Recombinant Lactobacillus johnsonii UDP-N-acetylglucosamine--N-acetylmuramyl- (pentapeptide) pyrophosphoryl-undecaprenol N-acetylglucosamine transferase (murG)

What is the role of MurG in Lactobacillus johnsonii peptidoglycan synthesis?

MurG in L. johnsonii functions as an essential UDP-N-acetylglucosamine transferase that catalyzes a critical step in peptidoglycan synthesis. Specifically, MurG transfers an N-acetylglucosamine (GlcNAc) residue from UDP-GlcNAc to undecaprenyl-pyrophosphoryl-MurNAc-pentapeptide (lipid intermediate I) to yield lipid intermediate II, which is GlcNAc-MurNAc(pentapeptide)-pyrophosphoryl-undecaprenol . This reaction is crucial for cell wall formation in L. johnsonii, similar to its role in other bacteria like E. coli.

The peptidoglycan layer in L. johnsonii, as in most gram-positive species, is characterized by a thick (20-100 nm) multilayer that provides structural integrity and protection against lysis. This multilayer is composed of glycan chains of repeating β-1,4-linked N-acetylglucosamine (GlcNAc) and N-acetylmuramic acid (MurNAc) residues, cross-linked by pentapeptide side chains . Unlike pharmaceutical applications, research on L. johnsonii MurG focuses on understanding bacterial cell wall biosynthesis and potential probiotic properties.

How do peptidoglycan structural variations in L. johnsonii affect experimental approaches to studying MurG?

Peptidoglycan in L. johnsonii shows specific structural variations that necessitate tailored experimental approaches. While the glycan chain chemistry is similar across bacteria, L. johnsonii exhibits variations in stem peptide composition, typically containing L-lysine rather than meso-diaminopimelic acid, which is common in other bacteria .

When studying MurG in L. johnsonii, researchers must account for these structural differences by:

• Using appropriate substrate analogs that match L. johnsonii's natural substrates

• Developing strain-specific antibodies for immunoblotting and localization studies

• Optimizing cell fractionation procedures for gram-positive bacteria to maintain membrane association

Modifications such as O-acetylation of MurNAc residues and D-asparagine cross-bridges between D-alanine and L-lysine present in L. johnsonii peptidoglycan require adjustment of enzymatic assays and substrate preparations when studying recombinant MurG . These variations directly impact substrate recognition by MurG and consequently influence experimental design.

What techniques are most effective for preliminary identification of MurG activity in L. johnsonii strains?

For initial identification of MurG activity in L. johnsonii strains, several complementary techniques have proven effective:

Genomic identification:

• PCR amplification using degenerate primers targeting conserved MurG regions

• Whole genome sequencing and comparative genomics with reference strains like L. johnsonii ZLJ010

Biochemical characterization:

• Scintillation proximity assay using radiolabeled UDP-[³H]N-acetylglucosamine to track MurG-catalyzed conversion of lipid I to lipid II

• In vitro assays for phospho-MurNAc-pentapeptide translocase and N-acetylglucosaminyl transferase activities

Morphological assessment:

• Phase contrast microscopy to observe cell shape alterations indicative of peptidoglycan synthesis disruption

• Transmission electron microscopy to visualize cell wall thickness and integrity

When implementing these techniques, researchers should maintain L. johnsonii under acidic conditions (pH 4.5-5.5) as this species is acidotolerant, which affects experimental outcomes and cell preparation protocols . Additionally, identification should include catalase testing, as most L. johnsonii strains are catalase-negative, showing no degradation of H₂O₂ to O₂ .

What expression systems yield optimal recombinant L. johnsonii MurG production and why?

Several expression systems have been evaluated for recombinant L. johnsonii MurG production, with E. coli-based systems demonstrating the highest yield and functional activity. The optimal expression system characteristics include:

For L. johnsonii MurG, plasmids for high-level overproduction of wild-type and His-tagged forms (C-terminal and N-terminal) have been successfully constructed, with all three forms demonstrating activity in vivo. After IPTG induction, growth, spheroplast formation, and lysis, the overproduced MurG proteins are predominantly (90%) present in the particulate fraction . The His-tagged forms can be purified to over 80% purity without detergent, while wild-type forms typically achieve only 20% purity, making tagged constructs preferable for most biochemical studies .

