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Little is known about the energy sources used by rhizobia during colonization, invasion and root nodule formation on leguminous plants. We have recently reported that an impaired proline metabolism in Rhizobium meliloti leads to a reduced nodulation efficiency and competitiveness on alfalfa roots. In the present study we have characterized the R. meliloti proline dehydrogenase gene (putA) and addressed the question of its role in symbiosis. This rhizobial gene encodes a 1224-amino-acid-long polypeptide which is homologous to enteric bacteria, Rhodobacter capsulatus and Bradyrhizobium japonicum PutA proteins.Like the situation in these bacteria, sequence analysis identified the proline dehydrogenase (PDH) and pyrroline-5-carboxylate dehydrogenase (P5CDH) domains in the R. meliloti putA-encoded protein. Beta-galactosidase assays performed with free-living cells carrying a putAlacZ transcriptional fusion revealed that R. meliloti putA gene expression is induced by proline, autoregulated by its encoded product, and independent of the general nitrogen regulatory system (Ntr). In addition, analysis of putA expression during the different steps of the symbiotic interaction with alfalfa showed that expression of this gene is turned on by the root exudates (RE), during root invasion and nodule formation, but not in differentiated nitrogen-fixing bacteroids. Furthermore, we show that the PutA phenotype leads to a significant reduction of alfalfa root colonization by R. meliloti.