Recombinant Lactobacillus plantarum UDP-N-acetylglucosamine 1-carboxyvinyltransferase 1 (murA1)

What is UDP-N-acetylglucosamine 1-carboxyvinyltransferase 1 (murA1) and what is its significance in Lactobacillus plantarum?

UDP-N-acetylglucosamine 1-carboxyvinyltransferase 1 (murA1) is an essential enzyme that catalyzes the first committed step in bacterial peptidoglycan biosynthesis. In L. plantarum, murA1 transfers the enolpyruvate moiety of phosphoenolpyruvate (PEP) to UDP-N-acetylglucosamine (UNAG), forming UDP-N-acetylglucosamine-enolpyruvate, which is critical for cell wall formation . MurA1 is particularly significant in L. plantarum as it has been identified as one of the key stress-responsive proteins that helps maintain cell wall integrity during environmental challenges such as cold and acid stress conditions . The enzyme is part of the broader Mur family proteins (including MurB, MurC, MurD, MurE1, and MurF) that collectively regulate peptidoglycan synthesis, which is essential for bacterial survival, growth, and resistance to external stressors.

How does murA1 function within the peptidoglycan synthesis pathway of L. plantarum?

MurA1 functions at the initial stage of the peptidoglycan synthesis pathway in L. plantarum. The complete pathway involves:

• MurA1 catalyzes the transfer of enolpyruvate from PEP to UNAG

• MurB reduces the enolpyruvate moiety to form UDP-N-acetylmuramic acid (UNAM)

• MurC adds L-alanine to UNAM

• MurD incorporates D-glutamate

• MurE1 adds meso-diaminopimelic acid or L-lysine

• MurF attaches D-alanyl-D-alanine

This sequential process forms the complete peptidoglycan precursor that is then incorporated into the cell wall structure . In response to stress conditions, L. plantarum has been observed to upregulate proteins including DacA1, DacB, MurA1, MurB, MurC, MurD, MurE1, and MurF to increase total peptidoglycan production, which enhances cellular protection against environmental challenges .

What biochemical characteristics distinguish murA1 from other enzymes in the peptidoglycan synthesis pathway?

MurA1 has several distinctive biochemical characteristics:

• It utilizes a unique catalytic mechanism involving the formation of a tetrahedral intermediate between PEP and UNAG

• Contains a critical cysteine residue (similar to Cys-115 in Enterobacter cloacae MurA) that is essential for catalytic activity and forms a covalent adduct with PEP

• Is specifically targeted by the antibiotic fosfomycin, which covalently modifies the active site cysteine residue

• Has structural and sequence similarity to 5-enolpyruvylshikimate 3-phosphate synthase, the only other known enzyme that catalyzes the transfer of enolpyruvate from PEP to a substrate

• Forms part of a feedback regulation system, where the enzyme can exist in a "dormant" complex with UNAM and covalently bound PEP, enabling efficient regulation of murein biosynthesis

What are the optimal expression systems for producing recombinant L. plantarum murA1?

For recombinant expression of L. plantarum murA1, several expression systems have proven effective:

Table 1: Comparison of Expression Systems for Recombinant L. plantarum murA1

The selection of an appropriate expression system depends on the research objectives. For structural and biochemical characterization, E. coli systems typically provide sufficient yields. For immunological applications and vaccine development, expression in L. plantarum itself using vectors like pSIP409 offers advantages of proper protein folding and potential for surface display .

What methodological approaches are most effective for purifying recombinant murA1?

Purification of recombinant murA1 requires a systematic approach:

• Cell Lysis: Optimized lysis buffers containing protease inhibitors to prevent degradation of the target protein

• Initial Capture:

  • Affinity chromatography using His-tagged constructs with Ni-NTA resin

  • Alternative affinity tags (GST, MBP) if solubility issues are encountered

• Intermediate Purification:

  • Ion exchange chromatography (typically DEAE or Q-Sepharose)

  • Optimization of pH and salt gradients based on murA1's theoretical pI

• Polishing:

  • Size exclusion chromatography to separate monomeric from aggregated forms

  • Typical yields of >95% purity can be achieved through this three-step process

• Quality Control:

  • SDS-PAGE and Western blotting to confirm identity

  • Activity assays to verify functional integrity

  • Mass spectrometry to confirm protein sequence and modifications

The purification strategy should be tailored to the intended application, with structural studies requiring higher purity than functional assays or immunological applications.

