Recombinant Escherichia coli O8 Monofunctional biosynthetic peptidoglycan transglycosylase (mtgA)

What is mtgA and what function does it serve in E. coli?

Monofunctional biosynthetic peptidoglycan transglycosylase (mtgA) is a 242-amino acid protein that participates in the formation of the peptidoglycan backbone in E. coli cell walls . Unlike some other cell wall components, mtgA is non-lethal when deleted, suggesting redundant pathways for peptidoglycan synthesis . MtgA belongs to a family of enzymes that catalyze the polymerization of lipid II to form the glycan strands of peptidoglycan, which provides structural integrity to the bacterial cell envelope. The protein functions as a glycan polymerase (also known as peptidoglycan glycosyltransferase) and is particularly important for maintaining proper cell morphology and division processes.

How is recombinant mtgA typically expressed and purified for research applications?

Recombinant mtgA is typically expressed in E. coli expression systems using vectors containing a His-tag for purification purposes. Based on established protocols, the full-length mtgA protein (1-242 amino acids) can be expressed in E. coli with an N-terminal His-tag . After cell cultivation and induction, cells are harvested and lysed, followed by purification using nickel-nitrilotriacetic acid affinity chromatography to capture the His-tagged protein . For higher purity, size-exclusion chromatography (SEC) can be employed as a secondary purification step to remove aggregates and contaminants. The purified protein is typically stored in Tris/PBS-based buffer at pH 8.0 with 6% trehalose as a stabilizer, and can be maintained at -20°C/-80°C for long-term storage . To prevent activity loss from repeated freeze-thaw cycles, the protein should be aliquoted before freezing, and working stocks can be kept at 4°C for up to one week.

What expression vectors and E. coli strains are most suitable for mtgA production?

For recombinant mtgA production, pET-series vectors (particularly pET28a) have proven effective due to their strong T7 promoter system and compatibility with His-tag purification . BL21(DE3) is the most commonly used E. coli strain for mtgA expression as it lacks certain proteases (lon and ompT) that might degrade the recombinant protein, and contains the λDE3 lysogen that carries the T7 RNA polymerase gene under control of the lacUV5 promoter . For applications requiring extracellular secretion of mtgA or other recombinant proteins, engineered BL21 strains with enhanced membrane permeability can be utilized. These include strains with engineered dacA expression as described in recent literature, which can increase extracellular production of recombinant proteins by up to 2-fold compared to standard BL21 strains .

What assays are used to measure mtgA enzymatic activity?

MtgA activity can be assessed using several complementary approaches:

• Peptidoglycan synthesis assay: This involves monitoring the polymerization of fluorescently labeled lipid II substrates and measuring the extension of glycan chains using high-performance liquid chromatography (HPLC) or mass spectrometry.

• Growth complementation assay: By expressing mtgA in strains deficient in multiple peptidoglycan synthesis enzymes, researchers can assess functional activity through restoration of normal growth and morphology.

• Microscopy-based morphological analysis: Since mtgA deletion leads to cell enlargement, microscopic examination of cell morphology before and after mtgA expression can provide qualitative assessment of enzyme activity .

• Polymer accumulation quantification: In specific applications focused on polymer production, mtgA activity can be indirectly assessed by measuring polymer accumulation using techniques like Nile red staining and subsequent HPLC analysis, as demonstrated in studies showing that mtgA deletion can increase poly(lactate-co-3-hydroxybutyrate) production from 2.9 g/l to 5.1 g/l .

How does mtgA deletion affect cell morphology and recombinant protein production in E. coli?

The deletion of mtgA in E. coli results in significant phenotypic changes that impact both cellular morphology and recombinant protein production capabilities. From a morphological perspective, mtgA deletion leads to cell enlargement due to alterations in peptidoglycan biosynthesis . This enlarged phenotype is consistent with compromised cell wall integrity, which paradoxically creates advantages for recombinant protein production.

Studies have demonstrated that mtgA-deleted strains can produce up to 1.8-fold higher quantities of polymers like poly(lactate-co-3-hydroxybutyrate) compared to wild-type strains . The specific production increase from 2.9 g/l to 5.1 g/l has been documented in transposon-based mutagenesis experiments . This enhancement occurs without severe growth disadvantages, suggesting that the metabolic burden of the deletion is manageable for the cells.

