Recombinant Pseudomonas putida Monofunctional biosynthetic peptidoglycan transglycosylase (mtgA)

What is monofunctional biosynthetic peptidoglycan transglycosylase (mtgA) and how does it differ from class A PBPs?

MtgA catalyzes glycan chain elongation of the bacterial cell wall by polymerizing peptidoglycan precursors. Unlike bifunctional class A Penicillin-Binding Proteins (PBPs) that possess both transglycosylase (TG) and transpeptidase (TP) activities, mtgA exclusively performs the transglycosylase function . This enzyme belongs to the GT51 family of glycosyltransferases and shares structural similarities with the lysozyme fold .

The monofunctionality of mtgA allows for specialized coordination with other peptidoglycan synthesis enzymes, potentially providing greater flexibility in cell wall assembly. This contrasts with class A PBPs, which coordinate both TG and TP activities within a single protein through distinct domains.

What is the genomic context of mtgA in Pseudomonas putida KT2440?

In P. putida KT2440, the mtgA gene is located adjacent to the rpoH gene (encoding heat shock sigma factor σ32), separated by a 198 bp intergenic region. Detailed analysis reveals this intergenic region contains:

• A 34 bp inverted repeat sequence with rho-independent terminator activity

• Three copies of a well-conserved 35 bp sequence (at positions 13, 57, and 102 bp from the terminator)

• These 35 bp sequences are organized as one direct unit followed by two copies in the opposite orientation

• Each unit contains a partially palindromic sequence with an internal 6 bp inverted repeat

This genomic organization suggests potential regulatory relationships between rpoH and mtgA expression, possibly linking cell wall synthesis to heat shock responses.

How does mtgA contribute to bacterial cell division?

MtgA plays a significant role in bacterial cell division through:

• Localization at the division site in cells deficient in PBP1b and expressing thermosensitive PBP1a

• Direct interaction with key divisome components including:

  • PBP3 (FtsI), an essential transpeptidase for septum formation

  • FtsW, a proposed lipid II flippase

  • FtsN, a late cell division protein

• Potential compensation for the absence of class A PBPs during septum formation

Experiments measuring protein-protein interactions showed that T18-(G4S)3-MtgA and T25-(G4S)3-MtgA interactions produced positive responses, indicating that MtgA can interact with itself in vivo, suggesting possible homodimerization during peptidoglycan synthesis .

What are the most effective expression systems for producing recombinant mtgA in P. putida?

Multiple expression systems have been successfully employed for recombinant mtgA production in P. putida:

For constitutive expression, the TREX-based transposon system has proven particularly effective, as demonstrated in the production of prodigiosin in P. putida, where the transposon allowed for stable, T7 RNA polymerase-independent expression .

How can researchers verify the enzymatic activity of recombinant mtgA?

The enzymatic activity of recombinant mtgA can be verified through multiple complementary approaches:

• Radiolabeled precursor incorporation assay:

  • Monitor incorporation of 14C-N-acetylglucosamine into trichloroacetic acid (TCA)-precipitable material

  • Typical reaction conditions include membrane fractions (e.g., from A. viridans), UDP-N-acetylglucosamine, UDP-N-acetylmuramylpentapeptide, MgCl2, and purified mtgA

  • Confirm specificity through:

    • Inhibition by moenomycin A (a specific transglycosylase inhibitor)

    • Sensitivity of the reaction product to lysozyme treatment

• Fusion protein activity assessment:

  • For GFP-MtgA fusion proteins, measure peptidoglycan polymerization using lipid II as substrate

  • A 2.4-fold increase in polymerization rate (compared to control) indicates functional activity

  • Complete digestion of polymerized material by lysozyme confirms product identity

• In vivo complementation:

  • Express recombinant mtgA in strains with compromised class A PBPs

  • Assess restoration of normal growth and morphology

  • Localization studies using fluorescent protein fusions

What genetic engineering approaches enable efficient modification of mtgA in P. putida?

Several genetic engineering strategies have been developed for efficient modification of mtgA in P. putida:

• I-SceI-mediated recombination with CRISPR-Cas9 counterselection:

  • Enables precise genome manipulation including insertions, deletions, or genetic part exchanges

  • Employs double-stranded DNA cuts by I-SceI endonuclease followed by CRISPR-Cas9 counterselection

  • This optimized workflow has been demonstrated for deleting selected genes and integrating fluorescent reporter genes in P. putida KT2440

• Expanded CRISPRi toolbox:

  • Single-plasmid CRISPR-interference system for tunable control of gene expression

  • Enables tightly controlled gene repression of chromosomally-expressed genes

  • Allows simultaneous suppression of multiple genes

  • Can target essential genes, resulting in altered growth rates or morphological changes

• Base-editing systems:

  • Unconstrained CRISPR-editing via cytidine and adenine base-substitution

  • Uses PAM-relaxed nCas9 variant making the majority of the bacterial genome targetable

  • Self-curing vectors enable efficient plasmid removal after editing

How do researchers investigate mtgA's contribution to P. putida's cell wall properties?

