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By hybridization and heteroduplex studies the fixABC and nifA genes of the Rhizobium leguminosarum symbiotic plasmid pRL6JI have been identified. DNA sequencing of the region containing nifA showed an open reading frame of 1557 bp encoding a protein of 56, 178 D. Based on sequence homology, this ORF was confirmed to correspond to the nifA gene. Comparison of three nifA proteins (Klebsiella pneumoniae, Rhizobium meliloti, Rhizobium leguminosarum) revealed only a weak relationship in their N-terminal regions, whereas the C-terminal parts exhibited strong homology. Sequence analysis also showed that the R. leguminosarum nifA gene is followed by nifB and preceded by fixC with an open reading frame inserted in between. This novel ORF of 294 bp was found to be highly conserved also in R. meliloti. No known promoter and termination signals could be defined on the sequenced R. leguminosarum fragment.

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Arch. Microbiol. 144:142-146, 1986) was used to study the relationship between dicarboxylic acid transport and nitrogen fixation in Bradyrhizobium japonicum. The expression of the dct sequences on plasmid pRK290:4:46 in B. japonicum CJ1 resulted in increased growth rates in media containing dicarboxyiic acids as the sole source of carbon. In addition, strain CJl(pRK290:4:46) exhibited enhanced succinate uptake activity when grown on dicarboxylic acids under aerobic conditions. Under free-living nitrogen-fixing conditions, strain CJt(pRK290:4:46) exhibited higher nitrogenase (acetylene reduction) activity co'mpared with that of the wild-type strain. This increase in nitrogenase activity also correlated with an enhanced dicarboxylic acid uptake rate under these microaerobic conditions. The regulation of dicarboxylic acid transport by factors such as metabolic inhibitors and the presence of additional carbon sources was similar in both the wild-type and the engineered strains. The implications of increasing nitrogenase activity through alterations in the dicarboxylic acid transport system are discussed.Nitrogen fixation by the Rhizobium-legume symbiosis is a high-energy-demanding process. The infected host plant supplies bacteroids with photosynthate, which is converted to ATP, and reducing power for the support of nitrogen fixation and the maintenance of nodule functions. The nitrogen-fixing capacity of the Rhizobium-legume symbiosis is thought to be limited by the amount of photosynthate available to the bacteroid (14,27). Therefore, knowledge of the carbon compounds supplied to bacteroids is a prerequisite step for formulating any strategy aimed at improving the nitrogen fixation process.Results of biochemical studies suggest that dicarboxylic acids (succinate, malate, and fumarate) play an important role in the symbiotic nitrogen fixation process. Dicarboxylic acids are the most effective substrates for respiration and promnoting nitrogen fixation by isolated bacteroids (2). The isolation of mutants defective in dicarboxylic acid transport (dct) in a number of Rhizobium strains has also indicated the vital role played by these compounds in symbiotic nitrogen fixation. Dicarboxylic acid transport mutants of Rhizobium leguminosarum (1, 10, 12), Rhizobium trifolii (25), and Rhizobium meliloti (3) that are unable to transport dicarboxylic acids but capable of utilizing other carbon sources form ineffective nodules on their respective host plants. Furthermore, Rhizobium strains that are defective in succinate dehydrogenase (11) and malate dehydrogenase (31) activity have also been found to be incapable of effective symbiotic nitrogen fixation. Taken together, these results indicate the essential nature of the transport and subsequent rnetabolism of dicarboxylic acids for the nitrogen fixation process.With regard to the regulation of the dicarboxylic acid transport system, evidence suggests that succinate, malate, and fumarate are transported via a common system in both cultured cells and bacteroids of Bradyrhizobi...

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