Recombinant Salmonella paratyphi B Monofunctional biosynthetic peptidoglycan transglycosylase (mtgA)

What is the biological function of mtgA in Salmonella paratyphi B?

Monofunctional biosynthetic peptidoglycan transglycosylase (mtgA) in Salmonella paratyphi B functions as a glycan polymerase that catalyzes the polymerization of glycan chains during peptidoglycan synthesis. The enzyme specifically transfers the growing glycan chain to N-acetylglucosamine (GlcNAc) residues in lipid II precursors, facilitating the formation of the bacterial cell wall. As suggested by the protein's nomenclature, mtgA is classified as a "monofunctional" enzyme because it exclusively performs transglycosylase activity without the bifunctional capabilities seen in some other peptidoglycan synthesis enzymes. The protein is encoded by the mtgA gene, which appears to be widely conserved among Salmonella species .

How should researchers store and reconstitute recombinant mtgA protein?

For optimal stability, recombinant mtgA protein should be stored at -20°C to -80°C immediately upon receipt. The lyophilized powder form requires proper reconstitution before experimental use. Researchers should briefly centrifuge the vial prior to opening to bring contents to the bottom, then reconstitute the protein in deionized sterile water to a concentration of 0.1-1.0 mg/mL. Addition of glycerol to a final concentration of 5-50% (with 50% being commonly recommended) is advised before aliquoting for long-term storage at -20°C/-80°C. Repeated freeze-thaw cycles significantly reduce protein activity and should be avoided. For short-term work, maintain working aliquots at 4°C for up to one week .

What expression systems are commonly used for recombinant mtgA production?

Multiple expression systems have been successfully employed for recombinant mtgA production, each offering distinct advantages depending on research requirements. E. coli expression systems are most commonly used due to their high yield, cost-effectiveness, and relatively straightforward protocols, as evidenced by commercially available recombinant proteins . For applications requiring post-translational modifications, yeast-based expression has been documented (Saccharomyces cerevisiae). More complex eukaryotic expression systems including baculovirus-infected insect cells and mammalian cell expression systems have also been utilized for specialized applications requiring specific modifications or when protein folding issues occur in prokaryotic systems . The choice of expression system should be determined by the intended experimental application, required protein modifications, and anticipated yield requirements.

What amino acid sequence characterizes Salmonella paratyphi B mtgA?

The full-length Salmonella paratyphi B mtgA protein consists of 242 amino acids, with high sequence conservation among Salmonella species. While the exact sequence for the B strain variant is not explicitly provided in the search results, the Salmonella paratyphi A mtgA sequence (which serves as a close reference) is: MSKRRIAPLTFLRRLLLRILAALAVFWGGGIALFSVVPVPFSAVMAERQISAWLGGEFGYVAHSDWVSMADISPWMGLAVITAEDQKFPEHWGFDVPAIEKALAHNERNESRIRGASTLSQQTAKNLFLWDGRSWVRKGLEAGLTLGIETVWSKKRILTVYLNIAEFGDGIFGVEAAAQRYFHKPASRLSVSEAALLAAVLPNPLRYKANAPSGYVRSRQAWIMRQMRQLGGESFMTRNQLN . Sequence analysis and comparison between different Salmonella serovars reveals high conservation of the mtgA gene, with similarities ranging from 99.31% to 99.88% observed in related genes across Salmonella strains .

What purification tags are commonly used for recombinant mtgA isolation?

Recombinant mtgA proteins are commonly produced with affinity tags to facilitate purification. Histidine (His) tags are most frequently utilized due to their small size and efficient purification via immobilized metal affinity chromatography (IMAC) . Avi-tag biotinylated variants have also been developed, which leverage the highly specific interaction between biotin and streptavidin for purification and detection purposes. The biotinylation process typically employs E. coli biotin ligase (BirA), which catalyzes the amide linkage between biotin and a specific lysine residue in the 15-amino acid AviTag peptide . Other tag types may be incorporated during the manufacturing process based on specific research requirements. The choice of tag should consider potential interference with protein structure or function, particularly for studies involving structural analysis or enzymatic activity.

What structural and functional differences exist between mtgA from Salmonella paratyphi B and other Salmonella serovars?

Detailed comparative studies of mtgA across Salmonella serovars reveal subtle but potentially significant structural and functional variations. While high sequence conservation (99.31-99.88%) exists among different Salmonella strains , even minor amino acid substitutions may impact enzyme kinetics, substrate specificity, or interaction with inhibitors. The Salmonella paratyphi A mtgA contains membrane-spanning regions and catalytic domains typical of transglycosylases, with conserved motifs for substrate binding and catalysis . Evolutionary analysis of Salmonella strains indicates that many genetic changes, including those affecting cell wall synthesis genes, have undergone transient Darwinian selection followed by purifying selection . This evolutionary pattern suggests that while mtgA variants may temporarily provide adaptive advantages in specific environments, conserved functionality remains critical for bacterial viability, resulting in limited divergence between serovars.

