Recombinant Yersinia pseudotuberculosis serotype O:1b Monofunctional biosynthetic peptidoglycan transglycosylase (mtgA)

How does mtgA differ structurally and functionally from bifunctional peptidoglycan synthases?

Unlike bifunctional PBPs such as PBP1A and PBP1B that possess both glycosyltransferase (GTase) and transpeptidase (TPase) domains, mtgA contains only the GTase domain. This fundamental difference affects how these enzymes participate in peptidoglycan synthesis:

The monofunctional nature of mtgA means it works processively to polymerize lipid II into glycan strands but requires cooperation with separate transpeptidases to form the complete cross-linked peptidoglycan structure .

What are the optimal conditions for expressing and purifying recombinant Y. pseudotuberculosis mtgA?

Based on established protocols for similar glycosyltransferases:

Expression System:

• Host: E. coli is the preferred expression system

• Vector: His-tagged expression vectors for simplified purification

• Induction: IPTG-inducible promoters at lower temperatures (16-25°C) to maximize protein solubility

Purification Protocol:

• Harvest cells and resuspend in buffer containing 50 mM Tris-HCl pH 8.0, 300 mM NaCl, 10% glycerol

• Cell lysis by sonication or mechanical disruption

• Centrifugation at high speed (>30,000 g) to separate membrane and soluble fractions

• Solubilization of membrane-associated mtgA using mild detergents (0.5-1% Triton X-100 or n-dodecyl-β-D-maltoside)

• Nickel affinity chromatography for His-tagged protein

• Size exclusion chromatography to obtain pure protein

For storage, the purified protein is stable in Tris/PBS-based buffer with 50% glycerol at pH 8.0, stored at -20°C/-80°C .

How can the enzymatic activity of mtgA be measured in laboratory settings?

Several complementary methods can be used to assess mtgA activity:

1. Fluorescence-Based GTase Assay:

• Utilize fluorescently labeled lipid II substrates (e.g., lipid II-Atto550 and lipid II-Atto647n)

• Monitor polymerization through Förster Resonance Energy Transfer (FRET)

• Calculate activity based on FRET efficiency changes during glycan strand formation

2. SDS-PAGE Analysis:

• Separate polymerized glycan products by size on SDS-PAGE

• Visualize bands using fluorescently labeled lipid II

• Quantify using densitometric analysis

3. HPLC Analysis of Reaction Products:

• Digest products with muramidase (cellosyl or mutanolysin)

• Analyze resulting muropeptides by HPLC

• Calculate average glycan strand length

Control Reactions:

• Negative control: Addition of moenomycin (GTase inhibitor)

• Positive control: Commercially available GTase enzymes

The FRET-based assay is particularly valuable as it allows real-time monitoring of enzyme activity and can be used to screen for inhibitors .

What are the recommended storage conditions to maintain mtgA stability and activity?

For optimal stability of recombinant Y. pseudotuberculosis mtgA:

• Short-term storage (1-2 weeks): Store working aliquots at 4°C

• Long-term storage: Store at -20°C/-80°C with 50% glycerol as cryoprotectant

• Avoid repeated freeze-thaw cycles as this significantly reduces activity

• For reconstituted lyophilized protein:

  • Reconstitute in deionized sterile water to 0.1-1.0 mg/mL

  • Add glycerol to 5-50% final concentration

  • Aliquot before freezing to minimize freeze-thaw cycles

The protein is typically stable for several months when stored properly in Tris/PBS-based buffer with 50% glycerol at pH 8.0 .

How does mtgA contribute to cell wall integrity and bacterial survival in Y. pseudotuberculosis?

MtgA plays a crucial role in bacterial cell wall biogenesis through its glycosyltransferase activity. Its importance stems from:

• Essential Peptidoglycan Synthesis: MtgA polymerizes lipid II precursors to form glycan strands, which are fundamental components of the bacterial cell wall.

• Growth and Division Support: The enzyme contributes to cell envelope integrity during growth and division phases, when new peptidoglycan must be synthesized rapidly.

• Stress Response: During stress conditions, proper peptidoglycan synthesis becomes even more critical for bacterial survival.

While not directly studied in Y. pseudotuberculosis, research on similar bacteria suggests that mtgA works cooperatively with other peptidoglycan synthases (PBPs) to maintain cell wall integrity. Proteomic studies have shown that mtgA can be selectively enriched in membrane vesicles formed during stress conditions, suggesting a role in bacterial adaptation .

What is the relationship between mtgA activity and virulence in pathogenic Yersinia species?

While direct evidence linking mtgA specifically to virulence is limited, peptidoglycan biosynthesis plays important indirect roles in pathogenesis:

• Cell Wall Integrity During Infection: Proper cell wall maintenance is essential for bacterial survival within host environments.

