Rhizobium meliloti genes required for C4-dicarboxylate transport and symbiotic nitrogen fixation are located on a megaplasmid

Abstract

A mutant of Rhizobium melioti unable to transport C4 dicarboxylates (dct) was isolated after TnS mutagenesis. The mutant, 4F6, could not grow on aspartate or the tricarboxylic acid cycle intermediates succinate, fumarate, or malate. It produced symbiotically ineffective nodules on Medicago sativa in which bacteroids appeared normal, but the symbiotic zone was reduced and the plant cells contained numerous starch granules at their peripheries. Cosmids containing the dct region were obtained by selecting those which restored the ability of 4F6 to grow on succinate. The Tn5 insertion in 4F6 was found to be within a 5.9-kilobase (kb) EcoRI fragment common to the complementing cosmids. Site-specific Tn5-mutagenesis revealed dct genes in a segment of DNA about 4 kb in size extending from within the 5.9-kb EcoRI fragment into an adjacent 2.9-kb EcoRI fragment. The 4F6 mutation was found to be in a complementation group in which mutations yielded a Fix-phenotype, whereas other dct mutations in the region resulted in mutants which produced effective nodules in most, although not all, plant tests (partially Fix-). The dct region was found to be located on a megaplasmid known to carry genes required for exopolysaccharide production.

The product of the Rhizobium meliloti ilvC gene is required for isoleucine and valine synthesis and nodulation of alfalfa

“…We are currently exploring this possibility. Overall, the present results represent another example in which R. meliloti utilizes the general metabolic pathways that are common to a larger number of bacteria for the active expression of specific symbiotic functions (15,25,36,38,48).…”

“…Mutants defective in exopolysaccharide synthesis (24,25) or synthesis and export of P-(1,2)-glucan (18,41) induce small bacterium-free nodules. Mutations affecting defined metabolic functions can cause a Fix phenotype (15,36,48).…”

The complete sequence of the 1,683-kb pSymB megaplasmid from the N 2 -fixing endosymbiont Sinorhizobium meliloti

“…Other predicted solutes include iron (4), amino acids (6), peptides and oligopeptides (6), spermidine/putrescine (2), sulfate (1), aliphatic sulfonate (1), phosphate (1), choline (1), glycerol-3-phosphate (1), rhizopine (1), and taurine (1). Although a number of genes show similarity to C 4 -dicarboxylate transporters, they are unlikely to transport C 4 -dicarboxylates because mutation of dctA (also located on pSymB) has been shown to abolish C 4 -dicarboxylate transport in S. meliloti (18).…”

“…This symbiotic defect was rescued by second-site mutations that increased the synthesis of a second galactoglucan EPS (EPS II), whose biosynthetic genes were also located on the pSymB megaplasmid (13,14). Other genes located on pSymB that are required for the formation of N 2 -fixing root nodules include the C 4 -dicarboxylate (dctA) and phosphate transport (phoCDET) genes and the bacA gene (15)(16)(17)(18). The presence of large plasmids in bacteria that form associations with plants was described over 20 years ago (19).…”

Factors contributing to the accumulation of glutamate in Bradyrhizobium japonicum bacteroids under microaerobic conditions

“…Various lines of evidence indicate that dicarboxylic acids are important sources of reduced carbon for N,-fixing bacteroids in legume nodules (Arwas et al, 1985 ;Ronson et al, 198 1 ;Saroso et al, 1984;Tuzimura & Meguro, 1960;Watson et al, 1988). However, we found that, when Bradyrhizobium japonicum bacteroids were isolated anaerobically and supplied with [ U-14C]succinate or [U-14C]malate under microaerobic conditions, 20 to 40% of the label taken up was converted to glutamate (Salminen & Streeter, 1987).…”