How can researchers effectively measure murA1 enzymatic activity in vitro?

MurA1 enzymatic activity can be measured through several complementary approaches:

• Spectrophotometric Coupled Assay:

  • Measures the release of inorganic phosphate during the transfer of enolpyruvate

  • Uses enzymes like pyruvate kinase and lactate dehydrogenase to couple PEP utilization to NADH oxidation

  • Monitored at 340 nm for NADH consumption

  • Advantages: Continuous monitoring, readily available reagents

  • Limitations: Indirect measurement, potential for interference

• Direct Product Formation Assay:

  • HPLC or LC-MS analysis of UDP-N-acetylglucosamine-enolpyruvate formation

  • Allows direct quantification of product formation

  • Advantages: Direct measurement, high specificity

  • Limitations: Equipment-intensive, discontinuous measurement

• Radioactive Assay:

  • Uses 14C or 3H-labeled PEP to track transfer to UNAG

  • Advantages: High sensitivity, direct measurement of transfer

  • Limitations: Requires radioisotope handling facilities

• Fluorescence-Based Assays:

  • Utilizes fluorescently-labeled substrate analogs

  • Advantages: High sensitivity, potential for high-throughput screening

  • Limitations: May alter enzyme kinetics, requires specialized substrates

For kinetic characterization, researchers should determine Km values for both PEP and UNAG substrates, as well as the catalytic efficiency (kcat/Km) under various pH and temperature conditions relevant to L. plantarum's natural environment.

What structural features of murA1 contribute to its catalytic mechanism?

Based on structural studies of MurA from related species, L. plantarum murA1 likely possesses the following key structural features:

• Domain Organization:

  • Two globular domains connected by a flexible hinge region

  • Substrate binding occurs in the cleft between domains

  • Conformational change from "open" to "closed" state upon substrate binding

• Active Site Architecture:

  • Contains a critical cysteine residue analogous to Cys-115 in E. cloacae MurA

  • Forms a covalent phospholactoyl adduct with PEP during catalysis

  • The addition of UNAG triggers enolpyruvate transfer

• Loop Regions:

  • Dynamic loops that participate in substrate recognition and binding

  • Contribute to specificity for UNAG over other UDP-sugars

• Regulatory Features:

  • Forms a "dormant" complex with UNAM (product of MurB reaction) with PEP covalently attached to the catalytic cysteine

  • This complex represents a feedback regulatory mechanism

Understanding these structural features is essential for rational design of inhibitors or engineering variant enzymes with modified properties for research applications.

How does expression of murA1 change under different environmental stress conditions in L. plantarum?

L. plantarum has evolved sophisticated regulatory mechanisms for murA1 expression under various stress conditions:

Table 2: Regulation of murA1 Expression Under Different Stress Conditions

During cold and acid stress, L. plantarum NMGL2 shows upregulation of murA1 along with other peptidoglycan synthesis genes (MurB, MurC, MurD, MurE1, and MurF) to increase total peptidoglycan production . This adaptation helps strengthen the cell wall, providing enhanced protection against environmental challenges. The upregulation of murA1 is part of a coordinated response involving multiple stress-response pathways, including proteins associated with cell repair systems and membrane modification.

What regulatory elements control murA1 expression in L. plantarum?

The expression of murA1 in L. plantarum is regulated through multiple mechanisms:

• Transcriptional Regulation:

  • Stress-responsive promoter elements that respond to environmental signals

  • Potential binding sites for global regulators that coordinate stress responses

• Post-Translational Regulation:

  • Feedback inhibition by peptidoglycan precursors

  • Formation of the "dormant" complex with UNAM and covalently bound PEP

  • Protein-protein interactions with other cell wall synthesis enzymes

• Metabolic Regulation:

  • Availability of substrates (PEP and UNAG) influences activity

  • Energy status of the cell affects resource allocation to cell wall synthesis

• Integration with Stress Response Pathways:

  • Coordination with heat shock proteins and cold shock proteins

  • Connection to global stress regulators that monitor membrane integrity

Understanding these regulatory elements provides potential targets for manipulating murA1 expression in recombinant systems for research applications.

How does recombinant L. plantarum expressing murA1 affect host immune responses?

