Recombinant Shigella boydii serotype 4 Monofunctional biosynthetic peptidoglycan transglycosylase (mtgA)
Description
Introduction to Recombinant Shigella boydii Serotype 4 Monofunctional Biosynthetic Peptidoglycan Transglycosylase (mtgA)
Recombinant Shigella boydii serotype 4 monofunctional biosynthetic peptidoglycan transglycosylase (mtgA) is a protein that plays a crucial role in the biosynthesis of peptidoglycan, a key component of bacterial cell walls. Peptidoglycan, also known as murein, is essential for maintaining bacterial cell integrity and providing structural support against osmotic pressure. The mtgA protein is specifically involved in the polymerization of peptidoglycan strands, which are composed of repeating disaccharides of N-acetylglucosamine (GlcNAc) and N-acetylmuramic acid (MurNAc) linked to short peptides.
Structure and Function of mtgA
The recombinant mtgA protein from Shigella boydii serotype 4 is a monofunctional biosynthetic enzyme, meaning it catalyzes a single step in the peptidoglycan biosynthesis pathway. It is expressed as a full-length protein with 242 amino acids and is often fused with an N-terminal His tag to facilitate purification and detection . The His tag allows for easy affinity chromatography, which is a common method used to purify recombinant proteins.
Expression and Purification
The recombinant mtgA protein is typically expressed in Escherichia coli (E. coli), a common host organism for recombinant protein production due to its well-understood genetics and efficient expression systems . The expression conditions can be optimized to prevent protein aggregation and ensure the production of soluble protein. For instance, lowering the temperature and reducing the concentration of inducers like IPTG can help in achieving high yields of soluble recombinant proteins .
Role in Peptidoglycan Biosynthesis
Peptidoglycan biosynthesis involves several steps, starting with the assembly of disaccharide subunits on the cytoplasmic side of the bacterial membrane. These subunits are then polymerized by transglycosylases, such as mtgA, and cross-linked by transpeptidases on the outer side of the membrane . The mtgA enzyme specifically catalyzes the polymerization step, which is crucial for the formation of a robust peptidoglycan layer.
Research Findings and Applications
Research on recombinant mtgA proteins can provide insights into the mechanisms of peptidoglycan biosynthesis and its role in bacterial pathogenicity. Understanding these processes can lead to the development of new antimicrobial strategies targeting peptidoglycan synthesis. For example, inhibitors of peptidoglycan biosynthesis are a major class of antibiotics, and studying enzymes like mtgA can help in designing more effective drugs .
Product Specs
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Generally, liquid formulations have a 6-month shelf life at -20°C/-80°C, while lyophilized formulations have a 12-month shelf life at -20°C/-80°C.
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Target Background
KEGG: sbo:SBO_3174
Q&A
What is Monofunctional biosynthetic peptidoglycan transglycosylase (mtgA) in Shigella boydii serotype 4?
Monofunctional biosynthetic peptidoglycan transglycosylase (mtgA) in Shigella boydii serotype 4 is an enzyme that catalyzes glycan chain elongation of the bacterial cell wall, specifically functioning in peptidoglycan assembly. This protein belongs to the glycosyltransferase family with EC classification 2.4.2.- and is encoded by the mtgA gene (locus SBO_3174) in the S. boydii genome . Unlike bifunctional penicillin-binding proteins (PBPs) that possess both transglycosylase and transpeptidase activities, mtgA exclusively performs the glycosyltransferase function, making it "monofunctional." The protein plays a crucial role in bacterial cell wall biosynthesis, potentially collaborating with other divisome proteins during cell division .
What are the optimal storage conditions for recombinant mtgA protein?
