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A screening method was used to identify Sinorhizobium meliloti mutants which are affected in stationaryphase survival. Of 20,000 individual colonies mutagenized with transposon Tn5-B20, 10 mutant strains which showed poor or no survival in the stationary phase were identified. Analyses of expression patterns of the promoterless lacZ genes in the mutant strains revealed individual induction patterns. Most strains were induced in stationary phase as well as under carbon limitation and in pure H 2 O, but none of the mutants was induced under heat, alkali stress conditions, or low oxygen tension. Plant inoculation tests revealed that the symbiotic proficiency of the mutants was not affected. Two mutants, however, showed gene induction not only in the stationary phase under free-living conditions but also in the bacteroid state. A long-term starvation test was carried out to examine the ability of the 10 mutants to survive prolonged stationary-phase conditions. All mutants showed a clear decrease in the colony-forming ability under the chosen experimental conditions. Staining with green and red fluorescent nucleic acid stain showed that the mutants fell into two different classes. Seven mutants died during stationary phase; the three other mutants remained viable but did not resume growth after prolonged starvation. Five of the ten Tn5-B20 insertions were cloned from the genomes of the mutant strains. Nucleotide sequence analyses established that the transposon had inserted in five distinctive genes. Database searches revealed that four of the tagged loci corresponded to already characterized genes whose gene products are involved in important cellular processes such as amino acid metabolism or aerobic respiration.

Abstract. The Rhizobium meliloti C4-dicarboxylate transport (Dct) system is essential for an effective symbiosis with alfalfa plants. C4-dicarboxylates are the major carbon source taken up by bacteroids. Genetic analysis of Dctmutant strains led to the isolation of the dct carrier gene dctA and the regulatory genes dctB and dctD. The carrier gene detA is regulated in free-living cells by the alternative sigma factor RpoN and the two-component regulatory system DctB/D. In addition, DctA is involved in its own regulation, possibly by interacting with DctB. In bacteroids, besides the DctB/DctD system an additional symbiotic activator is thought to be involved in dctA expression. Further regulation of detA in the free-living state is reflected by diauxic growth of rhizobia, with succinate being the preferred carbon source. The tight coupling of C4-dicarboxylate transport and nitrogen fixation is revealed by a reduced level of C4-dicarboxylate transport in nitrogenase negative bacteroids. Key words. C4-dicarboxylate transport; dot genes; energy source; regulation; Rhizobium meliloti; symbiosis. Energy and carbon metabolism of rhizobia under free-living and symbiotic conditionsThe symbiotic interaction between legumes and rhizobia is based on the development of root nodules. The root nodules are the sites of biological nitrogen fixation, which is known to be a highly energy-intensive process. Bacteroids thus profit from the plant compounds derived from photosynthetic activity and in response, reduced nitrogen is exported to the plant. The reduction of nitrogen to ammonia is catalyzed by the nitrogenase enzyme complex 4~ Under optimal conditions, nitrogen fixation requires 16 molecules of ATP per molecule of N 2 fixed, but the energy consumption can actually be as high as 42 molecules of ATP, according to growth yield measurements, as summarized recently 56. The significance of symbiotic nitrogen fixation is reflected in the fact that legumes are important crop plants. The terrestrial flux of nitrogen from biological fixation has been calculated to range from 100 to 120 x 106t N/year 5s. Fast growing Rhizobium species, including the alfalfanodulating Rhizobium meliloti, are capable of utilizing a wide range of carbon sources such as carbohydrates, organic alcohols, organic acids, and a variety of aromatic compounds (reviewed by Stowers72). Concerning the central catabolic pathways, it was reported that hexose degradation mainly proceeds through the Entner-Doudoroff (ED) pathway and that organic acids are directly metabolized via the tricarboxylic acid cycle 72. It has been observed that growth with succinate as sole carbon source leads to the fastest growth rate of R. meliloti 73. The generation time in minimal medium with succinate was determined to be 2.6 hours, compared to 3.2 hours in minimal medium with glucose ~5.In symbiosis, carbon metabolism and transport are considered to be of great importance in ensuring the tight physiological coupling between the two partners. At the end of the 1970s, the question of which...