Investigating mtgA's contribution to P. putida's cell wall properties requires multifaceted approaches:

• Comparative muropeptide analysis:

  • Isolate peptidoglycan from wild-type and mtgA-modified strains

  • Digest with muramidase to release muropeptides

  • Analyze by HPLC and mass spectrometry to determine:

    • Glycan chain length distribution

    • Cross-linking degree

    • Modifications in peptide stems

  • Quantify differences in muropeptide composition and abundance

• Microscopic analysis of cell morphology:

  • Phase contrast and electron microscopy to assess gross morphological changes

  • Fluorescent D-amino acid labeling to visualize sites of active peptidoglycan synthesis

  • Time-lapse microscopy to track cell division and elongation dynamics

  • Atomic force microscopy to measure cell wall mechanical properties

• Stress response and tolerance testing:

  • Challenge with osmotic stress (both hypotonic and hypertonic conditions)

  • Examine survival under cell wall-targeting antibiotics

  • Assess tolerance to mechanical stress

  • Measure response to detergents and membrane-perturbing agents

• Interaction studies with other cell wall synthesis proteins:

  • Bacterial two-hybrid assays to map protein interaction networks

  • Co-immunoprecipitation followed by mass spectrometry to identify binding partners

  • FRET or BiFC to visualize interactions in living cells

What mechanisms explain the effects of mtgA expression on P. putida morphology and stress responses?

Recombinant mtgA expression can significantly impact P. putida cell morphology and stress responses through several mechanisms:

• Altered peptidoglycan synthesis dynamics:

  • Imbalance between transglycosylation and transpeptidation rates

  • Changes in glycan chain length distribution

  • Modified cross-linking patterns

  • Altered localization of peptidoglycan synthesis machinery

• Integration with stress response pathways:

  • Connection to RpoH (σ32) regulation due to genomic proximity

  • Activation of envelope stress response systems

  • Alterations in cell division protein localization and function

  • Redistribution of peptidoglycan synthesis resources

• Effects on membrane vesicle formation:

  • P. putida produces outer membrane vesicles in response to stress

  • These vesicles increase cell surface hydrophobicity and enhance biofilm formation

  • mtgA-mediated changes in cell wall properties may influence vesicle formation

  • This potentially provides extracellular storage for hydrophobic compounds

• Impact on efflux systems:

  • P. putida's exceptional tolerance to various compounds relies on efficient efflux systems

  • Changes in cell wall structure can affect the anchoring and function of these systems

  • mtgA modifications may alter the expression or activity of efflux pumps

How can researchers differentiate the specific functions of mtgA from those of class A PBPs in P. putida?

Differentiating the specific functions of mtgA from class A PBPs requires sophisticated experimental designs:

• Genetic depletion strategies:

  • Create conditional mutants of mtgA and individual class A PBPs

  • Employ CRISPRi for tunable repression of target genes

  • Analyze phenotypic consequences of single and combined depletions

  • Use transcriptomic analysis to identify compensatory mechanisms

• Domain swap experiments:

  • Generate chimeric proteins by swapping the transglycosylase domain of class A PBPs with mtgA

  • Express these chimeras in appropriate deletion backgrounds

  • Assess functional complementation and localization patterns

  • Identify domain-specific contributions to cellular phenotypes

• Selective inhibition:

  • Utilize specific inhibitors of transglycosylase (moenomycin) versus transpeptidase (β-lactams) activities

  • Apply at sub-lethal concentrations to wild-type and mutant strains

  • Monitor compensatory changes in expression and localization

  • Quantify differential sensitivity patterns

• High-resolution localization studies:

  • Employ super-resolution microscopy with fluorescent protein fusions

  • Track dynamic localization during cell cycle progression

  • Compare co-localization patterns with other divisome components

  • Analyze differential recruitment to septation versus elongation complexes

How can researchers overcome common challenges in expressing functional recombinant mtgA?

