Karine Mandon

H2O2 Is Required for Optimal Establishment of the Medicago sativa/Sinorhizobium meliloti Symbiosis

The symbiotic interaction between Medicago sativa and Sinorhizobium meliloti RmkatB(++) overexpressing the housekeeping catalase katB is delayed, and this delay is combined with an enlargement of infection threads. This result provides evidence that H(2)O(2) is required for optimal progression of infection threads through the root hairs and plant cell layers.

Hydrogen peroxide and nitric oxide: key regulators of the Legume-Rhizobium and mycorrhizal symbioses.

SIGNIFICANCE During the Legume-Rhizobium symbiosis, hydrogen peroxide (H(2)O(2)) and nitric oxide (NO) appear to play an important signaling role in the establishment and the functioning of this interaction. Modifications of the levels of these reactive species in both partners impair either the development of the nodules (new root organs formed on the interaction) or their N(2)-fixing activity. RECENT ADVANCES NADPH oxidases (Noxs) have been recently described as major sources of H(2)O(2) production, via superoxide anion dismutation, during symbiosis. Nitrate reductases (NR) and electron transfer chains from both partners were found to significantly contribute to NO production in N(2)-fixing nodules. Both S-sulfenylated and S-nitrosylated proteins have been detected during early interaction and in functioning nodules, linking reactive oxygen species (ROS)/NO production to redox-based protein regulation. NO was also found to play a metabolic role in nodule energy metabolism. CRITICAL ISSUES H(2)O(2) may control the infection process and the subsequent bacterial differentiation into the symbiotic form. NO is required for an optimal establishment of symbiosis and appears to be a key player in nodule senescence. FUTURE DIRECTIONS A challenging question is to define more precisely when and where reactive species are generated and to develop adapted tools to detect their production in vivo. To investigate the role of Noxs and NRs in the production of H(2)O(2) and NO, respectively, the use of mutants under the control of organ-specific promoters will be of crucial interest. The balance between ROS and NO production appears to be a key point to understand the redox regulation of symbiosis.

BetS Is a Major Glycine Betaine/Proline Betaine Transporter Required for Early Osmotic Adjustment in Sinorhizobium meliloti

Hybridization to a PCR product derived from conserved betaine choline carnitine transporter (BCCT) sequences led to the identification of a 3.4-kb Sinorhizobium meliloti DNA segment encoding a protein (BetS) that displays significant sequence identities to the choline transporter BetT of Escherichia coli (34%) and to the glycine betaine transporter OpuD of Bacillus subtilis (30%). Although the BetS protein shows a common structure with BCCT systems, it possesses an unusually long hydrophilic C-terminal extension (169 amino acids). After heterologous expression of betS in E. coli mutant strain MKH13, which lacks choline, glycine betaine, and proline transport systems, both glycine betaine and proline betaine uptake were restored, but only in cells grown at high osmolarity or subjected to a sudden osmotic upshock. Competition experiments demonstrated that choline, ectoine, carnitine, and proline were not effective competitors for BetS-mediated betaine transport. Kinetic analysis revealed that BetS has a high affinity for betaines, with K(m)s of 16 +/- 2 microM and 56 +/- 6 microM for glycine betaine and proline betaine, respectively, in cells grown in minimal medium with 0.3 M NaCl. BetS activity appears to be Na(+) driven. In an S. meliloti betS mutant, glycine betaine and proline betaine uptake was reduced by about 60%, suggesting that BetS represents a major component of the overall betaine uptake activities in response to salt stress. beta-Galactosidase activities of a betS-lacZ strain grown in various conditions showed that betS is constitutively expressed. Osmotic upshock experiments performed with wild-type and betS mutant cells, treated or not with chloramphenicol, indicated that BetS-mediated betaine uptake is the consequence of immediate activation of existing proteins by high osmolarity, most likely through posttranslational activation. Growth experiments underscored the crucial role of BetS as an emerging system involved in the rapid acquisition of betaines by S. meliloti subjected to osmotic upshock.

