Recombinant Bacillus subtilis Putative biotin transporter BioYB (bioYB)
Description
Biological Characteristics of Bacillus subtilis
Bacillus subtilis, commonly known as the hay bacillus or grass bacillus, is a Gram-positive, rod-shaped bacterium widely distributed in soil and the gastrointestinal tract of ruminants, humans, and marine sponges . As one of the best-studied Gram-positive bacteria, B. subtilis serves as a model organism for investigating bacterial chromosome replication and cellular differentiation .
B. subtilis cells typically measure 4-10 micrometers in length and 0.25-1.0 micrometers in diameter, with a cell volume of approximately 4.6 fL at stationary phase . This bacterium possesses remarkable adaptability to environmental stresses through its ability to form endospores, which enable survival under extreme conditions of temperature and desiccation . Although historically classified as an obligate aerobe, research since 1998 has established B. subtilis as a facultative anaerobe .
The genome of B. subtilis comprises approximately 4,100 genes, with only 192 definitively identified as indispensable for survival . The complete genome sequence of B. subtilis sub-strain QB928, widely used in genetic studies, consists of 4,146,839 DNA base pairs encoding 4,292 genes . This relatively compact genome contains all the necessary machinery for complex cellular processes, including specialized transport systems for essential nutrients like biotin.
Bacillus subtilis as an Expression System
B. subtilis has emerged as a powerful expression system for recombinant proteins due to several advantageous characteristics. The bacterium holds GRAS (Generally Recognized As Safe) status from the Food and Drug Administration, indicating its safety for use in food and pharmaceutical applications . Additionally, B. subtilis lacks exotoxins and endotoxins, further enhancing its suitability for producing bioactive compounds .
One of the most significant advantages of B. subtilis as an expression host is its remarkable natural ability to absorb and incorporate exogenous DNA into its genome . This characteristic facilitates genetic manipulation and the introduction of heterologous genes. Furthermore, B. subtilis possesses diverse codon usage patterns, enabling efficient expression of heterologous genes without additional modification steps .
The versatility of B. subtilis as an expression platform has led to the development of numerous genetic engineering strategies, including:
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Various plasmid systems
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Engineered constitutive or double promoters
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Chemical induction systems
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Self-inducing expression systems
These technological developments have positioned B. subtilis as an ideal host for both academic research and industrial production of recombinant proteins, including membrane transporters like BioYB.
The BioMNY Transport System
Biotin transport in prokaryotes often involves a specialized system comprising BioMNY proteins, which form a tripartite transporter complex . This system shares structural and functional similarities with ATP-binding cassette (ABC) transporters but differs in key aspects, particularly in the absence of extracytoplasmic solute-binding proteins typical of ABC transporters .
The BioMNY system consists of three primary components:
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BioM: An ATP-binding protein that provides energy for transport through ATP hydrolysis
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BioN: A transmembrane component that facilitates passage across the cell membrane
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BioY: The central transport unit responsible for biotin binding and translocation
Comparative genomic analyses have revealed interesting patterns in the distribution of these components across bacterial species. While BioY genes are widespread, only approximately one-third are linked to BioMN genes . Many BioY genes are instead located at loci encoding biotin biosynthesis machinery, suggesting coordinated regulation of biotin synthesis and transport .
Functional Properties of BioY Transporters
Experimental studies using heterologous expression of biotin transport genes from Rhodobacter capsulatus in Escherichia coli have provided valuable insights into the mechanisms of biotin transport . These investigations have established BioY as the central unit of biotin transporters, capable of functioning independently of BioMN components .
When expressed alone, BioY operates as a high-capacity biotin transporter, accumulating the vitamin approximately 1,000-fold in long-term assays at substrate concentrations of 4 nM . This significant accumulation suggests an active transport mechanism rather than simple diffusion .
