Recombinant Yersinia pseudotuberculosis serotype IB Monofunctional biosynthetic peptidoglycan transglycosylase (mtgA)

What is the primary function of MtgA in Yersinia pseudotuberculosis?

MtgA (Monofunctional biosynthetic peptidoglycan transglycosylase) in Y. pseudotuberculosis, similar to its E. coli homolog, catalyzes glycan chain elongation during peptidoglycan synthesis in the bacterial cell wall. This enzyme performs the critical function of polymerizing lipid II precursors to form the glycan strands of peptidoglycan without the transpeptidase activity found in bifunctional penicillin-binding proteins (PBPs) . In vitro studies with E. coli MtgA have demonstrated a 2.4-fold increase in peptidoglycan polymerization when the enzyme is overexpressed, with complete product digestion by lysozyme confirming its glycosyltransferase activity . The enzyme's activity is essential for maintaining cell wall integrity and proper cell division, particularly in conditions where other peptidoglycan synthesis enzymes may be compromised.

How does MtgA localize within Yersinia pseudotuberculosis cells?

Based on studies of the E. coli homolog, MtgA likely localizes to the division site in Y. pseudotuberculosis, particularly under conditions where class A PBPs (Penicillin Binding Proteins) are absent or deficient. In E. coli, GFP-tagged MtgA has been observed to localize at mid-cell in strains deficient in PBP1b and expressing thermosensitive PBP1a . This localization pattern was reversed when PBP1b was reintroduced, suggesting competitive localization dynamics between MtgA and class A PBPs . In Y. pseudotuberculosis, MtgA would be expected to exhibit similar localization patterns, particularly during cell division when peptidoglycan synthesis is most active at the developing septum.

Which protein interactions are crucial for MtgA function in bacterial cell division?

For MtgA to properly function in bacterial cell division, it forms specific interactions with multiple divisome components. Studies in E. coli have demonstrated that MtgA interacts with:

These interactions suggest MtgA participates in a coordinated complex that synthesizes peptidoglycan at the division septum . The particularly strong interaction with FtsW is significant as FtsW is thought to transport lipid II, the substrate for MtgA, across the membrane. In Y. pseudotuberculosis, these interactions would likely be conserved given the fundamental nature of bacterial cell division processes.

What are the optimal conditions for expressing recombinant MtgA from Yersinia pseudotuberculosis?

For optimal recombinant expression of Y. pseudotuberculosis MtgA, consider the following methodological approach:

• Expression System Selection: E. coli BL21(DE3) with a pET-based vector containing the mtgA gene under control of a T7 promoter typically yields good results.

• Growth and Induction Parameters:

  • Culture medium: LB supplemented with appropriate antibiotics

  • Growth temperature: 30°C until OD600 reaches 0.6-0.8

  • Induction: 0.5 mM IPTG

  • Post-induction: Lower temperature to 18-20°C for 16-18 hours to enhance soluble protein production

• Buffer Composition for Purification:

  • Lysis buffer: 50 mM HEPES (pH 7.5), 300 mM NaCl, 10% glycerol, 10 mM MgCl2

  • Wash buffer: Same as lysis buffer with 20-40 mM imidazole

  • Elution buffer: Same as lysis buffer with 250-300 mM imidazole

• Protein Stability Considerations:

  • The addition of glycerol (10%) and MgCl2 (10 mM) significantly improves stability, as seen with other transglycosylases

  • Storage at -80°C in small aliquots with glycerol (15-20%) prevents activity loss during freeze-thaw cycles

This approach draws from established protocols for expressing related peptidoglycan synthesis enzymes, with modifications to account for the specific requirements of Y. pseudotuberculosis MtgA.

How can researchers effectively measure MtgA enzymatic activity in vitro?

To effectively measure Y. pseudotuberculosis MtgA enzymatic activity in vitro, implement the following methodological approach:

• Substrate Preparation: Use fluorescently-labeled or radiolabeled lipid II as substrate. For radiolabeled assays, UDP-[14C]GlcNAc-labeled lipid II (approximately 9,000-10,000 dpm/nmol) is effective based on E. coli MtgA assays .