For optimal stability and activity of recombinant Shigella boydii serotype 4 mtgA, the following storage conditions are recommended:
| Storage Purpose | Temperature | Duration | Buffer Composition |
|---|---|---|---|
| Standard storage | -20°C | Medium-term | Tris-based buffer with 50% glycerol |
| Extended storage | -20°C or -80°C | Long-term | Tris-based buffer with 50% glycerol |
| Working aliquots | 4°C | Up to one week | Same as original buffer |
It is important to note that repeated freezing and thawing cycles should be avoided as they can lead to protein denaturation and loss of enzymatic activity. Therefore, it is advisable to prepare working aliquots that can be stored at 4°C for experiments lasting up to one week .
How does mtgA contribute to bacterial cell wall synthesis?
The mtgA protein performs a critical catalytic function in bacterial cell wall synthesis by catalyzing the polymerization of glycan chains in peptidoglycan assembly. Specifically, mtgA utilizes lipid II as a substrate to form the glycosidic bonds that connect N-acetylglucosamine (GlcNAc) and N-acetylmuramic acid (MurNAc) units in the growing glycan chain. In vitro studies with E. coli mtgA, which shares high homology with S. boydii mtgA, demonstrate significant peptidoglycan polymerization activity, with a 2.4-fold increase in polymerization when GFP-mtgA is overexpressed compared to controls .
The enzyme has been shown to localize at the division site in cells deficient in certain penicillin-binding proteins (PBPs), suggesting a role in septal peptidoglycan synthesis during cell division. This localization pattern indicates that mtgA may collaborate with other divisome proteins to form the peptidoglycan of new poles during bacterial cell division .
What protein-protein interactions has mtgA been shown to participate in within the divisome complex?
Studies with E. coli mtgA, which is homologous to S. boydii mtgA, reveal significant protein-protein interactions within the bacterial divisome complex. Using bacterial two-hybrid systems, researchers have demonstrated that mtgA interacts with multiple divisome components:
| Interaction Partner | Fold Increase in β-galactosidase Activity | Biological Significance |
|---|---|---|
| PBP3 (FtsI) | 10-fold | Suggests coordinated action in peptidoglycan synthesis |
| FtsN | 20-fold | Potential regulation of peptidoglycan synthases |
| FtsW | 37-fold | Possible coordination of lipid II transport and polymerization |
| mtgA (self) | 37-fold | Indicates potential oligomerization during function |
These interactions require the transmembrane segment of PBP3, suggesting membrane-associated complex formation. The interaction with FtsN is particularly noteworthy as FtsN has been shown to stimulate the in vitro peptidoglycan synthesis activities of PBP1b, suggesting FtsN may coordinate the activities of multiple peptidoglycan synthases during cell division .
What methodologies can be used to assess mtgA glycosyltransferase activity in vitro?
To assess the glycosyltransferase activity of mtgA in vitro, researchers can employ the following methodological approach:
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Reaction Setup:
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Use radiolabeled lipid II substrate (e.g., [14C]GlcNAc-labeled lipid II)
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Reaction buffer composition: 15% dimethyl sulfoxide, 10% octanol, 50 mM HEPES (pH 7.0), 0.5% decyl-polyethylene glycol, and 10 mM CaCl₂
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Include purified mtgA or GFP-mtgA fusion protein
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Product Analysis:
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Separate reaction products via thin-layer chromatography or gel filtration
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Quantify radiolabeled material by scintillation counting
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Calculate percentage of lipid II incorporated into polymeric material
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Verification of Polymerization:
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Treat reaction products with lysozyme (which specifically digests peptidoglycan)
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Complete digestion confirms that the product is indeed polymerized peptidoglycan
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Using this methodology, researchers have demonstrated that GFP-mtgA fusion proteins retain glycosyltransferase activity, with a 2.4-fold increase in peptidoglycan polymerization compared to controls (26% versus 11% of lipid II utilized) .
How does mtgA localization differ between wild-type and PBP-deficient bacterial strains?