• Immune System Interaction: Peptidoglycan fragments can be recognized by host immune receptors (NOD1/NOD2), and alterations in peptidoglycan structure may affect immune recognition.

• Association with Virulence Mechanisms: In Y. pseudotuberculosis, virulence is primarily mediated by plasmid-encoded factors (e.g., Yop proteins and the T3SS), but cell wall integrity supported by mtgA is necessary for these systems to function properly.

Research on Y. pseudotuberculosis as a vaccine vector against Y. pestis has demonstrated that various attenuated strains (through deletion of virulence factors like YopK and YopJ) remain immunogenic while showing reduced pathogenicity, suggesting that basic cellular functions including cell wall maintenance remain intact in these modified strains .

How does mtgA activity in Y. pseudotuberculosis compare with other Yersinia species?

Comparative analysis of mtgA across Yersinia species reveals:

What approaches can be used to investigate mtgA as a potential antimicrobial target?

MtgA represents a promising antimicrobial target due to its essential role in peptidoglycan biosynthesis. Several research approaches can be employed:

1. Inhibitor Screening Strategies:

• High-throughput FRET-based assays using fluorescently labeled lipid II

• Competition assays with known GTase inhibitors like moenomycin

• Structure-based virtual screening using crystal structures of related GTases

2. Target Validation Methods:

• Conditional knockout or depletion studies in Y. pseudotuberculosis

• Overexpression studies to evaluate enzyme mechanism

• Point mutations to identify critical catalytic residues

3. Combination Therapy Exploration:

• Testing synergy between GTase inhibitors and β-lactam antibiotics

• Evaluation of membrane permeabilizers to enhance inhibitor access

4. Specificity Determination:

• Comparative analysis with human glycosyltransferases to ensure selectivity

• Broad-spectrum testing against different bacterial GTases

Given the emergence of multidrug-resistant bacteria, novel targets like monofunctional glycosyltransferases provide valuable opportunities for antimicrobial development .

How do environmental conditions affect mtgA expression and activity in Y. pseudotuberculosis?

Y. pseudotuberculosis encounters diverse environments during its lifecycle, and mtgA regulation is likely responsive to these changing conditions:

Temperature Effects:

• Expression may be differentially regulated between environmental (25°C) and host (37°C) temperatures

• Studies show that when grown at 37°C (host temperature), Y. pseudotuberculosis undergoes significant changes in cell envelope protein expression

Stress Response:

• Proteomic analyses indicate that cell wall synthesis enzymes, including some PG-modifying enzymes, can be upregulated during stress conditions

• MtgA has been detected among proteins enriched in membrane vesicles formed during stress responses

Host-Pathogen Interactions:

• When Y. pseudotuberculosis colonizes host tissues like Peyer's patches, the expression of cell wall synthesis genes may be modulated

• The ability to adjust peptidoglycan synthesis could be critical for surviving immune responses

Nutrient Availability:

• Y. pseudotuberculosis adapts to various nutrient sources in different environments

• These adaptations may include changes in cell wall metabolism to optimize resource allocation

Laboratory adaptation studies of the related Y. pestis have shown that bacteria can evolve modifications in cell envelope-related genes during serial passage, suggesting that peptidoglycan synthesis enzymes are subject to selection pressure under changing conditions .

What role does mtgA play in bacterial adaptation during laboratory domestication of Yersinia species?

Laboratory domestication represents a significant shift in selective pressures for bacteria adapted to complex natural environments. Research on Y. pestis laboratory adaptation provides insights relevant to Y. pseudotuberculosis:

• Evolution During Serial Passage:

  • Parallel serial passage experiments (PSPE) with Y. pestis revealed consistent mutations in cell envelope-related genes across independent populations

  • These adaptations likely reflect shifts toward optimizing growth in rich, constant laboratory media versus variable natural environments

• Metabolic Reprogramming:

  • Proteomic and metabolomic analyses of laboratory-adapted strains show changes in amino acid metabolism and cell envelope biogenesis

  • These shifts may include alterations in peptidoglycan synthesis regulation

• Virulence Attenuation:

  • Laboratory adaptation often leads to reduced virulence

  • Changes in cell wall composition may contribute to this attenuation by altering interactions with host immune systems

• Growth Optimization:

  • Domesticated strains typically optimize for rapid growth in rich media

  • This may involve changes in cell wall biosynthesis genes to support faster replication cycles

For Y. pseudotuberculosis, which shows greater genetic stability than Y. pestis due to fewer insertion sequences, adaptation may proceed more slowly but still involve adjustments in fundamental processes like peptidoglycan synthesis .