Recombinant L. plantarum strains can elicit specific immune responses influenced by murA1 expression:

• Mucosal Immunity:

  • Increased secretion of SIgA in intestinal mucosa, an important defense mechanism

  • Enhanced production of IgA and IgG in serum

• Cellular Immunity:

  • Activation of dendritic cells in Peyer's patches

  • Increased proliferation of CD4+ and CD8+ T cells

  • Significant proliferation of mesenteric lymph node (MLN) and spleen (SP) lymphocytes

• Cytokine Modulation:

  • Reduction in pro-inflammatory cytokines like TNF-α (mean difference of -2.34 pg/mL)

  • Enhancement of anti-inflammatory cytokine IL-10 (mean difference of 9.88 pg/mL)

  • Decreased levels of IL-17 and IL-22 from CD4+ T cells

• Innate Immunity Activation:

  • Significant activation of NK cells and macrophages

  • Increased levels of IL-6 and IL-1β in serum and colon

These immunomodulatory effects suggest recombinant L. plantarum expressing murA1 may have applications in vaccine development and as therapeutic agents for inflammatory conditions.

How can recombinant L. plantarum murA1 be utilized for vaccine development?

Recombinant L. plantarum expressing murA1, either alone or in combination with other antigens, shows promise for vaccine development through several mechanisms:

• Expression System Design:

  • Utilization of surface display systems like pSIP409-pgsA' or pSIP409-FnBPA-pgsA' for antigen presentation

  • Coexpression with adjuvant molecules like dendritic cell-targeting peptide (DCpep) to enhance immune response

• Immune Response Induction:

  • Proven capability to induce both mucosal and systemic immunity

  • Generation of specific antibodies with hemagglutination inhibition (HI) potency

  • Stimulation of memory lymphocytes in mesenteric lymph nodes

• Advantages Over Conventional Vaccines:

  • Effective in protecting against mucosal infection, where conventional vaccines often fail

  • Requires no purification of antigens, reducing production complexity

  • Room temperature stability, reducing cold chain requirements

  • Oral administration possibility, eliminating need for injections

• Experimental Validation:

  • Successful immunization of mice via oral administration

  • Demonstrated protection in animal models of infection

  • Favorable safety profile with no adverse effects observed in experimental models

The design of such vaccines requires careful optimization of antigen expression levels, selection of appropriate promoters, and validation of stability during gastric transit.

What are the methodological approaches for studying the covalent reaction of murA1 with its substrates?

Investigating the covalent reaction mechanism of murA1 requires sophisticated methodological approaches:

• X-ray Crystallography:

  • Capture of enzyme-substrate complexes in various reaction states

  • Visualization of the Cys-PEP adduct formation

  • Structural determination of the "dormant" complex with UNAM

  • Typical resolution requirement: ≤2.0 Å for detailed active site visualization

• Mass Spectrometry:

  • Identification of covalent adducts between the active site cysteine and PEP

  • Peptide mapping to confirm modification sites

  • Time-resolved analysis to track reaction intermediates

  • Techniques: MALDI-TOF, LC-MS/MS with electron transfer dissociation (ETD)

• NMR Spectroscopy:

  • Real-time monitoring of reaction progression

  • Structural dynamics information during catalysis

  • Chemical shift changes indicating covalent bond formation

  • Requires isotopic labeling (13C, 15N) of the enzyme or substrates

• Site-Directed Mutagenesis:

  • Systematic mutation of active site residues

  • Creation of catalytically inactive variants for trapping reaction intermediates

  • Development of cysteine-free variants to prevent covalent adduct formation

  • Comparative kinetic analysis of wild-type and mutant enzymes

These approaches have revealed that murA1 forms a covalent phospholactoyl adduct with PEP during catalysis, and this covalent reaction serves dual functions: tightening the complex with UNAM for feedback regulation and priming the PEP molecule for reaction with UNAG .

How can CRISPR-Cas9 technology be applied to modify murA1 expression in L. plantarum?

