Random transposon mutagenesis led to the isolation of a novel Mesorhizobium loti mutant that is defective in nitrogen fixation during symbiosis with Lotus japonicus. The mutated locus, designated cep, encodes a putative cell-envelope protein displaying no significant sequence similarity to proteins with known functions. This mutant elicits the formation of nodule-like bumps and root-hair curling, but not the elongation of infection threads, on L. japonicus roots. This is reminiscent of the phenotypes of rhizobial mutants impaired in cyclic b-glucan biosynthesis. The cep mutant exhibits partially reduced content of cell-associated glucans and intermediate deficiency of motility under hypo-osmotic conditions as compared to a glucan-deficient mutant. Second-site pseudorevertants of the cep mutant were isolated by selecting for restoration of symbiotic nitrogen fixation. A subset of pseudorevertants restored both symbiotic capability and glucan content to levels comparable to that of the wild-type. These results suggest that the Cep product acts on a successful symbiosis by affecting cell-associated glucan content.
INTRODUCTIONMesorhizobium loti is a symbiotic partner of Lotus japonicus, a model legume widely used for molecular genetic studies. It dwells in the soil and, on its host plant, elicits the formation of root nodules through a complex interaction between the two partners. Specific lipochitooligosaccharides produced by rhizobia, called Nod factors, have been well documented for many rhizobial species, including M. loti; these compounds trigger root-hair curling and nodule organogenesis in a host-specific manner (Hadri & Bisseling, 1998;Niwa et al., 2001). Rhizobia colonize the curled root hairs and invade the developing nodules via infection threads (ITs), which are formed by invagination of the root-hair cell membrane. Rhizobial cells are finally released into the host nodule cells, where they carry out symbiotic nitrogen fixation. The specificity between rhizobia and host legumes is also exhibited at stages later than those at which Nod factors act. This is exemplified in the symbiotic phenotype of Rhizobium etli on L. japonicus, in which early nodule senescence occurs despite the fact that the Nod factors from M. loti and R. etli have the same structure (Banba et al., 2001). To fully understand the molecular basis for the interaction between the model symbiotic pair M. loti and L. japonicus, additional symbiotic genes need to be identified extensively in the M. loti genome.In rhizobia, the cell envelope should first come to the bacterium-plant interface and mediate communication with the host through attachment or signal exchange. Also, the cell envelope contains essential machinery for nutrient uptake and electron transport, some components of which play specific roles in symbiosis (Delgado et al., 1998;Lodwig & Poole, 2003). As constituents of the cell envelope, cell-surface carbohydrates such as lipopolysaccharides, extracellular polysaccharides, capsular polysaccharides and cyclic b-glucans are known...