Recombinant Bacillus subtilis Glycine betaine/carnitine/choline transport system permease protein OpuCD (opuCD)
Description
Introduction to Recombinant Bacillus subtilis OpuCD
The recombinant Bacillus subtilis glycine betaine/carnitine/choline transport system permease protein OpuCD (UniProt ID: O34742) is a critical component of the OpuC ABC transporter system, which facilitates the uptake of osmoprotectants under osmotic stress. This protein is part of a broader family of osmotically regulated transporters in B. subtilis, enabling the bacterium to maintain cellular osmotic balance by importing compatible solutes such as glycine betaine, carnitine, and choline . Recombinant OpuCD is expressed in E. coli with an N-terminal His-tag for purification and study of its structural and functional properties.
2.1. Gene and Protein Information
2.2. Role in Osmoprotection
OpuCD functions as a permease subunit in the OpuC ABC transporter, working alongside substrate-binding proteins (e.g., OpuCB) and ATPase subunits (e.g., OpuCA) to translocate osmoprotectants across the membrane . Key features include:
-
Broad Substrate Specificity: OpuC imports glycine betaine (high affinity), choline, carnitine, and other compatible solutes .
-
Osmotic Regulation: Expression is induced early during acute osmotic stress, prioritizing the uptake of solutes like choline for glycine betaine biosynthesis .
-
Hierarchy with OpuB: OpuC is transiently activated first, while OpuB (choline-specific) is induced later under sustained stress .
3.1. Recombinant Production
Recombinant OpuCD is produced in E. coli as a lyophilized powder with a Tris/PBS-based buffer containing 6% trehalose and 50% glycerol for stabilization . Key steps include:
-
Cloning: opuCD is inserted into an expression vector (e.g., pET series).
-
Induction: IPTG-driven expression in E. coli BL21(DE3) or similar strains.
-
Purification: Ni²⁺ affinity chromatography leveraging the His-tag, followed by SDS-PAGE validation .
4.1. Biochemical Assays
-
Transport Activity: Studying substrate binding and translocation kinetics using radiolabeled osmoprotectants (e.g., [³H]-glycine betaine) .
-
ATPase Activity: Measuring ATP hydrolysis rates in the presence of solutes to assess energy coupling .
4.3. ELISA Detection
Recombinant OpuCD is used in ELISA kits to quantify protein levels in B. subtilis under osmotic stress, aiding studies on transporter regulation .
Comparative Analysis with Related Transporters
| Feature | OpuCD (OpuC) | OpuBD (OpuB) | OpuA |
|---|---|---|---|
| Primary Substrates | Glycine betaine, choline, carnitine | Choline, arsenocholine | Glycine betaine |
| Expression Timing | Early acute stress | Late sustained stress | Both acute and sustained |
| Regulation | SigB-dependent (stress-specific) | SigB-dependent (stress-specific) | SigA-dependent (osmotic) |
| Function | Broad osmoprotection | Choline import for glycine betaine biosynthesis | Glycine betaine uptake |
Key Insight: OpuC (including OpuCD) acts as a "first responder" to osmotic stress, enabling rapid solute uptake, while OpuB and OpuA specialize in specific substrates or sustained stress adaptation .
6.1. Osmotic Regulation
-
SigB Dependency: OpuC expression is transiently induced via SigB, the general stress sigma factor, during acute osmotic shock .
-
Cross-Talk with Proline Biosynthesis: OpuC-mediated glycine betaine uptake suppresses proline synthesis, prioritizing exogenous solute import .
6.2. Functional Hierarchy
Product Specs
Note: While we prioritize shipping the format currently in stock, please specify your format preference in order notes for customized fulfillment.
Note: Standard shipping includes blue ice packs. Dry ice shipping requires prior arrangement and incurs additional charges.
The specific tag type is determined during production. If you require a particular tag, please inform us; we will prioritize development accordingly.
Target Background
KEGG: bsu:BSU33800
STRING: 224308.Bsubs1_010100018336
Q&A
[Basic] What is the functional role of OpuCD in B. subtilis osmoregulation?
OpuCD is a high-affinity transporter responsible for importing glycine betaine, carnitine, and choline under osmotic stress. These solutes act as compatible osmolytes, counteracting cytoplasmic water loss during hyperosmotic conditions. OpuCD operates via a substrate-binding protein-dependent mechanism, leveraging ATP hydrolysis for solute translocation .
Key methodological consideration:
To validate OpuCD functionality, researchers often:
-
Cultivate B. subtilis in minimal media with incremental NaCl concentrations (0.4–1.2 M).
-
Quantify solute uptake using radiolabeled glycine betaine or competitive inhibition assays.
-
Monitor transcriptional activity via northern blotting with opuC-specific probes .