Researchers face several challenges when expressing functional recombinant mtgA, which can be addressed through specific strategies:

The case of prodigiosin production in P. putida demonstrates that chromosomal integration via transposition can significantly improve expression compared to plasmid-based systems .

What analytical approaches help resolve data inconsistencies in mtgA research?

When faced with inconsistent or contradictory data in mtgA research, these analytical approaches can help resolve discrepancies:

• Complementary activity assays:

  • Compare results from multiple independent assay methods

  • For example, combining radiolabeled substrate incorporation with detection of released pyrophosphate

  • Validate with both in vitro biochemical assays and in vivo phenotypic analyses

  • Apply controls including known transglycosylase inhibitors (moenomycin)

• Strain background considerations:

  • Thoroughly document genetic backgrounds of all strains

  • Note that the presence of additional mutations can significantly impact results:

    • The ponA(ts) ponB strain EJ801 also carries dacA and dacB mutations affecting peptidoglycan metabolism

    • These were not present in BW25113-derived strains, explaining differences in mtgA localization

• Protein verification:

  • Confirm proper expression using multiple detection methods:

    • Western blot analysis with specific antibodies

    • Mass spectrometry for protein identification

    • Activity assays to confirm functionality

  • Verify correct localization using fractionation and microscopy

• Control for experimental variables:

  • Standardize growth conditions, particularly temperature and media composition

  • Control induction timing and expression levels

  • Document buffer compositions, especially regarding divalent cations and pH

How should researchers interpret conflicting data between natively expressed and recombinant mtgA?

When interpreting conflicting data between native and recombinant mtgA, researchers should consider:

• Expression level disparities:

  • Recombinant systems often produce higher protein levels than native expression

  • Quantify expression levels using quantitative Western blot or mass spectrometry

  • Consider titrating expression to physiological levels using tunable promoters

• Post-translational modifications:

  • Native mtgA may undergo modifications absent in recombinant systems

  • Analyze both forms by mass spectrometry to identify differences

  • Consider host-specific factors that might affect protein processing

• Protein-protein interaction differences:

  • Native mtgA functions within established protein complexes

  • Recombinant expression may disrupt normal stoichiometry with partner proteins

  • Verify interactions using co-immunoprecipitation or bacterial two-hybrid assays

• Localization variations:

  • MtgA localization depends on cell cycle stage and growth conditions

  • Verify localization patterns using fluorescent protein fusions

  • Compare with established cell division markers (FtsZ)

  • Note that overexpression can lead to artificial localization patterns

• Genetic background effects:

  • The functional impact of mtgA can be masked by compensatory mechanisms

  • Consider the presence of other mutations affecting cell wall metabolism (as in the ponA(ts) ponB strain EJ801 which also carries dacA and dacB mutations)

How can mtgA engineering be used to enhance P. putida as a production host?

Engineering mtgA can significantly enhance P. putida's capabilities as a production host through several strategies:

• Cell morphology modification:

  • Engineering cell morphology by modulating mtgA and other cell wall synthesis genes

  • Increase cell size to improve accumulation of intracellular products

  • Research has shown that engineering P. putida cell morphology can significantly increase polyhydroxyalkanoate (PHA) accumulation

• Stress tolerance improvement:

  • Modify peptidoglycan structure through mtgA engineering to enhance:

    • Resistance to osmotic stress during high-density fermentation

    • Tolerance to toxic compounds including products and feedstocks

    • P. putida already exhibits tolerance to high concentrations of compounds like rhamnolipids (up to 90 g/l)

• Metabolic flux optimization:

  • Use CRISPRi to dynamically control mtgA expression

  • Coordinate with central metabolism to optimize precursor availability

  • Model-guided dynamic CRISPRi control has been shown to boost acetyl-CoA-dependent bioproduction in rewired P. putida strains

• Mini-cell production platforms:

  • Engineer mtgA to facilitate production of chromosome-free, catalytically active mini-cells

  • These mini-cells can maintain production capabilities with stable yields

  • Has been demonstrated for 2-pentanone production in P. putida

What methodologies enable the utilization of mtgA for antimicrobial research?

