activities became detectable above a C/N ratio of abo ut 8. Essentially the same C/N ratios were determined with batch cultures f or the occurrence of N-limitation of growth and hydrogen evolution. The exp erimentally determined C/N ratios for nitrogenase de-repression essentially agreed with C/N ratio of 5.8 and 7.8 calculated for the assimilation of am monium and either lactate or malate, into biomass of an elemental compositi on of CH1.83N0.183O0.5 This means that the occurrence of N-limitation and n itrogenase de-repression is defined by a threshold C/N ratio required for b iomass production. As experimentally and theoretically shown, this ratio de pends on the reduction state of the carbon source. It is concluded that the C/N ratio of nutrient consumption represents an intracellular signal which is directly translated into nitrogenase de-repression.