Geneviève Alloing

Proline Betaine Uptake in Sinorhizobium meliloti: Characterization of Prb, an Opp-Like ABC Transporter Regulated by both Proline Betaine and Salinity Stress

Sinorhizobium meliloti uses proline betaine (PB) as an osmoprotectant when osmotically stressed and as an energy source in low-osmolarity environments. To fulfill this dual function, two separate PB transporters, BetS and Hut, that contribute to PB uptake at high and low osmolarity, respectively, have been previously identified. Here, we characterized a novel transport system that mediates the uptake of PB at both high and low osmolarities. Sequence analysis of Tn5-luxAB chromosomal insertions from several PB-inducible mutants has revealed the presence of a four-gene locus encoding the components of an ABC transporter, Prb, which belongs to the oligopeptide permease (Opp) family. Surprisingly, prb mutants were impaired in their ability to transport PB, and oligopeptides were not shown to be competitors for PB uptake. Further analysis of Prb specificity has shown its ability to take up other quaternary ammonium compounds such as choline and, to a lesser extent, glycine betaine. Interestingly, salt stress and PB were found to control prb expression in a positive and synergistic way and to increase Prb transport activity. At low osmolarity, Prb is largely implicated in PB uptake by stationary-phase cells, likely to provide PB as a source of carbon and nitrogen. Furthermore, at high osmolarity, the analysis of prb and betS single and double mutants demonstrated that Prb, together with BetS, is a key system for protection by PB.

The Sinorhizobium meliloti ABC Transporter Cho Is Highly Specific for Choline and Expressed in Bacteroids from Medicago sativa Nodules

In Sinorhizobium meliloti, choline is the direct precursor of phosphatidylcholine, a major lipid membrane component in the Rhizobiaceae family, and glycine betaine, an important osmoprotectant. Moreover, choline is an efficient energy source which supports growth. Using a PCR strategy, we identified three chromosomal genes (choXWV) which encode components of an ABC transporter: ChoX (binding protein), ChoW (permease), and ChoV (ATPase). Whereas the best homology scores were obtained with components of betaine ProU-like systems, Cho is not involved in betaine transport. Site-directed mutagenesis of choX strongly reduced (60 to 75%) the choline uptake activity, and purification of ChoX, together with analysis of the ligand-binding specificity, showed that ChoX binds choline with a high affinity (KD, 2.7 microM) and acetylcholine with a low affinity (KD, 145 microM) but binds none of the betaines. Uptake competition experiments also revealed that ectoine, various betaines, and choline derivatives were not effective competitors for Cho-mediated choline transport. Thus, Cho is a highly specific high-affinity choline transporter. Choline transport activity and ChoX expression were induced by choline but not by salt stress. Western blotting experiments with antibodies raised against ChoX demonstrated the presence of ChoX in bacteroids isolated from nitrogen-fixing nodules obtained from Medicago sativa roots. The choX mutation did not have an effect on growth under standard conditions, and neither Nod nor Fix phenotypes were impaired in the mutant, suggesting that the remaining choline uptake system(s) still present in the mutant strain can compensate for the lack of Cho transporter.

Osmotically induced synthesis of the dipeptide N-acetylglutaminylglutamine amide is mediated by a new pathway conserved among bacteria

The dipeptide N-acetylglutaminylglutamine amide (NAGGN) was discovered in the bacterium Sinorhizobium meliloti grown at high osmolarity, and subsequently shown to be synthesized and accumulated by a few osmotically challenged bacteria. However, its biosynthetic pathway remained unknown. Recently, two genes, which putatively encode a glutamine amidotransferase and an acetyltransferase and are up-regulated by osmotic stress, were identified in Pseudomonas aeruginosa. In this work, a locus carrying the orthologous genes in S. meliloti, asnO and ngg, was identified, and the genetic and molecular characterization of the NAGGN biosynthetic pathway is reported. By using NMR experiments, it was found that strains inactivated in asnO and ngg were unable to produce the dipeptide. Such inability has a deleterious effect on S. meliloti growth at high osmolarity, demonstrating the key role of NAGGN biosynthesis in cell osmoprotection. β-Glucuronidase activity from transcriptional fusion revealed strong induction of asnO expression in cells grown in increased NaCl concentration, in good agreement with the NAGGN accumulation. The asnO–ngg cluster encodes a unique enzymatic machinery mediating nonribosomal peptide synthesis. This pathway first involves Ngg, a bifunctional enzyme that catalyzes the formation of the intermediate N-acetylglutaminylglutamine, and second AsnO, required for subsequent addition of an amide group and the conversion of N-acetylglutaminylglutamine into NAGGN. Interestingly, a strong conservation of the asnO–ngg cluster is observed in a large number of bacteria with different lifestyles, such as marine, symbiotic, and pathogenic bacteria, highlighting the ecological importance of NAGGN synthesis capability in osmoprotection and also potentially in bacteria host–cell interactions.

