Recombinant Escherichia coli O6:K15:H31 Monofunctional biosynthetic peptidoglycan transglycosylase (mtgA)

What is the primary function of MtgA in Escherichia coli?

MtgA (monofunctional peptidoglycan transglycosylase) is an enzyme that catalyzes the polymerization of lipid II molecules into glycan strands of peptidoglycans, which are essential components of the bacterial cell wall. Unlike bifunctional penicillin-binding proteins (PBPs), MtgA functions specifically as a monofunctional glycosyltransferase involved in cell wall synthesis. The protein plays a role in peptidoglycan assembly during the cell cycle, particularly in collaboration with other cell division proteins .

How does MtgA localize within E. coli cells?

MtgA has been shown to localize at the division site (midcell) of E. coli cells, particularly in strains that are deficient in PBP1b and produce a thermosensitive PBP1a. This localization pattern suggests that MtgA functions within the divisome complex during cell division. Interestingly, when PBP1b is reintroduced to these cells via plasmid expression, the specific midcell localization of MtgA is no longer observed, indicating a competitive relationship between MtgA and class A PBPs for the division site .

Which proteins interact with MtgA during cell division?

In vivo bacterial two-hybrid experiments have demonstrated that MtgA interacts specifically with three essential constituents of the divisome:

• PBP3 (FtsI): A transpeptidase essential for septum formation

• FtsW: A protein involved in peptidoglycan synthesis

• FtsN: A component required for cell division

These interactions require the transmembrane segment of PBP3 and suggest that MtgA collaborates with these proteins within the divisome to synthesize peptidoglycan at the new poles during cell division. MtgA also shows self-interaction, suggesting it may function as a multimer .

What phenotypes are observed in MtgA deletion mutants?

• Remarkable increase in cell size (approximately 1.4-fold larger)

• Specifically increased cell diameter without corresponding increases in polar axis length

• Cells that become "fat" rather than "tall"

• Increased production of polyhydroxyalkanoates such as P(LA-co-3HB)

These phenotypes can be reversed by complementation with the mtgA gene, confirming the direct relationship between mtgA deletion and the observed changes .

How can I design experiments to assess the compensatory role of MtgA in PBP-deficient strains?

To investigate MtgA's compensatory role when PBPs are deficient:

• Strain Construction:

  • Generate strains with various combinations of PBP deficiencies:

    • PBP1a thermosensitive mutant

    • PBP1b deletion mutant

    • Double mutant (thermosensitive PBP1a + PBP1b deletion)

  • Introduce plasmids expressing MtgA at different levels

• Localization Analysis:

  • Create GFP-MtgA fusion constructs for visualization

  • Use fluorescence microscopy to monitor MtgA localization at various growth phases

  • Compare localization patterns between wild-type and PBP-deficient backgrounds

• Viability Assessment:

  • Conduct growth curve analyses at permissive and non-permissive temperatures

  • Perform colony-forming unit (CFU) counts under various stress conditions

  • Assess morphological changes using microscopy

• Genetic Approach:

  • Create an inducible MtgA depletion system in PBP-deficient backgrounds

  • Monitor phenotypic changes upon MtgA depletion

  • Quantify peptidoglycan synthesis rates using radioactive precursors

• Data Analysis:

  • Compare growth rates, morphology, and viability across all strains

  • Determine threshold levels of MtgA required for compensation

  • Identify conditions where MtgA becomes essential

What methodologies can be used to study the enzymatic activity of MtgA in vitro?