How can recombinant mtgA be used in developing attenuated Yersinia vaccines?

Attenuated Y. pseudotuberculosis strains have shown promise as vaccine candidates against plague. Recombinant mtgA could contribute to vaccine development in several ways:

• Antigen Delivery Systems:

  • Engineered Y. pseudotuberculosis strains expressing modified mtgA could potentially serve as live attenuated vaccines

  • Modified mtgA could influence cell wall properties to enhance immunogenicity without increasing virulence

• Adjuvant Properties:

  • Peptidoglycan fragments are recognized by the immune system and can serve as natural adjuvants

  • Engineered mtgA with altered activity could generate modified peptidoglycan that enhances immune responses

• Vaccine Strain Stabilization:

  • Y. pseudotuberculosis has greater genetic stability than Y. pestis, making it attractive for vaccine development

  • Careful manipulation of cell wall synthesis genes could further enhance strain stability

• Combining with Virulence Attenuation:

  • Several attenuated Y. pseudotuberculosis vaccine strains have been developed through deletion of virulence factors:

    • ΔyopK ΔyopJ Δasd mutant strain χ10069

    • V674pF1 strain with deleted HPI, yopK, and psaA genes

  • These strains could be further modified through mtgA engineering

These approaches build on successful studies showing that oral immunization with attenuated Y. pseudotuberculosis strains can protect against both bubonic and pneumonic plague .

What are the key considerations when designing experiments to study mtgA-substrate interactions?

When investigating mtgA-substrate interactions, researchers should consider:

Substrate Preparation:

• Lipid II synthesis options:

  • Chemical synthesis (challenging but provides pure material)

  • Enzymatic synthesis using MraY and MurG

  • Extraction from bacterial membranes (less pure but more native)

• Fluorescent or radioactive labeling strategies:

  • Fluorescent lipid II (e.g., dansyl-lipid II, Atto-labeled lipid II)

  • [14C]-labeled lipid II for quantitative analysis

Reaction Conditions:

• Buffer composition:

  • Optimal pH range (typically 7.5-8.5)

  • Divalent cation requirements (Mg2+ or Mn2+)

  • Salt concentration effects

• Detergent selection:

  • Critical for maintaining enzyme activity while solubilizing lipid substrates

  • Common choices: Triton X-100, n-dodecyl-β-D-maltoside

  • Concentration optimization critical (typically 0.02-0.1%)

Analytical Methods:

• For monitoring substrate binding:

  • Surface plasmon resonance

  • Isothermal titration calorimetry

  • Fluorescence polarization

• For detecting catalytic activity:

  • FRET-based continuous assays

  • SDS-PAGE for product analysis

  • HPLC analysis of digested products

Controls and Validation:

• Essential controls:

  • Known GTase inhibitor (moenomycin)

  • Heat-inactivated enzyme

  • Substrate-only reactions

• Validation with orthogonal methods to confirm observations .

How can researchers differentiate between the activity of mtgA and other peptidoglycan synthases in complex bacterial systems?

Distinguishing mtgA activity from other peptidoglycan synthases requires specialized approaches:

1. Genetic Approaches:

• Gene deletion/complementation studies with mtgA knockout strains

• Conditional expression systems to regulate mtgA levels

• Introduction of point mutations in catalytic residues

2. Biochemical Discrimination:

• Utilize the differential sensitivity of enzymes to inhibitors:

  • mtgA (monofunctional GTase): Sensitive to moenomycin, resistant to β-lactams

  • PBP1A/1B (bifunctional): Sensitive to both moenomycin and β-lactams

  • Monofunctional transpeptidases: Sensitive only to β-lactams

3. Mass Spectrometry-Based Analysis:

• Use targeted proteomics to quantify specific enzymes

• Activity-based protein profiling with enzyme-specific probes

• Analysis of peptidoglycan structure to infer enzyme activities

4. Microscopy Techniques:

• Fluorescent D-amino acid labeling to visualize sites of peptidoglycan synthesis

• Localization studies using fluorescently tagged enzymes

• Super-resolution microscopy to differentiate spatial distribution

5. In vitro Reconstitution:

• Purify individual components and reconstitute with defined substrates

• Analyze product profiles to distinguish enzyme contributions

These approaches can be combined to build a comprehensive understanding of mtgA's specific contribution to peptidoglycan synthesis in complex bacterial systems .

What are the major challenges in studying mtgA function and how can they be overcome?

