Recombinant Bacillus cereus UDP-N-acetylglucosamine 1-carboxyvinyltransferase 1 (murA1)

What is the biological function of murA1 in Bacillus cereus?

UDP-N-acetylglucosamine 1-carboxyvinyltransferase 1 (murA1) catalyzes the transfer of an enolpyruvyl moiety from phosphoenolpyruvate (PEP) to UDP-N-acetylglucosamine (UDP-GlcNAc), forming UDP-N-acetylglucosamine-enolpyruvate. This reaction represents the first committed step in peptidoglycan biosynthesis, which is essential for bacterial cell wall formation and integrity.

The murA1 gene in B. cereus is part of the peptidoglycan synthesis pathway that interconnects with other carbohydrate metabolism pathways, including the UDP-GlcNAc biosynthetic pathway identified in B. cereus ATCC 14579 . While murA1 specifically acts on UDP-GlcNAc, it's worth noting that B. cereus possesses enzymes like UDP-GlcNAc C4,6-dehydratase (Pdeg) that can modify UDP-GlcNAc for alternative glycan biosynthesis pathways .

How does murA1 differ from murA2 in Bacillus cereus?

Bacillus cereus, like many other gram-positive bacteria, possesses two paralogs of the murA gene (murA1 and murA2) that encode UDP-N-acetylglucosamine 1-carboxyvinyltransferase. These paralogs exhibit:

• Sequence homology: Typically 55-65% amino acid sequence identity between murA1 and murA2 within the same B. cereus strain

• Functional redundancy: Both enzymes catalyze the same reaction, though often with different kinetic parameters

• Expression patterns: murA1 is generally constitutively expressed, while murA2 expression may be induced under specific stress conditions

• Antibiotic sensitivity: murA1 often shows greater sensitivity to fosfomycin compared to murA2

This gene duplication is believed to provide resilience under varying environmental conditions, consistent with B. cereus' ability to adapt to diverse ecological niches .

What are the optimal expression systems for recombinant B. cereus murA1 production?

For successful recombinant production of B. cereus murA1, several expression systems have been evaluated, with these methodological considerations:

When designing expression constructs, incorporating a His6-tag at the N-terminus rather than C-terminus typically produces higher yields of functionally active enzyme. Expression in E. coli should be performed at lower temperatures (16-20°C) after induction to minimize inclusion body formation.

How do mutations in murA1 affect antibiotic resistance in Bacillus cereus strains?

Mutations in murA1 can significantly alter B. cereus antibiotic susceptibility profiles through several mechanisms:

• Fosfomycin resistance: Point mutations in the active site cysteine (typically Cys117) can prevent fosfomycin binding while maintaining enzymatic activity.

• Substrate binding alterations: Mutations in the UDP-GlcNAc binding pocket can modify substrate affinity without eliminating catalytic function.

• Conformational changes: Distal mutations can induce allosteric effects that alter enzyme dynamics and inhibitor access.

Research methodology for investigating these mutations typically involves:

• Site-directed mutagenesis of recombinant murA1

• Enzymatic activity assays comparing wildtype and mutant proteins

• Minimum inhibitory concentration (MIC) determinations using isogenic B. cereus strains

• Structural analysis through X-ray crystallography or molecular dynamics simulations

Studies of mutations must consider the strain-specific genetic background, as B. cereus strains show considerable genomic diversity, particularly between those causing foodborne illness versus anthrax-like disease .

What are the structural determinants of murA1 substrate specificity and inhibitor binding?

The substrate specificity and inhibitor binding properties of B. cereus murA1 are governed by several structural elements:

Key structural regions affecting function:

• Loop I (residues 111-122): Contains the catalytic cysteine (Cys117) and undergoes conformational changes upon substrate binding

• Loop II (residues 156-164): Interacts with both the UDP moiety of UDP-GlcNAc and phosphoenolpyruvate

• C-terminal domain (residues 230-419): Forms part of the active site upon domain closure

Experimental approaches for structural studies:

• X-ray crystallography of murA1 in different conformational states

• Hydrogen-deuterium exchange mass spectrometry to identify flexible regions

• Site-directed mutagenesis coupled with enzymatic assays

• Molecular dynamics simulations to model substrate and inhibitor interactions

The substrate binding pocket of murA1 differs from UDP-GlcNAc modifying enzymes like UDP-GlcNAc C4,6-dehydratase (Pdeg) found in B. cereus, which acts on the same substrate but catalyzes different modifications for specialized glycan production .

How does murA1 activity differ between pathogenic and non-pathogenic Bacillus cereus strains?

B. cereus encompasses diverse strains ranging from foodborne pathogens to those causing anthrax-like disease. Analysis of murA1 across these strains reveals:

Methodologically, comparative studies of murA1 from different B. cereus isolates should:

• Sequence murA1 genes from diverse strain collections

• Quantify expression levels through RT-qPCR under standardized conditions

• Express and purify recombinant enzymes for kinetic characterization

• Correlate enzymatic properties with virulence using animal models

The varying murA1 properties between strains may reflect adaptations to different ecological niches and contribute to the spectrum of virulence observed across the B. cereus sensu lato group .

What is the optimal experimental design for assessing murA1 inhibitors against Bacillus cereus?

