Laurence Dupont

Crystal Structures of the Choline/Acetylcholine Substrate-binding Protein ChoX from Sinorhizobium meliloti in the Liganded and Unliganded-Closed States

The ATP-binding cassette transporter ChoVWX is one of several choline import systems operating in Sinorhizobium meliloti. Here fluorescence-based ligand binding assays were used to quantitate substrate binding by the periplasmic ligand-binding protein ChoX. These data confirmed that ChoX recognizes choline and acetylcholine with high and medium affinity, respectively. We also report the crystal structures of ChoX in complex with either choline or acetylcholine. These structural investigations revealed an architecture of the ChoX binding pocket and mode of substrate binding similar to that reported previously for several compatible solute-binding proteins. Additionally the ChoX-acetylcholine complex permitted a detailed structural comparison with the carbamylcholine-binding site of the acetylcholine-binding protein from the mollusc Lymnaea stagnalis. In addition to the two liganded structures of ChoX, we were also able to solve the crystal structure of ChoX in a closed, substrate-free conformation that revealed an architecture of the ligand-binding site that is superimposable to the closed, ligand-bound form of ChoX. This structure is only the second of its kind and raises the important question of how ATP-binding cassette transporters are capable of distinguishing liganded and unliganded-closed states of the binding protein.

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.

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.

Characterization of a Sinorhizobium meliloti ATP-binding cassette histidine transporter also involved in betaine and proline uptake.

The symbiotic soil bacterium Sinorhizobium meliloti uses the compatible solutes glycine betaine and proline betaine for both protection against osmotic stress and, at low osmolarities, as an energy source. A PCR strategy based on conserved domains in components of the glycine betaine uptake systems from Escherichia coli (ProU) and Bacillus subtilis (OpuA and OpuC) allowed us to identify a highly homologous ATP-binding cassette (ABC) binding protein-dependent transporter in S. meliloti. This system was encoded by three genes (hutXWV) of an operon which also contained a fourth gene (hutH2) encoding a putative histidase, which is an enzyme involved in the first step of histidine catabolism. Site-directed mutagenesis of the gene encoding the periplasmic binding protein (hutX) and of the gene encoding the cytoplasmic ATPase (hutV) was done to study the substrate specificity of this transporter and its contribution in betaine uptake. These mutants showed a 50% reduction in high-affinity uptake of histidine, proline, and proline betaine and about a 30% reduction in low-affinity glycine betaine transport. When histidine was used as a nitrogen source, a 30% inhibition of growth was observed in hut mutants (hutX and hutH2). Expression analysis of the hut operon determined using a hutX-lacZ fusion revealed induction by histidine, but not by salt stress, suggesting this uptake system has a catabolic role rather than being involved in osmoprotection. To our knowledge, Hut is the first characterized histidine ABC transporter also involved in proline and betaine uptake.

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).

The endobacterium of an arbuscular mycorrhizal fungus modulates the expression of its toxin–antitoxin systems during the life cycle of its host

Arbuscular mycorrhizal fungi (AMF) are widespread root symbionts that perform important ecological services, such as improving plant nutrient and water acquisition. Some AMF from the Gigasporaceae family host a population of endobacteria, Candidatus Glomeribacter gigasporarum (Cagg). The analysis of the Cagg genome identified six putative toxin–antitoxin modules (TAs), consisting of pairs of stable toxins and unstable antitoxins that affect diverse physiological functions. Sequence analysis suggested that these TA modules were acquired by horizontal transfer. Gene expression patterns of two TAs (yoeB/yefM and chpB/chpS) changed during the fungal life cycle, with the expression during the pre-symbiotic phase higher than during the symbiosis with the plant host. The heterologous expression in Escherichia coli demonstrated the functionality only for the YoeB–YefM pair. On the basis of these observations, we speculate that TA modules might help Cagg adapt to its intracellular habitat, coordinating its proliferation with the physiological state of the AMF host.