The mechanism behind this increased production capacity likely involves:

• Altered cell envelope permeability that facilitates protein secretion

• Changes in cellular resource allocation that favor recombinant protein synthesis

• Reduced competition between cell wall synthesis and recombinant protein production pathways

Complementation experiments confirm the specific role of mtgA in this phenotype, as reintroduction of the mtgA gene reverses the enhanced polymer production capability back to wild-type levels .

What are the optimal induction conditions for maximizing soluble mtgA expression?

Maximizing soluble mtgA expression requires careful optimization of multiple parameters:

Temperature: Lower induction temperatures (16-25°C) generally increase soluble mtgA yield by slowing protein synthesis and allowing proper folding, compared to standard 37°C conditions.

Inducer concentration: For IPTG-inducible systems, concentrations between 0.1-0.5 mM typically provide optimal balance between expression level and solubility for mtgA. Higher concentrations may lead to inclusion body formation.

Growth media: Rich media such as Terrific Broth (TB) significantly outperforms standard LB medium for mtgA expression, with documented cell pellet yields of 1.0-4.2 g (wet weight) from 250 mL cultures .

Induction timing: Initiating induction at mid-log phase (OD600 ~0.6-0.8) rather than early or late growth phases provides optimal results for mtgA.

Co-expression strategies: Similar to other challenging proteins, co-expression with chaperones (GroEL/GroES) or fusion partners (MBP) can dramatically improve soluble yields of mtgA.

The following table summarizes optimal induction parameters based on research findings:

What genetic engineering approaches can modify mtgA to enhance its stability and activity?

Several genetic engineering strategies have proven effective for enhancing mtgA stability and activity:

• Targeted mutagenesis: Similar to approaches used with other peptidoglycan-modifying enzymes, targeted amino acid substitutions at the catalytic domain can enhance thermal stability and specific activity. This approach has been demonstrated in related enzymes where mutations (like K10R and Y12A in the propeptide region of MTG) led to improved dissociation characteristics and higher specific activity .

• Fusion protein engineering: Creation of chimeric proteins combining mtgA with stability-enhancing partners has shown promise. For example, tobacco etch virus (TEV) protease fusions have been successfully used with related enzymes to produce active, soluble enzymes in E. coli . This approach could be adapted for mtgA to improve expression yields and stability.

• Promoter engineering: Modifying the native promoter structure by inserting one or two Shine-Dalgarno (SD) sequences between the promoter and the target gene has been shown to increase expression levels by enhancing transcription efficiency. This approach has resulted in up to 2.0-fold increases in extracellular recombinant protein activity in E. coli .

• Domain trimming: Structure-guided removal of non-essential or destabilizing domains can improve both expression and stability of mtgA while maintaining catalytic function.

• Codon optimization: Adapting the mtgA coding sequence to the codon usage bias of the expression host can substantially improve translation efficiency and protein yield.

Each engineering approach should be validated through activity assays to ensure that modifications preserve or enhance the native function of mtgA.

How can high-throughput screening methods be optimized to identify improved mtgA variants?

Developing effective high-throughput screening approaches for improved mtgA variants requires multiparametric optimization:

• Nile red-based plate assays: Nile red staining has proven effective for screening E. coli colonies with enhanced polymer accumulation, which indirectly identifies strains with altered mtgA function. This approach successfully identified mutant C21 with 1.8-fold higher polymer production from approximately 10,000 screened colonies . The method can be optimized by adjusting dye concentration, incubation time, and visualization parameters to maximize signal-to-noise ratio.

• Fluorescence-based reporter systems: Engineering fluorescent protein reporters linked to mtgA activity provides quantitative readouts for variant screening. This can be achieved by:

  • Developing peptidoglycan incorporation assays with fluorescent substrates

  • Creating transcriptional fusions where reporter expression correlates with cell wall modifications

  • Employing FRET-based sensors that respond to changes in cell wall structure

• Morphological screening: Automated microscopy coupled with image analysis algorithms can rapidly assess morphological changes (cell enlargement) associated with mtgA variants, enabling screening of thousands of colonies for desired morphological phenotypes.

• Growth-based selection: Designing selection conditions where cell survival depends on optimal mtgA activity can enable direct selection rather than screening. This approach typically yields fewer false positives than conventional screening methods.

• Multiplexed sequencing analysis: Deep mutational scanning combined with next-generation sequencing can comprehensively evaluate thousands of mtgA variants simultaneously, correlating sequence variations with function.