Expression optimization should include varying induction temperature (16-30°C), IPTG concentration (0.1-1.0 mM), and induction time (4-16 hours) to balance yield with proper folding and membrane association.

How can researchers overcome challenges in solubilizing and purifying membrane-associated MurG from L. johnsonii?

MurG from L. johnsonii presents purification challenges due to its tight membrane association, similar to that observed in E. coli MurG, which remains membrane-associated even without a transmembrane domain . Effective solubilization and purification strategies include:

• Optimized membrane extraction:

  • Prepare spheroplasts using lysozyme treatment in isotonic buffer

  • Disrupt cells using French press or sonication with protease inhibitors

  • Separate inner membrane fractions using sucrose density equilibrium centrifugation (45% sucrose layer)

• Solubilization approaches:

  • CHAPS detergent has proven effective for MurG solubilization without activity loss

  • Detergent screening panel: CHAPS (8-12 mM), DDM (0.5-1%), LDAO (1-2%), or Triton X-100 (0.5-1%)

  • Chemical extraction attempts with 2M NaCl are ineffective, confirming tight membrane association

• Purification strategies:

  • IMAC (immobilized metal affinity chromatography) for His-tagged constructs

  • Size exclusion chromatography to separate MurG from other membrane proteins

  • Ion exchange chromatography as a polishing step

• Activity preservation:

  • Include 10-15% glycerol in all buffers

  • Maintain reducing environment with 1-5 mM DTT or β-mercaptoethanol

  • Store at -80°C in small aliquots to prevent freeze-thaw cycles

Recent studies have identified that MurG may exist in a high-molecular-weight complex (approximately 250 kDa) with other proteins, including possible association with MreB . This complex formation necessitates gentle solubilization conditions to maintain protein-protein interactions when studying MurG in its native complex state.

What functional assays best validate the activity of purified recombinant L. johnsonii MurG?

Validating recombinant L. johnsonii MurG activity requires assays that can detect the specific transfer of GlcNAc from UDP-GlcNAc to lipid I. The following complementary approaches provide robust functional validation:

• Scintillation proximity assay (SPA):

  • Add UDP-[³H]N-acetylglucosamine to membranes with preformed lipid I

  • Capture product using wheat germ agglutinin scintillation proximity assay beads

  • Measure radioactive signal to quantify the conversion to lipid II

  • Can be adapted to high-throughput format for inhibitor screening

• Coupled enzyme assays:

  • MraY-MurG coupled assay detecting simultaneous activity of both enzymes

  • Useful for understanding the coordinated action of peptidoglycan synthesis enzymes

  • Can incorporate moenomycin to inhibit transglycosylase and prevent further conversion of lipid II

• Complementation of conditional murG mutants:

  • Transform temperature-sensitive MurG mutant strains with recombinant L. johnsonii MurG

  • Assess growth restoration at non-permissive temperatures

  • Provides in vivo validation of functional activity

• Mass spectrometry-based approaches:

  • LC-MS/MS analysis of reaction products

  • Provides direct evidence of lipid II formation without radioactive labeling

  • Can detect modifications in the product structure

When validating recombinant MurG activity, it's important to note that specific inhibitors like ramoplanin affect both wild-type and recombinant His-tagged forms to the same extent , providing a useful control for activity assays. Additionally, unexpected inhibitors like cephalosporin C have been identified, indicating secondary mechanisms that should be controlled for when developing new assays .

How does MurG's interaction with other proteins in the peptidoglycan synthesis pathway affect experimental design?

MurG functions within a complex network of protein interactions in the peptidoglycan synthesis pathway, which significantly impacts experimental design. Research has demonstrated that MurG forms a high-molecular-weight complex of approximately 250 kDa and 120 kDa, suggesting interaction with other proteins involved in cell wall synthesis .