The localization pattern of mtgA shows significant dependency on the genetic background, particularly regarding penicillin-binding proteins (PBPs). Research findings illustrate dramatic differences in mtgA localization patterns:
| Bacterial Strain | mtgA Localization Pattern | Notes |
|---|---|---|
| Wild-type E. coli | Diffuse distribution | No specific localization at division sites |
| PBP1b-deficient with thermosensitive PBP1a (ponA(ts) ponB) | Concentrated at division sites | Suggests compensation for missing class A PBPs |
| PBP1b-deficient with thermosensitive PBP1a, complemented with plasmid-expressed PBP1b | Diffuse distribution | Confirms that mtgA localization depends on PBP status |
These findings suggest that mtgA may play a compensatory role in septal peptidoglycan synthesis when class A PBPs (particularly PBP1a and PBP1b) are compromised. This localization pattern indicates potential functional redundancy in bacterial cell wall synthesis machinery, which may have implications for understanding antimicrobial resistance mechanisms .
What are the implications of mtgA being insensitive to penicillin for antimicrobial research?
The penicillin insensitivity of mtgA presents significant implications for antimicrobial research and development:
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Alternative Therapeutic Targets: Unlike penicillin-binding proteins (PBPs), mtgA maintains its glycosyltransferase activity in the presence of β-lactam antibiotics. This characteristic makes mtgA a potential target for developing novel antimicrobials that could be effective against penicillin-resistant bacteria.
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Persistent Cell Wall Synthesis: Research indicates that septal peptidoglycan synthesis can be divided into early and late steps, with the early step requiring penicillin-insensitive peptidoglycan synthesis before constriction. MtgA, being insensitive to penicillin, may be responsible for this early synthesis activity, which is later taken over by penicillin-sensitive proteins .
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Compensatory Mechanisms: When penicillin-sensitive PBPs are inhibited by antibiotics, bacteria may upregulate mtgA to partially compensate for lost cell wall synthesis activity. Understanding this compensatory mechanism could provide insights into bacterial adaptation to antimicrobial stress.
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Combination Therapy Approaches: Developing inhibitors specific to mtgA could potentially create synergistic effects when combined with traditional β-lactam antibiotics, targeting both penicillin-sensitive and penicillin-insensitive cell wall synthesis pathways simultaneously.
What experimental systems are available for studying mtgA function in cellular contexts?
Several experimental systems have been developed to study mtgA function in cellular contexts:
| Experimental System | Methodology | Applications | Considerations |
|---|---|---|---|
| GFP-mtgA Fusion | Construct GFP fusion proteins and visualize using fluorescence microscopy | Localization studies, protein dynamics | Ensure fusion does not disrupt protein function |
| Bacterial Two-Hybrid | Express fusion proteins with T18 and T25 fragments of adenylate cyclase | Protein-protein interaction studies | Requires proper controls (e.g., T18-T25 alone) |
| Gene Knockout/Complementation | Create mtgA deletion strains and complement with plasmid-expressed variants | Functional analysis | Single mutants may not show phenotypes due to redundancy |
| Conditional Expression | Use temperature-sensitive or inducible systems | Study effects of mtgA depletion/overexpression | Leaky expression can complicate interpretation |
| In Vitro Reconstitution | Purify components and reconstruct systems with defined components | Biochemical activity studies | May not fully recapitulate in vivo complexity |
When employing these systems for studying S. boydii mtgA, researchers should consider the genetic background, particularly the status of class A PBPs, as studies with E. coli have shown that mtgA localization and function can be influenced by the presence or absence of these proteins .
What are promising avenues for future research on Shigella boydii serotype 4 mtgA?
Based on current knowledge gaps and potential applications, several promising research directions for Shigella boydii serotype 4 mtgA include:
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Structural Characterization: Determining the three-dimensional structure of S. boydii mtgA through X-ray crystallography or cryo-electron microscopy would provide insights into its catalytic mechanism and potential inhibitor binding sites.
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Species-Specific Functions: Comparative analyses between mtgA from Shigella boydii and other bacterial species could reveal species-specific adaptations and functions. Current research with E. coli mtgA provides a foundation, but S. boydii-specific studies are needed .