Researching mtgA presents several technical challenges:

Challenge 1: Protein Solubility and Stability

• Problem: MtgA is membrane-associated and often difficult to maintain in soluble, active form

• Solutions:

  • Use mild detergents (DDM, CHAPS) at optimized concentrations

  • Consider fusion tags that enhance solubility (e.g., MBP, SUMO)

  • Nanodiscs or liposomes to provide membrane-like environment

  • Work at reduced temperatures (4-16°C) during purification and assays

Challenge 2: Substrate Accessibility

• Problem: Natural substrate (lipid II) is complex and difficult to synthesize

• Solutions:

  • Establish collaborations with specialized lipid II synthesis labs

  • Use commercially available fluorescent lipid II analogs

  • Develop simplified substrate mimics for initial screening

  • Consider enzymatic synthesis of lipid II in situ

Challenge 3: Assay Sensitivity and Specificity

• Problem: Distinguishing mtgA activity from other peptidoglycan synthases

• Solutions:

  • Combine genetic approaches (knockouts) with biochemical assays

  • Use specific inhibitors as controls (moenomycin for GTase activity)

  • Employ FRET-based assays for increased sensitivity

  • Analyze reaction products by HPLC or mass spectrometry

Challenge 4: Physiological Relevance

• Problem: In vitro conditions may not reflect the natural environment

• Solutions:

  • Validate findings with in vivo studies

  • Use membrane preparations rather than purified proteins

  • Reconstruct minimal synthetic systems with multiple components

  • Develop cell-based reporter systems

These methodological approaches can help overcome the inherent difficulties in studying membrane-associated enzymes like mtgA .

How can researchers troubleshoot common issues in recombinant mtgA expression and activity assays?

Expression Troubleshooting:

Activity Assay Troubleshooting:

Specific mtgA Considerations:

• Verify membrane association by fractionation experiments

• Test activity in the presence of potential activators

• Consider that mtgA may require specific lipid environments for optimal activity

• Ensure proper divalent cation (Mg2+, Mn2+) concentration in activity buffers

What are promising future research directions for understanding mtgA function in Yersinia pathogenesis?

Several high-potential research avenues include:

• Structural Studies:

  • Determine the crystal structure of Y. pseudotuberculosis mtgA to understand species-specific features

  • Investigate substrate binding through co-crystallization with moenomycin or lipid II analogs

  • Employ cryo-EM to visualize mtgA in membrane environments

• Systems Biology Approaches:

  • Map the interaction network of mtgA with other cell wall synthesis proteins

  • Investigate transcriptional regulation of mtgA during infection processes

  • Apply proteomics to identify post-translational modifications affecting activity

• Host-Pathogen Interaction:

  • Examine how host factors influence mtgA activity during infection

  • Investigate peptidoglycan fragments generated by mtgA as potential immune modulators

  • Study the impact of mtgA activity on immune recognition and evasion

• Synthetic Biology Applications:

  • Engineer Y. pseudotuberculosis strains with modified mtgA to develop improved vaccine vectors

  • Create synthetic peptidoglycan with defined properties for immunological studies

  • Develop mtgA-based biosensors for antimicrobial screening

• Ecological Adaptations:

  • Compare mtgA function across Yersinia species adapted to different ecological niches

  • Investigate temperature-dependent regulation relevant to environmental versus host conditions

  • Study how mtgA contributes to survival in diverse environments

These directions would significantly advance our understanding of bacterial cell wall biosynthesis in the context of Yersinia pathogenesis and could lead to novel therapeutic strategies .

How might CRISPR-Cas9 and other genetic engineering tools be applied to study mtgA function?

CRISPR-Cas9 and advanced genetic tools open new possibilities for mtgA research:

1. Precise Genome Editing:

• Generate clean mtgA deletions without polar effects

• Introduce point mutations to study structure-function relationships

• Create tagged versions at native chromosomal loci

• Engineer conditional expression systems for essential gene analysis

2. Regulatory Studies:

• CRISPR interference (CRISPRi) to tune down mtgA expression

• CRISPR activation (CRISPRa) to upregulate expression

• Promoter replacement to control expression patterns

• Targeted epigenetic modifications to study regulatory mechanisms

3. High-Throughput Screening:

• CRISPR library screens to identify genetic interactions

• Barcode-based competition assays to assess fitness contributions

• Multiplexed editing to study combinatorial effects with other genes

4. Live Cell Applications:

• CRISPR-based fluorescent tagging for real-time localization

• Optogenetic control of mtgA expression

• Development of biosensors to monitor peptidoglycan synthesis

5. In vivo Applications:

• Engineer Y. pseudotuberculosis strains with modified mtgA for vaccination studies

• Create reporter strains to visualize peptidoglycan synthesis during infection

• Generate complementation libraries to screen for functional domains

These genetic approaches, combined with biochemical and structural studies, would provide comprehensive insights into mtgA function in bacterial physiology and pathogenesis .