How can recombinant mtgA be incorporated into vaccine development strategies?

Although mtgA itself has not been directly validated as a vaccine antigen, research on Salmonella outer membrane components provides valuable insights for potential vaccine applications. Generalized Modules for Membrane Antigens (GMMA) approaches, which utilize outer membrane vesicles from genetically modified Salmonella to display multiple antigens in their native conformation, have shown promise in vaccine development . Rather than using mtgA alone, a more effective strategy might incorporate it within multivalent vaccine formulations. For comparison, co-immunization with recombinant antigens SpaO and H1a provided 75.0-91.7% protection in mouse models, significantly higher than either antigen alone (41.7-66.7%) . Researchers developing mtgA-based vaccine components should consider: (1) designing constructs that optimize epitope presentation, (2) evaluating adjuvant formulations that enhance immunogenicity, (3) assessing cross-protection against multiple Salmonella serovars, and (4) implementing rigorous challenge studies to validate protective efficacy.

What experimental approaches are most effective for studying mtgA enzymatic activity?

Investigating mtgA enzymatic activity requires specialized techniques addressing both the membrane-associated nature of the protein and its specific transglycosylase function. Researchers should implement a multi-faceted experimental approach including:

• In vitro transglycosylation assays: Using fluorescently labeled lipid II substrates to monitor glycan chain formation through changes in fluorescence intensity or FRET signals.

• Radioactive substrate incorporation: Tracking the transfer of radiolabeled precursors into growing peptidoglycan chains to quantify enzymatic activity under various conditions.

• Membrane reconstitution systems: Incorporating purified mtgA into liposomes or nanodiscs to study activity in a membrane-like environment that better mimics physiological conditions.

• Computational modeling: Employing molecular dynamics simulations to predict substrate binding sites, catalytic mechanisms, and potential inhibitor interactions.

• Site-directed mutagenesis: Systematically altering conserved residues to correlate sequence features with enzymatic function and substrate specificity.

The recombinant protein's high purity (>90% as determined by SDS-PAGE) makes it suitable for these specialized enzymatic assays, providing researchers with a reliable tool for mechanistic studies.

What are the optimal conditions for expression and purification of recombinant mtgA?

Successful expression and purification of recombinant mtgA requires careful optimization of multiple parameters. For E. coli-based expression systems, researchers should consider the following protocol elements:

• Strain selection: BL21(DE3) or Rosetta strains are preferable for membrane-associated proteins like mtgA, as they provide the reducing environment necessary for proper folding.

• Growth conditions: Initial culture in LB medium at 37°C until OD600 reaches 0.6-0.8, followed by temperature reduction to 16-25°C prior to induction minimizes inclusion body formation.

• Induction parameters: IPTG concentration should be optimized (typically 0.1-0.5 mM) with extended expression periods (12-24 hours) at reduced temperature.

• Cell lysis: Gentle disruption methods using enzymatic lysis combined with mild detergents (0.5-1% n-dodecyl-β-D-maltoside or CHAPS) better preserve membrane protein structure.

• Purification strategy: For His-tagged constructs, immobilized metal affinity chromatography using Ni-NTA resin with imidazole gradient elution (20-250 mM) provides high purity, followed by size exclusion chromatography to remove aggregates .

• Quality control: Final purity assessment via SDS-PAGE should exceed 85-90%, with additional verification by Western blotting using tag-specific antibodies .

How can researchers effectively analyze the interaction between mtgA and potential inhibitors?

Investigating interactions between mtgA and potential inhibitors requires a comprehensive approach combining biophysical, biochemical, and computational methods:

• Thermal shift assays (TSA): Measuring changes in protein thermal stability upon inhibitor binding provides initial screening data for potential interactions.

• Surface plasmon resonance (SPR): Quantifying binding kinetics (kon/koff) and affinity (KD) between immobilized mtgA and inhibitor compounds in real-time.

• Isothermal titration calorimetry (ITC): Determining thermodynamic parameters (ΔH, ΔG, ΔS) of inhibitor binding to understand interaction mechanisms.

• Enzymatic inhibition assays: Measuring dose-dependent inhibition of transglycosylase activity using fluorescently-labeled lipid II substrates with varying inhibitor concentrations to calculate IC50 values.

• Competitive binding assays: Using known mtgA ligands with displacement studies to identify binding site competition.

• In silico molecular docking: Computational prediction of binding modes and affinity, particularly valuable when structural data is available.

• Membrane permeability assessment: Evaluating inhibitor access to mtgA in its native membrane environment using liposome-reconstituted systems.

When validating hits from these assays, researchers should consider establishing structure-activity relationships through systematic modification of lead compounds and correlating molecular features with inhibitory potency.

What techniques are available for studying mtgA localization and dynamics in live bacteria?

