Identification of a system that allows a Rhizobium tropici dctA mutant to grow on succinate, but not on other C4-dicarboxylates.

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.

Methods to evaluate nodulation competitiveness between Sinorhizobium meliloti strains using melanin production as a marker.

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.

Isolation and characterization of alfalfa-nodulating rhizobia present in acidic soils of central argentina and uruguay

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.

Effect of divalent cations on succinate transport in Rhizobium tropici, R. leguminosarum bv phaseoli and R. loti.

Rhizobium tropici, R. leguminosarum bv phaseoli and R. loti each have an active C4-dicarboxylic acid transport system dependent on an energized membrane. Free thiol groups are probably involved at the active site. Since EDTA inhibited succinate transport in R. leguminosarum bv phaseoli and R. loti, divalent cations may participate in the process; the activity was reconstituted by the addition of Ca(2+) or Mg(2+). However, EDTA had no effect on succinate transport in R. tropici, R. meliloti or R. trifolii strains. Ca(2+) or Mg(2+) had a similar effect on the growth rates of R. tropici and R. leguminosarum bv phaseoli; R. tropici did not require Ca(2+) to grow on minimal medium supplemented with succinate but R. leguminosarum bv phaseoli required either or both of the divalent cations Ca(2+) and Mg(2+). A R. tropici Mu-dI (lacZ) mutant defective in dicarboxylic acid transport, was isolated and found unable to form effective bean nodules.

Induction of C4-dicarboxylate transport genes by external stimuli in Rhizobium meliloti.

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.

Carbohydrate Catabolism in Azospirillum amazonense.

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.

Catabolism of carbohydrates and organic acids and expression of nitrogenase by azospirilla.

Fructose, galactose, L-arabinose, gluconate, and several organic acids support rapid growth and N2 fixation of Azospirillum brasiliense ATCC 29145 (strain Sp7) as a sole source of carbon and energy. Growth of Azospirillum lipoferum ATCC 29707 (strain Sp59b) is also supported by glucose, mannose, mannitol, and alpha-ketoglutarate. Oxidation of fructose and gluconate by A. brasiliense Sp7 and of glucose, gluconate, and fructose by A. lipoferum Sp59b was achieved through inducible enzymatic mechanisms. Both strains exhibited all of the enzymes of the Embden-Meyerhof-Parnas pathway, and strain Sp59b also possesses all the enzymes of the Entner-Doudoroff pathway. Fluoride inhibited growth on fructose (strains Sp7 and Sp59b) or on glucose (strain Sp59b) but not on malate. There was no activity via the oxidative hexose monophosphate pathway in either strain. There was greater activity with 1-phosphofructokinase than with 6-phosphofructokinase in both strains. Strain Sp59b formed fructose-6-phosphate via hexokinase, an enzyme that is lacking in strain Sp7. A. brasiliense and A. lipoferum exhibited the enzymes both of the tricarboxylic acid cycle and of the glyoxylate shunt; iodoacetate, fluoropyruvate, and malonate were inhibitory. A. brasiliense Sp7 could not transport [14C]glucose and alpha-[14C]ketoglutarate into its cells.

Transport and catabolism of D-mannose in Rhizobium meliloti.

Rhizobium meliloti L5-30 grows on D-mannose as the sole carbon source. The catabolic pathway of D-mannose was characterized. The following activities were present: mannose transport system, mannokinase, and mannosephosphate isomerase. Several mannose-negative mutants were selected; they were classified into three functional groups: group I, mannokinase and mannosephosphate isomerase defective: group II, mannokinase defective; and group III, mannosephosphate isomerase defective. Mannose uptake was an active process, since it was inhibited by azide, dinitrophenol, and cyanide, but not by fluoride or arsenate. Growth on succinate repressed mannose uptake activity. The mannose transport system was present in all the mutants. Uptake studies showed that mannose-negative mutants did not metabolize this sugar.

Succinate dehydrogenase mutant of Rhizobium meliloti.

A succinate dehydrogenase mutant strain of Rhizobium meliloti was isolated after nitrosoguanidine mutagenesis. It failed to grow on succinate, glutamate, acetate, pyruvate, or arabinose but grew on glucose, sucrose, fructose, and other carbohydrates. The mutant strain showed delayed nodulation of lucerne plants, and the nodules were white and ineffective. A spontaneous revertant strain of normal growth phenotype induced red and effective nodules.

Biochemical characterization of a fructokinase mutant of Rhizobium meliloti.

A double mutant strain (UR3) of Rhizobium meliloti L5-30 was isolated from a phosphoglucose isomerase mutant (UR1) on the basis of its resistance to fructose inhibition when grown on fructose-rich medium. UR3 lacked both phosphoglucose isomerase and fructokinase activity. A mutant strain (UR4) lacking only the fructokinase activity was derived from UR3; it grew on the same carbon sources as the parent strain, but not on fructose, mannitol, or sorbitol. A spontaneous revertant (UR5) of normal growth phenotype contained fructokinase activity. A fructose transport system was found in L5-30, UR4, and UR5 grown in arabinose-fructose minimal medium. No fructose uptake activity was detected when L5-30 and UR5 were grown on arabinose minimal medium, but this activity was present in strain UR4. Free fructose was concentrated intracellularly by UR4 > 200-fold above the external level. A partial transformation of fructose into mannitol and sorbitol was detected by enzymatic analysis of the uptake products. Polyol dehydrogenase activity was detected in UR4 grown in arabinose-fructose minimal medium. The induction pattern of polyol dehydrogenase activities in this strain might be due to slight intracellular fructose accumulation.

Phosphoglucose isomerase mutant of Rhizobium meliloti.

A mutant strain of complex phenotype was selected in Rhizobium meliloti after nitrosoguanidine mutagenesis. It failed to grow on mannitol, sorbitol, fructose, mannose, ribose, arabitol, or xylose, but grew on glucose, maltose, gluconate, L-arabinose, and many other carbohydrates. Assay showed the enzyme lesion to be in phosphoglucose isomerase (pgi), and revertants, which were of normal growth phenotype, contained the enzyme again. Nonpermissive substrates such as fructose and xylose prevented growth on permissive ones such as L-arabinose, and in such situations there was high accumulation of fructose 6-phosphate. The mutant strain had about 20% as much exopolysaccharide as the parent. Nitrogen fixation by whole plants was low and delayed when the mutant strain was the inoculant.

6-Phospho-D-gluconate:NAD+ 2-oxidoreductase (decarboxylating) from slow-growing Rhizobia.

6-Phospho-D-gluconate:NAD+ 2-oxidoreductase (decarboxylating) (NAD+-6PGD) was detected in several slow-growing strains of rhizobia, and no activity involving NADP+ was found in the same extracts. By contrast, fast-growing strains of rhizobia had NADP+-6PGD activity; most of them also had NAD+-6PGD activity. NAD+-6PGD was partially purified from the slow-growing strain Rhizobium japonicum 5006. The reaction was shown to be an oxidative decarboxylation.

Glycerol metabolism in Rhizobium.

Four strains of Rhizobium japonicum and one strain of R. trifolii were grown on glycerol and found to contain a soluble ATP-glycerol kinase and a particulate glycerolphosphate dehydrogenase. Both enzymes are induced by glycerol. The presence of NAD+-or NADP+-glycerol dehydrogenase was not detected in any of the strains. No significant differences were found in the glycerol metabolic pathway between fast-and slow-growing rhizobia.