Constitutive nitrogenase synthesis from de novo transcribed mRNA in isolated Rhizobium leguminosarum bacteroids

Abstract

Nitrogenase synthesis was studied in Rhizobium leguminosarum by incubation of bacteroids isolated from Pisum sativum nodules in a medium containing succinate, myoglobin and [ 35S]methionine. Protein synthesis analysed by SDS-polyacrylamide gel electrophoresis and autoradiography revealed a strong nitrogenase synthesis in bacteroids isolated from root nodules 16 days after infection. The labeled proteins were identified as nitrogenase by immunoprecipitation with specific antisera. Nitrogenase synthesis appeared not to be repressed by oxygen concentrations up to 100 μM; neither was repression found using 25 mM NH + 4 or by NO − 2 or NO − 3 at 10 mM. The ionophores valinomycin, and triphenylphosphonium bromide do have an immediate effect on acetylene reduction by bacteroids but were found to have no repressive influence on nitrogenase structural gene expression. In bacteroids isolated 22 days after infection acetylene reduction under a low pO 2 was much stronger than at 15 days, but overall protein synthesis and label incorporated into nitrogenase proteins were reduced. Evidence is presented that synthesis of nitrogenase proteins in isolated bacteroids takes place by translation of de novo synthesized mRNA. By inhibiting RNA-transcription with rifampicin and assuming that the rate of nitrogenase synthesis at a given moment is proportional to the amount of nif mRNA present, the decay of nif mRNA was found to proceed with a biological half-life of approx. 1.6 min.