Recombinant Cronobacter sakazakii Monofunctional biosynthetic peptidoglycan transglycosylase (mtgA)

What is the function of mtgA in Cronobacter sakazakii?

MtgA (Monofunctional biosynthetic peptidoglycan transglycosylase) in Cronobacter sakazakii is a key enzyme involved in peptidoglycan synthesis, which is essential for bacterial cell wall formation. Unlike bifunctional penicillin-binding proteins (PBPs), mtgA specifically catalyzes the polymerization of lipid II to form peptidoglycan strands by creating glycosidic bonds between N-acetylmuramic acid and N-acetylglucosamine residues. This enzyme lacks transpeptidase activity, making it a specialized component in the bacterial cell wall biosynthesis machinery.

According to genomic analyses, mtgA is classified as a biosynthetic peptidoglycan transglycosylase with a molecular weight of approximately 26.5 kDa and is typically localized to the inner membrane of bacterial cells . The enzyme plays a crucial role in maintaining cell wall integrity, which directly impacts bacterial survival, growth, and pathogenicity.

How is recombinant Cronobacter sakazakii mtgA expressed and purified?

Recombinant C. sakazakii mtgA is typically expressed using E. coli expression systems. The standard protocol involves:

• Cloning the full-length mtgA gene (1-241 amino acids) into an expression vector with an N-terminal His-tag

• Transformation into E. coli expression hosts

• Induction of protein expression under optimized conditions

• Cell lysis followed by affinity chromatography using Ni-NTA resin

• Elution and collection of purified protein

The purified protein is generally stored in a Tris/PBS-based buffer with 6% trehalose at pH 8.0 . For long-term storage, glycerol is added to a final concentration of 50% . The purified protein typically achieves greater than 90% purity as determined by SDS-PAGE analysis.

To prevent protein degradation, it's recommended to avoid repeated freeze-thaw cycles and store working aliquots at 4°C for up to one week . For reconstitution, brief centrifugation is advised prior to opening the vial, with reconstitution in deionized sterile water to a concentration of 0.1-1.0 mg/mL .

How can transglycosylase activity of recombinant Cronobacter sakazakii mtgA be measured accurately?

Multiple methodologies exist for measuring transglycosylase activity of recombinant C. sakazakii mtgA, each with distinct advantages:

When selecting an appropriate assay, researchers should consider:

• Transglycosylase activity is highly sensitive to in vitro conditions including temperature, DMSO concentration, detergents, and divalent cations

• Full-length enzymes often demonstrate higher activities than truncated forms, highlighting the importance of the transmembrane portion in substrate binding

• There exists a significant gap between observed in vitro activity and that required to support bacterial growth, suggesting that other regulatory factors may be necessary for physiological activity

For high-throughput inhibitor screening, continuous fluorometric assays or moenomycin displacement assays are most suitable due to their sensitivity and real-time monitoring capabilities.

What are the effects of mtgA deletion on bacterial cell morphology and physiology?

While specific information about mtgA deletion in C. sakazakii is limited in the research literature, studies in related bacteria provide valuable insights:

• In E. coli, mtgA deletion triggers cell enlargement, which has been exploited for increased production of biopolymers like P(LA-co-3HB)

• Complementation experiments in E. coli confirm that mtgA deletion leads to increased P(LA-co-3HB) production, with the phenotype reverting to wild-type upon reintroduction of the mtgA gene

These findings suggest that mtgA plays a significant role in determining cell size and morphology, likely through its influence on peptidoglycan synthesis and cell wall architecture. The enlargement phenotype observed in mtgA-deficient cells may result from altered cell wall integrity or changes in the coordination between cell growth and division.

From a biotechnological perspective, mtgA deletion represents a potential strategy for enhancing production of biopolymers or other industrially relevant compounds, as demonstrated by the increased P(LA-co-3HB) production in mtgA-deleted E. coli strains . This suggests that mtgA could be a target for metabolic engineering approaches aimed at improving bacterial cell factories.

How is mtgA expression regulated in response to environmental stresses?

Research indicates that mtgA expression and activity in bacteria like Cronobacter sakazakii can be modulated by various environmental stresses, reflecting its role in maintaining cell wall integrity under challenging conditions.

Cronobacter species are known for their exceptional desiccation tolerance, which enables their survival in extremely dry conditions such as powdered infant formula . This tolerance likely involves adaptations in cell wall structure and dynamics, suggesting potential regulation of peptidoglycan synthesis enzymes including mtgA.