Several approaches can be employed to study MtgA enzymatic activity:

• Protein Purification:

  • Express recombinant MtgA with affinity tags (His-tag or GFP fusion)

  • Optimize purification conditions to maintain enzymatic activity

  • Verify purity using SDS-PAGE and Western blotting

• Glycosyltransferase Activity Assay:

  • Use radiolabeled lipid II substrate (e.g., [14C]GlcNAc-labeled lipid II)

  • Create reaction mixtures containing:

    • Purified MtgA (≈0.5-5 μg/reaction)

    • 15% dimethyl sulfoxide

    • 10% octanol

    • 50 mM HEPES (pH 7.0)

    • 0.5% decyl-polyethylene glycol

    • 10 mM CaCl2

  • Incubate at 30°C for 1 hour

  • Extract reaction products and analyze by thin-layer chromatography

  • Validate polymerization by lysozyme digestion (should result in complete digestion)

• Kinetic Analysis:

  • Determine Km and Vmax values for lipid II substrate

  • Assess the effects of various cofactors on enzyme activity

  • Study inhibition profiles using various antibiotics targeting peptidoglycan synthesis

• Structural Studies:

  • Use X-ray crystallography or cryo-EM to determine MtgA structure

  • Perform mutagenesis of key residues to identify catalytic site

  • Assess conformational changes during substrate binding using FRET technology

How does MtgA deletion affect polyhydroxyalkanoate (PHA) production in recombinant E. coli?

MtgA deletion significantly impacts polyhydroxyalkanoate production in recombinant E. coli:

• Production Enhancement:

  Strain	P(LA-co-3HB) Production (g/l)	Cell Size Increase	LA/3HB Ratio Change
  Parent recombinant	5.2	-	Baseline
  mtgA deletion (rJW)	7.0	1.4-fold	Minimal
  Complemented strain	5.2 (approx.)	None	Baseline

• Proposed Mechanism:

  • MtgA deletion affects cell wall integrity and flexibility

  • Altered peptidoglycan structure likely increases cell volume

  • Larger cells can accommodate more polymer accumulation

  • Cell enlargement occurs specifically in diameter rather than length

  • The effect is dependent on polymer-producing conditions (not observed under non-producing conditions)

• Experimental Verification:

  • Complementation with intact mtgA gene restores normal phenotype

  • Cell morphology analysis confirms the "fat" rather than "tall" appearance

  • Polymer production correlates directly with cell size changes

  • Transposon-inserted strain (C21) shows similar morphology changes

What techniques can be used to analyze MtgA interactions with divisome proteins?

Multiple complementary approaches can be employed to study MtgA interactions:

• Bacterial Two-Hybrid System:

  • Construct fusion proteins with T18 and T25 fragments of adenylate cyclase

  • Transform into DHM1 strain (cya-) and measure β-galactosidase activity

  • Quantify interaction strength by comparing to positive and negative controls

  • Use specific protein domains to map interaction interfaces

  • Example significant values: MtgA-PBP3 interaction shows approximately 10-13 fold higher signal than negative controls

• Co-immunoprecipitation:

  • Express tagged versions of MtgA and potential interaction partners

  • Perform pull-down experiments using antibodies against the tags

  • Analyze co-precipitated proteins by Western blotting

  • Confirm specificity using non-interacting protein controls

• Fluorescence Microscopy Colocalization:

  • Create fluorescent protein fusions (e.g., GFP-MtgA, mCherry-PBP3)

  • Express in E. coli and visualize by fluorescence microscopy

  • Analyze colocalization patterns during different cell cycle stages

  • Quantify colocalization using Pearson's correlation coefficient

• Surface Plasmon Resonance (SPR):

  • Immobilize purified MtgA on a sensor chip

  • Flow purified divisome proteins over the chip

  • Measure binding kinetics (kon and koff rates)

  • Determine affinity constants (KD) for each interaction

  • Compare binding strengths between different protein partners

How can genetic manipulation of the mtgA gene be optimized in E. coli strain O6:K15:H31?

