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Genetic control of osmoadaptive glycine betaine synthesis in Bacillus subtilis through the choline-sensing and glycine betaine-responsive GbsR repressor.

ABSTRACT: Synthesis of the compatible solute glycine betaine confers a considerable degree of osmotic stress tolerance to Bacillus subtilis. This osmoprotectant is produced through the uptake of the precursor choline via the osmotically inducible OpuB and OpuC ABC transporters and a subsequent two-step oxidation process by the GbsB and GbsA enzymes. We characterized a regulatory protein, GbsR, controlling the transcription of both the structural genes for the glycine betaine biosynthetic enzymes (gbsAB) and those for the choline-specific OpuB transporter (opuB) but not of that for the promiscuous OpuC transporter. GbsR acts genetically as a repressor and functions as an intracellular choline sensor. Spectroscopic analysis of the purified GbsR protein showed that it binds the inducer choline with an apparent K(D) (equilibrium dissociation constant) of approximately 165 ?M. Based on the X-ray structure of a protein (Mj223) from Methanococcus jannaschii, a homology model for GbsR was derived. Inspection of this GbsR in silico model revealed a possible ligand-binding pocket for choline resembling those of known choline-binding sites present in solute receptors of microbial ABC transporters, e.g., that of the OpuBC ligand-binding protein of the OpuB ABC transporter. GbsR was not only needed to control gbsAB and opuB expression in response to choline availability but also required to genetically tune down glycine betaine production once cellular adjustment to high osmolarity has been achieved. The GbsR regulatory protein from B. subtilis thus records and integrates cellular and environmental signals for both the onset and the repression of the synthesis of the osmoprotectant glycine betaine.

SUBMITTER: Nau-Wagner G

PROVIDER: S-EPMC3347207 | biostudies-literature | 2012 May

REPOSITORIES: biostudies-literature

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Genetic control of osmoadaptive glycine betaine synthesis in Bacillus subtilis through the choline-sensing and glycine betaine-responsive GbsR repressor.

Nau-Wagner Gabriele G Opper Daniela D Rolbetzki Anne A Boch Jens J Kempf Bettina B Hoffmann Tamara T Bremer Erhard E

Journal of bacteriology 20120309 10

Synthesis of the compatible solute glycine betaine confers a considerable degree of osmotic stress tolerance to Bacillus subtilis. This osmoprotectant is produced through the uptake of the precursor choline via the osmotically inducible OpuB and OpuC ABC transporters and a subsequent two-step oxidation process by the GbsB and GbsA enzymes. We characterized a regulatory protein, GbsR, controlling the transcription of both the structural genes for the glycine betaine biosynthetic enzymes (gbsAB) a ...[more]

PMID: 22408163

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Project description: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 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 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 sigma A-dependent promoters and was stimulated by the presence of choline in the growth medium.

Project description:Bacillus subtilis adjusts to high osmolarity surroundings through the amassing of compatible solutes. It synthesizes the compatible solute glycine betaine from prior imported choline and scavenges many pre-formed osmostress protectants, including glycine betaine, from environmental sources. Choline is imported through the substrate-restricted ABC transporter OpuB and the closely related, but promiscuous, OpuC system, followed by its GbsAB-mediated oxidation to glycine betaine. We have investigated the impact of two MarR-type regulators, GbsR and OpcR, on gbsAB, opuB, and opuC expression. Judging by the position of the previously identified OpcR operator in the regulatory regions of opuB and opuC [Lee et al. (2013) Microbiology 159, 2087-2096], and that of the GbsR operator identified in the current study, we found that the closely related GbsR and OpcR repressors use different molecular mechanisms to control transcription. OpcR functions by sterically hindering access of RNA-polymerase to the opuB and opuC promoters, while GbsR operates through a roadblock mechanism to control gbsAB and opuB transcription. Loss of GbsR or OpcR de-represses opuB and opuC transcription, respectively. With respect to the osmotic control of opuB and opuC expression, we found that this environmental cue operates independently of the OpcR and GbsR regulators. When assessed over a wide range of salinities, opuB and opuC exhibit a surprisingly different transcriptional profile. Expression of opuB increases monotonously in response to incrementally increase in salinity, while opuC transcription levels decrease after an initial up-regulation at moderate salinities. Transcription of the gbsR and opcR regulatory genes is up-regulated in response to salt stress, and is also affected through auto-regulatory processes. The opuB and opuC operons have evolved through a gene duplication event. However, evolution has shaped their mode of genetic regulation, their osmotic-stress dependent transcriptional profile, and the substrate specificity of the OpuB and OpuC ABC transporters in a distinctive fashion.

Project description:The accumulation of the osmoprotectant glycine betaine from exogenous sources provides a high degree of osmotic tolerance to Bacillus subtilis. We have identified, through functional complementation of an Escherichia coli mutant defective in glycine betaine uptake, a new glycine betaine transport system from B. subtilis. The DNA sequence of a 2,310-bp segment of the cloned region revealed a single gene (opuD) whose product (OpuD) was essential for glycine betaine uptake and osmoprotection in E. coli. The opuD gene encodes a hydrophobic 56.13-kDa protein (512 amino acid residues). OpuD shows a significant degree of sequence identity to the choline transporter BetT and the carnitine transporter CaiT from E. coli and a BetT-like protein from Haemophilus influenzae. These membrane proteins form a family of transporters involved in the uptake of trimethylammonium compounds. The OpuD-mediated glycine betaine transport activity in B. subtilis is controlled by the environmental osmolarity. High osmolarity stimulates de novo synthesis of OpuD and activates preexisting OpuD proteins to achieve maximal glycine betaine uptake activity. An opuD mutant was constructed by marker replacement, and the OpuD-mediated glycine betaine uptake activity was compared with that of the previously identified multicomponent OpuA and OpuC (ProU) glycine betaine uptake systems. In addition, a set of mutants was constructed, each of which synthesized only one of the three glycine betaine uptake systems. These mutants were used to determine the kinetic parameters for glycine betaine transport through OpuA, OpuC, and OpuD. Each of these uptake systems shows high substrate affinity, with Km values in the low micromolar range, which should allow B. subtilis to efficiently acquire the osmoprotectant from the environment. The systems differed in their contribution to the overall glycine betaine accumulation and osmoprotection. A triple opuA, opuC, and opuD mutant strain was isolated, and it showed no glycine betaine uptake activity, demonstrating that three transport systems for this osmoprotectant operate in B. subtilis.

Dataset Information

Genetic control of osmoadaptive glycine betaine synthesis in Bacillus subtilis through the choline-sensing and glycine betaine-responsive GbsR repressor.

Publications

Genetic control of osmoadaptive glycine betaine synthesis in Bacillus subtilis through the choline-sensing and glycine betaine-responsive GbsR repressor.

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