In membrane vesicles (MVs) of Pseudomonas aeruginosa, mtgA shows significant enrichment (11.2-fold increase) compared to whole cells , suggesting specific regulation of this enzyme during MV formation. This comparative analysis is presented in the following table:

While direct evidence for specific regulatory mechanisms of mtgA in C. sakazakii is limited, research in related bacteria suggests that its expression may be influenced by:

• Nutrient availability

• Growth phase

• Cell envelope stress responses

• Exposure to antimicrobial compounds

Understanding these regulatory mechanisms could provide insights into C. sakazakii adaptability and pathogenicity, particularly in the context of food production environments and neonatal infections.

What role does mtgA play in Cronobacter sakazakii biofilm formation and environmental persistence?

While direct experimental evidence specifically linking mtgA to C. sakazakii biofilm formation is limited, several lines of evidence suggest it likely plays an important role:

• Cronobacter sakazakii is known to produce capsular or biofilm materials that protect it from extremely dry conditions, enabling its high survival in milk powder and infant formula manufacturing environments

• As a peptidoglycan synthesis enzyme, mtgA contributes to cell wall structure, which influences bacterial surface properties and cell-to-cell interactions critical for biofilm development

• Deletion of cell wall synthesis genes often affects biofilm formation capacity in various bacterial species

The potential mechanisms by which mtgA may influence biofilm formation include:

• Modulation of cell surface hydrophobicity and charge through its effects on cell wall architecture

• Influence on cell shape and size, affecting spatial arrangements within biofilms

• Potential interactions with extracellular polymeric substance production pathways

• Contribution to stress responses that trigger biofilm formation

Research methodologies to investigate these connections could include:

• Comparative analysis of biofilm formation between wild-type and mtgA mutant strains

• Transcriptomic profiling of mtgA expression during different stages of biofilm development

• Microscopic analysis of cell morphology within biofilms using fluorescent reporters

• Testing the effects of subinhibitory concentrations of transglycosylase inhibitors on biofilm dynamics

How do assay conditions affect the measurement of mtgA transglycosylase activity?

The transglycosylase activity of purified recombinant mtgA is highly sensitive to in vitro conditions, which can significantly impact measurement accuracy and reproducibility. Key considerations include:

• Temperature effects: Optimal temperature for activity must be determined empirically, as it may differ from the physiological temperature of C. sakazakii growth

• DMSO sensitivity: Organic solvents like DMSO, often used to solubilize substrates or inhibitors, can significantly affect enzyme activity

• Detergent requirements: As a membrane-associated enzyme, mtgA activity is influenced by detergent type and concentration, which affect protein folding and substrate accessibility

• Divalent cation dependence: Metal ions such as Mg²⁺ or Mn²⁺ can modulate transglycosylase activity

• Substrate modifications: While studies indicate that modifications to the lysine/DAP position of lipid II substrates do not significantly affect kinetic parameters, fluorophores on the lipid chain are less likely to interfere with enzyme-substrate recognition

• Enzyme form: Full-length enzymes including transmembrane domains often demonstrate significantly higher activity than truncated forms

• Multi-enzyme coordination: In vivo, transglycosylases function in coordination with other peptidoglycan synthesis enzymes, which may be important for achieving physiologically relevant activity levels

To obtain reliable measurements, researchers should systematically optimize these parameters and consider using multiple complementary assay methods to validate results.

How can site-directed mutagenesis be used to investigate critical residues in Cronobacter sakazakii mtgA?

Site-directed mutagenesis offers a powerful approach to identify and characterize functionally important residues in C. sakazakii mtgA. A comprehensive mutagenesis strategy would include:

• Sequence alignment analysis with homologous transglycosylases to identify conserved residues likely to be functionally important

• Tertiary structure prediction to map potential catalytic and substrate-binding sites

• Systematic mutation of:

  • Conserved acidic residues potentially involved in catalysis

  • Aromatic residues that might participate in substrate binding through stacking interactions

  • Residues in the transmembrane domain that may affect membrane anchoring and orientation

  • Interface residues potentially involved in protein-protein interactions

• Functional characterization of mutants using transglycosylase activity assays as described in Question 4

• Stability analysis to distinguish between mutations affecting catalytic activity versus protein folding

A methodical mutagenesis workflow would include:

• PCR-based site-directed mutagenesis to generate mutant constructs

• Expression and purification of mutant proteins using the same protocol as for wild-type

• Comparative enzymatic assays under standardized conditions

• Structural characterization using techniques such as circular dichroism or differential scanning fluorimetry

This approach would provide insights into the catalytic mechanism of C. sakazakii mtgA and potentially identify residues that could be targeted for inhibitor design.

