Recombinant Pseudomonas mendocina Monofunctional biosynthetic peptidoglycan transglycosylase (mtgA)

What is the general function of mtgA in bacterial physiology?

Monofunctional biosynthetic peptidoglycan transglycosylase (mtgA) plays a crucial role in bacterial cell wall biosynthesis. It functions as a glycan polymerase (also known as peptidoglycan glycosyltransferase) that catalyzes the polymerization of lipid II precursors to form the glycan strands of peptidoglycan, which is essential for maintaining cell wall integrity and bacterial survival . Unlike bifunctional PBPs (Penicillin-Binding Proteins), mtgA lacks transpeptidase activity and exclusively performs transglycosylation reactions in peptidoglycan assembly.

How should recombinant mtgA be stored and handled for optimal stability?

For optimal stability and activity retention, recombinant mtgA should be:

• Initially stored as a lyophilized powder at -20°C/-80°C upon receipt

• Reconstituted in deionized sterile water to a concentration of 0.1-1.0 mg/mL

• Supplemented with glycerol (recommended final concentration: 50%) for long-term storage

• Aliquoted to avoid repeated freeze-thaw cycles

• Working aliquots can be stored at 4°C for up to one week

• Long-term storage requires -20°C/-80°C conditions

The protein is supplied in a Tris/PBS-based buffer containing 6% Trehalose at pH 8.0 . Repeated freeze-thaw cycles should be avoided as they can compromise protein integrity and enzymatic activity.

How should researchers design experiments to study mtgA enzymatic activity?

When designing experiments to study mtgA enzymatic activity, researchers should follow a structured experimental design approach:

• Question Formulation: Define specific aspects of mtgA activity to investigate (e.g., substrate specificity, reaction kinetics, inhibition patterns)

• Variable Identification:

  • Independent variables: enzyme concentration, substrate concentration, temperature, pH, cofactors

  • Dependent variables: reaction rate, product formation, enzyme stability

• Hypothesis Development: Formulate a testable hypothesis about mtgA activity (e.g., "mtgA exhibits optimal transglycosylase activity at pH 7.0-7.5")

• Controls: Include appropriate controls, such as:

  • Negative controls lacking enzyme or substrate

  • Positive controls using characterized transglycosylases

  • Heat-inactivated enzyme controls

• Data Collection Planning: Determine appropriate methods to monitor transglycosylase activity, such as:

  • HPLC analysis of reaction products

  • Fluorescent or radiolabeled substrate assays

  • Colorimetric assays for released products

What are the optimal conditions for expressing recombinant mtgA in E. coli?

The optimal expression conditions for recombinant mtgA in E. coli typically involve:

For purification, the N-terminal His tag allows for efficient immobilized metal affinity chromatography (IMAC) using Ni-NTA or similar matrices, typically yielding >90% purity after a single purification step .

How can researchers assess the substrate specificity of mtgA?

To comprehensively assess the substrate specificity of mtgA, researchers should implement a multi-faceted analytical approach:

• Synthetic Substrate Analysis:

  • Utilize chemically defined lipid II variants with modifications in:

    • Stem peptide composition

    • Glycan structure

    • Lipid carrier length

  • Monitor product formation via:

    • HPLC analysis with UV detection

    • Mass spectrometry for product identification

    • Real-time kinetic measurements using fluorescent substrates

• Competition Assays:

  • Perform reactions with mixtures of potential substrates

  • Analyze preferential utilization through:

    • Thin-layer chromatography (TLC)

    • Liquid chromatography-mass spectrometry (LC-MS)

• Structural Modeling and Docking:

  • Create homology models based on known transglycosylase structures

  • Perform in silico docking studies with various substrates

  • Identify key residues involved in substrate recognition

• Site-Directed Mutagenesis:

  • Target conserved residues in the active site

  • Assess impact on substrate specificity through:

    • Steady-state kinetics (Km, kcat, kcat/Km)

    • Binding affinity measurements (ITC, SPR)

• Comparative Analysis:

  • Benchmark against other characterized transglycosylases

  • Evaluate evolutionary conservation across bacterial species

This methodological framework provides a comprehensive assessment of mtgA substrate preferences and catalytic parameters.

What techniques are available for studying mtgA interactions with other cell wall biosynthesis proteins?