Optimizing genetic manipulation of mtgA in E. coli O6:K15:H31 requires specialized approaches:

• Transposon Mutagenesis Strategy:

  • Use mini-Tn5 transposon carried on pUTmini-Tn5 Km

  • Employ conjugative transfer using E. coli S17-1 λ-pir as donor strain

  • Perform conjugation on LB agar plates at 30°C for 16 hours

  • Select transconjugants on media containing appropriate antibiotics:

    • Chloramphenicol: Eliminates donor S17-1 cells

    • Kanamycin: Selects for transposon insertion

    • Ampicillin: Maintains recombinant plasmids

  • Screen for desired phenotypes (e.g., increased polymer production, altered morphology)

• Targeted Gene Deletion:

  • Implement λ Red recombineering system for scarless deletions

  • Design primers with 40-50bp homology arms flanking mtgA

  • Replace mtgA with antibiotic resistance cassette

  • Verify deletion by PCR and sequencing

  • Remove resistance marker using FLP recombinase if necessary

• Complementation Analysis:

  • Clone wild-type mtgA gene into compatible expression vectors

  • Transform into deletion mutants

  • Verify expression levels by RT-PCR or Western blotting

  • Assess restoration of wild-type phenotypes

  • Use inducible promoters to titrate mtgA expression levels

• Strain-Specific Considerations:

  • E. coli O6:K15:H31 (strain 536) harbors pathogenicity islands

  • These genomic regions may affect transformation efficiency

  • Higher voltage may be required for electroporation

  • Consider using plasmids with origin compatible with strain 536

  • Monitor possible instability of introduced genetic elements

How can I differentiate between direct and indirect effects of mtgA deletion on cell physiology?

To distinguish direct from indirect effects of mtgA deletion:

• Time-Course Analysis:

  • Monitor phenotypic changes immediately after mtgA inactivation

  • Use inducible Cas9-sgRNA system for rapid mtgA knockout

  • Track changes in cell morphology, polymer production, and gene expression

  • Early effects are more likely to be direct consequences

• Multi-Omics Approach:

  • Perform RNA-seq to identify differentially expressed genes

  • Use proteomics to detect changes in protein abundance

  • Employ metabolomics to characterize metabolic shifts

  • Integrate data to build regulatory networks and identify key nodes

• Genetic Suppressor Analysis:

  • Screen for secondary mutations that restore wild-type phenotype

  • Identify genetic pathways that interact with mtgA function

  • Construct double mutants with genes in related pathways

  • Analyze epistatic relationships to determine pathway hierarchy

• Controlled Complementation:

  • Create mtgA variants with specific domain mutations

  • Express these variants in deletion strains

  • Identify which protein domains are responsible for specific phenotypes

  • Use domain swap experiments with related glycosyltransferases

• In vitro Reconstitution:

  • Purify components of peptidoglycan synthesis machinery

  • Reconstruct glycan strand formation with and without MtgA

  • Analyze product differences using mass spectrometry

  • Compare to in vivo peptidoglycan structure changes

What controls should be included when studying MtgA localization via fluorescence microscopy?

Essential controls for MtgA localization studies include:

• Expression Level Controls:

  • Western blot to verify GFP-MtgA fusion is expressed at near-native levels

  • Growth curve analysis to ensure fusion protein doesn't impair viability

  • RT-qPCR to quantify mRNA levels of native vs. fusion constructs

• Fusion Protein Functionality:

  • In vitro glycosyltransferase activity assay using purified GFP-MtgA

  • Complementation test in mtgA deletion strain

  • Verification data: GFP-MtgA shows 2.4-fold increase in peptidoglycan polymerization compared to control (26% versus 11% of lipid II used)

• Microscopy Controls:

  • Free GFP expression to control for non-specific localization

  • Fixed cells with immunofluorescence against MtgA to validate GFP fusion pattern

  • Co-imaging with membrane dye (FM4-64) to verify division site localization

  • z-stack acquisition to eliminate artifacts from different focal planes

• Genetic Background Controls:

  • Wild-type strain expressing GFP-MtgA

  • PBP1b deletion strain expressing GFP-MtgA

  • thermosensitive PBP1a strain expressing GFP-MtgA

  • Double mutant (thermosensitive PBP1a + PBP1b deletion) expressing GFP-MtgA

  • Each strain with complementing plasmid expressing PBP1b

• Data Analysis Controls:

  • Blinded scoring of localization patterns by multiple researchers

  • Quantitative image analysis with appropriate statistical tests

  • Clear criteria for defining "localized" versus "diffuse" signals

  • Minimum sample size of 200-300 cells per condition

How can contradictory results between in vitro and in vivo studies of MtgA be reconciled?

