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.

Several reactive oxygen and nitrogen species (ROS/RNS) are continuously produced in plants as by-products of aerobic metabolism or in response to stresses. Depending on the nature of the ROS and RNS, some of them are highly toxic and rapidly detoxified by various cellular enzymatic and non-enzymatic mechanisms. Whereas plants have many mechanisms with which to combat increased ROS/RNS levels produced during stress conditions, under other circumstances plants appear to generate ROS/RNS as signalling molecules to control various processes encompassing the whole lifespan of the plant such as normal growth and development stages. This review aims to summarize recent studies highlighting the involvement of ROS/RNS, as well as the low molecular weight thiols, glutathione and homoglutathione, during the symbiosis between rhizobia and leguminous plants. This compatible interaction initiated by a molecular dialogue between the plant and bacterial partners, leads to the formation of a novel root organ capable of fixing atmospheric nitrogen under nitrogen-limiting conditions. On the one hand, ROS/RNS detection during the symbiotic process highlights the similarity of the early response to infection by pathogenic and symbiotic bacteria, addressing the question as to which mechanism rhizobia use to counteract the plant defence response. Moreover, there is increasing evidence that ROS are needed to establish the symbiosis fully. On the other hand, GSH synthesis appears to be essential for proper development of the root nodules during the symbiotic interaction. Elucidating the mechanisms that control ROS/RNS signalling during symbiosis could therefore contribute in defining a powerful strategy to enhance the efficiency of the symbiotic interaction.

The deduced amino acid sequences of four open reading frames identified upstream of thefixGHI region in Azorhizobium caulinodans are very similar to the putative terminal oxidase complex coded by the fixNOQP operons from Rhizobium meliloti and Bradyrhizobium japonicum. The expression of the A. caulinodansfixNOQP genes, which was maximal under microaerobiosis, was positively regulated by FixK and independent of NifA. In contrast to the Fix-phenotype of B.japonicum and R. melilotifixN mutants, anA. caulinodansfixNO-deleted mutant strain retained 50%o of the nitrogenase activity of the wild type in the symbiotic state. In addition, the nitrogenase activity was scarcely reduced under free-living conditions. Analysis of membrane fractions of A.caulinodans wild-type and mutant strains suggests that the fixNOQP region encodes two proteins with covalently bound hemes, tentatively assigned tofixO andfixP. Spectral analysis showed a large decrease in the c-type cytochrome content of thefixN mutant compared with the wild type. These results provide evidence for the involvement of FixNOQP proteins in a respiratory process. The partial impairment in nitrogen fixation of thefixN mutant in planta may be due to the activity of an alternative terminal oxidase compensating for the loss of the oxidase complex encoded byfixNOQP.

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 (K D , 2.7 M) and acetylcholine with a low affinity (K D , 145 M) 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.Choline is a common constituent of eukaryotic membranes in the form of phosphatidylcholine (PC) and therefore should be widespread in different environments, including the soil and the rhizosphere. Indeed, significant amounts of choline are readily liberated into the environment from plant and animal residues (15). Sinorhizobium meliloti, a plant root-associated bacterium, possesses distinct transport activities for choline uptake (27) and has the ability to oxidize choline to glycine betaine via the bet operon (34,24). In contrast to Escherichia coli and Bacillus subtilis (25, 2), S. meliloti can use choline for growth. This depends on a functional bet locus (34, 24) associated with catabolism of glycine betaine which is absent in E. coli and B. subtilis. This catabolism is reduced under hyperosmotic conditions, and under these conditions glycine betaine accumulation is favored (34). Moreover, due to the presence of a PC synthase in S. meliloti, which directly condenses choline to CDP-diacylglyceride, choline is a direct precursor of PC, as recently demonstrated for other bacteria, including Agrobacterium, Brucella, and Pseudomonas (6,19). In addition to this PC synthase pathway, S. meliloti possesses a methylation pathway for PC biosynthesis which functions by threefold methylation of phosphatidylethanolamine with S-adenosylmethionine as a methy...

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.Leguminous plants can engage in a symbiotic interaction with Rhizobia and form new root organs, the nodules. The nodulation process is initiated by a complex signal exchange between both partners (5). Rhizobial invasion in the symbiotic model Medicago sativa/Sinorhizobium meliloti occurs via root hairs. The perception of bacterial nodulation factors by the host plant leads to cell division in the root pericycle and in the root cortex, where the nodule primordium forms. Simultaneously, root hairs deform and curl. The bacteria are entrapped in this curl, local cell wall is hydrolyzed, and a plasma membrane invagination occurs, leading to the formation of an infection thread (IT) (3). This plant-derived tubule filled with dividing and growing bacteria first progresses through the infected root hair and then traverses several cell layers toward the nodule primordium.

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 M and 56 ؎ 6 M 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. ␤-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 nucleotide sequence of a 1 kb fragment upstream of Azorhizobium caulinodans fixL was established. An open reading frame of 744 bp was identified as a fixK homologue. A kanamycin cartridge was inserted into the cloned fixK-like gene and recombined into the host genome. The resulting mutant was Nif-Fix-, suggesting that FixK was required for nitrogen fixation both in symbiotic conditions and in the free-living state. Using a pfixK-lacZ fusion, the FixLJ products were shown to control the expression of fixK. Using a pnifA-lacZ fusion, the FixK product was shown to regulate positively the transcription of nifA in bacteria grown in the free-living state. In addition, a double ntrC-fixL mutant was constructed and was shown to be completely devoid of nitrogenase activity. A model of regulation, based on these data, is presented and might explain the unusual ability of A. caulinodans to fix nitrogen both under symbiotic conditions and in the free-living state.

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.