Expression of the nodulation gene nodA in Rhizobium meliloti and localization of the gene product in the cytosol

Abstract

The nodA gene of Rhizobium meliloti encodes a 21.8-kDa protein, which is conserved in several Rhizobium species. We overproduced the nodA protein as a fusion product with a portion of the .lambda. cI repressor in Escherichia coli. This fusion protein was purified from inclusion bodies by gel and hydroxylapatite chromatography in the presence of NaDodSO4. Monospecific polyclonal antibodies against the hybrid protein were used to detect the nodA protein in the cytosol of E. coli and R. meliloti by immunoblotting. In contrast to experiments with antibodies against the R. meliloti nodC membrane protein, the alfalfa-R. meliloti nodulation was not affected by the addition of anti-nodA antibodies to medium and inoculum. This suggests that the nodA protein is located within the cell and is therefore not accessible to antibodies. The expression of the nodA gene is induced in R. meliloti by various compounds present in the exudate of leguminous plants, particularly by the flavone luteolin. We show that the plant hormone trigonelline also has some inducing activity. The nodC protein was further localized in the membrane fraction of R. meliloti. Our experiments demonstrate that the nodC transmembrane protein is not necessary for the uptake of the compounds inducing the synthesis of the nodA protein. The nodA and the nodC proteins were also detected in mature nodules. During nodule development, the nodC protein may be processed to a 34-kDa protein.