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.

The Sinorhizobium meliloti glycine betaine biosynthetic genes (betICBA) are induced by choline and highly expressed in bacteroids

The symbiotic soil bacterium Sinorhizobium meliloti has the capacity to synthesize the osmoprotectant glycine betaine from choline-O-sulfate and choline. This pathway is encoded by the betICBA locus, which comprises a regulatory gene, betI, and three structural genes, betC (choline sulfatase), betB (betaine aldehyde dehydrogenase), and betA (choline dehydrogenase). Here, we report that betICBA genes constitute a single operon, despite the existence of intergenic regions containing mosaic elements between betI and betC, and betB and betA. The regulation of the bet operon was investigated by using transcriptional lacZ (beta-galactosidase) fusions and has revealed a strong induction by choline at concentrations as low as 25 microM and to a lesser extent by choline-O-sulfate and acetylcholine but not by osmotic stress or oxygen. BetI is a repressor of the bet transcription in the absence of choline, and a nucleotide sequence of dyad symmetry upstream of betI was identified as a putative betI box. Measurements of intracellular pools of choline, well correlated with beta-galactosidase activities, strongly suggested that BetI senses the endogenous choline pool that modulates the intensity of BetI repression. In contrast to Escherichia coli, BetI did not repress choline transport. During symbiosis with Medicago sativa, S. meliloti bet gene expression was observed within the infection threads, in young and in mature nodules. The existence of free choline in nodule cytosol, peribacteroid space, and bacteroids was demonstrated, and the data suggest that bet regulation in planta is mediated by BetI repression, as in free-living cells. Neither Nod nor Fix phenotypes were significantly impaired in a betI::omega mutant, indicating that glycine betaine biosynthesis from choline is not crucial for nodulation and nitrogen fixation.

Fructose Uptake in Sinorhizobium meliloti Is Mediated by a High-Affinity ATP-Binding Cassette Transport System

By transposon mutagenesis, we have isolated a mutant of Sinorhizobium meliloti which is totally unable to grow on fructose as sole carbon source as a consequence of its inability to transport this sugar. The cloning and sequencing analysis of the chromosomal DNA region flanking the TnphoA insertion revealed the presence of six open reading frames (ORFs) organized in two loci, frcRS and frcBCAK, transcribed divergently. The frcBCA genes encode the characteristic components of an ATP-binding cassette transporter (FrcB, a periplasmic substrate binding protein, FrcC, an integral membrane permease, and FrcA, an ATP-binding cytoplasmic protein), which is the unique high-affinity (K(m) of 6 microM) fructose uptake system in S. meliloti. The FrcK protein shows homology with some kinases, while FrcR is probably a transcriptional regulator of the repressor-ORF-kinase family. The expression of S. meliloti frcBCAK in Escherichia coli, which transports fructose only via the phosphotransferase system, resulted in the detection of a periplasmic fructose binding activity, demonstrating that FrcB is the binding protein of the Frc transporter. The analysis of substrate specificities revealed that the Frc system is also a high-affinity transporter for ribose and mannose, which are both fructose competitors for the binding to the periplasmic FrcB protein. However, the Frc mutant was still able to grow on these sugars as sole carbon source, demonstrating the presence of at least one other uptake system for mannose and ribose in S. meliloti. The expression of the frcBC genes as determined by measurements of alkaline phosphatase activity was shown to be induced by mannitol and fructose, but not by mannose, ribose, glucose, or succinate, suggesting that the Frc system is primarily targeted towards fructose. Neither Nod nor Fix phenotypes were impared in the TnphoA mutant, demonstrating that fructose uptake is not essential for nodulation and nitrogen fixation, although FrcB protein is expressed in bacteroids isolated from alfalfa nodulated by S. meliloti wild-type strains.

