Recombinant Pseudomonas entomophila Monofunctional biosynthetic peptidoglycan transglycosylase (mtgA)

What is Monofunctional Biosynthetic Peptidoglycan Transglycosylase (mtgA)?

Monofunctional biosynthetic peptidoglycan transglycosylase (mtgA) is an enzyme that catalyzes the polymerization of lipid II molecules into glycan strands of peptidoglycans in bacterial cell walls . Unlike bifunctional penicillin-binding proteins (PBPs), mtgA exclusively performs the glycosyltransferase function without transpeptidase activity. In Pseudomonas entomophila, mtgA (UniProt accession: Q1IGD6) is a 236-amino acid protein that plays a critical role in cell wall biosynthesis .

The enzyme's primary function is to catalyze glycan chain elongation, which is essential for maintaining cell wall integrity and facilitating proper cell division. Research has demonstrated that mtgA interacts with other cell division proteins, suggesting its collaborative role in peptidoglycan assembly during the bacterial cell cycle .

How does mtgA contribute to bacterial cell wall synthesis?

The bacterial cell wall synthesis process involves multiple enzymes working in concert to build and maintain peptidoglycan architecture. mtgA specifically contributes to this process by:

• Catalyzing the polymerization of GlcNAc-labeled lipid II into peptidoglycan strands

• Working in coordination with penicillin-binding proteins (PBPs)

• Interacting with divisome proteins during cell division

• Contributing to peptidoglycan assembly at cell division sites

Experimental evidence shows that mtgA can increase peptidoglycan polymerization by up to 2.4-fold when overexpressed, demonstrating its significant role in cell wall synthesis . The enzyme's activity can be verified through in vitro assays where the polymerized material is completely digested by lysozyme, confirming the glycosidic linkages formed by mtgA .

What is the structure and localization pattern of mtgA in bacterial cells?

The mtgA protein contains a transmembrane segment that anchors it to the bacterial membrane, with the catalytic domain positioned to interact with cell wall precursors. In Escherichia coli, mtgA has been shown to localize specifically at the division site in cells deficient in PBP1b and producing thermosensitive PBP1a .

The localization pattern of mtgA is particularly interesting as it suggests a specialized role during cell division. Fluorescence microscopy studies with GFP-tagged mtgA have demonstrated that:

• mtgA concentrates at the division septum during cell division

• This localization is dependent on the divisome assembly

• mtgA interacts with at least three divisome components: PBP3, FtsW, and FtsN

These interaction patterns suggest that mtgA collaborates with PBP3 to synthesize at least part of the peptidoglycan at the new poles during cell division .

How do protein-protein interactions influence mtgA function in the divisome?

The functional capacity of mtgA is significantly enhanced through its interactions with other divisome proteins. Bacterial two-hybrid analyses have demonstrated that mtgA interacts specifically with PBP3, FtsW, and FtsN in vivo, with interaction strengths varying between these proteins .

The β-galactosidase activity measurements from these interaction studies reveal:

These interactions suggest that mtgA functions within a protein complex during cell division. Notably, the transmembrane segment of PBP3 is required for interaction with mtgA, as demonstrated by the lack of interaction with Lpp-PBP3 fusion proteins lacking this domain . The strong self-interaction of mtgA (37-fold increase in activity) suggests potential dimerization or oligomerization that may be functionally significant for its glycosyltransferase activity .

What are the experimental approaches for measuring mtgA enzymatic activity?

Researchers studying mtgA activity employ several methodological approaches:

• In vitro glycosyltransferase assays: These assays measure the polymerization of radiolabeled lipid II substrates into peptidoglycan products. A typical reaction mixture contains:

  • GlcNAc-labeled lipid II (9,180 dpm/nmol)

  • 15% dimethyl sulfoxide

  • 10% octanol

  • 50 mM HEPES (pH 7.0)

  • 0.5% decyl-polyethylene glycol

  • 10 mM CaCl₂

• Polymerization product analysis: The peptidoglycan products are separated and quantified, with activity indicated by the percentage of lipid II incorporated into polymer. GFP-MtgA overexpression has been shown to increase peptidoglycan polymerization 2.4-fold compared to controls (26% versus 11% of lipid II used) .

