SITE-DIRECTED MUTAGENESIS OF THE BETA-(1-]3),BETA-(1-]6)-D-GLUCAN SYNTHESIS LOCUS OF BRADYRHIZOBIUM-JAPONICUM

Bradyrhizobium japonicum produces beta-(1-->3),beta-(1-->6)-D-glucans, which appear to be functionally equivalent to beta-(1-->2)-D-glucans produced by Rhizobium and Agrobacterium spp. These compounds are involved in osmoregulation and may play an important role in symbiosis and tumorigenesis. We previously isolated a cosmid clone from a Bradyrhizobium japonicum USDA 110 gene Library coding for proteins involved in the synthesis of beta-(1-->3),beta-(1-->6)-D-glucans (Bhagwat et al. FEMS Microbiol, Lett. 114:139-144, 1993), Site-directed Tn5 mutagenesis was used to delimit the region coding for beta-(1-->3),beta-(1-->6)-D-glucan synthesis, The Tn5 insertion and flanking DNA region was homogenotized into the chromosome of B. japonicum USDA 110. A recombinant strain was isolated which was defective in synthesis of beta-(1-->3),beta-(1-->6)-D-glucans, The membrane fraction also failed to incorporate UDP-(C-14)glucose into soluble, neutral glucans, The mutant was defective in motility and growth under conditions of low osmolarity and formed ineffective nodules on Glycine mar (soybean), These results indicate that the cyclic beta-(1-->3),beta-(1-->6)-D-glucans may be required for an effective symbiosis in B. japonicum.