The efficiency of these screening approaches can be enhanced by implementing robotic liquid handling systems and automated analysis workflows to process large variant libraries.

What are the molecular mechanisms by which mtgA deletion enhances recombinant protein production?

The enhancement of recombinant protein production following mtgA deletion involves complex cellular adaptations at multiple levels:

• Cell envelope reorganization: Deletion of mtgA, which participates in peptidoglycan backbone formation, compromises cell wall integrity and increases membrane permeability . This structural change facilitates protein secretion by creating additional pathways for proteins to traverse the cell envelope, effectively creating a "leakier" cell that allows enhanced protein export.

• Metabolic redirection: The absence of mtgA-mediated peptidoglycan synthesis likely redirects cellular resources (energy, precursors, translation machinery) away from cell wall biosynthesis toward recombinant protein production. This metabolic redirection may involve altered expression of hundreds of genes involved in central carbon metabolism and protein synthesis.

• Stress response activation: The cell wall defect introduced by mtgA deletion triggers multiple stress responses that paradoxically enhance protein production capacity. These include the envelope stress response (Rse/σE pathway) and the Cpx two-component system, which upregulate chaperones and folding factors that improve recombinant protein folding and stability.

• Reduced growth rate compensation: The slightly reduced growth rate observed in mtgA deletion strains may reduce the metabolic burden of rapid division, allowing more resources to be allocated to recombinant protein synthesis. This phenomenon has been observed in the production of poly(lactate-co-3-hydroxybutyrate), where the mtgA deletion strain showed enhanced polymer accumulation despite slightly compromised growth .

• Altered gene expression profiles: Transcriptomic analyses have revealed that cell wall defects trigger comprehensive rewiring of gene expression patterns that can be beneficial for recombinant protein production. These changes include upregulation of transport systems and downregulation of competing biosynthetic pathways.

Understanding these molecular mechanisms provides opportunities for rational design of superior production strains beyond simple mtgA deletion.

How does the interaction between mtgA and other peptidoglycan synthesis enzymes affect cell wall formation?

MtgA functions within a complex network of enzymes involved in peptidoglycan synthesis, and its interactions with other components significantly influence cell wall architecture:

Understanding these complex interactions provides opportunities for more sophisticated engineering approaches beyond single-gene deletions to optimize cellular phenotypes for biotechnological applications.

What protocols optimize the purification of high-quality recombinant mtgA protein?

Purification of high-quality recombinant mtgA requires a multi-step approach to ensure maximal purity, yield, and activity:

• Cell lysis optimization: Gentle lysis methods like enzymatic treatment with lysozyme (100 μg/ml) combined with mild sonication (6 cycles of 10s on/30s off at 40% amplitude) preserve mtgA structural integrity better than harsh mechanical disruption.

• Affinity chromatography: For His-tagged mtgA, nickel-nitrilotriacetic acid affinity chromatography using imidazole gradients (20-250 mM) provides efficient capture while minimizing non-specific binding . Critical wash steps with 20-50 mM imidazole remove weakly bound contaminants without significant mtgA loss.

• Size-exclusion chromatography: SEC effectively separates active mtgA from aggregates and highly cross-linked proteins . A Superdex 200 column equilibrated with PBS buffer (pH 7.4) with flow rates of 0.5 ml/min provides optimal separation.

• Buffer optimization: Tris/PBS-based buffers (pH 8.0) containing 6% trehalose have been identified as optimal for maintaining mtgA stability . Addition of reducing agents (1-5 mM DTT or 2-10 mM β-mercaptoethanol) prevents oxidation of critical cysteine residues.

• Concentration and storage: Gentle concentration methods using 10 kDa molecular weight cutoff centrifugal filters at 4°C prevent aggregation. Aliquoting and flash-freezing in liquid nitrogen before storage at -80°C maintains activity through multiple experiments .

• Quality control: SDS-PAGE analysis to confirm >90% purity and activity assays to verify functionality are essential quality control steps before using purified mtgA in experiments .

For researchers working with mtgA variants that tend to co-purify with cleaved propeptide domains, introducing specific mutations (similar to K10R and Y12A used with related enzymes) can facilitate separation and yield higher purity protein preparations .

How can CRISPR-Cas9 genome editing be applied to modify mtgA in E. coli?

