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The Rhizobium meliloti C-dicarboxylate transport (Dct) system is essential for an effective symbiosis with alfalfa plants. C-dicarboxylates are the major carbon source taken up by bacteroids. Genetic analysis of Dct mutant strains led to the isolation of the dct carrier gene dctA and the regulatory genes dctB and dctD. The carrier gene dctA 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 dctA in the free-living state is reflected by diauxic growth of rhizobia, with succinate being the preferred carbon source. The tight coupling of C-dicarboxylate transport and nitrogen fixation is revealed by a reduced level of C-dicarboxylate transport in nitrogenase negative bacteroids. [ABSTRACT FROM AUTHOR]

Immunoelectron microscopy (IEM) and enzyme-linked immunosorbent assay (ELISA) studies with polyclonal antibodies (PAbs) indicated that the isolates 1A, Nsaba, and Kpeve of cacao swollen shoot virus (CSSV; genus Badnavirus) are serologically distinct. Out of these isolates, only 1A was reliably detected from infected cacao trees with the homologous PAbs. To improve serological detection of the isolates Nsaba and Kpeve by the application of monoclonal antibodies (MAbs), virion preparations of CSSV-1A, -Nsaba and -Kpeve were used to immunize mice. Although immunization with CSSV-1A yielded a large number of 1A-specific MAbs, no Nsaba- or Kpeve-specific MAbs were obtained after immunization with these isolates. Instead, the latter exclusively led to the production of MAbs which reacted only with isolate 1A. Epitope characterization of 1A-specific MAbs using different ELISA procedures, IEM and Western blot experiments showed that three of the 1A-specific MAbs detected a continuous epitope [sodium dodecyl sulphate (SDS)-stable] and nine MAbs reacted with conformation-dependent epitopes (SDS-labile). Direct adsorption of virus particles to microtitre plates in antigen-coated plate ELISA affected epitope stability in a similar fashion as SDS treatment of virions. When comparing the sensitivity of CSSV detection by MAbs and PAbs in various ELISA formats, MAbs were slightly more sensitive than PAbs in CSSV detection from plant sap. Using the panel of available 1A-specific MAbs for triple antibody sandwich-ELISA analysis of 31 CSSV samples from different cacao growing areas of Ghana revealed the occurrence of four serotypes in the samples containing CSSV-1A isolates. In addition, the serological reaction patterns of the MAbs correlated well with the geographic origin of the isolates, thus giving further indications about the regional distribution of CSSV 1A-like isolates in Ghana. [ABSTRACT FROM AUTHOR]

A screening method was used to identify Sinorhizobium meliloti mutants which are affected in stationary-phase 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 H2O, 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.