SYNTHESIS OF THE OSMOPROTECTANT GLYCINE BETAINE IN BACILLUS-SUBTILIS - CHARACTERIZATION OF THE GBSAB GENES

Synthesis of the osmoprotectant glycine betaine from the exogenously p rovided precursor choline or glycine betaine aldehyde confers consider able osmotic stress tolerance to Bacillus subtilis in high-osmolarity media. Using an Escherichia coli mutant (betBA) defective in the glyci ne betaine synthesis enzymes, we cloned by functional complementation the genes that are required for the synthesis of the osmoprotectant gl ycine betaine in B. subtilis. The DNA sequence of a 4.1-kb segment fro m the cloned chromosomal B. subtilis DNA was established, and two gene s (gbsA and gbsB) whose products were essential for glycine betaine bi osynthesis and osmoprotection were identified. The gbsA and gbsB genes are transcribed in the same direction, are separated by a short inter genic region, and are likely to form an operon. The deduced gbsA gene product exhibits strong sequence identity with members of a superfamil y of specialized and nonspecialized aldehyde dehydrogenases. This supe rfamily comprises glycine betaine aldehyde dehydrogenases from bacteri a and plants with known involvement in the cellular adaptation to high -osmolarity stress and drought. The deduced gbsB gene product shows si gnificant similarity to the family of type III alcohol dehydrogenases. B. subtilis mutants with defects in the chromosomal gbsAB genes were constructed by marker replacement, and the growth properties of these mutant strains in high-osmolarity medium were analyzed. Deletion of th e gbsAB genes destroyed the choline-glycine betaine synthesis pathway and abolished the ability of B. subtilis to deal effectively with high -osmolarity stress in choline- or glycine betaine aldehyde-containing medium. Uptake of radiolabelled choline was unaltered in the gbsAB mut ant strain. The continued intracellular accumulation of choline or gly cine betaine aldehyde in a strain lacking the glycine betaine-biosynth etic enzymes strongly interfered with the growth of B. subtilis, even in medium of moderate osmolarity. A single transcription initiation si te for gbsAB was detected by high-resolution primer extension analysis . gbsAB transcription was initiated from a promoter with close homolog y to sigma(A)-dependent promoters and was stimulated by the presence o f choline in the growth medium.