Thiol-based redox signaling in the nitrogen-fixing symbiosis

In nitrogen poor soils legumes establish a symbiotic interaction with rhizobia that results in the formation of root nodules. These are unique plant organs where bacteria differentiate into bacteroids, which express the nitrogenase enzyme complex that reduces atmospheric N 2 to ammonia. Nodule metabolism requires a tight control of the concentrations of reactive oxygen and nitrogen species (RONS) so that they can perform useful signaling roles while avoiding nitro-oxidative damage. In nodules a thiol-dependent regulatory network that senses, transmits and responds to redox changes is starting to be elucidated. A combination of enzymatic, immunological, pharmacological and molecular analyses has allowed us to conclude that glutathione and its legume-specific homolog, homoglutathione, are abundant in meristematic and infected cells, that their spatio-temporally distribution is correlated with the corresponding (homo)glutathione synthetase activities, and that they are crucial for nodule development and function. Glutathione is at high concentrations in the bacteroids and at moderate amounts in the mitochondria, cytosol and nuclei. Less information is available on other components of the network. The expression of multiple isoforms of glutathione peroxidases, peroxiredoxins, thioredoxins, glutaredoxins and NADPH-thioredoxin reductases has been detected in nodule cells using antibodies and proteomics. Peroxiredoxins and thioredoxins are essential to regulate and in some cases to detoxify RONS in nodules. Further research is necessary to clarify the regulation of the expression and activity of thiol redox-active proteins in response to abiotic, biotic and developmental cues, their interactions with downstream targets by disulfide-exchange reactions, and their participation in signaling cascades. The availability of mutants and transgenic lines will be crucial to facilitate systematic investigations into the function of the various proteins in the legume-rhizobial symbiosis.

Two Sinorhizobium meliloti glutaredoxins regulate iron metabolism and symbiotic bacteroid differentiation

Legumes interact symbiotically with bacteria of the Rhizobiaceae to form nitrogen-fixing root nodules. We investigated the contribution of the three glutaredoxin (Grx)-encoding genes present in the Sinorhizobium meliloti genome to this symbiosis. SmGRX1 (CGYC active site) and SmGRX3 (CPYG) recombinant proteins displayed deglutathionylation activity in the 2-hydroethyldisulfide assay, whereas SmGRX2 (CGFS) did not. Mutation of SmGRX3 did not affect S. meliloti growth or symbiotic capacities. In contrast, SmGRX1 and SmGRX2 mutations decreased the growth of free-living bacteria and the nitrogen fixation capacity of bacteroids. Mutation of SmGRX1 led to nodule abortion and an absence of bacteroid differentiation, whereas SmGRX2 mutation decreased nodule development without modifying bacteroid development. The higher sensitivity of the Smgrx1 mutant strain as compared with wild-type strain to oxidative stress was associated with larger amounts of glutathionylated proteins. The Smgrx2 mutant strain displayed significantly lower levels of activity than the wild type for two iron-sulfur-containing enzymes, aconitase and succinate dehydrogenase. This lower level of activity could be associated with deregulation of the transcriptional activity of the RirA iron regulator and higher intracellular iron content. Thus, two S. meliloti Grx proteins are essential for symbiotic nitrogen fixation, playing independent roles in bacterial differentiation and the regulation of iron metabolism.

Redox regulation of differentiation in symbiotic nitrogen fixation

BACKGROUND Nitrogen-fixing symbiosis between Rhizobium bacteria and legumes leads to the formation of a new organ, the root nodule. The development of the nodule requires the differentiation of plant root cells to welcome the endosymbiotic bacterial partner. This development includes the formation of an efficient vascular tissue which allows metabolic exchanges between the root and the nodule, the formation of a barrier to oxygen diffusion necessary for the bacterial nitrogenase activity and the enlargement of cells in the infection zone to support the large bacterial population. Inside the plant cell, the bacteria differentiate into bacteroids which are able to reduce atmospheric nitrogen to ammonia needed for plant growth in exchange for carbon sources. Nodule functioning requires a tight regulation of the development of plant cells and bacteria. SCOPE OF THE REVIEW Nodule functioning requires a tight regulation of the development of plant cells and bacteria. The importance of redox control in nodule development and N-fixation is discussed in this review. The involvement of reactive oxygen and nitrogen species and the importance of the antioxidant defense are analyzed. MAJOR CONCLUSIONS Plant differentiation and bacterial differentiation are controlled by reactive oxygen and nitrogen species, enzymes involved in the antioxidant defense and antioxidant compounds. GENERAL SIGNIFICANCE The establishment and functioning of nitrogen-fixing symbiosis involve a redox control important for both the plant-bacteria crosstalk and the consideration of environmental parameters. This article is part of a Special Issue entitled Redox regulation of differentiation and de-differentiation.