Studying protein-protein interactions involving mtgA requires sophisticated biochemical and biophysical approaches:

• Co-Immunoprecipitation (Co-IP):

  • Use antibodies against mtgA or potential interacting partners

  • Analyze precipitated complexes by SDS-PAGE and Western blotting

  • Identify novel interactions through mass spectrometry analysis

• Pull-Down Assays:

  • Utilize the His-tagged mtgA as bait protein

  • Incubate with cell lysates or purified potential partners

  • Identify interactions by mass spectrometry or immunoblotting

• Surface Plasmon Resonance (SPR):

  • Immobilize mtgA on sensor chips

  • Measure real-time binding kinetics (kon, koff, KD)

  • Generate detailed binding profiles for different partners

• Förster Resonance Energy Transfer (FRET):

  • Label mtgA and potential partners with compatible fluorophores

  • Monitor proximity-dependent energy transfer in vitro or in vivo

  • Quantify interaction strength and dynamics

• Bacterial Two-Hybrid System:

  • Fuse mtgA and potential partners to complementary reporter domains

  • Screen for interactions based on reporter activation

  • Validate positive hits through orthogonal methods

• Cross-Linking Mass Spectrometry:

  • Utilize chemical cross-linkers to stabilize transient interactions

  • Identify cross-linked peptides by tandem mass spectrometry

  • Map interaction interfaces at amino acid resolution

This multi-technique approach enables robust characterization of mtgA's protein interaction network within the peptidoglycan biosynthesis machinery.

How can researchers design inhibitor screening assays for mtgA?

To design effective inhibitor screening assays for mtgA, researchers should implement the following methodological framework:

• Primary High-Throughput Screening (HTS):

  • Fluorescence-Based Assays:

    • Utilize dansylated or fluorescently-labeled lipid II substrates

    • Monitor decrease in fluorescence anisotropy upon polymerization

    • Measure in 384-well format for high throughput

  • FRET-Based Assays:

    • Design dual-labeled substrates with donor-acceptor pairs

    • Monitor changes in FRET signal upon polymerization

    • Allows real-time kinetic measurements

• Secondary Validation Assays:

  • Radiochemical Assays:

    • Use [14C]-labeled lipid II substrates

    • Quantify incorporation into polymeric peptidoglycan

    • Offers high sensitivity and specificity

  • Mass Spectrometry-Based Assays:

    • Monitor substrate depletion and product formation

    • Provides detailed structural information on reaction products

    • Useful for mechanism-of-action studies

• Counter-Screening and Specificity Assessment:

  • Test hits against other glycosyltransferases to confirm specificity

  • Evaluate potential off-target effects on related enzymes

  • Assess membrane permeability using bacterial penetration assays

• Structure-Activity Relationship (SAR) Analysis:

  • Systematically modify hit compounds to improve potency and specificity

  • Correlate structural features with inhibitory activity

  • Guide rational design of optimized inhibitors

• Mechanism of Inhibition Studies:

  • Perform enzyme kinetics with varying substrate and inhibitor concentrations

  • Determine inhibition type (competitive, non-competitive, uncompetitive)

  • Calculate Ki values and evaluate inhibition constants

Implementation of this comprehensive screening cascade will facilitate the identification and characterization of specific mtgA inhibitors with potential as research tools or antimicrobial lead compounds.

What methodologies are appropriate for studying mtgA function in intact bacterial cells?

Investigating mtgA function in intact bacterial cells requires specialized approaches that bridge in vitro biochemistry with cellular physiology:

• Genetic Manipulation Strategies:

  • Gene Deletion/Knockdown:

    • Create mtgA deletion mutants in Pseudomonas mendocina

    • Use CRISPR-Cas9 or homologous recombination techniques

    • Assess cellular phenotypes (growth, morphology, stress resistance)

  • Complementation Studies:

    • Express wild-type or mutant mtgA in deletion strains

    • Use inducible promoters for controlled expression

    • Quantify restoration of normal phenotypes

  • Depletion Systems:

    • Place mtgA under controllable promoters (e.g., tet-regulated)

    • Monitor cellular changes during protein depletion

    • Establish temporal sequence of phenotypic alterations

• Fluorescent Protein Fusions:

  • Generate functional mtgA-fluorescent protein fusions

  • Track subcellular localization during:

    • Different growth phases

    • Cell division

    • Antibiotic stress responses

  • Perform time-lapse microscopy to monitor dynamic behavior

• Peptidoglycan Analysis Techniques:

  • Muropeptide Profiling:

    • Isolate peptidoglycan and digest with muramidases

    • Analyze muropeptide composition by HPLC

    • Compare profiles between wild-type and mtgA-modified strains

  • Cell Wall Labeling:

    • Use fluorescent D-amino acids (FDAAs) for nascent peptidoglycan labeling

    • Employ click chemistry with azide-modified cell wall precursors

    • Visualize peptidoglycan synthesis patterns by microscopy

• Biochemical Approaches in Cellular Context:

  • Chemical Genetics:

    • Apply specific mtgA inhibitors to intact cells

    • Monitor phenotypic changes and compensatory responses

    • Compare with genetic manipulation outcomes

  • Activity-Based Protein Profiling:

    • Utilize activity-based probes targeting transglycosylases

    • Assess mtgA activity in living cells under various conditions

    • Identify potential regulatory mechanisms

This integrated approach provides a comprehensive framework for elucidating mtgA function within its native cellular environment.