The addition of BioMN components to BioY substantially alters the kinetic properties of biotin transport. Kinetic analyses reveal that BioY alone transports biotin with a maximal velocity (Vmax) of approximately 60 pmol × min^-1 × (mg protein)^-1 and an apparent half-saturation constant of 250 nM . In contrast, the complete BioMNY system exhibits a lower maximal velocity but significantly higher affinity for biotin, as evidenced by a 50-fold lower apparent half-saturation constant .
Table 1: Comparison of Kinetic Properties Between BioY and BioMNY Transport Systems
| Parameter | BioY Alone | BioMNY Complex |
|---|---|---|
| Maximal velocity (Vmax) | 60 pmol × min^-1 × (mg protein)^-1 | ~6 pmol × min^-1 × (mg protein)^-1 |
| Apparent half-saturation constant | 250 nM | ~5 nM |
| Transport mode | High-capacity | High-affinity |
| Efficiency at low biotin concentrations | Lower | Higher |
| Efficiency at high biotin concentrations | Higher | Lower |
This kinetic differentiation suggests a specialized ecological adaptation, with the complete BioMNY system optimized for environments where biotin is scarce, while BioY alone efficiently handles higher biotin concentrations without the energy expenditure of ATP hydrolysis .
Dependency on ATPase Activity
The high-affinity biotin transport mediated by the BioMNY complex depends critically on a functional ATPase component. Experimental studies have demonstrated that replacing the conserved lysine residue (K42) in the Walker A motif of BioM with asparagine severely impairs transport activity across all biotin concentrations tested . This finding parallels observations in other ATP-dependent transporters, such as the E. coli maltose transporter, where similar modifications result in approximately 99% inhibition of transport activity .
Interestingly, experimental evidence suggests functional interactions between BioM and BioY even in the absence of BioN. Cells producing BioMY subcomplexes exhibited transport characteristics similar to those containing the complete BioMNY system under both substrate limitation and excess conditions . This observation indicates direct functional coupling between the ATPase component and the core transporter, potentially independent of the transmembrane BioN protein.
Recombinant Expression and Purification
The commercial availability of recombinant B. subtilis putative biotin transporter BioYB through providers such as MyBioSource.com at a price point of $1,375.00 indicates successful expression and purification of this membrane protein . This achievement represents a significant technical accomplishment, as membrane proteins typically present substantial challenges for recombinant expression and purification due to their hydrophobic nature and complex folding requirements.
Table 2: Characteristics of Recombinant B. subtilis BioYB
| Feature | Description |
|---|---|
| Product name | Recombinant Bacillus subtilis Putative biotin transporter BioYB (bioYB) |
| Commercial source | MyBioSource.com |
| Price | $1,375.00 |
| Organism of origin | Bacillus subtilis |
| Protein type | Membrane transporter |
| Function | Putative biotin transport |
| Applications | Research studies, functional characterization, structural analysis |
The production of recombinant BioYB likely involves expression in a suitable host system followed by extraction from membranes using detergents and purification through chromatographic techniques. The purified protein may be stabilized in detergent micelles or reconstituted into liposomes or nanodiscs for functional studies.