What is the relationship between mtgA and antimicrobial resistance in Cronobacter sakazakii?

The relationship between mtgA and antimicrobial resistance in C. sakazakii represents an important but understudied area of research. Several potential connections can be hypothesized:

• Cell wall thickness and permeability: mtgA contributes to peptidoglycan synthesis, which influences cell wall architecture and potentially the penetration of antimicrobial agents, particularly those targeting intracellular processes

• Stress response coordination: Genome analysis of C. sakazakii SP291 has identified genes related to bacterial stress response and resistance to antimicrobial and toxic compounds , suggesting potential regulatory networks that might include cell wall synthesis enzymes

• Biofilm-associated resistance: If mtgA influences biofilm formation as discussed in Question 7, this could indirectly contribute to the increased antimicrobial tolerance typically observed in biofilm communities

• Compensatory mechanisms: Alterations in mtgA expression or activity might serve as compensatory mechanisms in response to other cell wall-targeting antimicrobials

Research approaches to investigate these connections could include:

• Comparative genomics of mtgA sequences across antimicrobial-resistant and susceptible C. sakazakii isolates

• Transcriptomic analysis of mtgA expression following exposure to various antimicrobials

• Generation of mtgA overexpression and knockout strains to assess changes in minimum inhibitory concentrations

• Testing potential synergistic effects between transglycosylase inhibitors and conventional antibiotics

How does the interaction between mtgA and other peptidoglycan synthesis enzymes coordinate cell wall assembly?

In bacterial cell wall biosynthesis, mtgA functions within a complex network of enzymes that must be spatially and temporally coordinated. Based on research in related bacteria, we can infer several coordination mechanisms:

• Protein-protein interactions: mtgA likely interacts directly with other peptidoglycan synthesis enzymes, creating functional complexes that enhance efficiency and specificity

• Spatial co-localization: Peptidoglycan synthesis machinery components, including mtgA, are likely positioned at specific subcellular locations, particularly during cell division

• Substrate channeling: Intermediates in peptidoglycan synthesis may be transferred directly between enzymes without diffusing into the bulk medium

• Shared regulatory control: Expression and activity of mtgA and other cell wall synthesis enzymes may be co-regulated in response to growth conditions and stresses

The table below, adapted from research on Pseudomonas aeruginosa, illustrates various peptidoglycan synthesis enzymes that potentially interact with mtgA:

Understanding these interactions is critical for developing a complete model of bacterial cell wall assembly and identifying potential targets for antimicrobial intervention.

Can recombinant Cronobacter sakazakii mtgA be used as a potential diagnostic marker?

The potential use of recombinant C. sakazakii mtgA as a diagnostic marker presents both opportunities and challenges:

Potential advantages:

• The mtgA protein is specifically expressed in Cronobacter species

• Recombinant protein can be produced with high purity (>90%) for assay development

• The full-length sequence is well-characterized, enabling design of specific detection reagents

• Antibody-based detection methods (e.g., ELISA) have already been developed for this protein

Challenges:

• Cronobacter detection in infant formula typically requires enrichment steps due to low bacterial numbers and uneven distribution

• As an inner membrane protein, mtgA may be less accessible in intact bacterial cells compared to surface proteins

• Cross-reactivity with mtgA from related Enterobacteriaceae must be assessed

• Sensitivity requirements for clinical and food safety applications are stringent

Diagnostic applications could include:

• Species-specific identification in clinical isolates

• Detection in food production environments to identify contamination risks

• Monitoring in powdered infant formula manufacturing

• Differentiation from other Enterobacteriaceae in mixed samples

Current detection methods for Cronobacter include pre-enrichment followed by selective enrichment, chromogenic media, immunomagnetic separation, and molecular methods . An mtgA-based assay would need to demonstrate advantages over these established techniques in terms of specificity, sensitivity, speed, or cost to gain adoption in diagnostic settings.

How does mtgA contribute to the pathogenesis of Cronobacter sakazakii infections?