CRISPR-Cas9 technology offers powerful approaches for precise manipulation of murA1 in L. plantarum:

• Gene Knockout Studies:

  • Design of specific sgRNAs targeting murA1

  • Integration of CRISPR-Cas9 components via transformation of appropriate vectors

  • Selection of knockout mutants through appropriate markers

  • Phenotypic characterization of murA1 deficiency on cell wall integrity and stress response

• Promoter Modification:

  • Targeted modification of native murA1 promoter to alter expression levels

  • Integration of inducible or constitutive promoters to control murA1 expression

  • Creation of reporter fusions to monitor expression under different conditions

  • Protocol adaptation: lower transformation temperature (30°C) and extended recovery times for L. plantarum

• Site-Specific Mutations:

  • Introduction of specific mutations in the catalytic site

  • Creation of variants resistant to specific inhibitors

  • Engineering of MurA1 with altered substrate specificity

  • Homology-directed repair with carefully designed donor templates

• Regulatory Element Engineering:

  • Modification of regulatory elements controlling murA1 expression

  • Creation of stress-responsive expression systems

  • Implementation of tunable expression systems for detailed functional studies

  • Utilization of base editing approaches for precise nucleotide modifications

Implementation of CRISPR-Cas9 in L. plantarum requires optimization of several parameters including transformation efficiency, guide RNA design specific to L. plantarum genomic context, and appropriate selection methods for identifying successful transformants.

What are the implications of murA1 inhibition on L. plantarum viability and probiotic properties?

Inhibition of murA1 has multifaceted implications for L. plantarum:

• Cell Viability and Morphology:

  • Reduced peptidoglycan synthesis leading to compromised cell wall integrity

  • Potential cell elongation or abnormal morphology due to incomplete cell division

  • Increased susceptibility to osmotic stress and mechanical damage

  • Possible induction of cell wall stress response pathways

• Stress Response Capabilities:

  • Diminished ability to adapt to acid and cold stress conditions

  • Altered expression of stress response proteins including heat shock proteins

  • Compromised survival during gastrointestinal transit

  • Modified resistance to environmental challenges

• Probiotic Functions:

  • Potentially altered immunomodulatory properties, affecting IL-10 and TNF-α regulation

  • Changed adhesion capabilities to intestinal epithelial cells

  • Modified interaction with host immune cells

  • Possible alterations in competitive exclusion of pathogens

• Applications in Research:

  • Controllable inhibition provides a tool for studying cell wall biosynthesis

  • Potential for creating attenuated strains for vaccine development

  • Model system for investigating bacterial adaptation to cell wall stress

  • Platform for screening novel peptidoglycan-targeting antimicrobials

The selective inhibition of murA1 through chemical or genetic approaches provides valuable insights into the relationship between cell wall integrity, stress response, and the probiotic properties of L. plantarum.

What are the best approaches for analyzing proteomics data related to murA1 expression under stress conditions?

Analysis of proteomics data for murA1 expression requires comprehensive methodological approaches:

• Data Acquisition Strategies:

  • Data-independent acquisition (DIA) proteomics for comprehensive protein profiling

  • Multiple reaction monitoring (MRM) for targeted quantification of murA1 and related proteins

  • Label-free quantification for broad coverage of the proteome

  • Tandem mass tag (TMT) labeling for multiplexed comparative analysis

• Statistical Analysis Pipeline:

  • Normalization methods accounting for global protein changes

  • Statistical significance determination using appropriate tests (t-test, ANOVA)

  • Multiple testing correction (Benjamini-Hochberg, Bonferroni)

  • Fold change thresholds (typically >1.5-fold) for biological relevance

• Functional Data Integration:

  • Pathway enrichment analysis to identify coordinated responses

  • Protein-protein interaction network analysis to identify functional modules

  • Integration with transcriptomics data for multi-omics perspective

  • Correlation analysis between murA1 expression and phenotypic stress resistance

• Visualization Approaches:

  • Heat maps of differentially expressed proteins across conditions

  • Volcano plots highlighting statistical and biological significance

  • Protein interaction networks with color-coded expression changes

  • Time-course visualizations for temporal dynamics of response

This systematic approach has been successfully applied to understand how L. plantarum NMGL2 responds to combinational cold and acid stresses, revealing that murA1 is part of a coordinated stress response involving multiple cellular systems .

How should researchers design experiments to evaluate the effect of murA1 modifications on L. plantarum stress resistance?

Designing robust experiments to evaluate murA1 modifications requires a comprehensive approach:

• Strain Construction Strategy:

  • Generation of murA1 overexpression strains using controlled promoters

  • Creation of murA1 knockout or knockdown strains using CRISPR-Cas9 or antisense RNA

  • Development of strains expressing modified murA1 (site-directed mutants)

  • Inclusion of appropriate control strains (empty vector, wild-type, etc.)

