Characterization of ndvD,the third gene involved in the synthesis of cyclic beta-(1 --> 3),(1 --> 6)-D-glucans in Bradyrhizobium japonicum

Previously, we identified two genes in Bradyrhizobium japonicum (ndvB, ndvC) that are required for cyclic beta-(1 --> 3),(1 --> 6)-D-glucan synthesis and successful symbiotic interaction with soybean (Glycine max). In this study, we report a new open reading frame (ORF1) located in the intergenic region between ndvB and ndvC, which is essential for beta-glucan synthesis and effective nodulation of G. max. This new gene is designated ndvD (nodule development). The ndvD translation product has a predicted molecular mass of 26.4 kDa with one transmembrane domain. Genetic experiments involving gene deletion, Tn5 insertion, and gene complementation revealed that the mutation of ndvD generated pleiotropic phenotypes, including hypoosmotic sensitivity, reduced motility, and defects in conjugative gene transfer, in addition to symbiotic ineffectiveness. Although deficient in in vivo beta-glucan synthesis, membrane preparations from the ndvD mutant synthesized neutral beta-glucans in vitro. Therefore, ndvD does not appear to be a structural gene for beta-glucan synthesis. Our hypothesis for the mechanism of beta-(1 --> 3),(1 --> 6)-D-glucan synthesis is presented.