How can researchers design experiments to study the role of mtgA in antimicrobial resistance?

To investigate mtgA's potential role in antimicrobial resistance, researchers should implement a multi-faceted experimental approach:

• Expression Analysis Under Antibiotic Stress:

  • Transcriptomics:

    • Perform RNA-Seq or qRT-PCR on bacteria exposed to cell wall-targeting antibiotics

    • Monitor mtgA expression changes in response to sub-inhibitory concentrations

    • Identify co-regulated genes in resistance pathways

  • Proteomics:

    • Quantify mtgA protein levels using targeted mass spectrometry (MRM/PRM)

    • Analyze post-translational modifications under antibiotic stress

    • Assess protein-protein interaction networks using pull-down/MS

• Genetic Manipulation Studies:

  • Overexpression Analysis:

    • Generate strains with controlled mtgA overexpression

    • Determine minimum inhibitory concentrations (MICs) for various antibiotics

    • Assess changes in resistance profiles and cross-resistance patterns

  • Resistance Selection Experiments:

    • Perform serial passage in increasing antibiotic concentrations

    • Sequence mtgA in resistant isolates to identify mutations

    • Introduce identified mutations using site-directed mutagenesis to confirm causality

• Structural and Biochemical Approaches:

  • Antibiotic Binding Studies:

    • Perform binding assays between mtgA and cell wall antibiotics

    • Use techniques like isothermal titration calorimetry (ITC) or microscale thermophoresis (MST)

    • Determine binding constants and thermodynamic parameters

  • Enzyme Kinetics:

    • Measure mtgA activity in presence of antibiotics

    • Determine inhibition constants and mechanisms

    • Compare wild-type and mutant enzymes from resistant strains

• Systems Biology Integration:

  • Construct mathematical models of cell wall biosynthesis including mtgA

  • Simulate antibiotic effects and potential resistance mechanisms

  • Validate model predictions with experimental data

This comprehensive experimental design framework enables systematic investigation of mtgA's contributions to antimicrobial resistance mechanisms.

What methodological approaches are available for studying mtgA in biofilm formation?

Investigating the role of mtgA in biofilm formation requires specialized methodologies spanning molecular, cellular, and community levels:

• Genetic and Molecular Approaches:

  • Mutant Construction and Phenotyping:

    • Generate mtgA deletion, point mutation, and controlled expression strains

    • Assess biofilm formation using:

      • Crystal violet staining (biomass quantification)

      • Confocal laser scanning microscopy (structure analysis)

      • Flow cell systems (dynamic biofilm development)

    • Compare phenotypes under various environmental conditions

  • Complementation Studies:

    • Express mtgA variants in deletion backgrounds

    • Quantify restoration of biofilm formation capacity

    • Identify critical functional domains through truncation/mutation analysis

• Advanced Microscopy Techniques:

  • Super-Resolution Microscopy:

    • Localize fluorescently-tagged mtgA within biofilm structures

    • Track dynamic redistribution during biofilm development

    • Correlate localization with extracellular matrix components

  • Correlative Light-Electron Microscopy:

    • Combine fluorescence imaging of mtgA with ultrastructural analysis

    • Visualize mtgA in relation to cell wall architecture

    • Map enzyme distribution across biofilm regions

• Biochemical and Matrix Analysis:

  • Extracellular Matrix Characterization:

    • Compare matrix composition between wild-type and mtgA-modified strains

    • Analyze polysaccharide, protein, and eDNA content

    • Assess cell wall fragment incorporation into matrix

  • Cell Wall Integrity Assessment:

    • Measure peptidoglycan crosslinking in biofilm vs. planktonic cells

    • Analyze muropeptide profiles from different biofilm regions

    • Correlate modifications with mtgA activity levels

• Systems-Level Approaches:

  • Transcriptomics/Proteomics:

    • Compare expression profiles between biofilms with varying mtgA levels

    • Identify co-regulated genes and potential regulatory networks

    • Track temporal expression patterns during biofilm maturation

  • Metabolomics:

    • Analyze metabolite profiles in mtgA-modified biofilms

    • Focus on cell wall precursor pools and turnover products

    • Identify metabolic signatures associated with altered mtgA function

This methodological framework provides a comprehensive approach to understanding mtgA's role in the complex process of biofilm formation and maintenance.

