MOLECULAR CHARACTERIZATION OF THE BET GENES ENCODING GLYCINE BETAINE SYNTHESIS IN SINORHIZOBIUM-MELILOTI 102F34

As a first step towards the elucidation of the molecular mechanisms re sponsible for the utilization of choline and glycine betaine (betaine) either as carbon and nitrogen sources or as osmoprotectants in Sinorh izobium meliloti, we selected a Tn5 mutant, LTS23-1020, which failed t o grow on choline but grew on betaine. The mutant was deficient in cho line dehydrogenase (CDH) activity, failed to oxidize [methyl-C-14]chol ine to [methyl-C-14]betaine, and did not use choline, but still used b etaine, as an osmoprotectant. The Tn5 mutation in LTS23-1020 was compl emented by plasmid pCHO34, isolated from a genomic bank of S. meliloti 102F34. Subcloning and DNA sequencing showed that pCHO34 harbours two ORFs which showed 60% and 57% identity with the Escherichia coil betB gene encoding betaine-aldehyde dehydrogenase (BADH) and betA gene enc oding CDH, respectively. In addition to the homology with 5. coil gene s, the deduced sequence of the sinorhizobial BADH protein displays con sensus sequences also found in plant BADHs. The deduced sequence of th e sinorhizobial CDH protein shares only 21% identical residues with ch oline oxidase from Arthrobacter globiformis. The structural organizati on of the betBA genes in 5. meliloti differs from that described in E. coil: (i) the two ORFs are separated by a 210 bp sequence containing inverted repeats ressembling a putative rho-independent transcription terminator, and (ii) no sequence homologous to betT (high-affinity cho line transport system) or betI (regulator) was found in the vicinity o f the sinorhizobial betBA genes. Evidence is also presented that the 5 . meliloti betBA genes are not located on the megaplasmids.