Recombinant Escherichia coli O157:H7 Monofunctional biosynthetic peptidoglycan transglycosylase (mtgA)

What is the primary function of monofunctional peptidoglycan transglycosylase (MtgA) in Escherichia coli?

MtgA is a specialized glycosyltransferase that catalyzes glycan chain elongation of the bacterial cell wall peptidoglycan. Unlike bifunctional penicillin-binding proteins (PBPs), MtgA exclusively performs the transglycosylase function without transpeptidase activity. The enzyme facilitates the polymerization of lipid II precursors, transferring the growing glycan chain to the 4-OH group of the incoming N-acetylglucosamine (GlcNAc) moiety. This reaction is critical for maintaining cell wall integrity and proper bacterial morphology. In in vitro studies, MtgA has demonstrated significant glycosyltransferase activity, as evidenced by a 2.4-fold increase in peptidoglycan polymerization when GFP-MtgA is overexpressed compared to control conditions .

How does MtgA localize within the bacterial cell during different growth phases?

MtgA exhibits specific subcellular localization patterns dependent on the genetic background of the host cell. When fused with green fluorescent protein (GFP), MtgA has been observed to localize at the division site of E. coli cells under specific conditions, particularly in strains deficient in PBP1b and expressing a thermosensitive PBP1a. This localization pattern suggests MtgA plays a specialized role during septum formation in the absence of functional class A PBPs. Notably, when PBP1b is reintroduced via plasmid transformation (pDML924), the midcell localization of MtgA disappears, indicating competitive recruitment between these peptidoglycan synthases at the division site .

What protein interactions does MtgA form within the divisome complex?

MtgA forms specific protein-protein interactions with multiple divisome components, demonstrating its integration into the cell division machinery. Bacterial two-hybrid system analyses have revealed that MtgA interacts with at least three critical divisome proteins:

Additionally, MtgA demonstrates self-interaction capacity, suggesting it may function as a dimer or higher-order oligomer during peptidoglycan synthesis. These interaction profiles support the hypothesis that MtgA collaborates with other divisome components to synthesize peptidoglycan at the new poles during cell division .

What are the established methods for assaying MtgA glycosyltransferase activity in vitro?

The glycosyltransferase activity of MtgA can be assessed through multiple complementary approaches:

• Radiolabeled lipid II incorporation assay: This method employs [14C]GlcNAc-labeled lipid II (9,180 dpm/nmol) as substrate in a reaction buffer containing 15% dimethyl sulfoxide, 10% octanol, 50 mM HEPES (pH 7.0), 0.5% decyl-polyethylene glycol, and 10 mM CaCl2. The polymerized products are separated and quantified by scintillation counting, with activity expressed as percentage of lipid II substrate utilized .

• Lysozyme sensitivity test: Following the polymerization reaction, treatment with lysozyme should completely digest the synthesized peptidoglycan material, confirming authentic glycosyltransferase activity rather than non-specific aggregation .

• Fluorescent substrate analogs: More advanced approaches employ fluorescently labeled lipid II analogs, which can be monitored in real-time to assess enzyme kinetics and inhibition patterns. This approach has been successfully adapted for high-throughput screening applications .

Researchers should consider that the transmembrane segment of MtgA significantly influences enzymatic activity, with full-length protein showing higher activity than truncated forms lacking this domain .

How can protein-protein interactions of MtgA be effectively studied?

Several complementary techniques can be employed to investigate MtgA's interactions with divisome components:

• Bacterial two-hybrid system: The adenylate cyclase-based bacterial two-hybrid system represents a powerful approach for detecting in vivo protein interactions. This system allows for quantitative measurement of interaction strength through β-galactosidase activity assays. When implementing this methodology, researchers should:

  • Create fusion constructs with adenylate cyclase fragments (e.g., T18 and T25)

  • Include flexible linkers (such as (G4S)3) to minimize steric hindrance

  • Use appropriate negative controls (empty vectors) and positive controls (known interacting pairs)

  • Express the constructs in a cya-deficient strain like DHM1

• Co-immunoprecipitation with tagged proteins: This approach can validate interactions in native membrane environments.

• Fluorescence microscopy co-localization: Using fluorescently tagged proteins to visualize potential interaction partners at the division site.

When interpreting interaction data, it's important to consider that overexpression of interaction partners may lead to detection of interactions that may also occur outside the septal region in experimental conditions .

What are the considerations for generating MtgA mutants to analyze structure-function relationships?

When designing mutational studies of MtgA, researchers should consider several key factors:

How does MtgA contribute to peptidoglycan synthesis during different phases of cell division?

MtgA appears to have a specialized role in peptidoglycan synthesis during the cell division cycle, particularly during septum formation. Research suggests that septal peptidoglycan synthesis occurs in two distinct phases:

• Early phase (Z-ring assembly dependent): This initial phase requires penicillin-insensitive peptidoglycan synthesis before constriction begins. Since MtgA is naturally insensitive to penicillin (as it lacks transpeptidase activity), it may be responsible for this early synthetic activity in concert with PBP1c. This hypothesis is supported by biochemical evidence, though single and double mutants lack obvious phenotypes .

• Late phase (mature divisome dependent): Once the divisome is fully assembled, MtgA colocalizes with PBP3, FtsW, and FtsN at the division site, suggesting it contributes to septal peptidoglycan synthesis during constriction. The interaction with FtsN is particularly significant, as FtsN has been shown to stimulate peptidoglycan synthesis activities of PBP1b in vitro and may coordinate the activities of multiple peptidoglycan synthases during division .