When facing contradictions between in vitro and in vivo MtgA studies:

• Identify Sources of Discrepancy:

  • Protein conformation differences in purified versus cellular environment

  • Missing cofactors or interaction partners in in vitro systems

  • Non-physiological buffer conditions affecting enzyme activity

  • Differences in substrate accessibility or concentration

• Experimental Approaches to Reconcile Contradictions:

  • Membrane Environment Reconstitution:

    • Use liposomes containing lipid II substrates

    • Incorporate purified MtgA into lipid bilayers

    • Create more physiologically relevant reaction conditions

  • Cell-Free Expression Systems:

    • Express MtgA in E. coli extract systems

    • Include relevant divisome components

    • Bridge gap between purified protein studies and in vivo experiments

  • Spheroplast-Based Assays:

    • Create spheroplasts with accessible peptidoglycan synthesis sites

    • Add purified or labeled components to monitor incorporation

    • Provide semi-in vivo environment with better experimental control

  • Correlation Analysis:

    • Create series of MtgA mutants with varying in vitro activity

    • Test same mutants for in vivo function

    • Establish quantitative relationship between in vitro activity and in vivo function

• Case Study Example:

  • Single MtgA mutants show no obvious phenotype in vivo

  • Yet, in vitro studies demonstrate clear glycosyltransferase activity

  • Resolution: Functional redundancy with PBPs in vivo masks MtgA contribution

  • Evidence: MtgA localization and function become apparent only in PBP-deficient backgrounds

How can MtgA function be leveraged for biotechnological applications?

MtgA's unique properties can be exploited for several biotechnological applications:

• Enhanced Biopolymer Production:

  • Utilize mtgA deletion to increase polyhydroxyalkanoate production

  • Optimize cell morphology for maximum polymer accumulation

  • Engineer strains with controlled expression of mtgA for tunable cell size

  • Data shows mtgA deletion increases P(LA-co-3HB) production from 5.2 g/l to 7.0 g/l

• Novel Antimicrobial Target:

  • Design inhibitors specific to MtgA glycosyltransferase activity

  • Target MtgA-divisome protein interactions as a novel approach

  • Screen for compounds that affect MtgA localization

  • Develop combination therapies targeting both MtgA and PBPs

• Cell Factory Optimization:

  • Manipulate cell volume through mtgA modifications

  • Create larger E. coli cells for increased production capacity

  • Engineer strains with enhanced cell envelope properties

  • Combine with other cell wall modifications for synergistic effects

• Synthetic Biology Toolkit:

  • Use MtgA localization as a divisome targeting module

  • Create fusion proteins that deliver cargo to the division site

  • Develop biosensors based on MtgA interactions

  • Engineer orthogonal cell wall synthesis pathways

What are the current knowledge gaps regarding MtgA function and regulation?

Despite significant progress, several important knowledge gaps remain:

• Regulatory Mechanisms:

  • How is mtgA expression regulated throughout the cell cycle?

  • What signals trigger MtgA recruitment to the division site?

  • Are there post-translational modifications affecting MtgA activity?

  • How does MtgA activity coordinate with other peptidoglycan synthases?

• Structural Determinants:

  • What are the key residues for MtgA glycosyltransferase activity?

  • Which domains mediate interaction with divisome proteins?

  • How does MtgA self-interaction contribute to function?

  • What structural changes occur upon substrate binding?