• Lysozyme sensitivity confirmation: Addition of lysozyme to reaction products results in complete digestion of the polymerized material, confirming that the products contain the β-1,4-glycosidic linkages characteristic of peptidoglycan .

• Protein-protein interaction assays: Bacterial two-hybrid systems can be used to measure interactions between mtgA and other cell division proteins. The complementation of adenylate cyclase fragments leads to cAMP production and activation of reporter genes such as β-galactosidase .

How does deletion of mtgA affect bacterial cell morphology and physiology?

The deletion of mtgA has been shown to have significant effects on bacterial cell morphology, particularly under specific conditions. While mtgA deletion alone does not alter cell morphology under standard growth conditions, it can cause dramatic changes when combined with specific stressors or genetic backgrounds .

Research on E. coli has revealed that:

These morphological changes suggest that mtgA plays a role in maintaining proper cell shape during stress conditions or when the cell is engaged in enhanced biosynthetic activities. The mechanism may involve compensatory changes in other cell wall synthesizing enzymes or altered peptidoglycan architecture.

What genetic modification strategies can enhance recombinant mtgA expression?

Optimizing recombinant mtgA expression requires sophisticated genetic engineering approaches. Based on related research with the XylS/Pm expression system, several strategies can be applied:

• Promoter engineering: The Pm promoter and its regulatory elements can be modified through random mutagenesis to enhance expression levels. Libraries of promoter variants can be generated using:

  • Doped oligonucleotides

  • Error-prone PCR

  • DNA shuffling

  • Combinations of these approaches

• 5'-UTR optimization: The 5'-untranslated mRNA region (5'-UTR) derived from the Pm transcript can significantly impact translation efficiency and can be modified to enhance expression .

• Selection strategies: Modified expression elements can be cloned into plasmid vectors harboring the bla gene (encoding β-lactamase) under control of Pm. Mutants with enhanced expression can then be selected by plating on medium with increasing ampicillin concentrations, as ampicillin tolerance correlates with bla expression levels .

• Fusion partners: Various types of 5'-terminal fusion partners that stimulate expression of recombinant genes can be employed to enhance mtgA expression and solubility .

The combination of these approaches can lead to substantial improvements in expression levels, potentially increasing yields of functional recombinant mtgA protein for research applications.

What are the optimal conditions for expressing and purifying recombinant P. entomophila mtgA?

Based on the available commercial preparations of recombinant P. entomophila mtgA and research on similar proteins, the following conditions are recommended for expression and purification:

• Expression system: E. coli expression systems are commonly used, with BL21(DE3) or similar strains being preferred for membrane-associated proteins like mtgA.

• Construct design: The full-length sequence (amino acids 1-236) of the mtgA gene (PSEEN0304) from Pseudomonas entomophila (strain L48) should be optimized for the expression host .

• Purification approach: A two-step purification is typically effective:

  • Initial capture using affinity chromatography (His-tag or fusion partner tag)

  • Secondary purification by size exclusion or ion-exchange chromatography

• Buffer composition: Optimal storage conditions include:

  • Tris-based buffer

  • 50% glycerol for stability

  • Storage at -20°C for short-term and -80°C for long-term storage

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

• Handling precautions: Repeated freezing and thawing should be avoided to maintain enzyme activity .

The amino acid sequence of P. entomophila mtgA (UniProt: Q1IGD6) is:
mLSSLLRRLSRALLWFAAGSIAVVLVLRWVPPPGTALMVERKVESWFNGEPIDLQRDWTP
WEDISDELKVAVIAGEDQKFASHWGFDIPAIQAALAYNERGGKVRGASTLTQQVAKNMFL
WSGRSWLRKGLEAWFTALIELFWSKERILEVYLNSAEWGKGVFGAQAAARYHFGVDASRL
SRQQAAQLAAVLPSPIKWSASRPSAYVASRAGWIRRQMSQLGGPSYLMQLDASRKL

How can researchers effectively design mtgA mutation studies to investigate structure-function relationships?