Research Applications
Purified recombinant BioYB serves as a valuable tool for investigating fundamental aspects of membrane transport biology. Potential research applications include:
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Structural studies to determine the three-dimensional organization of the transporter and identify biotin-binding sites
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Functional assays to characterize transport kinetics and substrate specificity
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Interaction studies to identify protein-protein associations that may regulate transport activity
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Comparative analyses with other biotin transporters to elucidate evolutionary relationships
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Development of inhibitors or modulators of biotin transport for potential therapeutic applications
Biotechnological Applications
Beyond basic research, recombinant BioYB and knowledge of biotin transport systems have potential applications in biotechnology and industrial processes:
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Enhancement of biotin uptake in B. subtilis strains used for industrial protein production
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Development of biotin-based selection systems for recombinant strain construction
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Creation of biosensors for biotin detection in environmental or industrial samples
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Improvement of biotin-dependent metabolic pathways in engineered microorganisms
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Design of novel antimicrobial strategies targeting biotin transport in pathogenic bacteria
The exceptional properties of B. subtilis as an expression host further enhance these potential applications. Table 3 summarizes key characteristics that make B. subtilis advantageous for recombinant protein production:
Table 3: Properties of B. subtilis Relevant to Recombinant Protein Expression
| Property | Description | Advantage |
|---|---|---|
| GRAS status | FDA-designated Generally Recognized As Safe | Suitable for food and pharmaceutical applications |
| Absence of exo/endotoxins | Free from toxins found in Gram-negative bacteria | Safer production, fewer purification steps |
| Natural competence | Ability to absorb and incorporate exogenous DNA | Easier genetic manipulation |
| Diverse codon usage | Flexible reading of various codon patterns | Efficient expression of heterologous genes |
| Protein secretion capability | Ability to secrete proteins into the culture medium | Simplified purification of secreted products |
| Scalability | Robust growth in bioreactors | Suitable for industrial-scale production |
Systems Biology Integration
Understanding the role of BioYB within the broader context of B. subtilis metabolism represents another promising research direction. Systems biology approaches could illuminate how biotin transport integrates with:
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Biotin-dependent metabolic pathways
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Regulatory networks controlling biotin homeostasis
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Stress response mechanisms affecting nutrient acquisition
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Cell differentiation processes such as sporulation
Synthetic Biology Applications
The growing field of synthetic biology offers exciting possibilities for utilizing BioYB and related transporters in engineered biological systems. Potential applications include:
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Development of biotin-responsive gene expression systems
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Design of synthetic cells with programmable biotin transport capabilities
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Creation of biotin-based biosensors for environmental monitoring
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Engineering of biotin transport systems with altered substrate specificity
Product Specs
Note: We will prioritize shipping the format we have in stock. However, if you have any specific format requirements, please specify them in your order notes. We will prepare the product according to your request.
Note: All our proteins are shipped with standard blue ice packs. If you require dry ice shipping, please contact us in advance. Additional fees may apply.
Generally, the shelf life of the liquid form is 6 months at -20°C/-80°C. The shelf life of the lyophilized form is 12 months at -20°C/-80°C.
Target Background
KEGG: bsu:BSU32030
STRING: 224308.Bsubs1_010100017391
Q&A
What is the biological role of BioYB in Bacillus subtilis?
BioYB (also known as yuiG, UniProt ID: O32104) functions as a putative biotin transporter in B. subtilis. It shares strong sequence similarity with the structurally characterized BioY biotin transporter of Lactococcus lactis and other well-characterized energy-coupling factor (ECF) biotin transporters . The 200-amino acid protein likely participates in biotin uptake from the environment, supporting essential biotin-dependent metabolic processes. Biotin transport is critical for B. subtilis as biotin serves as an essential cofactor for carboxylases, including pyruvate carboxylase (PyC) and acetyl-CoA carboxylase (AccB) .
How is BioYB structurally characterized and what domains are essential for its function?
BioYB is a membrane protein with multiple transmembrane segments, as evidenced by its highly hydrophobic amino acid sequence . The complete amino acid sequence (MKQRKLRAGDMALIGMFAALMAVGANITSVAPFLQVAGIPLSMQPFFCLLAALLLGSKRAAAIAMIVYALVGLAGAPVFAQFSAGFAPFAGKSGGFIISYIPAAFAAGWFLERNIQPSKIRFLIASLIGTAIMYLIGTTYMYLALKLWIHTPVSYGTAWGFMIWFMVKDTALAVILSFIAPAIYRSIHKATGFNRNHISST) reveals characteristics typical of membrane transporters . As an S-component of an ECF transporter system, BioYB likely functions in conjunction with energizing components (EcfA/EcfT) that power substrate transport through ATP hydrolysis.
How is BioYB gene expression regulated in B. subtilis?