• Reaction Conditions:

  • Buffer composition: 50 mM HEPES (pH 7.0), 10-15% dimethyl sulfoxide, 10% octanol, 0.5% decyl-polyethylene glycol, and 10 mM CaCl2

  • Temperature: 30°C

  • Reaction time: 30-60 minutes

  • Enzyme concentration: Titrate to determine linear range (typically 0.1-1 μM)

• Product Analysis Options:

  Method	Advantages	Limitations	Key Parameters
  Radiolabeled assay	High sensitivity, quantitative	Requires radioisotope handling	Separate products by paper chromatography; quantify by scintillation counting
  HPLC analysis	No radioisotopes, good resolution	Lower sensitivity	C18 reverse-phase column; UV/fluorescence detection
  Lysozyme sensitivity test	Confirms polymerized product	Qualitative only	Add lysozyme post-reaction; complete digestion confirms glycan chain formation
  Fluorescence-based assay	Real-time kinetics, high-throughput	Requires specialized substrates	Use dansylated lipid II; monitor decrease in fluorescence during polymerization

• Controls and Validation:

  • Positive control: Known active transglycosylase (e.g., PBP1b)

  • Negative control: Heat-inactivated enzyme

  • Specificity control: Moenomycin inhibition (selective transglycosylase inhibitor)

  • Validation: Lysozyme digestion of products confirms glycan chain formation

This approach builds on established methods for E. coli MtgA while incorporating modifications to optimize for Y. pseudotuberculosis MtgA characteristics.

What genetic tools are available for studying mtgA function in Yersinia pseudotuberculosis?

Several genetic tools and approaches are available for studying mtgA function in Y. pseudotuberculosis:

• Gene Deletion Systems:

  • Lambda Red recombination system: Allows precise in-frame deletion of mtgA

  • Suicide vector approach: pDM4 or pDS132 vectors carrying flanking regions of mtgA for homologous recombination and sacB for counter-selection

  • CRISPR-Cas9: Emerging method for precise genomic modifications in Yersinia species

• Complementation Strategies:

  • Chromosomal integration: Using Tn7-based systems for single-copy complementation

  • Plasmid-based expression: low-copy (pWSK29) or arabinose-inducible (pBAD) vectors

  • Promoter considerations: Native promoter for physiological expression or inducible promoters for controlled expression

• Reporter Fusions for Localization and Expression Studies:

  Reporter Type	Applications	Considerations for Y. pseudotuberculosis
  GFP fusion	Protein localization, as demonstrated with E. coli MtgA	Temperature-sensitive variants necessary for 37°C studies
  mCherry fusion	Alternative for localization with different spectral properties	Less photobleaching than GFP; good for longer observations
  LacZ fusion	Promoter activity and transcriptional regulation	Colorimetric assay compatible with Yersinia culture conditions
  Luciferase fusion	Real-time expression monitoring	Sensitive detection in living cells

• Protein-Protein Interaction Tools:

  • Bacterial two-hybrid system: Demonstrated effective for studying MtgA interactions in E. coli

  • Pull-down assays: Using His-tagged or other affinity-tagged versions of MtgA

  • Cross-linking coupled with mass spectrometry: For identifying interaction partners in vivo

When implementing these tools, researchers should consider the growth temperature dependence of Y. pseudotuberculosis virulence factors and adjust experimental protocols accordingly. For temperature-shift experiments, pre-growing cultures at 26°C followed by a shift to 37°C can help investigate MtgA's role under conditions mimicking host infection.

How does MtgA activity relate to the Type III Secretion System (T3SS) in Yersinia pseudotuberculosis?

While the direct relationship between MtgA and the T3SS in Y. pseudotuberculosis remains to be fully elucidated, several lines of evidence suggest potential connections:

• Temporal and Spatial Coordination: Both peptidoglycan synthesis and T3SS assembly require precise coordination during bacterial growth and infection. The T3SS apparatus must traverse the peptidoglycan layer, necessitating controlled cell wall remodeling in which MtgA may participate.

