Summary auto-generated
This study characterized the molecular basis of betaine biosynthesis in Sinorhizobium meliloti, a rhizobial bacterium that can utilize glycine betaine both as a carbon/nitrogen source and as an osmoprotectant. Researchers isolated a Tn5 mutant (LTS23-1020) deficient in choline dehydrogenase (CDH) activity, preventing the conversion of choline to betaine. Using complementation with a genomic library, they cloned and sequenced the bet genes from S. meliloti. The bacteria possess betB and betA genes encoding betaine-aldehyde dehydrogenase (BADH) and CDH respectively, showing 60% and 57% nucleotide identity to E. coli homologs. The S. meliloti BADH shares structural features with plant BADHs, while the CDH differs significantly from Arthrobacter choline oxidase. Notably, the sinorhizobial bet genes are organized differently from E. coli: they are separated by a 210 bp sequence containing putative transcription termination signals, and lack homologs of the E. coli betT (choline transporter) and betI (regulatory) genes. Southern blot analysis confirmed that betBA genes are chromosomally located, not on the megaplasmids.
Key findings
- S. meliloti possesses chromosomal betB and betA genes encoding BADH and CDH, with 60% and 57% identity respectively to E. coli genes
- The sinorhizobial bet genes have a different organization than E. coli, separated by a putative rho-independent transcription terminator and lacking betT and betI homologs
- The S. meliloti BADH protein shares conserved domains with plant BADHs, while the CDH shares minimal identity with Arthrobacter choline oxidase
- The CDH-deficient mutant LTS23-1020 cannot oxidize choline to betaine but retains betaine transport and can use betaine as an osmoprotectant
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Abstract
JA Pocard, N Vincent, E Boncompagni, LT Smith, MC Poggi and D Le Rudulier
Laboratoire de Biologie Vegetale et Microbiologie, URA CNRS 1114, Universite de Nice-Sophia Antipolis, France.
As a first step towards the elucidation of the molecular mechanisms responsible for the utilization of choline and glycine betaine (betaine) either as carbon and nitrogen sources or as osmoprotectants in Sinorhizobium meliloti, we selected a Tn5 mutant, LTS23-1020, which failed to grow on choline but grew on betaine. The mutant was deficient in choline dehydrogenase (CDH) activity, failed to oxidize [methyl- 14C]choline to [methyl-14C]betaine, and did not use choline, but still used betaine, as an osmoprotectant. The Tn5 mutation in LTS23-1020 was complemented 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 coli betB gene encoding betaine-aldehyde dehydrogenase (BADH) and betA gene encoding CDH, respectively. In addition to the homology with E. coli genes, the deduced sequence of the sinorhizobial BADH protein displays consensus sequences also found in plant BADHs. The deduced sequence of the sinorhizobial CDH protein shares only 21% identical residues with choline oxidase from Arthrobacter globiformis. The structural organization of the betBA genes in S. meliloti differs from that described in E. coli: (i) the two ORFs are separated by a 210 bp sequence containing inverted repeats resembling a putative rho- independent transcription terminator, and (ii) no sequence homologous to betT (high-affinity choline transport system) or betI (regulator) was found in the vicinity of the sinorhizobial betBA genes. Evidence is also presented that the S. meliloti betBA genes are not located on the megaplasmids.