Enhanced nitrogenase activity in strains of Rhodobacter capsulatus that overexpress the rnf genes

In the photosynthetic bacterium Rhodobacter capsulatus, a putative membrane -bound complex encoded by the rnfABCDGEH operon is thought to be dedicated to electron transport to nitrogenase, In this study, the whole rnf operon w as cloned under the control of the nifH promoter in plasmid pNR117 and expr essed in several rnf mutants. Complementation analysis demonstrated that tr ansconjugants which integrated plasmid pNR117 directed effective biosynthes is of a functionally competent complex in R. capsulatus. Moreover, it was f ound that strains carrying pNR117 displayed nitrogenase activities 50 to 10 0% higher than the wild-type level. The results of radioactive labeling exp eriments indicated that the intracellular content of nitrogenase polypeptid es was marginally altered in strains containing pNR117,whereas the levels o f the RnfB and RnfC proteins present in the membrane were four- and twofold , respectively, higher than the wild-type level. Hence, the enhancement of in vivo nitrogenase activity was correlated with a commensurate overproduct ion of the Rnf polypeptides. In vitro nitrogenase assays performed in the p resence of an artificial electron donor indicated that the catalytic activi ty of the enzyme was not increased in strains overproducing the Rnf polypep tides. It is proposed that the supply of reductants through the Rnf complex might be rate limiting for nitrogenase activity in vivo. Immunoprecipitati on experiments performed on solubilized membrane proteins revealed that Rnf B and RnfC are associated with each other and with additional polypeptides which may be components of the membrane-bound complex.