• Physiological Role:

  • Why does mtgA deletion alone show minimal phenotype?

  • What is the exact contribution of MtgA to septal peptidoglycan synthesis?

  • How does MtgA function differ between growth conditions?

  • What is the evolutionary significance of monofunctional glycosyltransferases?

• Strain-Specific Differences:

  • How does MtgA function differ in pathogenic versus non-pathogenic E. coli?

  • Are there strain-specific interaction partners?

  • Do virulence factors affect MtgA activity or localization?

  • Is MtgA activity modulated during host infection?

How can advanced imaging techniques be applied to study MtgA dynamics in live cells?

Advanced imaging approaches offer powerful tools for studying MtgA:

• Super-Resolution Microscopy:

  • Implement PALM/STORM for nanoscale localization precision

  • Use structured illumination microscopy (SIM) for improved resolution

  • Apply STED microscopy to visualize MtgA within the divisome complex

  • Achieve sub-diffraction resolution of 20-50 nm to resolve divisome substructures

• Single-Molecule Tracking:

  • Tag MtgA with photoactivatable fluorescent proteins

  • Track individual molecules to determine diffusion coefficients

  • Identify confined motion at the division site

  • Measure residence times at the septum versus cytoplasm

  • Calculate binding/unbinding kinetics in vivo

• FRET-Based Approaches:

  • Create MtgA-FP donor and divisome protein-FP acceptor pairs

  • Measure protein-protein interactions in real time

  • Detect conformational changes during enzyme activity

  • Monitor interaction dynamics throughout the cell cycle

• Correlative Light and Electron Microscopy (CLEM):

  • Combine fluorescence imaging of MtgA-FP with electron microscopy

  • Visualize MtgA localization in the context of cell envelope ultrastructure

  • Directly observe peptidoglycan synthesis sites

  • Link MtgA position to nascent peptidoglycan architecture

• Experimental Design Considerations:

  • Use photobleaching-resistant fluorophores for extended imaging

  • Implement microfluidic devices for long-term single-cell tracking

  • Apply deconvolution algorithms to improve image quality

  • Develop automated tracking software for unbiased analysis

How conserved is MtgA across different bacterial species and what does this suggest about its evolutionary importance?

Evolutionary analysis of MtgA reveals important patterns:

• Conservation Analysis:

  • MtgA homologs are widely distributed across Gram-negative bacteria

  • Core catalytic domain is highly conserved

  • N-terminal transmembrane segment shows greater variability

  • Firmicutes possess functionally analogous monofunctional glycosyltransferases

• Evolutionary Significance:

  • Conservation suggests fundamental role in peptidoglycan synthesis

  • Variable regions may reflect adaptation to different cell envelope architectures

  • Presence of compensatory mechanisms explains viability of deletion mutants

  • Co-evolution with divisome components indicates integrated functional network

• Pathogen-Specific Adaptations:

  • Uropathogenic E. coli strains like O6:K15:H31 may show specific variations

  • Potential interactions with virulence factors encoded on pathogenicity islands

  • Possible role in pathogen fitness during host colonization

  • Strain 536 (O6:K15:H31) contains large unstable DNA regions termed "pathogenicity islands" that encode various virulence factors

• Methodological Approaches:

  • Conduct phylogenetic analysis of MtgA sequences across bacterial phyla

  • Perform selection pressure analysis (dN/dS ratios)

  • Identify co-evolving residues using mutual information analysis

  • Use ancestral sequence reconstruction to trace evolutionary history

What genetic regulatory networks control mtgA expression in E. coli?

