Rhizobium NGR234 Research Articles

Plasmid replicons of Rhizobium

Rhizobium spp. are found in soil. They are both free-living and found symbiotically associated with the nodules of leguminous plants. Traditionally, studies have focused on the association of these organisms with plants in nitrogen-fixing nodules, since this is regarded as the most important role of these bacteria in the environment. Rhizobium sp. are known to possess several replicons. Some, like the Rhizobium etli symbiotic plasmid p42d and the plasmid pNGR234b of Rhizobium NGR234, have been sequenced and characterized. The plasmids from these organisms are the focus of this short review.

Multiple genetic controls on Rhizobium meliloti syrA, a regulator of exopolysaccharide abundance.

Exopolysaccharides (EPS) are produced by a wide assortment of bacteria including plant pathogens and rhizobial symbionts. Rhizobium meliloti mutants defective in EPS production fail to invade alfalfa nodules. Production of EPS in R. meliloti is likely controlled at several levels. We have characterized a new gene of this regulatory circuit. syrA was identified by its ability to confer mucoid colony morphology and by its ability to suppress the colonial phenotype of an exoD mutant. Here we show that syrA encodes a 9-kD hydrophobic protein that has sequence similarity to two other EPS regulatory proteins: ExoX of Rhizobium NGR234 and R. meliloti, and Psi of R. leguminosarum bv. phaseoli. The syrA transcription start site lies 522 nucleotides upstream of a non-canonical TTG start codon. The syrA promoter region is similar to the promoter region of the nodulation regulatory protein, nodD3. We found that in free-living bacteria, syrA expression is activated by the regulatory locus, syrM, but not by nodD3. In planta, syrM is not required for expression of syrA. Instead, expression of the nitrogen fixation (nifHDKE) genes upstream of syrA plays a role. Specific and distinct sets of genetic controls may operate at different times during nodule invasion.

Evaluation of N 2 fixation and agroforestry potential in selected tree legumes for sustainable use in South Africa

Seven tree legumes with agroforestry potential were tested for their capacity to nodulate with indigenous rhizobia or bradyrhizobia in soils collected from different ecological zones in South Africa. Our findings show that all the tested species, except Acacia senegal, could effectively nodulate with root-nodule bacteria native to Venda and Klipspruit soils. Since Acacia seyal, Acacia galpinii, Albizia lebbeck and Acacia auriculiformis exhibited, in that order, a greater level of growth and symbiotic performance, they manifest themselves as suitable species for agroforestry programs in Venda and Klipspruit soils. One indigenous species, Acacia ataxancantha, was assessed to be a non-nodulating legume even when inoculated with a promiscuous strain of Rhizobium NGR234.

Exopolysaccharide-deficient mutants of Astragali rhizobia are symbiotically effective on Astragalus sinicus, an indeterminate nodulating host

Five exopolysaccharide-deficient mutants were isolated after rhizobial strain 107 was subjected to transposon Tn5 mutagenesis. The amount of EPS produced by the mutants was dramatically decreased to between 3% and 6% of wild-type level. All mutants carried a singel copy of Tn5. Two mutants (NA3 and NA10) were complemented by the R. meliloti exoA gene and the functionally equivalent exoD gene of Rhizobium sp. strain NGR234. Two other mutants (NA7 and NA8) were complemented by the R. meliloti exoB gene and the functionally equivalent NGR234 exoC gene. The remaining mutant (NA11) was not complemented by any exo genes of R. meliloti or Rhizobium NGR234. All mutants induced normal nitrogen-fixing nodules on Astragalus sinicus, an indeterminate nodulating host.

Siderophore and organic acid production in root nodule bacteria

Nineteen strains of root nodule bacteria were grown under various iron regimes (0.1, 1.0 and 20 microM added iron) and tested for catechol and hydroxamate siderophore production and the excretion of malate and citrate. The growth response of the strains to iron differed markedly. For 12 strains (Bradyrhizobium strains NC92B and 32H1, B. japonicum USDA110 and CB1809, B. lupini WU8, cowpea Rhizobium NGR234, Rhizobium meliloti strains U45 and CC169, Rhizobium leguminosarum bv viciae WU235 and Rhizobium leguminosarum bv trifolii strains TA1, T1 and WU95) the mean generation time showed no variation with the 200-fold increase in iron concentration. In contrast, in Bradyrhizobium strains NC921, CB756 and TAL1000, B. japonicum strain 61A76 and R. leguminosarum bv viciae MNF300 there was a 2-5 fold decrease in growth rate at low iron. R. meliloti strains WSM419 and WSM540 showed decreased growth at high iron. All strains of root nodule bacteria tested gave a positive CAS (chrome azurol S) assay for siderophore production. No catechol-type siderophores were found in any strain, and only R. leguminosarum bv trifolii T1 and bv viciae WU235 produced hydroxamate under low iron (0.1 and 1.0 microM added iron). Malate was excreted by all strains grown under all iron regimes. Citrate was excreted by B. japonicum USDA110 and B. lupini WU8 in all iron concentrations, while Bradyrhizobium TAL1000, R. leguminosarum bv viciae MNF300 and B. japonicum 61A76 only produced citrate under low iron (0.1 and/or 1.0 microM added iron) during the stationary phase of growth.