• Regulatory Networks: Y. pseudotuberculosis demonstrates a massive transcriptional shift from chromosomal to virulence plasmid-encoded genes during T3SS/Yop secretion . This reprogramming affects multiple cellular processes, potentially including peptidoglycan metabolism where MtgA functions.

• Growth Phenotype Correlation: Y. pseudotuberculosis exhibits growth reduction during Yop secretion , a phenotype that may involve changes in cell wall synthesis. Since MtgA is involved in peptidoglycan synthesis, its activity might be modulated during this process.

• RNA-Mediated Control: The control of virulence factors in Y. pseudotuberculosis involves complex RNA-mediated processes . Similar post-transcriptional regulation might affect mtgA expression during infection, connecting it to the broader virulence program.

To investigate these potential relationships, researchers could implement:

• Transcriptomic analysis comparing mtgA expression levels before and after T3SS induction

• Microscopy studies examining MtgA localization during T3SS assembly

• Construction of mtgA conditional mutants to assess effects on T3SS functionality

• Protein-protein interaction studies to identify potential connections between MtgA and T3SS components

This research direction could reveal important insights into how fundamental cellular processes like peptidoglycan synthesis are integrated with specialized virulence mechanisms in pathogenic bacteria.

What role does MtgA play in Yersinia pseudotuberculosis virulence and intracellular survival?

The role of MtgA in Y. pseudotuberculosis virulence likely involves several dimensions, drawing parallels with related systems:

While Y. pseudotuberculosis possesses multiple peptidoglycan synthesis enzymes that may provide functional redundancy, the specialized role of MtgA during infection could be revealed through careful experimentation under conditions that closely mimic the host environment.

How do environmental factors affect MtgA expression and activity in Yersinia pseudotuberculosis?

MtgA expression and activity in Y. pseudotuberculosis are likely modulated by several environmental factors that the bacterium encounters during its lifecycle:

• Temperature Regulation:

  • Y. pseudotuberculosis transitions between environmental temperatures (approximately 25°C) and mammalian host temperature (37°C)

  • This temperature shift triggers extensive transcriptional reprogramming that may include changes in mtgA expression

  • Research approach: qRT-PCR analysis of mtgA transcription at different temperatures and Western blot analysis of protein levels

• Magnesium Concentration Effects:

  • Y. pseudotuberculosis virulence is significantly influenced by magnesium concentration via the PhoPQ two-component system

  • Since MgtB (magnesium transporter) affects virulence , there may be regulatory connections between magnesium sensing and cell wall synthesis genes including mtgA

  • Research approach: Analyze mtgA expression in wild-type vs. ΔphoP mutants under varying magnesium concentrations

• pH Adaptation:

  • During infection, Y. pseudotuberculosis encounters varying pH environments, from acidic phagolysosomes to near-neutral intestinal lumen

  • These pH changes may affect both mtgA expression and the enzymatic activity of MtgA protein

  • Research approach: Measure MtgA enzymatic activity across pH range 5.0-8.0 using in vitro assays

• Nutritional Stress Response:

  Nutrient Stress	Potential Impact on MtgA	Experimental Approach
  Carbon limitation	May alter cell wall composition and thickness	Chemostat cultures with limiting carbon; measure mtgA expression
  Iron restriction	Often coordinates with virulence expression	Iron chelation with dipyridyl; assess mtgA response
  Amino acid starvation	Triggers stringent response affecting cell wall	Measure mtgA expression during serine hydroxamate treatment
  Host-derived bile salts	Represent intestinal environment	Expose to physiological concentrations of bile; measure MtgA activity

• Growth Phase Dependency:

  • MtgA requirements may differ between exponential growth and stationary phase

  • Y. pseudotuberculosis modifies its cell wall throughout its growth cycle

  • Research approach: Time-course experiments with synchronized cultures to determine growth phase-specific expression patterns

Understanding these environmental influences on MtgA would provide insights into how Y. pseudotuberculosis integrates environmental sensing with cell wall modifications during host adaptation and pathogenesis.

What are common challenges in generating mtgA knockout mutants in Yersinia pseudotuberculosis and how can they be overcome?