Cronobacter sakazakii is an opportunistic pathogen associated with life-threatening infections including meningitis, necrotizing enterocolitis, and sepsis in neonates . The role of mtgA in C. sakazakii pathogenesis, while not directly addressed in the search results, can be inferred from its function in peptidoglycan synthesis and bacterial cell wall integrity:

• Survival during infection: The peptidoglycan layer provides structural integrity that helps bacteria withstand host defense mechanisms, including osmotic stress and antimicrobial peptides

• Immune recognition and evasion: Peptidoglycan fragments are recognized by host pattern recognition receptors (PRRs) such as NOD1 and NOD2, triggering inflammatory responses; modifications in peptidoglycan structure influenced by mtgA activity might affect this recognition

• Growth and division during infection: As a key enzyme in peptidoglycan synthesis, mtgA enables bacterial replication within host environments

• Adaptation to host environments: C. sakazakii must adapt to various host niches during infection; cell wall remodeling through the action of enzymes like mtgA may facilitate this adaptation

• Persistence in hostile environments: The genome of C. sakazakii contains genes related to stress response and antimicrobial resistance , which may act in concert with cell wall synthesis machinery to enable survival

Research approaches to investigate mtgA's role in pathogenesis could include:

• Virulence studies comparing wild-type and mtgA-deficient strains in appropriate infection models

• Analysis of peptidoglycan composition during different stages of infection

• Evaluation of immune responses to wild-type versus mtgA-mutant strains

• Testing the effect of transglycosylase inhibitors on C. sakazakii virulence

How does mtgA from Cronobacter sakazakii compare to homologous enzymes in other bacterial species?

Comparative analysis of mtgA across different bacterial species reveals both conservation of core functions and species-specific adaptations:

• Sequence conservation: The core catalytic domain of transglycosylases is generally conserved across Gram-negative bacteria, reflecting the essential nature of peptidoglycan synthesis

• Size comparison: C. sakazakii mtgA is 241 amino acids long , comparable to the 26.5 kDa mtgA described in Pseudomonas aeruginosa

• Localization: Like its homologs in other bacteria, C. sakazakii mtgA is localized to the inner membrane (IM)

• Functional conservation: The fundamental transglycosylase function of mtgA - polymerizing lipid II to form peptidoglycan strands - is conserved across species

• Species-specific adaptations: The complete genome sequence of C. sakazakii reveals adaptations that may influence cell wall architecture and consequently mtgA function, including genes related to stress response that could be relevant to its survival in dry conditions

Understanding these similarities and differences is important for:

• Developing species-specific inhibitors

• Predicting functional conservation across pathogens

• Understanding evolutionary adaptations in cell wall synthesis

• Designing broad-spectrum antimicrobials targeting conserved features

A comprehensive comparative analysis would require structural studies, enzymatic characterization, and in vivo functional assessment across multiple bacterial species.

What methodological approaches can be used to express and purify active recombinant mtgA for crystallographic studies?

Obtaining high-quality, active recombinant mtgA suitable for crystallographic studies requires specialized approaches due to its membrane-associated nature:

• Expression system optimization:

  • E. coli is the established system for C. sakazakii mtgA expression

  • Consider specialized strains optimized for membrane protein expression (C41/C43)

  • Evaluate codon optimization for improved expression yields

  • Test different promoter systems for optimal expression levels

• Fusion tag strategies:

  • N-terminal His-tag has been successfully used

  • Consider testing additional fusion partners (MBP, SUMO) to enhance solubility

  • Evaluate the impact of tag position (N- vs. C-terminal) on activity and crystallizability

• Membrane protein extraction:

  • Optimize detergent selection for efficient extraction while maintaining activity

  • Consider bicelle or nanodisc reconstitution for maintaining native-like environment

  • Evaluate lipid supplementation to stabilize the protein during purification

• Purification protocol refinement:

  • Multi-step purification combining affinity chromatography with size exclusion

  • Buffer optimization to identify stabilizing conditions

  • Addition of substrate analogs or inhibitors to stabilize specific conformations

• Quality control assessments:

  • Activity assays using methods described in Question 4

  • Thermal stability analysis using differential scanning fluorimetry

  • Size exclusion chromatography with multi-angle light scattering to confirm monodispersity

• Crystallization screening:

  • Lipidic cubic phase crystallization for membrane proteins

  • In situ proteolysis to remove flexible regions

  • Surface entropy reduction to promote crystal contacts

These methodological refinements should be guided by activity measurements to ensure that the purified protein maintains its native conformation and catalytic functionality throughout the process.