Key considerations for experimental design include:

• Protein complex preservation:

  • Use membrane-permeable cross-linkers like dithiobis(succinimidyl propionate) (DSP) to stabilize protein-protein interactions

  • Perform non-reducing SDS-PAGE (without β-mercaptoethanol, incubation at 37°C) to maintain cross-linked complexes

  • Implement blue native PAGE to analyze intact membrane protein complexes

• Co-immunoprecipitation strategies:

  • Use affinity-purified anti-MurG IgG-coated magnetic beads for targeted pull-down

  • Analyze by immunoblotting to identify interacting partners

  • Consider proximity-dependent biotin identification (BioID) to capture transient interactions

• Localization dependencies:

  • MurG localization depends on other divisome proteins like FtsQ

  • In E. coli, MurG follows a specific localization pattern even when the divisome is not functional (with aztreonam treatment)

  • Similar dependencies likely exist in L. johnsonii and require investigation using fluorescent protein fusions or immunofluorescence

• Reconstitution experiments:

  • Stepwise addition of purified components to identify minimal functional units

  • Liposome reconstitution to provide membrane environment for proper function

  • Use of nanodiscs to study individual complexes in a membrane-like environment

Research has shown that MurG association with MreB in E. coli affects its localization and function . Future experiments should investigate whether similar interactions occur in L. johnsonii, particularly since the cytoskeletal components may differ between these species. These protein-protein interactions are crucial for understanding the coordinated assembly of the peptidoglycan layer in L. johnsonii.

What genomic and proteomic approaches can identify strain-specific variations in L. johnsonii MurG that affect peptidoglycan synthesis?

Strain-specific variations in L. johnsonii MurG can significantly impact peptidoglycan synthesis and probiotic function. Comprehensive genomic and proteomic approaches to identify these variations include:

• Comparative genomics:

  • Whole genome sequencing of multiple L. johnsonii strains (as done with strains like ZLJ010, BS15, DPC6026)

  • Phylogenomic analysis based on single-copy genes (1,288 genes used in previous analyses)

  • Identification of strain-specific genes (ranges from 42 to 185 unique genes across strains)

• Advanced proteomic techniques:

  • Quantitative proteomics (iTRAQ or TMT labeling) to compare MurG expression levels

  • Phosphoproteomics to identify regulatory post-translational modifications

  • Protein-protein interaction mapping using proximity labeling methods

  • Structural proteomics via hydrogen-deuterium exchange mass spectrometry

• Functional genomics:

  • RNA-seq to determine expression patterns under different growth conditions

  • ChIP-seq to identify regulatory elements controlling murG expression

  • Tn-seq to identify genes that synthetic interactions with murG

• Metabolomic profiling:

  • Analysis of peptidoglycan precursor pools

  • Quantification of UDP-GlcNAc and UDP-MurNAc-pentapeptide levels

  • Monitoring lipid intermediate dynamics

Recent comparative genomic analysis of L. johnsonii strains revealed 2,732 pan-genome orthologous gene clusters and 1,324 core-genome orthologous gene clusters . Strain ZLJ010 contained 219 unique genes primarily involved in replication, recombination, repair, defense mechanisms, transcription, and amino acid and carbohydrate transport/metabolism . These genetic differences likely influence MurG function and peptidoglycan synthesis across strains.

How can researchers design inhibition studies of L. johnsonii MurG to understand its role in probiotic properties?

Designing inhibition studies of L. johnsonii MurG requires careful consideration of both the enzyme's biochemical properties and the bacterial physiology. Effective approaches include:

• Selective inhibitor development:

  • Known inhibitors: ramoplanin affects both wild-type and His-tagged recombinant MurG

  • Unexpected inhibitors: cephalosporin C inhibits MurG and MraY-MurG assays, suggesting secondary mechanisms

  • Virtual screening against L. johnsonii MurG structural models to identify novel inhibitors

  • Fragment-based drug design targeting the UDP-GlcNAc binding site

• Genetic approaches:

  • CRISPR interference (CRISPRi) for partial repression of murG expression

  • Temperature-sensitive murG mutants for conditional inhibition

  • Promoter replacement with inducible systems for titrated expression

• Correlation with probiotic properties:

  • Measure changes in acid resistance (relevant as L. johnsonii maintains higher viability at lower pH compared to other tested strains)

  • Assess alterations in pathogen inhibition capacity (L. johnsonii shows contact-dependent and independent inhibition of pathogen growth)

  • Evaluate impact on host cell adhesion (modified peptidoglycan may affect surface properties)

  • Monitor anti-inflammatory and anti-genotoxic effects in cellular and animal models

• Experimental design considerations:

  • Use sub-lethal inhibitor concentrations to avoid general growth inhibition

  • Monitor peptidoglycan precursor accumulation to confirm target engagement

  • Include controls for off-target effects, especially with β-lactams which may have secondary mechanisms

A controlled inhibition study should examine how varying degrees of MurG inhibition affect different probiotic properties, such as acid and bile resistance, host attachment, and pathogen inhibition. This approach can elucidate which probiotic functions are most dependent on proper peptidoglycan synthesis and MurG activity.

What genetic modification strategies can enhance recombinant L. johnsonii MurG expression while maintaining probiotic characteristics?

Enhancing recombinant L. johnsonii MurG expression while preserving probiotic properties requires balanced genetic modification strategies:

• Promoter optimization:

  • Use of native strong promoters like P₁₆s (16S rRNA promoter)

  • Synthetic promoters with optimized -10 and -35 regions for L. johnsonii

  • Inducible promoters like nisin-inducible promoter for controlled expression

  • Constitutive promoters derived from highly expressed genes in L. johnsonii genomes

• Codon optimization:

  • Adjust codon usage based on L. johnsonii genomic GC content (34.91%)

  • Consider tRNA availability in L. johnsonii (encoded by 77 tRNA genes)

  • Balance expression level with proper folding by selective codon optimization

• Vector system selection:

  • Integration vectors for stable chromosomal expression

  • Expression cassettes targeted to neutral genomic sites

  • Use of native L. johnsonii plasmids as backbone for expression vectors

• Post-translational considerations:

  • Signal peptides for proper membrane targeting or secretion

  • Fusion tags that minimize interference with membrane association

  • Chaperone co-expression to facilitate proper folding

When implementing these strategies, it's important to monitor how genetic modifications affect key probiotic properties. For instance, L. johnsonii strains show notable properties like resistance to gastric and bile acids, pathogen inhibition, and adherence to host mucosa , which should be preserved in engineered strains. Additionally, genomic analysis has revealed strain-specific features, such as the presence of ermB gene (position 1,623,992–1,624,729) conferring erythromycin resistance in some L. johnsonii strains , which should be considered when selecting markers for genetic modification.

How can gene knockout and complementation studies be designed to elucidate MurG function in L. johnsonii?

Designing effective gene knockout and complementation studies for MurG in L. johnsonii requires specialized approaches due to the essential nature of the murG gene:

• Conditional knockout strategies:

  • CRISPR-based inducible repression using dCas9 system

  • Tetracycline-responsive promoter replacement at the native locus

  • Temperature-sensitive murG allele generation through targeted mutagenesis

  • Depletion strategy using degron tags for post-translational control

• Complementation system design:

  • Integration of complementing gene at a neutral site (e.g., between convergent genes)

  • Trans-complementation using stable plasmids with compatible replicons

  • Use of different inducible promoters for knockout and complementation

  • Wild-type and mutant allele complementation panels to assess structure-function relationships

• Phenotypic assessment:

  • Growth rate and morphology analysis under depletion conditions

  • Peptidoglycan precursor accumulation analysis by LC-MS

  • Measurement of UDP-GlcNAc and UDP-MurNAc-pentapeptide pools

  • Electron microscopy to assess cell wall thickness and morphology

  • Fluorescent D-amino acid incorporation to visualize peptidoglycan synthesis

• Controls and validation:

  • Complementation with E. coli murG as heterologous control

  • Partial complementation with related glycosyltransferases

  • Quantitative RT-PCR to confirm depletion efficiency

  • Western blotting to track protein levels during depletion

Since MurG inactivation in E. coli leads to rapid inhibition of peptidoglycan synthesis, cell shape alterations, and eventual cell lysis when peptidoglycan content decreases by 40% , similar effects are expected in L. johnsonii. The experimental timeline should be carefully designed to capture changes before complete lysis occurs, typically within 2-3 generations after effective depletion.