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Role in Virulence: Investigating potential connections between mtgA activity and Shigella virulence could reveal whether this enzyme contributes to pathogenesis beyond basic cell wall synthesis.
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Development of Specific Inhibitors: Given mtgA's penicillin insensitivity, developing specific inhibitors targeting this enzyme could lead to novel antimicrobial strategies, particularly for treating infections caused by β-lactam-resistant Shigella strains.
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Integration with Vaccine Development: Understanding the role of mtgA in cell wall composition may provide insights relevant to vaccine development, potentially complementing current Shigella vaccine approaches that focus on O-antigen components .
How might recombinant mtgA be utilized in vaccine development research?
While current Shigella vaccine development primarily focuses on O-antigen components (as seen in bioconjugate and GMMA approaches), recombinant mtgA could potentially contribute to vaccine research in several ways:
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Adjuvant Properties: Peptidoglycan components have known immunostimulatory properties. Recombinant mtgA could be used to generate defined peptidoglycan structures with potential adjuvant activities for Shigella vaccines.
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Understanding Vaccine Stability: Cell wall modifications can affect outer membrane vesicle formation and stability. Studying mtgA's role in this process could help optimize GMMA-based vaccine platforms, which utilize outer membrane vesicles as vaccine components .
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Alternative Antigenic Targets: While O-antigen-based vaccines show promise (with 50-70% efficacy against moderate to severe diarrhea in some studies), exploring conserved proteins like mtgA could potentially lead to broader protection across Shigella serotypes .
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Marker for Attenuated Strains: Modifying mtgA expression or activity could contribute to the development of live-attenuated vaccine strains with altered cell wall properties that maintain immunogenicity while reducing virulence.
What are the optimal conditions for expressing and purifying recombinant Shigella boydii serotype 4 mtgA?
Based on available information about recombinant S. boydii mtgA and similar proteins, the following methodological approach is recommended:
Expression Conditions:
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Expression System: E. coli BL21(DE3) or similar expression strain
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Vector: pET-based expression vector with appropriate tag (His-tag often preferred)
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Induction: 0.5-1 mM IPTG when culture reaches OD600 of 0.6-0.8
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Temperature: Consider lower temperature (16-25°C) post-induction to improve solubility
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Duration: 4-16 hours depending on expression temperature
Purification Protocol:
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Cell lysis in Tris-based buffer (typically 50 mM Tris-HCl, pH 7.5-8.0, 300 mM NaCl, 10% glycerol)
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Addition of appropriate protease inhibitors to prevent degradation
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Affinity chromatography using the chosen tag (e.g., Ni-NTA for His-tagged proteins)
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Optional tag removal depending on downstream applications
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Size exclusion chromatography for final purification
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Storage in Tris-based buffer with 50% glycerol at -20°C or -80°C
How can researchers validate the functional activity of purified recombinant mtgA?
To ensure that purified recombinant mtgA maintains its functional activity, researchers should perform the following validation tests:
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In Vitro Glycosyltransferase Assay:
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Protein-Protein Interaction Analysis:
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Complementation Studies:
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Test ability of recombinant mtgA to complement phenotypes in mtgA-deficient strains
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Most effective in strains with compromised class A PBPs where mtgA function becomes more critical
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Structural Integrity Assessment:
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Circular dichroism spectroscopy to confirm proper secondary structure
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Thermal shift assays to assess protein stability
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Limited proteolysis to verify correct folding
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A functional recombinant mtgA should demonstrate glycosyltransferase activity in vitro, maintain expected protein-protein interactions, and possess the proper structural characteristics necessary for enzymatic function.
How does mtgA from Shigella boydii compare with homologs from other bacterial species?
While specific comparative data for S. boydii mtgA is limited in the provided search results, we can make informed comparisons based on known information about E. coli mtgA and general principles of bacterial evolution:
