Site-Directed Mutagenesis of the Beta-(1®3), Beta-(1®6)-D-Glucan Synthesis Locus of Bradyrhizobium japonicum. Arvind A. Bhagwat. (1)Soybean and Alfalfa Research Laboratory, USDA, Agricultural Research Service, Bldg. 011, HH-19, BARC-W, Beltsville, MD 20705 U.S.A. (2)Department of Agronomy, University of Maryland, College Park, MD 20742 U.S.A. Donald L. Keister (1). (1) Soybean and Alfalfa Research Laboratory, USDA, Agricultural Research Service, Bldg. 011, HH-19, BARC-W, Beltsville, MD 20705 U.S.A. MPMI 8: 366-370. Accepted 2 February 1995. This article is in the public domain and not copyrightable. It may be freely reprinted with customary crediting of the source. The American Phytopathological Society, 1995.

Bradyrhizobium japonicum produces Beta-(1®3), Beta (1®6)-D-glucans, which appear to be functionally equivalent to Beta(1®2)-D-gIucans produced by Rhizobium and Agrobacte-rium spp. These compounds are involved in osmoregula-lion 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-(l®3), Beta-(l®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-(l4C)gIucose into soluble, neutral glucans. The mutant was defective in motility and growth under conditions of low osmolarity and formed ineffective nodules on Glycine max (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.