What advanced methods can detect structural changes in peptidoglycan when MurG activity is altered in L. johnsonii?

Detecting structural changes in L. johnsonii peptidoglycan resulting from altered MurG activity requires sophisticated analytical techniques:

• Mass spectrometry-based approaches:

  • Liquid chromatography-tandem mass spectrometry (LC-MS/MS) of muropeptides

  • MALDI-TOF analysis of enzymatically digested peptidoglycan

  • Ion mobility mass spectrometry to distinguish structural isomers

  • Quantitative comparison of cross-linking patterns and modifications

• Nuclear magnetic resonance (NMR) spectroscopy:

  • Solid-state NMR of intact peptidoglycan sacculi

  • Solution NMR of enzymatically digested fragments

  • Real-time NMR to monitor peptidoglycan synthesis with isotopically labeled precursors

• Advanced microscopy techniques:

  • Atomic force microscopy (AFM) to measure cell wall rigidity and topography

  • Super-resolution microscopy (STORM/PALM) with fluorescent D-amino acids

  • Cryo-electron tomography to visualize peptidoglycan architecture in near-native state

  • Correlative light and electron microscopy for targeted structural analysis

• Biochemical characterization:

  • Differential sensitivity to peptidoglycan hydrolases

  • Analysis of O-acetylation patterns, which can be altered in L. casei and potentially L. johnsonii

  • Assessment of D-asparagine cross-bridge formation and amidation

  • Quantification of peptide cross-linking patterns using specific enzymes

When analyzing peptidoglycan from L. johnsonii with altered MurG activity, researchers should pay particular attention to:

• Changes in glycan chain length distribution

• Alterations in peptide stem composition (typically L-alanine-D-glutamate-L-lysine-D-alanine-D-alanine in L. johnsonii)

• Modifications in cross-bridge formation (often D-asparagine in lactic acid bacteria)

• Variations in resistance to lysozyme and other cell wall-targeting compounds

These methods can provide detailed insights into how MurG activity affects not only the presence of peptidoglycan but also its fine structure and resulting cellular properties.

How does MurG activity in L. johnsonii contribute to its documented probiotic properties?

MurG activity in L. johnsonii contributes significantly to its probiotic properties through several mechanisms:

• Acid and bile resistance:

  • Properly synthesized peptidoglycan provides structural integrity under acidic conditions

  • L. johnsonii maintains higher viability at lower pH compared to other tested strains

  • Structurally sound cell wall helps resist bile salt detergent effects

• Host adhesion and colonization:

  • Peptidoglycan and associated surface molecules mediate attachment to intestinal mucosa

  • Genome analysis of L. johnsonii reveals genetic elements involved in host attachment

  • Lipoteichoic acids anchored in the cell membrane but extending through peptidoglycan contribute to adhesion

• Pathogen inhibition mechanisms:

  • Cell wall integrity enables production and release of inhibitory compounds

  • Contact-dependent inhibition requires proper cell surface architecture

  • L. johnsonii alters pathogen adhesion to cell monolayers and demonstrates both contact-dependent and independent inhibition of pathogen growth

• Immunomodulatory effects:

  • Peptidoglycan fragments serve as microbe-associated molecular patterns (MAMPs)

  • L. johnsonii suppresses intestinal and systemic pro-inflammatory responses

  • Enhances anti-inflammatory immune responses, likely through controlled release of peptidoglycan fragments

L. johnsonii strain 456 (LBJ 456) demonstrates persistent viability even beyond the period of initial ingestion, unlike many other probiotic lactic acid bacteria . This persistence depends on proper cell wall synthesis maintained by MurG activity. Studies with mice demonstrated that L. johnsonii administration (both prophylactic and therapeutic) significantly suppressed intestinal and systemic pro-inflammatory responses while enhancing anti-inflammatory immune responses in Campylobacter jejuni infections .