Researchers may encounter several challenges when generating mtgA knockout mutants in Y. pseudotuberculosis:

• Potential Essentiality or Growth Defects:

  • Challenge: If mtgA plays a crucial role in peptidoglycan synthesis under certain conditions, knockout attempts may fail or produce severe growth defects.

  • Solution: Implement conditional knockout strategies using:

    • Inducible promoter systems (tetracycline-responsive or arabinose-inducible)

    • Temperature-sensitive plasmid complementation

    • Depletion approaches with degradation tags (e.g., SsrA tag)

• Functional Redundancy:

  • Challenge: Y. pseudotuberculosis likely possesses multiple enzymes with transglycosylase activity (including bifunctional PBPs), potentially masking phenotypes of single mtgA knockouts.

  • Solution: Consider:

    • Generating double or triple mutants targeting multiple transglycosylases

    • Creating mutations under conditions where other transglycosylases are inhibited or downregulated

    • Using specific growth conditions where MtgA function becomes more critical, similar to observations in E. coli with deficient PBP1a/PBP1b

• Technical Difficulties with Yersinia Genetic Manipulation:

  Challenge	Solution	Technical Details
  Restriction barriers	Use conjugation rather than transformation	Employ E. coli S17-1 or SM10 donor strains; optimize conjugation temperature (25-28°C)
  Low recombination efficiency	Enhanced recombineering approaches	Express phage recombination proteins (Redα/β/γ) from temperature-controlled promoter
  Plasmid instability	Optimize selection pressure	Maintain consistent antibiotic selection; consider chromosomal integration for stable complementation
  Non-specific phenotypes	Proper complementation controls	Use both in trans (plasmid) and cis (chromosomal) complementation to verify phenotypes

• Verification Challenges:

  • Challenge: Confirming complete deletion and absence of polar effects on neighboring genes.

  • Solution:

    • Perform RT-PCR on adjacent genes to confirm normal transcription

    • Use multiple primer pairs flanking the deletion for PCR verification

    • Sequence the deletion junction and surrounding regions

    • Consider whole genome sequencing to rule out secondary mutations

• Phenotypic Analysis Complications:

  • Challenge: Subtle phenotypes that may only manifest under specific conditions.

  • Solution:

    • Test growth under various stress conditions (temperature shifts, antimicrobial peptides, cell wall stressors)

    • Examine multiple parameters (growth rate, cell morphology, peptidoglycan composition)

    • Use electron microscopy to detect subtle cell wall architecture changes

Successfully addressing these challenges requires a multifaceted approach combining conditional gene expression systems, careful phenotypic analysis under various conditions, and appropriate complementation strategies.

How can researchers differentiate between MtgA activity and other transglycosylases in experimental systems?

Differentiating MtgA activity from other transglycosylases in Y. pseudotuberculosis requires targeted approaches:

• Differential Inhibition Profiles:

  • Moenomycin inhibits both bifunctional PBPs and monofunctional transglycosylases but with different sensitivities

  • Research approach: Compare inhibition curves of purified MtgA versus other transglycosylases to identify concentration windows where differential inhibition occurs

  • Create a standardized inhibition profile table for reference:

  Transglycosylase	Moenomycin IC50 (μM)	Other Specific Inhibitors	Penicillin Sensitivity
  MtgA	To be determined	None known	Insensitive
  PBP1a	Typically 0.1-1.0	Various β-lactams	Sensitive
  PBP1b	Typically 0.1-1.0	Various β-lactams	Sensitive
  Other TGs	Various	Various	Various

• Enzyme-Specific Assay Conditions:

  • Optimize buffer conditions (pH, salt concentration, metal ions) that preferentially support MtgA activity

  • MtgA from E. coli shows specific activity requirements including dimethyl sulfoxide (15%), octanol (10%), and CaCl2 (10 mM)

  • Research approach: Systematically vary reaction conditions to identify MtgA-favoring parameters

• Genetic Approaches:

  • Generate strains with combinations of transglycosylase gene deletions

  • Create conditional expression systems where MtgA is the only active transglycosylase

  • Research approach: Engineer strains with temperature-sensitive mutations in bifunctional PBPs while maintaining wild-type mtgA

