Molecular Characterization of a Binding Protein-Dependent-Transport System for Glycine Betaine in Sinorhizobium meliloti

S. meliloti can use glycine betaine as an effective osmoprotectant at high osmolarity and a glycine betaine-binding protein has been identified in the periplasmic fraction (Le Rudulier et al., 1991). The proU locus encoding a high-affinity glycine betaine transport system has been characterized in E. coli and B. subtilis (Gowrishankar 1989; Kempf et al., 1995; Kin et al., 1995). In S. meliloti, glycine betaine transport activity shows some strong biochemical homologies with the ProU system from E. coli. Three primers corresponding to conserved DNA boxes within the proV gene were synthesized and used in a PCR cloning strategy. This approach combined with a colony screening of a S. meliloti total DNA bank allowed us to identify a cosmid containing a 27-kb DNA insert with the entire S. meliloti proU locus. The proX (glycine betaine-binding protein) was the first gene followed by proW (inner membrane protein), and proV (membrane bound cytoplasmic ATPase). The deduced sequence proteins showed, respectively, 26%, 46% and 55% identity between S. meliloti and E. coli. The corresponding protein molecular weights are 37, 30 and 30 kDa, respectively. In addition to these 3 genes, one ORF (ORF1) is present upstream of proX and another ORF (hutH) is located downstream of proV. The function of the ORF1-product is unknown, whereas the hutH-product shows significant homology with histidases, enzymes involved in the first step of histidine catabolism. The proU locus was located on the bacterial chromosome. Construction of site-directed Ω interposon mutants has been done in order to determine the function of the different genes. These mutants are not affected in the infection process of the plant host and they induced fully functional nitrogen fixation activity. Their uptake capacities are currently tested.