How can researchers correlate peptidoglycan structural variations with specific health-promoting functions of L. johnsonii?

Correlating peptidoglycan structural variations with specific health-promoting functions requires multidisciplinary approaches:

• Strain library with defined peptidoglycan variations:

  • Generate murG point mutants affecting catalytic efficiency

  • Create strains with altered peptidoglycan hydrolase activity

  • Develop strains with modifications in peptidoglycan decoration enzymes

  • Engineer strains with varied cross-linking patterns

• Functional characterization paradigms:

  • Epithelial barrier function assays (transepithelial electrical resistance)

  • Immune cell stimulation profiles (dendritic cells, macrophages, T cells)

  • Anti-inflammatory cytokine induction capacity

  • Pathogen inhibition zones and mechanisms (contact-dependent vs. secreted factors)

  • Quantitative adhesion to intestinal cell lines and mucins

• Correlative analytical techniques:

  • Multi-omics integration (transcriptomics, proteomics, metabolomics)

  • Machine learning approaches to identify structure-function relationships

  • Principal component analysis to cluster strains by peptidoglycan structure and function

  • Network analysis to reveal associations between structural features and probiotic effects

• In vivo validation:

  • Animal models of intestinal inflammation

  • Pathogen challenge studies with defined L. johnsonii variants

  • Colonization persistence measurement in healthy and disease models

  • Immunomodulation assessment through cytokine profiling

Research has demonstrated that L. johnsonii can ameliorate intestinal and extra-intestinal inflammation in C. jejuni-infected mice . While the mechanisms aren't fully elucidated, it's likely that peptidoglycan structure plays a key role in these effects through pattern recognition receptor engagement. Similar studies with defined murG variants could establish direct correlations between MurG activity, resulting peptidoglycan structures, and specific health outcomes.

What experimental models best demonstrate the functional impact of recombinant MurG expression on L. johnsonii probiotic efficacy?

To effectively demonstrate how recombinant MurG expression affects L. johnsonii probiotic efficacy, researchers should employ a spectrum of complementary experimental models:

• In vitro cellular models:

  • Polarized intestinal epithelial cell monolayers (Caco-2, HT-29, LS 174T)

  • Co-culture systems with immune cells (dendritic cells, macrophages)

  • Organoid cultures derived from intestinal stem cells

  • Ex vivo intestinal tissue explants maintaining mucosal architecture

• Intermediate complexity models:

  • Gut-on-a-chip microfluidic devices with mechanical peristalsis

  • Multi-compartment fermenter systems simulating different GI regions

  • Biofilm formation assays on mucin-coated surfaces

  • Competition assays with pathogens in controlled environments

• Animal models with defined microbiota:

  • Secondary abiotic mice (generated through broad-spectrum antibiotic treatment)

  • Gnotobiotic animals with defined bacterial consortia

  • Humanized microbiome mouse models

  • Specific disease models (colitis, infection challenges)

• Assessment parameters:

  • Colonization efficiency and persistence

  • Mucosal immune response profiles

  • Pathogen inhibition capacity in vivo

  • Barrier function and epithelial integrity markers

  • Systemic immunomodulatory effects

Previous research has successfully used secondary abiotic mice subjected to broad-spectrum antibiotics for eight weeks to study L. johnsonii effects. These mice were then challenged with 10⁸ CFU of L. johnsonii either 14 days before (prophylactic) or 7 days after (therapeutic) C. jejuni infection . This model allowed researchers to demonstrate that L. johnsonii treatment did not lower intestinal C. jejuni colonization but significantly suppressed pro-inflammatory responses and enhanced anti-inflammatory immune responses .

When designing such studies with recombinant MurG-expressing L. johnsonii strains, researchers should include appropriate controls:

• Wild-type L. johnsonii strain

• Strain with empty vector

• Strains expressing catalytically inactive MurG mutants

• Strains with varying levels of MurG expression