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.
Dupont
Laurence
L
Unité Interactions Plantes-Microorganismes et Santé Végétale, UMR6192 CNRS-INRA-Universitéde Nice Sophia Antipolis, Centre INRA Agrobiotech, 400 Route des Chappes, BP167, 06903 Sophia Antipolis Cédex, France.
Garcia
Isabelle
I
Poggi
Marie-Christine
MC
Alloing
Geneviève
G
Mandon
Karine
K
Le Rudulier
Daniel
D
D018528:ATP-Binding Cassette Transporters / Q000235:genetics / Q000378:metabolism*; D000109:Acetylcholine / Q000378:metabolism; D000222:Adaptation, Physiological; D000251:Adenosine Triphosphatases / Q000235:genetics / Q000502:physiology; D001426:Bacterial Proteins / Q000235:genetics / Q000502:physiology; D001693:Biological Transport, Active / Q000235:genetics; D002352:Carrier Proteins / Q000235:genetics / Q000502:physiology; D002794:Choline / Q000378:metabolism*; D004269:DNA, Bacterial / Q000737:chemistry; D015964:Gene Expression Regulation, Bacterial; D023061:Gene Order; D005798:Genes, Bacterial; D000455:Medicago sativa / Q000382:microbiology*; D026901:Membrane Transport Proteins / Q000235:genetics / Q000502:physiology; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional; D016297:Mutagenesis, Site-Directed; D016366:Open Reading Frames; D009876:Operon; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D016962:Sinorhizobium meliloti / Q000235:genetics* / Q000378:metabolism*; D013379:Substrate Specificity
D018528:ATP-Binding Cassette Transporters; D001426:Bacterial Proteins; D002352:Carrier Proteins; D004269:DNA, Bacterial; D026901:Membrane Transport Proteins; D000251:Adenosine Triphosphatases; D002794:Choline; D000109:Acetylcholine