CRISPR-Cas9 genome editing provides precise tools for mtgA modification in E. coli:

• Complete mtgA gene knockout: Design sgRNAs targeting the mtgA coding sequence, introducing non-frameshift deletions or insertions to completely abolish gene function. Multiple sgRNA targets should be evaluated for efficiency, with optimal targets typically having NGG PAM sites and GC content between 40-60%.

• Targeted mutations: For introducing specific mutations (e.g., catalytic site modifications), design sgRNAs cutting near the target site and provide a repair template with desired mutations plus silent mutations to prevent re-cutting. Homology arms of 50-80 bp on each side of the cut site typically provide efficient recombination.

• Promoter engineering: CRISPR-Cas9 can precisely modify the native mtgA promoter to alter expression levels. This approach allows introduction of additional Shine-Dalgarno sequences between the promoter and gene, which has been shown to increase gene expression up to 2-fold .

• Domain swapping: Engineer chimeric forms of mtgA containing domains from related transglycosylases by precisely defining fusion junctions via CRISPR-Cas9 editing, potentially creating enzymes with novel properties.

• Multiplex modifications: Target mtgA along with related peptidoglycan synthesis genes in a single experiment using multiple sgRNAs to create strains with combined modifications, exploring synergistic effects.

A typical CRISPR-Cas9 workflow for mtgA modification includes:

• Designing and cloning sgRNAs into a CRISPR-Cas9 vector (e.g., pCas9)

• Creating repair templates with desired modifications

• Co-transforming E. coli with both components

• Screening transformants via colony PCR and sequencing

• Curing cells of CRISPR plasmids once modifications are confirmed

This approach has distinct advantages over traditional recombineering methods, including higher efficiency, reduced off-target effects, and the ability to create scarless modifications.

What are the best approaches for analyzing mtgA-dependent changes in cell wall structure?

Understanding mtgA-dependent changes in cell wall structure requires multiple complementary analytical techniques:

• Electron microscopy: Transmission electron microscopy (TEM) provides high-resolution images of peptidoglycan architecture, revealing alterations in cell wall thickness and density in mtgA-deleted strains. Sample preparation should include fixation with 2.5% glutaraldehyde and 2% paraformaldehyde, followed by OsO4 post-fixation and embedding in epoxy resin.

• Atomic force microscopy (AFM): AFM provides quantitative assessment of cell surface topography and mechanical properties like elasticity and rigidity, which often change significantly with mtgA deletion. Analysis of force-distance curves can quantify cell wall stiffness changes with nanometer precision.

• Muropeptide analysis: HPLC separation of digested peptidoglycan components allows quantification of specific muropeptide species, revealing detailed chemical changes in the cell wall. For E. coli, digestion with mutanolysin followed by reduction with sodium borohydride and separation on a C18 reverse-phase column provides comprehensive muropeptide profiles.

• Fluorescent D-amino acid labeling: Incorporation of fluorescent D-amino acids (FDAAs) like NADA or HADA into growing peptidoglycan allows visualization of active peptidoglycan synthesis sites and can reveal altered patterns in mtgA mutants.

• Cell wall isolation and compositional analysis: Purification of sacculi followed by colorimetric assays for peptidoglycan content (muramic acid determination) and cross-linking index measurement quantifies bulk changes in cell wall composition.

• Antibiotic susceptibility profiling: Systematic testing of β-lactam and glycopeptide antibiotic sensitivity can indirectly assess cell wall alterations, as mtgA-deleted strains often show altered minimum inhibitory concentrations (MICs) to cell wall-targeting antibiotics.

• Super-resolution microscopy: Techniques like STORM (Stochastic Optical Reconstruction Microscopy) or PALM (Photoactivated Localization Microscopy) can visualize peptidoglycan architecture with resolution below the diffraction limit, revealing nanoscale changes in cell wall organization.

By combining these approaches, researchers can comprehensively characterize how mtgA modifications affect cell wall structure at molecular, chemical, and physical levels.

How can transcriptomic and proteomic approaches reveal global cellular responses to mtgA modification?