• Biochemical Separation and Detection:

  • Use affinity-tagged versions of MtgA for specific isolation from cellular extracts

  • Implement activity-based protein profiling with transglycosylase-specific probes

  • Research approach: Develop chemoenzymatic labeling strategies specific for monofunctional transglycosylases

• Product Analysis:

  • Different transglycosylases may produce glycan chains with subtle structural differences

  • Research approach: Use mass spectrometry and specialized HPLC methods to detect MtgA-specific products

  • Analyze glycan chain length distribution patterns which may differ between enzymes

By combining these approaches, researchers can develop a comprehensive strategy to distinguish MtgA activity from other transglycosylases, enabling more precise characterization of its specific role in Y. pseudotuberculosis cell wall biosynthesis and virulence.

How might MtgA be exploited as a potential therapeutic target against Yersinia infections?

MtgA represents a promising therapeutic target against Yersinia infections for several reasons:

• Essential Cellular Function:

  • Peptidoglycan synthesis is critical for bacterial survival and growth

  • While redundancy exists among transglycosylases, specific inhibition of MtgA could still effectively compromise cell wall integrity under infection conditions

  • By targeting a monofunctional transglycosylase, novel inhibitors could complement existing β-lactam antibiotics that primarily target transpeptidase functions

• Structural Targeting Opportunities:

  • The transglycosylase domain offers a distinct structural target separate from transpeptidases

  • Research approach: Perform structure-based drug design using homology models or resolved structures of MtgA

  • Consider the following targeting strategies:

  Targeting Approach	Advantages	Challenges	Research Methods
  Active site inhibitors	Direct interference with catalytic function	Conservation with human glycosyltransferases	High-throughput screening against purified MtgA
  Allosteric inhibitors	Potentially higher specificity	More difficult to identify	Fragment-based screening approaches
  Protein-protein interaction disruptors	Novel mechanism of action	Complex interfaces to target	Bacterial two-hybrid screens to identify interaction sites
  Substrate analogs	Mechanistic understanding exists	Potential membrane permeability issues	Lipid II analogs with modified structures

• Virulence-Specific Effects:

  • If MtgA plays a specialized role during infection (similar to MgtB in Yersinia virulence ), inhibitors might attenuate pathogenicity without driving resistance through growth inhibition

  • Research approach: Screen for compounds that specifically block infection-relevant functions rather than purely growth inhibitory effects

• Considerations for Antimicrobial Development:

  • Bacterial specificity: Target regions unique to bacterial transglycosylases

  • Spectrum of activity: Determine conservation among Yersinia species and other Enterobacteriaceae

  • Resistance development: Assess potential compensatory mechanisms in Yersinia when MtgA is inhibited

  • Delivery challenges: Consider strategies to enhance permeability through the Gram-negative cell envelope

• Experimental Approaches:

  • Implement cell-based screening in conditions where MtgA function becomes more critical

  • Develop fluorescence-based assays with purified MtgA for high-throughput inhibitor screening

  • Use transposon sequencing (Tn-seq) to identify genetic interactions that enhance MtgA essentiality, revealing potential combination therapies

By focusing on the unique aspects of MtgA function in Y. pseudotuberculosis, researchers could develop targeted antimicrobial strategies that effectively control Yersinia infections while minimizing impacts on beneficial microbiota.

What are emerging technologies and approaches for studying MtgA function in the context of host-pathogen interactions?

Several emerging technologies offer powerful new approaches for studying MtgA function in Y. pseudotuberculosis during host-pathogen interactions:

• Advanced Imaging Techniques:

  • Super-resolution microscopy: Techniques like PALM and STORM can visualize MtgA localization with nanometer precision during infection

  • Time-lapse microscopy: Track dynamic changes in MtgA distribution during different infection stages

  • Correlative light and electron microscopy (CLEM): Combine fluorescence imaging of tagged MtgA with ultrastructural analysis of the bacterial cell wall

• Single-Cell Analysis Technologies:

  • Single-cell RNA-seq: Profile transcriptional changes in mtgA expression at the individual bacterial cell level during infection