A vapBC-type toxin–antitoxin module of Sinorhizobium meliloti influences symbiotic efficiency and nodule senescence of Medicago sativa

The symbiotic nitrogen-fixing soil bacterium Sinorhizobium meliloti carries a large number of toxin-antitoxin (TA) modules both on the chromosome and megaplasmids. One of them, the vapBC-5 module that belongs to the type II systems was characterized here. It encodes an active toxin vapC-5, and was shown to be controlled negatively by the complex of its own proteins. Different mutants of the vapBC-5 genes exhibited diverse effects on symbiotic efficiency during interaction with the host plant Medicago sativa. The absence of the entire vapBC-5 region had no influence on nodule formation and nitrogen fixation properties. The strain carrying an insertion in the antitoxin gene showed a reduced nitrogen fixation capacity resulting in a lower plant yield. In contrast, when the toxin gene was mutated, the strain developed more efficient symbiosis with the host plant. The nitrogen fixing root nodules had a delayed senescent phenotype and contained elevated level of plant-derived molecules characteristic of later steps of nodule development. The longer bacteroid viability and abundance of active nitrogen fixing zone resulted in increased production of plant material. These data indicate that modification of the toxin/antitoxin production may influence bacteroid metabolism and may have an impact on the adaptation to changing environmental conditions.

Physiological Roles of Betaines in Sinorhizobium meliloti and other Members of the Rhizobiaceae Subjected to Salt Stress

Sinorhizobium meliloti is able to survive osmotically stressful environments by the intracellular accumulation of compatible solutes, which restore the osmotic balance between the cell and the environment. Among these solutes, glycine betaine (the N-trimethyl derivative of glycine) has been shown to be a very efficient osmoprotective compound which strongly stimulates the growth rate of this bacterium in high-salt medium (Le Rudulier, Bernard, 1986). Unlike enteric bacteria, S. meliloti also catabolizes glycine betaine and uses it both as a carbon and a nitrogen source for growth. Glycine betaine either can be taken up directly from the environment by specific transport systems or synthesized from choline-O-sulfate or choline (Osteras et al., 1998; Smith et al., 1988). The osmoadaptative responses of other rhizobial species have not been thoroughly studied in comparison to those of S. meliloti. However, we have recently analyzed choline and glycine betaine uptake capacities and investigated the occurrence of glycine betaine biosynthesis pathway in various Rhizobiaceae strains which display a large variation in salt tolerance (Boncompagni et al., 1999).

Isolation and molecular characterization of theSinorhizobium meliloti bet locus encoding glycine betaine biosynthesis

To cope with osmotic stress,Sinorhizobium meliloti accumulates organic compatible solutes such as glutamate, trehalose, N-acetylglutaminylglutamine amide, and the most potent osmoprotectant glycine betaine. In order to study the regulation of the glycine betaine biosynthetic pathway, a genetic and molecular analysis was performed. We have selected a Tn5 mutant ofS. meliloti which was deficient in choline dehydrogenase activity. The mutation was complemented using a genomic bank ofS. meliloti. Subcloning and DNA sequencing of a 8-6 kb region from the complemented plasmid showed four open reading frames with an original structural organization of thebet locus compared to that described inE. coli. (i) ThebetB and thebetA genes which encode a glycine betaine aldehyde dehydrogenase, and a choline dehydrogenase, respectively, are separated from thebetI gene (regulatory protein) by an additional gene namedbetC. The BetC protein shares about 30% identity with various sulphatases and is involved in the conversion of choline-O-sulphate into choline. Choline-O-sulphate is used as an osmoprotectant, or as a carbon or sulphur source and this utilization is dependent on a functionalbet locus. (ii) No sequence homologous tobetT (encoding a high-affinity choline transport system inE. coli) was found in the vicinity of thebet locus. (iii) ThebetB and thebetA genes, as well as thebetI and thebetC genes are, respectively, separated by 211 and 167 bp sequences containing inverted repeats. Southern blot analysis indicated that thebet locus is located on the chromosome, and not on the megaplasmids.