Multi-omics approaches provide comprehensive insights into cellular adaptations following mtgA modification:

• RNA-Seq analysis: Transcriptome profiling via RNA-Seq reveals genome-wide expression changes in mtgA-modified strains. Key analytical steps include:

  • Extraction of total RNA using hot phenol or commercial kits optimized for bacterial samples

  • rRNA depletion to enrich for mRNA

  • Library preparation and deep sequencing (30-50 million reads per sample)

  • Differential expression analysis using DESeq2 or edgeR software

  • Pathway enrichment analysis using KEGG or GO databases

• Quantitative proteomics: LC-MS/MS-based proteomics quantifies protein-level changes, which may differ significantly from transcriptomic patterns:

  • Sample preparation using bacterial protein extraction buffer (B-PER) supplemented with protease inhibitors

  • Protein digestion with trypsin following reduction and alkylation

  • TMT or iTRAQ labeling for multiplexed quantitative comparison

  • Fractionation via high-pH reversed-phase chromatography

  • LC-MS/MS analysis on a high-resolution mass spectrometer

  • Protein identification and quantification using MaxQuant or Proteome Discoverer

• Phosphoproteomics: Analysis of protein phosphorylation states reveals changes in signaling networks triggered by mtgA modification:

  • Phosphopeptide enrichment using titanium dioxide (TiO2) or immobilized metal affinity chromatography (IMAC)

  • LC-MS/MS analysis with neutral loss scanning

  • Phosphosite localization and quantification

  • Kinase activity prediction using NetworKIN or similar tools

• Metabolomics: Targeted or untargeted metabolite profiling identifies changes in central carbon metabolism and cell wall precursor pools:

  • Rapid quenching of metabolism using cold methanol

  • Extraction of polar and non-polar metabolites

  • Analysis via LC-MS or GC-MS

  • Metabolite identification against standard libraries

  • Pathway mapping using MetaboAnalyst

• Integrated multi-omics analysis: Correlation of transcriptomic, proteomic, and metabolomic data sets reveals:

  • Discordant mRNA-protein relationships indicating post-transcriptional regulation

  • Regulatory networks activated in response to cell wall stress

  • Metabolic flux redistributions following mtgA modification

  • Potential compensatory mechanisms that maintain cell viability

A typical experimental design should include biological triplicates, appropriate controls (wild-type and complemented strains), and time-course sampling to capture dynamic responses to mtgA modification.

How can mtgA modification be leveraged to enhance recombinant protein production in E. coli?

The strategic modification of mtgA offers multiple approaches to enhance recombinant protein production:

• Targeted mtgA deletion: Complete knockout of mtgA using CRISPR-Cas9 or recombineering approaches can increase recombinant protein yields by up to 1.8-fold, as demonstrated with poly(lactate-co-3-hydroxybutyrate) production (increasing from 2.9 g/l to 5.1 g/l) . This approach is particularly effective for proteins that benefit from enhanced cell permeability.

• Conditional mtgA expression systems: Rather than complete deletion, placing mtgA under inducible control allows dynamic regulation of cell permeability. Using systems like arabinose-inducible (PBAD) or tetracycline-responsive promoters enables researchers to initiate protein production in cells with intact cell walls, then reduce mtgA expression to enhance secretion during production phases.

• Synergistic gene modifications: Combining mtgA deletion with other cell envelope modifications can produce additive or synergistic effects. For example, simultaneous modification of mtgA and dacA expression has been shown to significantly enhance extracellular protein production through complementary mechanisms .

• Custom promoter engineering: Modifying the promoters of both mtgA and target recombinant proteins can optimize production dynamics. The insertion of additional Shine-Dalgarno sequences between promoters and coding sequences has increased protein production by up to 2.0-fold .

• Strain-specific optimization: The effects of mtgA modification vary between E. coli strains and with different recombinant proteins. A systematic screening approach testing mtgA modifications in multiple strain backgrounds (BL21(DE3), JM109, W3110) can identify optimal host-modification combinations for specific target proteins.

The following table summarizes reported production enhancements through different mtgA modification strategies:

These approaches can be particularly valuable for difficult-to-express proteins and those requiring extracellular secretion for proper folding or function.

What potential does mtgA have as a target for developing novel antibacterial compounds?

MtgA represents a promising target for novel antibacterial development for several reasons:

Development approaches should include:

• High-throughput screening of chemical libraries against purified recombinant mtgA

• Fragment-based drug discovery targeting the mtgA active site

• Structure-based design using crystallographic data of mtgA-substrate complexes

• Whole-cell phenotypic screening for compounds that mimic mtgA deletion phenotypes

• Medicinal chemistry optimization of lead compounds for improved pharmacokinetic properties

The greatest challenge will be achieving selectivity for bacterial transglycosylases while maintaining adequate spectrum of activity across multiple bacterial species.