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Journal of Bacteriology, September 2004, p. 5988-5996, Vol. 186, No. 18
0021-9193/04/$08.00+0     DOI: 10.1128/JB.186.18.5988-5996.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

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

Laurence Dupont, Isabelle Garcia, Marie-Christine Poggi, Geneviève Alloing, Karine Mandon, and Daniel Le Rudulier*

Unité Interactions Plantes-Microorganismes et Santé Végétale, UMR6192 CNRS-INRA-Université de Nice Sophia Antipolis, Centre INRA Agrobiotech, Sophia Antipolis, France

Received 2 April 2004/ Accepted 9 June 2004

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 µM) and acetylcholine with a low affinity (KD, 145 µM) 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.

* Corresponding author. Mailing address: 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. Phone: (33) 492 386 630. Fax: (33) 492 386 587. E-mail: leruduli{at}unice.fr.

Journal of Bacteriology, September 2004, p. 5988-5996, Vol. 186, No. 18
0021-9193/04/$08.00+0     DOI: 10.1128/JB.186.18.5988-5996.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.



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