Designing effective mutation studies for mtgA requires a systematic approach to target specific domains and functional residues:

• Sequence conservation analysis: Compare mtgA sequences across bacterial species to identify highly conserved residues likely to be functionally important. Focus particularly on residues in the active site or protein-protein interaction interfaces.

• Domain-specific mutations: Based on the functional domains of mtgA:

  • Transmembrane domain: Mutations here could affect membrane anchoring and localization

  • Catalytic domain: Mutations in conserved glycosyltransferase motifs would affect enzymatic activity

  • Interaction domains: Mutations in regions that interact with PBP3, FtsW, or FtsN would affect divisome integration

• Mutation types to consider:

  • Alanine scanning of conserved regions

  • Conservative vs. non-conservative substitutions

  • Domain deletions or truncations

  • Site-directed mutagenesis of specific residues

• Functional assays: After generating mtgA mutants, evaluate:

  • Glycosyltransferase activity using in vitro lipid II polymerization assays

  • Protein-protein interactions using bacterial two-hybrid systems

  • Localization patterns using fluorescence microscopy with GFP-tagged mutants

  • Complementation of mtgA deletion phenotypes

• Controls: Include wild-type mtgA and established inactive mutants (if known) as positive and negative controls in all assays.

What are the recommended approaches for studying mtgA interactions with divisome proteins?

Studying mtgA interactions with divisome proteins requires multiple complementary techniques:

• Bacterial two-hybrid (BTH) analysis: This method has proven effective for studying mtgA interactions in vivo. The approach involves:

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

  • Co-expressing these fusion proteins in an adenylate cyclase-deficient strain (e.g., DHM1)

  • Measuring complementation through β-galactosidase activity

  • Using appropriate controls (T18-T25, T18-T25-X, T25-T18-X)

• Co-immunoprecipitation (Co-IP): This technique can validate interactions identified in BTH:

  • Express epitope-tagged mtgA and potential interacting partners

  • Lyse cells under conditions that preserve protein-protein interactions

  • Immunoprecipitate using antibodies against one protein

  • Detect co-precipitated proteins by Western blotting

• Fluorescence microscopy co-localization: This approach examines whether proteins co-localize in vivo:

  • Create fluorescent protein fusions (e.g., GFP-mtgA and RFP-PBP3)

  • Observe localization patterns in living cells

  • Quantify co-localization using image analysis software

• Biolayer interferometry or surface plasmon resonance: These techniques can measure binding kinetics between purified proteins:

  • Immobilize one purified protein on a sensor

  • Measure real-time binding of the second protein at various concentrations

  • Calculate association and dissociation rate constants

• Crosslinking mass spectrometry: This advanced approach can identify specific residues involved in protein-protein interactions:

  • Treat intact cells or purified protein complexes with crosslinking agents

  • Digest crosslinked proteins and analyze by mass spectrometry

  • Identify crosslinked peptides to map interaction interfaces

How can researchers integrate mtgA studies with broader peptidoglycan synthesis research?

Integrating mtgA studies with broader peptidoglycan synthesis research requires multidisciplinary approaches:

• Comprehensive deletion studies: Generate combination mutants lacking mtgA along with other peptidoglycan synthases (PBPs) to understand compensatory mechanisms and functional redundancy. This approach has revealed that mtgA becomes particularly important in strains deficient in PBP1b and with thermosensitive PBP1a .

• Cell wall labeling techniques: Use fluorescent D-amino acids (FDAAs) or clickable D-amino acid dipeptides to visualize patterns of peptidoglycan insertion in wild-type versus mtgA mutant strains.

• Quantitative peptidoglycan analysis: Employ muropeptide analysis by HPLC and mass spectrometry to determine if mtgA deletion or overexpression alters:

  • Glycan strand length distribution

  • Cross-linking density

  • Muropeptide composition

• Super-resolution microscopy: Apply techniques like STORM or PALM to visualize the nanoscale organization of mtgA and other peptidoglycan synthesis proteins during the cell cycle.