MtgA can be leveraged for antimicrobial research through several methodologies:

• Transglycosylase inhibitor screening platforms:

  • Develop high-throughput assays based on recombinant mtgA activity

  • Screen for inhibitors targeting the transglycosylase activity

  • Use in vitro biochemical assays with purified components

  • Validate hits through whole-cell activity testing

• Peptidoglycan biosynthesis pathway elucidation:

  • Employ recombinant mtgA in reconstituted systems

  • Study interactions with other cell wall synthesis proteins

  • Use analytical methods including:

    • Radioactive labeling

    • Gel electrophoresis

    • Mass spectrometry

    • Fluorescence labeling

    • Fluorescence anisotropy

    • Surface plasmon resonance

• Heterologous production of cell wall-active compounds:

  • Utilize P. putida as a host for biosynthetic gene clusters encoding antimicrobials

  • Engineer mtgA to enhance resistance to these compounds during production

  • P. putida has already been successfully used for heterologous production of:

    • Myxothiazol A (an inhibitor of the respiratory chain with antifungal and insecticidal activities)

    • Prodigiosin (possessing antimicrobial, anticancer, and immunosuppressive properties)

• Structure-based drug design:

  • Generate structural models of mtgA for in silico screening

  • Design peptidomimetics targeting the transglycosylase active site

  • Develop dual-action inhibitors targeting both transglycosylase and transpeptidase activities

What unresolved questions remain regarding mtgA function in P. putida?

Despite significant advances, several fundamental questions about mtgA function in P. putida remain unanswered:

• Regulatory networks:

  • How is mtgA expression regulated in response to different environmental conditions?

  • What is the significance of the genomic proximity to rpoH (heat shock sigma factor)?

  • How do the REP (repetitive extragenic palindromic) sequences in the mtgA-rpoH intergenic region influence regulation?

• Functional redundancy:

  • What is the degree of functional overlap between mtgA and the transglycosylase domains of class A PBPs?

  • Under what conditions does mtgA become essential for P. putida survival?

  • How do compensatory mechanisms operate when mtgA function is compromised?

• Divisome integration:

  • What is the precise timing of mtgA recruitment to the divisome?

  • Which protein-protein interactions are critical for proper localization and function?

  • How is mtgA activity coordinated with that of other peptidoglycan synthesis enzymes?

• Contribution to environmental adaptation:

  • How does mtgA contribute to P. putida's exceptional tolerance to various stressors?

  • What role does it play in biofilm formation and colonization of plant roots?

  • How does mtgA function change when P. putida transitions between different lifestyles?

What emerging technologies will advance mtgA research in P. putida?

Emerging technologies poised to transform mtgA research in P. putida include:

• Cryo-electron tomography:

  • Visualize native cell wall architecture at molecular resolution

  • Track changes in peptidoglycan organization during cell division

  • Map the spatial arrangement of peptidoglycan synthesis machinery in situ

• Advanced genome editing approaches:

  • Base editing systems for precise nucleotide substitutions

  • Prime editing for targeted insertions and deletions

  • PAM-relaxed Cas variants allowing targeting of previously inaccessible genomic regions

• Single-cell technologies:

  • Single-cell transcriptomics to capture cell-to-cell variability in mtgA expression

  • Microfluidic approaches to track individual cell responses to perturbations

  • High-throughput phenotypic screening of mutant libraries

• Synthetic cell wall engineering:

  • Designer peptidoglycan with non-canonical building blocks

  • Engineered peptidoglycan with novel cross-linking chemistries

  • Orthogonal cell wall synthesis pathways

• Computational approaches:

  • Molecular dynamics simulations of transglycosylase activity

  • Systems biology models integrating cell wall synthesis with central metabolism

  • Machine learning for predicting phenotypic outcomes of mtgA modifications

How might mtgA research contribute to broader understanding of bacterial cell biology?

Research on mtgA in P. putida has implications that extend beyond this specific system to broader bacterial cell biology:

• Evolution of cell wall synthesis pathways:

  • Comparative genomics of monofunctional transglycosylases across bacterial species

  • Understanding the evolutionary pressures driving the maintenance of both bifunctional PBPs and monofunctional enzymes

  • Elucidating how different bacteria balance transglycosylase and transpeptidase activities

• Principles of bacterial morphogenesis:

  • Insights into how peptidoglycan synthesis is spatially and temporally coordinated

  • Understanding the molecular mechanisms controlling bacterial cell shape

  • Clarifying how the cell cycle is integrated with cell wall synthesis

• Stress response integration:

  • Revealing connections between cell envelope integrity and other stress response pathways

  • Understanding how bacteria maintain cell wall homeostasis under changing conditions

  • Exploring the coordination between peptidoglycan synthesis and outer membrane biogenesis in Gram-negative bacteria

• Synthetic biology design principles:

  • Establishing rules for engineering bacterial morphology

  • Developing strategies for improving robustness of engineered bacteria

  • Creating modular, orthogonal systems for controlled cell growth and division