Understanding mtgA localization and dynamics within bacterial cells requires specialized microscopy and molecular techniques:

• Fluorescent protein fusions: Generating C-terminal or N-terminal fusions with fluorescent proteins (GFP, mCherry) while ensuring minimal disruption to mtgA function and localization.

• Super-resolution microscopy: Employing techniques such as PALM, STORM, or STED microscopy to visualize mtgA distribution with nanometer precision, particularly important for monitoring its association with the cell division apparatus.

• FRAP (Fluorescence Recovery After Photobleaching): Measuring protein mobility and exchange rates within the membrane to understand dynamic behavior during cell growth and division.

• Single-molecule tracking: Following individual mtgA molecules to characterize diffusion rates and potential confinement zones within the membrane.

• Correlative light-electron microscopy (CLEM): Combining fluorescence localization data with ultrastructural context provided by electron microscopy.

• Proximity labeling approaches: Using BioID or APEX2 fusions to identify proteins in close proximity to mtgA within the native cellular environment.

• Time-lapse microscopy: Monitoring changes in mtgA localization throughout the cell cycle, particularly during septum formation and cell division.

These approaches allow researchers to correlate mtgA localization with its functional roles in peptidoglycan synthesis during bacterial growth and division.

How does mtgA from Salmonella paratyphi B compare with similar enzymes from other pathogenic bacteria?

Peptidoglycan transglycosylases like mtgA are widely distributed across bacterial species, with notable structural and functional variations that reflect evolutionary adaptations to different cellular environments. While Salmonella paratyphi B mtgA shares the core catalytic mechanism of other transglycosylases, comparative analysis reveals several distinguishing features:

The Salmonella paratyphi B mtgA contains characteristic structural motifs including membrane-spanning regions and conserved catalytic residues essential for glycosyltransferase activity. Unlike some other bacterial species where transglycosylases are essential single-copy genes, Salmonella shows functional redundancy that may contribute to robustness during infection and environmental stress.

What distinguishes recombinant mtgA production strategies for research applications versus vaccine development?

The production strategies for recombinant mtgA differ significantly depending on whether the protein is intended for basic research or vaccine development applications:

For vaccine applications specifically, researchers should consider membrane-derived vesicle approaches such as Generalized Modules for Membrane Antigens (GMMA), which have shown promise for presenting multiple Salmonella antigens simultaneously in their native conformations . When designing recombinant mtgA for potential vaccine use, attention to proper folding and epitope presentation is critical for inducing protective immune responses.

What are the most promising approaches for developing mtgA-targeted antimicrobials?

The essential role of transglycosylases in bacterial cell wall synthesis makes mtgA an attractive target for novel antimicrobial development. Future research should focus on several promising approaches:

• Natural product derivatives: Expanding upon known transglycosylase inhibitors like moenomycin through structural modifications to improve pharmacokinetic properties while maintaining target specificity.

• Fragment-based drug discovery: Using biophysical screening methods to identify small molecule fragments that bind to distinct pockets within mtgA, followed by medicinal chemistry optimization.

• Peptide inhibitors: Developing peptide-based molecules that interfere with the protein-protein or protein-substrate interactions critical for mtgA function.

• Allosteric inhibitors: Identifying compounds that bind outside the active site but induce conformational changes that impair catalytic activity.

• Combination approaches: Exploring synergistic effects between mtgA inhibitors and other cell wall-targeting antibiotics to enhance efficacy and reduce resistance development.

• Structure-guided design: Leveraging structural information about mtgA to design compounds that precisely complement binding pocket topography and electronic properties.

Given the evolutionary conservation of mtgA across Salmonella strains (99.31-99.88% similarity) , inhibitors developed against one serovar would likely demonstrate broad activity against multiple pathogenic variants.

How might systems biology approaches enhance our understanding of mtgA function in bacterial physiology?

Systems biology approaches offer powerful frameworks for contextualizing mtgA within broader bacterial physiological networks:

• Multi-omics integration: Combining transcriptomics, proteomics, and metabolomics data to understand how mtgA expression correlates with other cell wall synthesis genes under various growth conditions and stresses.

• Interaction networks: Using techniques like protein-protein interaction screening and genetic epistasis analysis to map mtgA's functional connections with other cellular components.

• Mathematical modeling: Developing quantitative models of peptidoglycan synthesis incorporating enzymatic parameters of mtgA to predict cellular responses to genetic or environmental perturbations.

• Evolutionary systems biology: Analyzing selective pressures on mtgA across Salmonella strains and related species to understand functional constraints and adaptability of peptidoglycan synthesis pathways.

• Single-cell analysis: Employing microfluidics and time-lapse microscopy to characterize cell-to-cell variability in mtgA activity and its consequences for bacterial growth and division.

These integrated approaches would extend beyond studying mtgA in isolation, revealing how this enzyme functions within the complex cellular ecosystem and potentially identifying non-obvious pathways for therapeutic intervention.