• Systems biology approaches: Integrate mtgA studies with:

  • Transcriptomics to identify gene expression changes in response to mtgA deletion

  • Proteomics to map the complete divisome interaction network

  • Metabolomics to track changes in cell wall precursor pools

• Computational modeling: Develop models of peptidoglycan synthesis that incorporate mtgA activity to predict the effects of mutations or inhibitors on cell wall architecture.

How might mtgA be exploited as a target for novel antimicrobial development?

As a critical enzyme in bacterial cell wall synthesis, mtgA represents a potential target for antimicrobial development:

• Target validation: The essential nature of peptidoglycan synthesis makes mtgA an attractive target, particularly in bacteria where it may compensate for the loss of other glycosyltransferases. Research shows that mtgA becomes critically important in cells deficient in other peptidoglycan synthases, suggesting that mtgA inhibitors could be particularly effective in combination with other cell wall-targeting antibiotics .

• Inhibitor design approaches:

  • Structure-based design targeting the active site of mtgA

  • Fragment-based screening to identify initial binding molecules

  • Natural product screening, as many existing cell wall antibiotics are natural products

  • Peptidomimetic approaches based on substrate analogs

• Screening methodologies:

  • In vitro enzyme assays using purified recombinant mtgA

  • Whole-cell assays to identify compounds that cause morphological changes similar to mtgA deletion

  • Synergy testing with existing β-lactam antibiotics

• Advantages over traditional targets:

  • As a monofunctional enzyme, mtgA may offer greater specificity than bifunctional PBPs

  • Targeting protein-protein interactions between mtgA and divisome components could provide a novel mechanism of action

  • The self-interaction of mtgA suggests potential for disrupting oligomerization

What are the potential biotechnological applications of recombinant mtgA beyond basic research?

Recombinant mtgA has several potential biotechnological applications:

• Cell factory engineering: The observation that mtgA deletion triggers cell enlargement in E. coli under polymer-producing conditions suggests applications in metabolic engineering. Larger cells can accommodate increased production of intracellular products . Specific applications include:

  • Enhanced biopolymer production

  • Increased recombinant protein yields

  • Improved biocatalysis capacity

• Synthetic peptidoglycan production: Recombinant mtgA could be used in vitro to synthesize defined peptidoglycan structures for:

  • Vaccine development

  • Immunological studies

  • Material science applications

• Diagnostic tool development: Given its specific interactions with divisome proteins, labeled mtgA could potentially serve as a probe for divisome assembly in bacterial cytological studies .

• Drug screening platform: Recombinant mtgA activity assays could form the basis of high-throughput screening platforms for identifying novel cell wall-targeting antimicrobials.

• Protein engineering: The glycosyltransferase activity of mtgA could potentially be engineered to accept modified substrates, creating novel glycan structures with biotechnological applications.

How does comparative genomic analysis of mtgA across bacterial species inform evolutionary understanding?

Comparative genomic analysis of mtgA provides valuable insights into bacterial evolution:

• Conservation patterns: mtgA is widely distributed across bacterial species, but with varying levels of sequence conservation. Analysis of these patterns can reveal:

  • Core functional domains essential to glycosyltransferase activity

  • Species-specific adaptations in non-catalytic regions

  • Horizontal gene transfer events

• Functional adaptation: Differences in mtgA sequences between species like P. entomophila and E. coli may reflect adaptations to different ecological niches or cell wall architectures. For instance, Antarctic Pseudomonas isolates might show adaptations related to functioning at lower temperatures .

• Co-evolution with interaction partners: Examining how mtgA co-evolves with its interaction partners (PBP3, FtsW, FtsN) across species can reveal constraints on protein-protein interaction networks and divisome assembly.

• Evolutionary significance of redundancy: The functional overlap between mtgA and bifunctional PBPs suggests evolutionary pressure to maintain peptidoglycan synthesis capability even when primary enzymes are compromised .

• Metabolic integration: Comparative genomics approaches can reveal how mtgA is integrated into different metabolic networks across species, potentially identifying novel connections between cell wall synthesis and other cellular processes .