Synthesis of the osmoprotectant glycine betaine in Bacillus subtilis: characterization of the gbsAB genes

Abstract

Synthesis of the osmoprotectant glycine betaine from the exogenously provided precursor choline or glycine betaine aldehyde confers considerable osmotic stress tolerance to Bacillus subtilis in high-osmolarity media. Using an Escherichia coli mutant (betBA) defective in the glycine betaine synthesis enzymes, we cloned by functional complementation the genes that are required for the synthesis of the osmoprotectant glycine betaine in B. subtilis. The DNA sequence of a 4.1-kb segment from the cloned chromosomal B. subtilis DNA was established, and two genes (gbsA and gbsB) whose products were essential for glycine betaine biosynthesis and osmoprotection were identified. The gbsA and gbsB genes are transcribed in the same direction, are separated by a short intergenic region, and are likely to form an operon. The deduced gbsA gene product exhibits strong sequence identity with members of a superfamily of specialized and nonspecialized aldehyde dehydrogenases. This superfamily comprises glycine betaine aldehyde dehydrogenases from bacteria and plants with known involvement in the cellular adaptation to high-osmolarity stress and drought. The deduced gbsB gene product shows significant 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 the gbsAB genes destroyed the cholineglycine 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 mutant strain. The continued intracellular accumulation of choline or glycine betaine aldehyde in a strain lacking the glycine betaine-biosynthetic enzymes strongly interfered with the growth of B. subtilis, even in medium of moderate osmolarity. A single transcription initiation site for gbsAB was detected by high-resolution primer extension analysis. gbsAB transcription was initiated from a promoter with close homology to A -dependent promoters and was stimulated by the presence of choline in the growth medium.

Extreme Halophiles Synthesize Betaine from Glycine by Methylation

“…It is based on selective detection and analysis of radioactive reaction products, N-monomethylglycine (sarcosine), N,N-dimethylglycine, and betaine, after a methyl group transfer from [methyl- 14 C]S-adenosylmethionine (AdoMet) 1 to substrate. 25 l of 0.1 M substrate (glycine, sarcosine, or dimethylglycine), 25 l of Reaction Buffer (560 mM Tris-HCl, pH 7.5, 4 mM 2-mercaptoethanol, 50 M MgCl 2 , 160 M EDTA), 25 l of 4 mM…”

“…B, synthesis of methylation products in a reaction mixture containing 0.53 mg/ml purified E. halochloris GSMT and 0.7 mg/ml purified A. halophila SDMT. 1.25 mM glycine (white circle) or 20 mM glycine (black square) and 8 mM S-adenosyl-L-[methyl- 14 C]methionine were used as substrates. The relative amount of products is dependent on the concentration of glycine in the reaction mixture.…”

“…A choline dehydrogenase has been shown to catalyze the two-step reaction of choline to betaine in many microbes (13)(14)(15). Alternatively, the oxidation of choline to betaine can be catalyzed by a choline oxidase (16,17).…”

Purification and Characterization of Two Novel Halotolerant Extracellular Proteases fromBacillus subtilisStrain FP-133

“…24) This is the first report describing neutral halotolerant proteases produced by a B. subtilis strain, because expro-I and II had an optimal pH between 7.5 and 8, which is favorable for industrial use.…”

Osmoregulation of the opuE proline transport gene from Bacillus subtilis: contributions of the sigma A‐ and sigma B‐dependent stress‐responsive promoters

“…In addition to the potent osmoprotectant glycine betaine (Kempf and Bremer, 1995;Lin and Hansen, 1995;Boch et al, 1996;Kappes et al, 1996), a variety of structurally related trimethylammonium compounds and the cyclic amino acid derivative ectoine (Jebbar et al, 1997;Kappes and Bremer, 1998), proline plays a very important role in the adaptation reaction of B. subtilis to high osmolarity. Large amounts of proline are accumulated by the cells through de novo synthesis in response to growth in high osmolarity environments .…”