  • CyTOF (mass cytometry): Measure multiple parameters simultaneously in bacteria recovered from infection models

  • Microfluidics: Study bacterial responses to changing environmental conditions mimicking different host compartments

• Advanced Genetic Tools:

  Technology	Application to MtgA Research	Advantages
  CRISPRi	Tunable knockdown of mtgA expression	Allows titration of MtgA levels without complete deletion
  CRISPR-Cas base editors	Introduce specific amino acid changes in MtgA	Create subtle mutations without selection markers
  Proximity labeling (BioID, APEX)	Identify transient MtgA interaction partners in vivo	Captures weak or temporal interactions during infection
  Optogenetics	Light-controlled MtgA activity or expression	Precise temporal control during infection process

• Structural Biology Advances:

  • Cryo-electron microscopy: Determine high-resolution structures of MtgA alone and in complex with interaction partners

  • Hydrogen-deuterium exchange mass spectrometry: Map dynamic regions and binding interfaces of MtgA

  • AlphaFold2/RoseTTAFold: Predict structural features of Y. pseudotuberculosis MtgA and its complexes

• Host-Pathogen Interface Technologies:

  • Organoids: Study MtgA function during infection of physiologically relevant 3D tissue models

  • Intravital microscopy: Visualize MtgA-expressing Y. pseudotuberculosis during real-time infection in animal models

  • Dual RNA-seq: Simultaneously profile bacterial and host transcriptomes during infection

  • MALDI-imaging mass spectrometry: Spatially resolve peptidoglycan modifications in infected tissues

• Systems Biology Approaches:

  • Multi-omics integration: Combine transcriptomics, proteomics, and metabolomics data to place MtgA in broader cellular networks

  • Network analysis: Map genetic and physical interactions of MtgA to understand its contextual role

  • Computational modeling: Predict the impact of MtgA perturbation on cell wall architecture and bacterial fitness

These emerging technologies offer unprecedented resolution and systems-level understanding of MtgA function within the complex environment of host-pathogen interactions, potentially revealing new aspects of Y. pseudotuberculosis pathogenesis and identifying novel intervention strategies.

What are the key unresolved questions about MtgA in Yersinia pseudotuberculosis?

Despite advances in understanding bacterial cell wall biosynthesis, several critical questions about MtgA in Y. pseudotuberculosis remain unresolved:

• Specialized Functions During Infection:

  • Does MtgA play a specific role during different stages of Y. pseudotuberculosis infection?

  • How does MtgA activity change when bacteria transition from environmental reservoirs to mammalian hosts?

  • Is MtgA activity differentially regulated in various host tissues or cellular locations?

• Regulatory Networks:

  • How is mtgA expression integrated into the complex virulence regulatory networks of Y. pseudotuberculosis?

  • Does the global reprogramming observed during T3SS activation affect mtgA expression or activity?

  • Are there post-transcriptional regulatory mechanisms controlling MtgA synthesis or function?

• Structural and Mechanistic Questions:

  • What are the unique structural features of Y. pseudotuberculosis MtgA compared to homologs in other bacteria?

  • How does substrate recognition and processing differ between MtgA and bifunctional PBPs?

  • What determines the localization pattern of MtgA during different growth phases?

• Evolutionary Considerations:

  • Why have bacteria maintained monofunctional transglycosylases alongside bifunctional PBPs?

  • Has MtgA evolved specific features in pathogenic Yersinia compared to environmental species?

  • What selective pressures maintain mtgA in the Y. pseudotuberculosis genome?

• Clinical Relevance:

  • Could MtgA inhibition effectively attenuate Y. pseudotuberculosis infections?

  • How would targeting MtgA compare to targeting other aspects of cell wall synthesis?

  • What is the potential for resistance development against MtgA-targeted therapeutics?

Addressing these questions will require integrative approaches combining structural biology, advanced genetics, infection models, and systems-level analyses. The answers will not only advance our understanding of Y. pseudotuberculosis pathogenesis but may also reveal broadly applicable principles about bacterial cell wall biosynthesis during host-pathogen interactions.