What approaches can researchers use to study evolutionary conservation of mtgA across bacterial species?

To investigate the evolutionary conservation of mtgA across bacterial species, researchers should implement a comprehensive comparative genomics and phylogenetics approach:

• Sequence-Based Phylogenetic Analysis:

  • Dataset Construction:

    • Collect mtgA homologs across diverse bacterial phyla

    • Include related transglycosylases for outgroup comparison

    • Verify functional annotation through conserved domain analysis

  • Multiple Sequence Alignment:

    • Generate alignments using MUSCLE, MAFFT, or T-Coffee algorithms

    • Refine alignments to focus on conserved catalytic domains

    • Identify invariant residues critical for function

  • Phylogenetic Tree Construction:

    • Apply maximum likelihood or Bayesian inference methods

    • Calculate bootstrap values to assess branch support

    • Correlate branching patterns with bacterial taxonomy

• Structural Conservation Analysis:

  • Homology Modeling:

    • Generate structural models for mtgA across diverse species

    • Superimpose structures to identify conserved structural elements

    • Map sequence conservation onto structural features

  • Active Site Comparison:

    • Analyze conservation of catalytic residues across homologs

    • Identify species-specific variations in substrate-binding regions

    • Correlate structural differences with enzymatic properties

• Functional Complementation Assays:

  • Express mtgA homologs from diverse bacteria in P. mendocina mtgA deletion strains

  • Quantify degree of functional restoration

  • Correlate complementation efficiency with sequence/structural divergence

• Selective Pressure Analysis:

  • Calculate dN/dS ratios to identify regions under purifying or positive selection

  • Perform branch-site tests to detect lineage-specific selective pressures

  • Correlate selection patterns with bacterial lifestyle and environmental adaptations

• Comparative Genomic Context Analysis:

  • Examine gene neighborhood conservation across species

  • Identify co-evolved gene clusters and potential functional associations

  • Map genomic rearrangements affecting mtgA organization

This comprehensive analytical framework enables systematic characterization of mtgA evolutionary patterns and identification of conserved functional elements across bacterial diversity.

How can researchers develop robust experimental designs to compare mtgA function between different bacterial species?

Developing robust experimental designs for cross-species comparison of mtgA function requires careful consideration of standardization, controls, and species-specific factors:

• Standardized Expression and Purification:

  • Expression System Optimization:

    • Use identical expression vectors and host systems for all mtgA variants

    • Standardize induction conditions and purification protocols

    • Verify comparable protein folding using circular dichroism

  • Protein Quantification:

    • Employ multiple quantification methods (Bradford, BCA, A280)

    • Confirm active site titration using activity-based probes

    • Ensure equal active enzyme concentrations in comparative assays

• Parallel Biochemical Characterization:

  • Enzyme Kinetics:

    • Determine Km, kcat, and substrate specificity in identical reaction conditions

    • Use standardized substrates across all enzyme variants

    • Perform assays at multiple temperatures to account for thermal adaptation

  • Thermal and pH Stability:

    • Measure thermal denaturation profiles using DSF or nanoDSF

    • Determine pH-activity profiles under standardized buffer conditions

    • Correlate stability parameters with source organism environment

  Parameter	Measurement Method	Control Variables	Output Metrics
  Activity	Fluorescent substrate assay	Temperature, pH, ionic strength	kcat, Km, kcat/Km
  Thermal stability	Differential scanning fluorimetry	Scan rate, protein concentration	Tm, ΔH, ΔS
  pH profile	Activity measurements at pH intervals	Buffer system, ionic strength	pH optimum, stability range
  Inhibitor sensitivity	Dose-response curves	Inhibitor solubility, binding kinetics	IC50, Ki

• Heterologous Expression Studies:

  • Cross-Complementation:

    • Express each mtgA variant in deletion strains of multiple species

    • Quantify growth rates, morphology, and cell wall properties

    • Analyze muropeptide profiles to assess functional restoration

  • Chimeric Enzyme Analysis:

    • Construct domain-swapped variants between divergent mtgA proteins

    • Map functional domains responsible for species-specific properties

    • Identify critical regions for host adaptation

• Multivariate Data Analysis:

  • Apply principal component analysis to identify patterns in functional parameters

  • Perform hierarchical clustering to group enzymes by functional similarity

  • Correlate functional clusters with phylogenetic relationships

• Environmental Adaptation Analysis:

  • Test enzyme activity under conditions mimicking natural habitats

  • Compare performance under stress conditions (osmotic, temperature, pH)

  • Correlate specialized adaptations with ecological niche

This systematic experimental framework enables robust, standardized comparison of mtgA function across bacterial diversity while accounting for species-specific adaptations and evolutionary context.