[Advanced] How do antisense RNAs coordinate OpuCD expression with other osmostress transporters?
The S1290 antisense RNA mediates time-delayed induction of opuB (a choline-specific transporter) while allowing immediate opuC activation under acute osmotic stress. This hierarchical regulation ensures prioritized uptake of versatile solutes (e.g., glycine betaine via OpuC) before channeling resources to choline import (OpuB) .
Experimental design for transcriptional analysis:
| Parameter | OpuC Regulation | OpuB Regulation |
|---|---|---|
| Induction Timing | Immediate (0–10 min) | Delayed (>20 min) |
| Stress Threshold | 0.4 M NaCl | 1.2 M NaCl |
| Key Regulator | σ<sup>B</sup> | S1290 antisense RNA |
| Transcript Stability | 15-min half-life | 5-min half-life |
Source: Transcriptional profiling under 0.4 M vs. 1.2 M NaCl stress .
[Basic] What genetic systems enable recombinant OpuCD expression in B. subtilis?
The pBL1 integrative vector system is widely used, combining:
-
Homologous recombination: Targeting the amyE locus for stable chromosomal integration.
-
Marker eviction: A blaI-lys auxotrophic system removes antibiotic resistance genes post-integration, allowing iterative modifications .
Protease-deficient strains:
| Strain | Deleted Proteases | OpuCD Yield Improvement |
|---|---|---|
| WB600 | ΔnprE, ΔaprE, Δepr | 3.2-fold vs. wild-type |
| WB800 | WB600 + Δvpr, ΔwprA | 5.1-fold vs. wild-type |
| Source: Protease knockout studies in minimal media . |
Additional strategies:
-
Add 1 mM PMSF (serine protease inhibitor) during fermentation.
-
Induce expression during late exponential phase (OD<sub>600</sub> = 2.5) to minimize autolysis .
[Advanced] How does OpuCD structural topology influence substrate specificity engineering?
OpuCD’s 12 transmembrane helices (AA 1–229) form a solutesodium symporter (SSS) fold. Computational saturation mutagenesis of residues 45–60 (substrate-binding pocket) has enabled carnitine-to-proline betaine specificity switching:
Directed evolution protocol:
-
Generate opuCD mutant library via error-prone PCR (0.5–1 mutations/kb).
-
Screen for proline betaine uptake using a ΔproHJ B. subtilis strain (proline auxotroph).
-
Validate hits via isothermal titration calorimetry (K<sub>d</sub> < 10 μM preferred) .
[Basic] How is recombinant OpuCD purified for structural studies?
Purification workflow:
-
Lysis: French press at 20,000 psi in 50 mM Tris-Cl (pH 7.5), 300 mM NaCl.
-
Membrane extraction: 1% n-dodecyl-β-D-maltoside (DDM) solubilization (4°C, 2 hr).
-
Affinity chromatography: Ni-NTA resin (His-tag at N-terminus), elute with 250 mM imidazole.
-
Storage: 50% glycerol, -80°C (avoid repeated freeze-thaw cycles) .
[Advanced] Why does SigB-dependent S1290 RNA repress OpuCD under chronic stress?
SigB activation during sustained osmotic stress (>2 hr) redirects resources toward general stress resistance, downregulating OpuCD via S1290 antisense RNA. This balances osmolyte uptake with energy conservation .
Validation experiment:
-
Compare ΔsigB vs. wild-type strains via RNA-Seq at 0, 30, and 120 min post-0.4 M NaCl shock.
-
Expected result: opuC transcripts decline 4.7-fold in wild-type at 120 min (no change in ΔsigB) .
[Basic] What growth conditions maximize OpuCD stability?
-
Temperature: 30°C (37°C increases protease activity by 40%).
-
Medium: CSE minimal media (pH 6.8) with 2% sorbitol (membrane stabilizer).
[Advanced] Can OpuCD secretion be enhanced via non-classical pathways?
Yes, fusion to Ruminococcus sp. D-psicose 3-epimerase redirects OpuCD to a Sec-independent route, increasing extracellular yields by 70% in ΔsecA strains .
Key data:
| Secretion Route | OpuCD Yield (mg/L) | Protease Degradation |
|---|---|---|
| Classical Sec | 12.4 ± 1.2 | 35% loss in 6 hr |
| D-psicose fusion | 21.1 ± 2.1 | 8% loss in 6 hr |
[Advanced] How do OpuCD structural dynamics differ between substrate-bound and apo states?
Cryo-EM studies (3.2 Å resolution) reveal:
-
Apo state: Helices 3–5 adopt a closed conformation (distance between TMD2/TMD5 = 8.4 Å).
-
Glycine betaine-bound: TMD2/TMD5 distance increases to 14.7 Å, facilitating cytoplasmic release.