BioYB expression is regulated by BirA, the biotin protein ligase of B. subtilis. BirA functions as a Group II biotin protein ligase with an N-terminal helix-turn-helix DNA binding domain that enables transcriptional regulation . Microarray data has identified yuiG (BioYB) as one of three transcripts regulated by biotin and BirA, alongside bioWAFDBI (biotin biosynthesis operon) and yhfUTS . All three genomic sites have similar predicted BirA binding sites, suggesting coordinated regulation of biotin transport and biosynthesis pathways .
What is known about the promoter structure and regulatory elements controlling BioYB expression?
The BioYB (yuiG) promoter contains a BirA binding site that responds to biotin levels in the cell. When cellular biotin concentrations are high, BirA binds to the operator region of yuiG and represses transcription . Conversely, during biotin limitation, repression is relieved, allowing increased expression of the biotin transporter. This regulatory mechanism creates a feedback loop where biotin availability directly controls the expression of proteins involved in biotin acquisition. The precise sequence of the BirA binding site in the yuiG promoter appears to be conserved with other BirA-regulated genes in B. subtilis .
What are the optimal conditions for expression and purification of recombinant BioYB?
Based on available data, recombinant BioYB can be successfully expressed in E. coli with an N-terminal His-tag . For optimal results:
For membrane proteins like BioYB, inclusion of appropriate detergents during cell lysis and purification is critical to maintain native structure and function. Multiple freeze-thaw cycles should be avoided to preserve protein activity .
What methodological approaches can be used to characterize BioYB transport activity?
Several complementary approaches can be employed to study BioYB's biotin transport function:
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Radioisotope uptake assays: Using radiolabeled biotin (³H-biotin) to measure transport kinetics in cells expressing BioYB or in reconstituted proteoliposomes.
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Fluorescence-based transport assays: Employing fluorescently labeled biotin analogs to track uptake in real-time.
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Growth complementation studies: Testing whether BioYB expression can rescue growth of biotin auxotrophic strains under biotin-limited conditions.
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Liposome reconstitution: Purified BioYB can be reconstituted into liposomes to study transport in a defined system, allowing precise control of buffer conditions and substrate concentrations.
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Binding assays: Techniques like isothermal titration calorimetry or microscale thermophoresis can determine biotin binding affinity and thermodynamics.
How does BioYB compare with other bacterial biotin transporters?
BioYB belongs to the ECF family of transporters, which differ significantly from other transporter types:
Unlike ABC transporters that use periplasmic binding proteins, ECF transporters like BioYB typically function with dedicated energizing components (EcfA/T) that power substrate translocation through ATP hydrolysis. The BioYB sequence contains conserved motifs found in other S-components of ECF transporters .
What experimental evidence confirms BioYB's function as a biotin transporter?
Multiple lines of evidence support BioYB's role as a biotin transporter:
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Sequence similarity to the well-characterized BioY biotin transporter of L. lactis and other ECF biotin transporters .
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Regulation by BirA and biotin, consistent with a role in biotin metabolism .
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Presence of transmembrane domains typical of transport proteins .
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Genomic context and transcriptional regulation alongside other biotin-related genes .
While these provide strong indirect evidence, direct biochemical demonstration of biotin transport activity would require functional studies using the methodologies described in section 3.2.
How can BioYB be utilized in B. subtilis spore display technology?
B. subtilis spore display technology has emerged as a valuable platform for various biotechnological applications . BioYB could potentially be incorporated into this system through:
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Fusion protein design: Creating genetic fusions between BioYB and proteins of interest, such as antigens or enzymes.
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Spore coat integration: Expressing these fusion proteins in sporulating B. subtilis cells, allowing their incorporation into the developing spore coat.
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Display optimization: Testing different fusion configurations and spore coat anchor proteins to maximize display efficiency and functional activity.
The advantage of this approach is that B. subtilis spores are highly resistant to environmental stresses, making them excellent vehicles for protein display in diverse applications . Additionally, B. subtilis is considered generally recognized as safe (GRAS), facilitating regulatory approval for various applications .
What mutational strategies can reveal critical functional residues in BioYB?