Two C4-dicarboxylate transport systems in Rhizobium sp. NGR234: rhizobial dicarboxylate transport is essential for nitrogen fixation in tropical legume symbioses.

To investigate the role of dicarboxylate transport in nitrogen-fixing symbioses between Rhizobium and tropical legumes, we made a molecular genetic analysis of the bacterial transport system in Rhizobium sp. NGR234. This braod host range strain fixes nitrogen in association with evolutionarily divergent legumes. Two dicarboxylate transport systems were cloned from Rhizobium NGR234. One locus was chromosomally located, whereas the other was carried on the symbiotic plasmid (pSym) and contained a dctA carrier protein gene, which was analyzed in detail. Although the DNA and derived amino acid sequences of the structural gene were substantially homologous to that of R. meliloti, its promoter sequences was quite distinct, and the upstream sequence also exhibited no homology to dctB, which is found at this position in R. meliloti. A site-directed internal deletion mutant in dctA of NGR234 exhibited a (unique) exclusively symbiotic phenotype that could grow on dicarboxylates ex planta, but could not fix nitrogen in planta. This phenotype was found for tested host plants of NGR234 with either determinate- or indeterminate-type nodules, confirming for the first time that symbiosis-specific uptake of dicarboxylates is a prerequisite for nitrogen fixation in tropical legume symbioses.

Site-directed mutagenesis and DNA sequence of pckA of Rhizobium NGR234, encoding phosphoenolpyruvate carboxykinase: gluconeogenesis and host-dependent symbiotic phenotype

We have cloned and sequenced the pckA gene of Rhizobium sp. NGR234, a broad host-range strain. The gene encodes phosphoenolpyruvate carboxykinase (PEPCK), a key enzyme of gluconeogenesis. The locus was isolated and subcloned from a genomic library of NGR234 employing hybridization with an R. meliloti pck gene probe and complementation of a Tn5 mutant in this species. The DNA sequence of pckA (NGR234) was determined and encoded a PEPCK protein of 535 amino acids with a molecular weight of 58.4 kDa. The deduced polypeptide sequence was compared to those of three known ATP-dependent PEPCKs. Slightly higher homology was observed with yeast and trypanosome polypeptides than with that of Escherichia coli. We have identified several regions that are conserved in all four PEPCK proteins. A mutant constructed in the pck gene by site-directed mutagenesis with interposon omega failed to grow on succinate, malate and arabinose but grew on glucose and glycerol as sole carbon sources. These data show that NGR234 requires PEPCK-driven gluconeogenesis to grow on TCA cycle intermediates. A host-dependent effect of the pckA mutation was observed on nodule development and nitrogen fixation. Nodules formed by the site-directed mutant on Leucaena leucocephala and Macroptilium atropurpureum were FixRed, but on Vigna unguiculata were Fix-. The expression of the gene was positively regulated in free-living cells of NGR234 by either succinate or host-plant exudates, and was subject to catabolite repression by glucose.

The transport and catabolism of l-proline by cowpeaRhizobiumNGR 234

Cowpea Rhizobium NGR234 grows with a mean generation time of 3.5 h on L-proline; intracellular catabolism via proline oxidase and pyrroline 5-carboxylate dehydrogenase is induced by proline. In contrast, cells have some capacity to take up proline whatever the growth medium. Uptake of proline is active and transmembrane gradients of approximately 290-fold can be generated. The proline transporter(s) appear to carry a range of amino acids and intracellular [14C]proline can be exchanged with extracellular valine or histidine or isoleucine but not glutamate. Snake bean bacteroids are unable to transport or catabolize L-proline, consistent with the lack of detectable proline in snakebean nodule cytosol. In this symbiosis at least, proline is not an important carbon source for bacteroids.

Pentose Metabolism in Rhizobium leguminosarum MNF300 and in Cowpea Rhizobium NGR234

SUMMARY: L-Arabinose is broken down by Rhizobium leguminosarum MNF300 via 2-oxoglutarate semialdehyde. Enzyme activities in cells grown on succinate, mannitol or arabinose indicated much greater modulation of arabinonate dehydratase, 2-keto-3-deoxyarabinonate dehydratase and 2-oxoglutarate semialdehyde dehydrogenase than of arabinose dehydrogenase or of arabinono-γ-lactonase. In cowpea Rhizobium NGR234, all the enzymes of L-arabinose metabolism except L-arabinono-γ-lactonase were inducible. Assays for such enzymes in snake bean bacteroids indicated that L-arabinose did not reach the bacteroids in large quantities. The Tn5-induced mutant MNF3045 of R. leguminosarum was unable to grow on L-arabinose and accumulated L-arabinono-γ-lactone and L-arabinonate. Product accumulation and enzyme assays suggested that this mutant was defective in L-arabinonate dehydratase. It nodulated peas and the nodules fixed N2, indicating that the supply of L-arabinose is not essential for bacteroid function. Another Tn5-induced mutant of R. leguminosarum, MNF3041, lacked ribokinase and was unable to grow on D-ribose; this mutant was also able to nodulate peas and fix N2.