The regulation of mtgA involves complex genetic networks:

• Transcriptional Regulation:

  • Expression likely coordinated with cell cycle progression

  • Potential regulation by stress response pathways

  • Cell wall stress stimulon may upregulate mtgA under certain conditions

  • Possible control by global regulators (e.g., RpoS, CRP)

• Post-Transcriptional Mechanisms:

  • Small RNAs may modulate mtgA mRNA stability

  • RNA-binding proteins could affect translation efficiency

  • Potential for transcriptional attenuation or riboswitches

  • Differential regulation under various growth conditions

• Experimental Approaches:

  • Promoter Analysis:

    • Create transcriptional fusions with reporter genes

    • Identify transcription start sites using 5' RACE

    • Perform ChIP-seq to identify transcription factor binding

    • Use deletion analysis to map regulatory elements

  • Expression Profiling:

    • Monitor mtgA expression across growth phases

    • Compare expression in wild-type vs. regulatory mutants

    • Assess changes under cell wall stress conditions

    • Quantify mRNA and protein levels in parallel

• Cross-Regulation with Virulence:

  • In pathogenic strains like O6:K15:H31, potential regulatory cross-talk between adhesin determinants has been observed

  • This specific mode of virulence regulation is absent in mutant strains

  • Research indicates complex regulatory networks linking virulence and cell wall metabolism

How does MtgA function compare to bifunctional penicillin-binding proteins (PBPs) in E. coli?

A detailed comparison between MtgA and bifunctional PBPs reveals:

• Functional Comparison:

  Feature	MtgA	Bifunctional PBPs (e.g., PBP1a, PBP1b)
  Enzymatic activities	Glycosyltransferase only	Glycosyltransferase + Transpeptidase
  β-lactam sensitivity	Insensitive	Sensitive
  Essential for viability	No (single mutant viable)	Conditional (double mutants lethal)
  Divisome localization	Only in PBP-deficient backgrounds	Primary divisome components
  Polymer production impact	Deletion increases production	Complex, often detrimental effects

• Complementary Roles:

  • MtgA may serve as a backup system for glycan strand synthesis

  • Becomes important when bifunctional PBPs are compromised

  • Provides glycosyltransferase activity independent of transpeptidation

  • May contribute to specific aspects of peptidoglycan architecture

• Interaction Patterns:

  • MtgA interacts with divisome proteins (PBP3, FtsW, FtsN)

  • Different interaction network compared to bifunctional PBPs

  • Competition with PBP1a for division site localization

  • PBP1b presence prevents MtgA localization at division site

• Evolutionary Perspective:

  • Separation of glycosyltransferase and transpeptidase activities

  • Potentially allows for specialized regulation of each function

  • Provides redundancy in glycan strand synthesis

  • May reflect adaptation to specific environmental challenges

What is the relationship between MtgA and cell division proteins in E. coli O6:K15:H31?

The interaction between MtgA and the divisome in E. coli O6:K15:H31:

• Protein-Protein Interactions:

  • MtgA interacts with key divisome proteins:

    • PBP3 (FtsI): Essential septal transpeptidase

    • FtsW: Lipid II flippase candidate

    • FtsN: Late divisome protein, triggers constriction

  • These interactions suggest integration into the divisome complex

  • Bacterial two-hybrid assays confirm these interactions in vivo

  • The transmembrane segment of PBP3 is required for interaction with MtgA

• Spatial and Temporal Coordination:

  • MtgA localization at division site depends on cell's genetic background

  • Visible midcell localization in PBP1b-deficient, thermosensitive PBP1a strains

  • Suggests temporal regulation during the cell division process

  • May contribute to specific phases of septum formation

• Functional Significance:

  • MtgA may collaborate with PBP3 to synthesize peptidoglycan at new poles

  • Could provide penicillin-insensitive glycosyltransferase activity during early division

  • Potential role in discrete modification of septal peptidoglycan

  • May contribute to cell wall remodeling during constriction

• Strain-Specific Considerations:

  • E. coli O6:K15:H31 (strain 536) contains pathogenicity islands

  • These genomic elements may influence cell division processes

  • Potential integration of virulence factor expression with cell division

  • Mutants like 536-21 show altered virulence properties, indicating possible connections between cell wall synthesis and pathogenicity