Functional Expression of Sinorhizobium meliloti BetS, a High-Affinity Betaine Transporter, in Bradyrhizobium japonicum USDA110

ABSTRACT Among the Rhizobiaceae, Bradyrhizobium japonicum strain USDA110 appears to be extremely salt sensitive, and the presence of glycine betaine cannot restore its growth in medium with an increased osmolarity (E. Boncompagni, M. Østerås, M. C. Poggi, and D. Le Rudulier, Appl. Environ. Microbiol. 65:2072-2077, 1999). In order to improve the salt tolerance of B. japonicum, cells were transformed with the betS gene of Sinorhizobium meliloti. This gene encodes a major glycine betaine/proline betaine transporter from the betaine choline carnitine transporter family and is required for early osmotic adjustment. Whereas betaine transport was absent in the USDA110 strain, such transformation induced glycine betaine and proline betaine uptake in an osmotically dependent manner. Salt-treated transformed cells accumulated large amounts of glycine betaine, which was not catabolized. However, the accumulation was reversed through rapid efflux during osmotic downshock. An increased tolerance of transformant cells to a moderate NaCl concentration (80 mM) was also observed in the presence of glycine betaine or proline betaine, whereas the growth of the wild-type strain was totally abolished at 80 mM NaCl. Surprisingly, the deleterious effect due to a higher salt concentration (100 mM) could not be overcome by glycine betaine, despite a significant accumulation of this compound. Cell viability was not significantly affected in the presence of 100 mM NaCl, whereas 75% cell death occurred at 150 mM NaCl. The absence of a potential gene encoding Na+/H+ antiporters in B. japonicum could explain its very high Na+ sensitivity.

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.

Overexpression of BetS, a Sinorhizobium meliloti high-affinity betaine transporter, in bacteroids from Medicago sativa nodules sustains nitrogen fixation during early salt stress adaptation.

Sinorhizobium meliloti possesses several betaine transporters to cope with salt stress, and BetS represents a crucial high-affinity glycine and proline betaine uptake system involved in the rapid acquisition of betaines by cells subjected to osmotic upshock. Using a transcriptional lacZ (beta-galactosidase) fusion, we showed that betS is expressed during the establishment of the symbiosis and in mature nitrogen-fixing nodules. However, neither Nod nor Fix phenotypes were impaired in a betS mutant. BetS is functional in isolated bacteroids, and its activity is strongly activated by high osmolarity. In bacteroids from a betS mutant, glycine betaine and proline betaine uptake was reduced by 85 to 65%, indicating that BetS is a major component of the overall betaine uptake activity in bacteroids in response to osmotic stress. Upon betS overexpression (strain UNA349) in free-living cells, glycine betaine transport was 2.3-fold higher than in the wild-type strain. Interestingly, the accumulation of proline betaine, the endogenous betaine synthesized by alfalfa plants, was 41% higher in UNA349 bacteroids from alfalfa plants subjected to 1 week of salinization (0.3 M NaCl) than in wild-type bacteroids. In parallel, a much better maintenance of nitrogen fixation activity was observed in 7-day-salinized plants nodulated with the overexpressing strain than in wild-type nodulated plants. Taken altogether, these results are consistent with the major role of BetS as an emergency system involved in the rapid uptake of betaines in isolated and in planta osmotically stressed bacteroids of S. meliloti.

ROS in the Legume- Rhizobium Symbiosis

Plants appear to generate reactive oxygen species (ROS) as signaling molecules to control various fundamental processes. With this background, this review aims to highlight the involvement of ROS, and their possible interactions with nitric oxide (NO) and glutathione (GSH) in the symbiosis between rhizobia and leguminous plants. This compatible interaction, which is very important for sustainable agriculture, leads to the formation of a novel organ capable of fixing atmospheric nitrogen. ROS are involved in the early steps of the symbiotic interaction: their presence is essential for the development of optimal symbiosis and points to a signaling role for ROS during the symbiotic process. ROS may also regulate nodule function by interacting with NO.