A continuous culture study of the phosphorus nutrition of Rhizobium trifollii WU95, Rhizobium NGR234 and Bradyrhizobium CB756

With continuous cultures in a fully defined minimal salts medium steady states were achieved at both limiting and non-limiting concentrations of phosphate in the inflowing medium for Rhizobium trifolii WU95, cowpea Rhizobium NGR234, and Bradyrhizobium CB756.

Effect of phosphorus supply on phosphate uptake and alkaline phosphatase activity in Rhizobia

The effect of P nutrition on phosphate uptake and alkaline phosphatase activity was studied in chemostat culture for four rhizobial and three bradyrhizobial species. Phosphate-limited cells took up phosphate 10- to 180-fold faster than phosphate-rich cells. The four fast-growing rhizobial strains contained high levels of alkaline phosphatase activity under P-limited conditions compared to the repressed levels found in P-rich cells; alkaline phosphatase activity could not be detected in three slow-growing rhizobial strains, regardless of their P-status. Glycerol 1-phosphate-uptake in the cowpea Rhizobium NGR234 was derepressed over 50-fold under P-limited conditions, and appeared to be co-regulated with phosphate uptake. The phosphate-uptake system appeared similar in all strains with apparent K m values ranging from 1.6 μM to 6.0 μM phosphate and maximum activities from 17.2 to 126 nmol · min-1 · (mg dry weight of cells)-1. Carbonyl cyanide m-chlorophenyl hydrazone strongly inhibited phosphate uptake in all strains and a number of other metabolic inhibitors also decreased phosphate uptake in the cowpea Rhizobium NGR234. The phosphate uptake system in all strains failed to catalyse exchange of 32P label in preloaded cells or efflux of phosphate. The results suggest a single, repressible, unidirectional and energy-dependent system for the transport of phosphate into rhizobia.

Effect of phosphorus supply on periplasmic protein profiles in Rhizobia

Lysozyme/EDTA treatment of four fast-growing rhizobia released repeatable protein profiles after polyacrylamide slab gel electrophoresis. Similar treatment of slow-growing rhizobia failed to release such periplasmic proteins. For the four-fast-growing rhizobia, both P-repressible and P-inducible protein bands occurred. The only P-repressible protein identified was alkaline phosphatase, which showed strain differences in both electrophoretic mobility and activation by Mg2+. The derepression of the P-repressible periplasmic proteins in cowpea Rhizobium NGR234 correlated with derepression of both phosphate and glycerol 1-phosphate uptake.

Carbon Utilization by Free-living and Bacteroid Forms of Cowpea Rhizobium Strain NGR234

Free-living cells of the fast-growing cowpea Rhizobium NGR234 were able to grow on a variety of carbon substrates at growth rates varying from 2·5 h on glucose or fumarate to 15·6 h on p-hydroxybenzoate. Free-living cells constitutively oxidized glucose, glutamate and aspartate but were inducible for all the other systems investigated. Bacteroids from root nodules of snake bean, however, were only capable of oxidizing C4-dicarboxylic acids and failed to oxidize any other carbon sources. Free-living cells of NGR234 possess inducible fructose and succinate uptake systems. These substrates are accumulated by active processes since accumulation is inhibited by azide, 2,4-dinitophenol and carbonyl cyanide m-chlorophenyl hydrazone. Bacteroids failed to take up fructose although they actively accumulated succinate, suggesting that the latter substrate is significant in the development of an effective symbiosis.

The uptake and hydrolysis of disaccharides by fast-and slow-growing species of Rhizobium

Slow growing strains of rhizobia appear to lack both uptake systems and catabolic enzymes for disaccharides. In the fast-growing strains of rhizobia there are uptake mechanisms and catabolic enzymes for disaccharide metabolism. In Rhizobium leguminosarum WU 163 and WU235 and R. trifolii WU290, sucrose and maltose uptake appears to be constitutive whereas in R. meliloti WU60 and in cowpea Rhizobium NGR234 uptake of these disaccharides is inducible. There is evidence that there are at least two distinct disaccharide uptake systems in fast-growing rhizobia, one transporting sucrose, maltose and trehalose and the other, lactose. Disaccharide uptake is via an active process since uptake is inhibited by azide, dinitrophenol and carbonyl cyanide m-chlorophenylhydrazone but not by arsenate. Bacteroids of R. leguminosarum WU235 and R. lupini WU8 are unable to accumulate disaccharides.