ISOLATION AND MOLECULAR CHARACTERIZATION OF THE SINORHIZOBIUM-MELILOTI BET LOCUS ENCODING GLYCINE BETAINE BIOSYNTHESIS

To cope with osmotic stress, Sinorhizobium meliloti accumulates organi c compatible solutes such as glutamate, trehalose, N-acetylglutaminylg lutamine amide, and the most potent osmoprotectant glycine betaine. In order to study the regulation of the glycine betaine biosynthetic pat hway, a genetic and molecular analysis was performed. We have selected a Tn5 mutant of S. meliloti which was deficient in choline dehydrogen ase activity. The mutation was complemented using a genomic bank of S. meliloti. Subcloning and DNA sequencing of a 8.6 kb region from the c omplemented plasmid showed four open reading frames with an original s tructural organization of the bet locus compared to that described in E. coli. (i) The betB and the betA genes which encode a glycine betain e aldehyde dehydrogenase, and a choline dehydrogenase, respectively, a re separated from the berl gene (regulatory protein) by an additional gene named betC. The BetC protein shares about 30% identity with vario us 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 func tional bet locus. (ii) No sequence homologous to betT (encoding a high -affinity choline transport system in E. coli) was found in the vicini ty of the bet locus. (iii) The betB and the betA genes, as well as the betI and the betC genes are, respectively, separated by 211 and 167 b p sequences containing inverted repeats. Southern blot analysis indica ted that the ber locus is located on the chromosome, and not on the me gaplasmids.