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Two MarR-Type Repressors Balance Precursor Uptake and Glycine Betaine Synthesis in Bacillus subtilis to Provide Cytoprotection Against Sustained Osmotic Stress.

ABSTRACT: 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.

SUBMITTER: Warmbold B

PROVIDER: S-EPMC7396694 | biostudies-literature | 2020

REPOSITORIES: biostudies-literature

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Two MarR-Type Repressors Balance Precursor Uptake and Glycine Betaine Synthesis in <i>Bacillus subtilis</i> to Provide Cytoprotection Against Sustained Osmotic Stress.

Warmbold Bianca B Ronzheimer Stefanie S Freibert Sven-Andreas SA Seubert Andreas A Hoffmann Tamara T Bremer Erhard E

Frontiers in microbiology 20200723

<i>Bacillus subtilis</i> 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 inv ...[more]

PMID: 32849357

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Project description:Hybridization to a PCR product derived from conserved betaine choline carnitine transporter (BCCT) sequences led to the identification of a 3.4-kb Sinorhizobium meliloti DNA segment encoding a protein (BetS) that displays significant sequence identities to the choline transporter BetT of Escherichia coli (34%) and to the glycine betaine transporter OpuD of Bacillus subtilis (30%). Although the BetS protein shows a common structure with BCCT systems, it possesses an unusually long hydrophilic C-terminal extension (169 amino acids). After heterologous expression of betS in E. coli mutant strain MKH13, which lacks choline, glycine betaine, and proline transport systems, both glycine betaine and proline betaine uptake were restored, but only in cells grown at high osmolarity or subjected to a sudden osmotic upshock. Competition experiments demonstrated that choline, ectoine, carnitine, and proline were not effective competitors for BetS-mediated betaine transport. Kinetic analysis revealed that BetS has a high affinity for betaines, with K(m)s of 16 +/- 2 microM and 56 +/- 6 microM for glycine betaine and proline betaine, respectively, in cells grown in minimal medium with 0.3 M NaCl. BetS activity appears to be Na(+) driven. In an S. meliloti betS mutant, glycine betaine and proline betaine uptake was reduced by about 60%, suggesting that BetS represents a major component of the overall betaine uptake activities in response to salt stress. beta-Galactosidase activities of a betS-lacZ strain grown in various conditions showed that betS is constitutively expressed. Osmotic upshock experiments performed with wild-type and betS mutant cells, treated or not with chloramphenicol, indicated that BetS-mediated betaine uptake is the consequence of immediate activation of existing proteins by high osmolarity, most likely through posttranslational activation. Growth experiments underscored the crucial role of BetS as an emerging system involved in the rapid acquisition of betaines by S. meliloti subjected to osmotic upshock.

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: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

Two MarR-Type Repressors Balance Precursor Uptake and Glycine Betaine Synthesis in Bacillus subtilis to Provide Cytoprotection Against Sustained Osmotic Stress.

Publications

Two MarR-Type Repressors Balance Precursor Uptake and Glycine Betaine Synthesis in <i>Bacillus subtilis</i> to Provide Cytoprotection Against Sustained Osmotic Stress.

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