RESUMEN

A defined insertion mutant of a gene encoding a homolog of the rhizobial C4-dicarboxylate permease (dctA) was constructed in Rhizobium tropici strain CIAT899. This mutant (GA1) was unable to grow on fumarate or malate; however, in contrast with other rhizobial dctA mutants, it retained a limited ability to grow on succinate with ammonia as a nitrogen source. Our results suggest the presence of a novel succinate-specific transport system in R. tropici. Biochemical characterization indicated that this alternative transport system in GAI is active and dependent on an energized membrane. It was also induced by succinate and aspartate, and was repressed by glucose and glycerol. Bean plants inoculated with GA1 showed a reduced nitrogen-fixing ability, achieving only 29% of the acetylene reduction activity determined in CIAT899 strain nodules, 33 days after inoculation. Also, bean plants inoculated with GA1 had reduced shoot dry weight compared with plants inoculated with the wild-type strain.

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RESUMEN

Three methods to evaluate the relative ability of different strains of Sinorhizobium meliloti to occupy nodules formed on alfalfa after co-inoculation were compare in this study. Results obtained using the three methods of evaluation together, provided insight into the relative nodulation competitiveness between two given sinorhizobial strains. A simple visual phenotypic marker, i.e., melanin production was used to distinguish individual strains in a given assay. As such, melanin producing strains were compared with melanin non-producing strains throughout this study. Method 1 required the use of an ELISA plate, took 35 min for the analysis of 40 nodules, and allowed strain identification by melanin production 2 days after nodule harvest. Method 2 required 3 h for the analysis of 40 nodules, used an ELISA plate, growth of bacteria on Petri dishes, and melanin production was analysed after 48 h of cell culture. Finally, method 3 involved the whole nodulated plant root, required less material than the above methods, and results were obtained after 24 h. Only method 2 was useful in determining if both a melanin producing strain and a melanin non-producing strain had occupied an individual nodule. Each of the three methods represented a rapid way of studying strain competition for field studies, using a natural trait as a marker.

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We describe the isolation and characterization of alfalfa-nodulating rhizobia from acid soils of different locations in Central Argentina and Uruguay. A collection of 465 isolates was assembled, and the rhizobia were characterized for acid tolerance. Growth tests revealed the existence of 15 acid-tolerant (AT) isolates which were able to grow at pH 5.0 and formed nodules in alfalfa with a low rate of nitrogen fixation. Analysis of those isolates, including partial sequencing of the genes encoding 16S rRNA and genomic PCR-fingerprinting with MBOREP1 and BOXC1 primers, demonstrated that the new isolates share a genetic background closely related to that of the previously reported Rhizobium sp. Or191 recovered from an acid soil in Oregon (B. D. Eardly, J. P. Young, and R. K. Selander, Appl. Environ. Microbiol. 58:1809-1815, 1992). Growth curves, melanin production, temperature tolerance, and megaplasmid profiles of the AT isolates were all coincident with these characteristics in strain Or191. In addition to the ability of all of these strains to nodulate alfalfa (Medicago sativa) inefficiently, the AT isolates also nodulated the common bean and Leucaena leucocephala, showing an extended host range for nodulation of legumes. In alfalfa, the time course of nodule formation by the AT isolate LPU 83 showed a continued nodulation restricted to the emerging secondary roots, which was probably related to the low rate of nitrogen fixation by the largely ineffective nodules. Results demonstrate the complexity of the rhizobial populations present in the acidic soils represented by a main group of N2-fixing rhizobia and a second group of ineffective and less-predominant isolates related to the AT strain Or191.

RESUMEN

We have characterized two mutants of Rhizobium meliloti L5-30 obtained by random mutagenesis using Mu-lacZ that were defective in transport of C4-dicarboxylic acids. These mutants induced ineffective nodules on alfalfa. Mutations in the two strains appeared to be located in a dctA gene. Levels of dctA gene expression were determined under different environmental conditions using dctA-lacZ fusions, and beta-galactosidase activities increased in response to osmotic stress and also when the cells were incubated in medium low in calcium. The transcriptional induction of the dctA gene by environmental signals was decreased by DNA gyrase inhibitors such as novobiocin and coumermycin.

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The nitrogen fixer Azospirillum amazonense grew on the various disaccharides, hexoses, and pentoses tested in this study but not on polyols and on some tricarboxylic acid cycle intermediates. An active transport system was detected for sucrose and glucose but not for mannitol and 2-ketoglutarate. Six A. amazonense strains were examined for 16 carbon-metabolizing enzymes, and the results indicate that these strains employ the Entner-Doudoroff pathway to catabolize sucrose, fructose, and glucose. The hexose monophosphate and Embden-Meyerhof-Parnas pathways were not detectable.