The Legume Root Nodule: From Symbiotic Nitrogen Fixation to Senescence

Biological nitrogen fixation (BNF) is the biological process by which the atmospheric nitrogen (N2) is converted to ammonia by an enzyme called nitrogenase. It is the major source of the biosphere nitrogen and as such has an important ecological and agronomical role, accounting for 65 % of the nitrogen used in agriculture worldwide. The most important source of fixed nitrogen is the symbiotic association between rhizobia and legumes. The nitrogen fixation is achieved by bacteria inside the cells of de novo formed organs, the nodules, which usually develop on roots, and more occasionally on stems. This mutualistic relationship is beneficial for both partners, the plant supplying dicarboxylic acids as a carbon source to bacteria and receiving, in return, ammonium. Legume symbioses have an important role in environment-friendly agriculture. They allow plants to grow on nitrogen poor soils and reduce the need for nitrogen inputs for leguminous crops, and thus soil pollution. Nitrogen-fixing legumes also contribute to nitrogen enrichment of the soil and have been used from Antiquity as crop-rotation species to improve soil fertility. They produce high protein-containing leaves and seeds, and legumes such as soybeans, groundnuts, peas, beans, lentils, alfalfa and clover are a major source of protein for human and animal consumption. Most research concentrates on the two legume-rhizobium model systems Lotus-Mesorhizobium loti and Medicago-Sinorhizobium meliloti, with another focus on the economically-important Glycine max (soybean) -Bradyrhizobium japonicum association. The legume genetic models Medicago truncatula and Lotus japonicus have a small genome size of ca. 450 Mbp while Glycine max has a genome size of 1,115 Mbp, and all are currently targets of large-scale genome sequencing projects (He et al., 2009; Sato et al., 2008; Schmutz et al., 2010). The complete genome sequence of their bacterial partners has been established (Galibert et al., 2001; Kaneko et al., 2000; Kaneko et al., 2002; Schneiker-Bekel et al., 2011).

Synthesis and Roles of Glutathione and Homoglutathione in the Nitrogen-Fixing Symbiosis

Glutathione (GSH) is a major antioxidant molecule in plants. It is involved in regulating plant development and responses to abiotic and biotic environment changes. In leguminous plants, a GSH homolog, homoglutathione is also found. Most legumes can develop a symbiotic interaction with soil bacteria of the rhizobium family under nitrogen deficiency. This symbiosis allows the reduction of atmospheric nitrogen by the bacteria in plant organs called root nodules. In this chapter, we summarize studies that describe the synthesis and the roles of GSH and hGSH in the nitrogen-fixing symbiosis.

Identification of BetX, a Periplasmic Protein Involved in Binding and Uptake of Proline Betaine and Glycine Betaine in Sinorhizobium meliloti

An osmotic stress, such as excessive salinity or drought, has deleterious effects on the establishment and maintenance of symbiosis between Sinorhizobium meliloti and its host plant alfalfa. Bacteria protect themselves against high external osmolarity by accumulating osmoprotective organic compounds in their cytoplasm, the so-called compatible solutes. Although glycine betaine (GB) is one of the most effective osmoprotectant used by bacteria, proline betaine (PB) is the major betaine produced by alfalfa. PB is secreted by germinating seedlings of many Medicago species and is found in roots, nodules, and bacteroids. S. meliloti has the capacity to use betaines as compatible solutes but also as carbon and nitrogen sources. Three different transport systems (two ABC transporters and one BCCT transporter) are involved in the uptake of PB and GB in S. meliloti. BetX, a periplasmic binding protein encoded by a gene located in a PB catabolic locus, has been characterized. Although the betX gene does not belong to an ABC transporter operon, BetX contributes to the binding and uptake of PB and GB. Further, BetX is not associated with the already characterised betaine ABC transporters and the proteinmembrane complex with which it is associated is unknown. Expression analysis of a betX-lacZ fusion revealed induction by PB, mono-methyl-proline (MMP, an intermediate in PB catabolism), and salt stress, suggesting that BetX has a catabolic and osmoprotection role. BetR, a TetR-like regulator is the betX expression repressor in the absence of ligand, and the repression is abolished by addition of substrate (MMP or PB). However, betX induction by osmotic stress is independent of BetR, suggesting an additional level of regulation. A betX mutant shows a defect in growth capacity but osmoprotection by PB and GB was unchanged due to the presence of other betaine transporters in S. meliloti. Nodulation capacity and nitrogen-fixation activity of a betX mutant was not affected.