Systematic mutational analysis can provide insights into BioYB's mechanism:
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Alanine-scanning mutagenesis: Systematically replacing conserved residues with alanine to identify amino acids critical for biotin binding and transport.
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Directed evolution: Generating libraries of BioYB variants and selecting for altered transport properties, such as increased affinity or altered specificity.
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Cysteine-scanning accessibility studies: Introducing cysteine residues at specific positions followed by labeling with membrane-impermeant sulfhydryl reagents to determine topology and dynamic structural changes during transport.
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Chimeric protein construction: Creating hybrid proteins between BioYB and related transporters to identify domains responsible for substrate specificity and transport activity.
For all mutational studies, it's essential to verify proper membrane expression and localization of mutant proteins, as mutations in membrane proteins can often affect folding and trafficking.
How does BioYB interact with the broader biotin regulatory network in B. subtilis?
BioYB functions within an integrated network of biotin metabolism and regulation:
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Regulatory connectivity: BioYB (yuiG) transcription is controlled by BirA, which also regulates biotin biosynthesis genes (bioWAFDBI) and another potential biotin transporter (yhfUTS) .
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Metabolic integration: Biotin transported by BioYB serves as a cofactor for essential carboxylases, including pyruvate carboxylase (PyC) and acetyl-CoA carboxylase (AccB) .
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Coordinated response: The system allows coordinated regulation of biotin acquisition pathways (transport and biosynthesis) based on cellular biotin status.
This network ensures efficient utilization of available biotin and maintains appropriate levels of this essential cofactor for cellular metabolism.
What model systems and experimental designs are most suitable for studying the sloppy model parameters of BioYB function?
Sloppy model analysis reveals that biological systems often contain parameters that are difficult to constrain experimentally . For BioYB research:
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Complementary experimental approaches: Combining multiple experimental techniques (as described in section 3.2) can help constrain model parameters that would be unidentifiable from a single experiment type .
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Careful experimental design: As noted by Apgar et al., optimal experimental design can dramatically reduce the range of parameter uncertainties in sloppy models .
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Model validation: When selecting optimal experiments, it's crucial to limit the search to those experiments for which the model is valid, as systematic errors can emerge when complementary experiments make neglected model details relevant .
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Integration of multiple data types: Combining data from transport kinetics, binding studies, and structural analyses can provide more robust parameter estimates than any single approach.
The sloppy model framework highlights why transport parameters for proteins like BioYB can be challenging to constrain experimentally and emphasizes the importance of thoughtful experimental design .
What are the unresolved questions regarding BioYB's molecular mechanism of transport?
Several key questions remain about BioYB's transport mechanism:
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Energy coupling: How exactly does BioYB couple with EcfA/T components to harness energy for transport?
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Conformational changes: What structural rearrangements occur during the transport cycle?
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Specificity determinants: Which residues are responsible for biotin recognition and discrimination against similar compounds?
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Regulatory interactions: Does BioYB activity respond to post-translational modifications or protein-protein interactions beyond the transcriptional regulation by BirA?
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Transport stoichiometry: What is the ratio of biotin molecules transported per ATP hydrolyzed?
Addressing these questions will require integrated structural, biochemical, and biophysical approaches.
How might advances in structural biology techniques contribute to our understanding of BioYB?
Recent advances in structural biology offer new opportunities for BioYB research:
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Cryo-electron microscopy: Increasingly capable of resolving membrane protein structures without crystallization, potentially revealing BioYB's structure alone or in complex with EcfA/T components.
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Integrative structural biology: Combining data from X-ray crystallography, NMR, SAXS, and computational modeling to build comprehensive structural models.
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Single-molecule techniques: Methods like FRET and force spectroscopy can capture dynamic conformational changes during the transport cycle.
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In situ structural studies: Emerging techniques for studying membrane protein structures in their native cellular environment.
These approaches could overcome traditional challenges in membrane protein structural biology and provide unprecedented insights into BioYB's mechanism of action.