• Stress Challenge Parameters:

  • Acid stress: pH gradients (3.0-5.5) with varied exposure times

  • Cold stress: Temperature ranges (4-15°C) with monitoring over extended periods

  • Combined stresses: Factorial design incorporating multiple stressors

  • Recovery conditions: Standard and stress-relief media

• Assessment Metrics:

  • Viability measurements: Colony forming units, fluorescent viability indicators

  • Growth kinetics: Lag phase duration, growth rate, maximum cell density

  • Cell morphology: Phase contrast microscopy, transmission electron microscopy

  • Membrane integrity: Fluorescent dye exclusion assays

• Molecular Analysis:

  • Proteomics: Global protein expression changes

  • Transcriptomics: RNA-seq for genome-wide expression patterns

  • Metabolomics: Changes in key metabolic pathways

  • Cell wall composition: Peptidoglycan structure and modifications

• Statistical Design Considerations:

  • Minimum of biological triplicates for all experiments

  • Power analysis to determine appropriate sample size

  • Blocking factors to account for batch effects

  • Appropriate statistical tests based on data distribution

This experimental framework allows for comprehensive evaluation of how murA1 modifications affect L. plantarum's ability to withstand environmental challenges, with implications for both basic research and applied probiotic development.

What are the emerging technologies that could advance our understanding of murA1 function in L. plantarum?

Several cutting-edge technologies show promise for advancing murA1 research:

• Cryo-Electron Microscopy:

  • Near-atomic resolution structures of murA1 in different conformational states

  • Visualization of murA1 within the context of multiprotein peptidoglycan synthesis complexes

  • Time-resolved structural changes during catalysis

  • Advantages: No crystallization required, native-like conditions

• Single-Cell Technologies:

  • Analysis of cell-to-cell variation in murA1 expression

  • Correlation between murA1 levels and individual cell stress resistance

  • Real-time monitoring of murA1 activity using fluorescent reporters

  • Applications: Understanding heterogeneous responses in bacterial populations

• Synthetic Biology Approaches:

  • Creation of murA1 variants with non-canonical amino acids for mechanistic studies

  • Development of orthogonal translation systems in L. plantarum

  • Design of synthetic regulatory circuits controlling murA1 expression

  • Potential: Precise control over murA1 function and regulation

• Advanced Computational Methods:

  • Molecular dynamics simulations of murA1-substrate interactions

  • Machine learning algorithms to predict murA1 expression under various conditions

  • Systems biology models integrating murA1 into whole-cell metabolic networks

  • Benefits: In silico prediction of experimental outcomes, hypothesis generation

These technologies promise to provide unprecedented insights into murA1 function and regulation, potentially leading to novel applications in biotechnology and medicine.

What unresolved questions remain regarding recombinant L. plantarum murA1 in immunomodulation?

Despite significant advances, several critical questions remain unanswered:

• Mechanistic Understanding:

  • How does murA1 expression level correlate with specific immunomodulatory effects?

  • What is the precise mechanism by which recombinant L. plantarum murA1 influences cytokine production?

  • Are there strain-specific differences in immunomodulatory effects related to murA1 variants?

  • How do post-translational modifications of murA1 affect immunogenic properties?

• Host-Microbe Interactions:

  • Does murA1 directly interact with host pattern recognition receptors?

  • How does the host microbiome influence the immunomodulatory effects of recombinant L. plantarum murA1?

  • Are there host genetic factors that influence response to L. plantarum murA1?

  • What is the durability of immune responses elicited by recombinant L. plantarum murA1?

• Clinical Applications:

  • Can recombinant L. plantarum murA1 be effective in treating specific inflammatory conditions?

  • What dosing regimens optimize immunomodulatory effects while minimizing potential adverse reactions?

  • How does recombinant L. plantarum murA1 interact with conventional immunomodulatory therapies?

  • What biomarkers can predict individual response to recombinant L. plantarum murA1 treatment?

• Technical Challenges:

  • How can expression stability be maintained in vivo over extended periods?

  • What delivery systems optimize the presentation of recombinant murA1 to the immune system?

  • How can murA1 expression be targeted to specific gastrointestinal regions?

  • What quality control metrics ensure consistent immunomodulatory properties?