MtgA appears to act more prominently when competition with class A PBPs (particularly PBP1a and PBP1b) for the division site is reduced, suggesting a potential compensatory or specialized role when these major synthases are compromised .

What substrate analogs have been developed to study MtgA function and inhibition?

Several substrate and inhibitor analogs have been developed to study glycosyltransferases like MtgA:

These analogs have significantly advanced our understanding of glycosyltransferase mechanisms and provided templates for inhibitor design. Notably, the complete synthesis of moenomycin has been achieved, and synthetic genes have been identified and expressed in heterologous strains to produce defined fragments of the molecule .

What expression systems are optimal for producing recombinant MtgA for structural and functional studies?

Several expression systems can be considered for recombinant MtgA production, each with specific advantages:

• E. coli-based expression:

  • BL21(DE3) derivatives with T7 promoter systems offer high-yield expression

  • C41/C43 strains are optimized for membrane protein expression

  • Fusion tags (His6, MBP, GST) facilitate purification while potentially enhancing solubility

  • Inducible promoters allow tight regulation of potentially toxic membrane protein expression

• Cell-free expression systems:

  • Bypass toxicity issues associated with membrane protein overexpression

  • Allow direct incorporation of modified amino acids or unnatural lipids

  • Provide rapid protein production for screening studies

• Considerations for transmembrane domain:

  • Full-length MtgA with its transmembrane segment shows higher enzymatic activity than truncated forms

  • Expression as a GFP fusion protein has successfully yielded active enzyme, as demonstrated by a 2.4-fold increase in peptidoglycan polymerization compared to control conditions

For structural studies, researchers should consider detergent selection carefully, as inappropriate detergents may destabilize the protein or interfere with activity assays.

How can inhibitor screening assays be developed for MtgA-targeted antimicrobial discovery?

Development of robust screening assays for MtgA inhibitors requires careful consideration of several factors:

• High-throughput primary screens:

  • Fluorescently labeled moenomycin derivatives have been successfully used to develop displacement-based high-throughput screening assays

  • These assays can identify compounds that compete with moenomycin for binding to glycosyltransferases

• Secondary functional assays:

  • Radiolabeled or fluorescently labeled lipid II incorporation assays to directly measure inhibition of glycosyltransferase activity

  • Lysozyme sensitivity tests to confirm authentic peptidoglycan synthesis inhibition

• Tertiary cellular assays:

  • Bacterial growth inhibition in wild-type versus MtgA-overexpressing strains

  • Morphological analysis to detect cell division defects characteristic of peptidoglycan synthesis inhibition

  • Synergy testing with β-lactams or other cell wall-targeting antibiotics

• Structure-activity relationship development:

  • The transmembrane domain influences substrate and moenomycin binding

  • Full-length MtgA shows higher affinity for moenomycin than truncated versions lacking the transmembrane segment

High-quality recombinant enzyme preparations and appropriate positive controls (such as moenomycin) are essential for assay development and validation.

How might study of MtgA contribute to understanding antimicrobial resistance mechanisms?

Investigation of MtgA offers several promising avenues for addressing antimicrobial resistance challenges:

• Novel target identification: As a monofunctional glycosyltransferase, MtgA represents a distinct target from bifunctional PBPs that are inhibited by β-lactams. This distinction offers potential for developing antibiotics that could remain effective against β-lactam-resistant pathogens.

• Compensatory mechanisms: Understanding how MtgA compensates for deficiencies in PBP1a and PBP1b may provide insights into bacterial adaptability to cell wall-targeting antibiotics. The observation that MtgA localizes to the division site when PBP1b is absent and PBP1a is thermosensitive suggests potential redundancy mechanisms that could contribute to resilience against peptidoglycan synthesis inhibitors .

• Synergistic drug combinations: The interaction network of MtgA with divisome components suggests potential for developing combination therapies targeting multiple peptidoglycan synthesis enzymes simultaneously, potentially overcoming resistance mechanisms.

• Structure-based drug design: Detailed structural understanding of MtgA's catalytic mechanism and substrate binding could facilitate rational design of inhibitors that are less susceptible to existing resistance mechanisms.

Research into these areas could potentially yield new strategies for combating antimicrobial resistance in pathogenic E. coli O157:H7 and related organisms.

What is the significance of MtgA self-interaction and how might it affect enzyme function?

The observation that MtgA can interact with itself, as demonstrated through bacterial two-hybrid analysis, has several potential implications for enzyme function:

• Quaternary structure: Self-interaction suggests MtgA may function as a dimer or higher-order oligomer. This quaternary structure could influence:

  • Catalytic efficiency through cooperativity

  • Processivity during glycan chain elongation

  • Stability of the enzyme complex at the division site

• Spatial organization: Self-interaction might facilitate the formation of organized MtgA clusters at the division site, potentially creating specialized domains for coordinated peptidoglycan synthesis during septum formation.

• Regulatory implications: Oligomerization state could serve as a regulatory mechanism, responding to environmental conditions or cell cycle cues to modulate activity.

• Therapeutic targeting: The self-interaction interface represents a potential target for inhibitor development, distinct from the catalytic site. Disrupting oligomerization could offer an alternative approach to inhibiting MtgA function.

Further structural and functional studies are needed to characterize the precise nature of MtgA self-interaction and its impact on enzyme dynamics and activity in the context of the bacterial divisome .