A comprehensive assessment of potential murA1 inhibitors requires a multi-tiered experimental approach:

Stage 1: In vitro enzyme inhibition studies

• Assay principle: Coupled spectrophotometric assay measuring pyruvate release

• Controls: Fosfomycin (positive control), buffer only (negative control)

• Data collection: IC50 determination with ≥8 inhibitor concentrations

• Analysis: Nonlinear regression analysis with appropriate enzyme inhibition models

Stage 2: Mechanism of inhibition studies

• Michaelis-Menten kinetics with varying substrate and inhibitor concentrations

• Dixon and Lineweaver-Burk plot analysis for inhibition type determination

• Residence time measurements for slow-binding inhibitors

• Thermal shift assays to assess protein stabilization upon inhibitor binding

Stage 3: Cellular studies

• Minimum inhibitory concentration (MIC) determination against:

  • Laboratory B. cereus strains (ATCC 14579)

  • Clinical isolates

  • Anthrax-like B. cereus strains (e.g., G9241)

• Cell wall integrity assays (osmotic stability testing)

• Assessment of synergy with other antibiotics

Stage 4: In vivo validation

• Mouse infection models with appropriate B. cereus strains

• Pharmacokinetic/pharmacodynamic analyses

• Efficacy against different routes of infection (systemic, gastrointestinal)

This systematic approach ensures comprehensive characterization of inhibitor properties while accounting for the biological complexity of different B. cereus strains .

How can isothermal titration calorimetry (ITC) be optimized for studying murA1-ligand interactions?

Isothermal titration calorimetry provides valuable thermodynamic information about murA1 interactions with substrates and inhibitors. Optimal experimental design includes:

Sample preparation considerations:

• Protein concentration: 20-50 μM murA1 in cell, thoroughly dialyzed

• Ligand concentration: 10-20× protein concentration in syringe

• Buffer composition: 50 mM HEPES pH 7.5, 150 mM NaCl, 5 mM MgCl2

• Temperature control: Maintain at 25°C with 1°C stability

Experimental parameters:

• Injection schedule: 25-30 injections of 1-2 μL each

• Spacing between injections: 180-240 seconds for complete equilibration

• Stirring speed: 750-850 rpm for optimal mixing without protein denaturation

Data analysis approach:

• Subtract reference injections (ligand into buffer)

• Apply appropriate binding model (one-site, two-site, sequential)

• Extract thermodynamic parameters (ΔH, ΔS, ΔG, Kd)

• Validate with orthogonal techniques (e.g., surface plasmon resonance)

Common pitfalls and solutions:

• Heat of dilution artifacts: Ensure identical buffer composition in cell and syringe

• Protein instability: Add 5% glycerol to buffer and verify stability by dynamic light scattering

• Aggregation issues: Filter samples immediately before experiment (0.22 μm filter)

This methodology allows precise determination of binding constants and associated thermodynamic parameters, enabling discrimination between different binding mechanisms.

What are the key considerations for developing a high-throughput screening assay for murA1 inhibitors?

Developing a robust high-throughput screening (HTS) assay for B. cereus murA1 inhibitors requires careful optimization of multiple parameters:

Assay format selection:

• Phosphate release assays: Malachite green detection of released phosphate

  • Sensitivity: Detection limit ~1 μM phosphate

  • Z' factor: Typically 0.7-0.8 when optimized

  • Interference: Phosphate contaminants in buffers can increase background

• Fluorescence-based assays: NADH-coupled detection systems

  • Sensitivity: Detection limit ~0.1 μM NADH

  • Z' factor: 0.8-0.9 under optimal conditions

  • Interference: Compound autofluorescence can cause false positives/negatives

Critical optimization variables:

• Enzyme concentration: Typically 10-50 nM (determined by activity titration)

• Substrate concentrations: UDP-GlcNAc at Km (~150 μM), PEP at 1.5× Km

• DMSO tolerance: Validate linearity up to 2% DMSO

• Assay stabilizers: BSA (0.01%) to prevent surface adsorption

• Incubation time: 30-60 minutes at 30°C for optimal signal:noise ratio

Controls and validation:

• Positive controls: Fosfomycin at multiple concentrations

• Negative controls: DMSO only, heat-inactivated enzyme

• Counter-screen: Test hits against downstream pathway enzymes to confirm specificity

• Orthogonal validation: Confirm hits using a secondary assay with different detection method

Data analysis approach:

• Normalization: Convert raw data to percent inhibition relative to controls

• Hit selection criteria: ≥50% inhibition at 10 μM with Z-score ≥3

• Dose-response characterization: 8-point curves with 3-fold dilutions

This methodological framework provides a foundation for identifying murA1-specific inhibitors with potential antimicrobial activity against B. cereus.

How should kinetic data for recombinant B. cereus murA1 be analyzed to determine mechanism of action?

Accurate kinetic analysis of B. cereus murA1 requires systematic data collection and appropriate mathematical models:

Experimental design for mechanism determination:

• Initial velocity experiments varying both UDP-GlcNAc and PEP concentrations

• Product inhibition studies with UDP-GlcNAc-enolpyruvate

• Dead-end inhibitor studies with substrate analogs

• Pre-steady-state kinetics to identify rate-limiting steps

Data analysis framework:

• Primary plot analysis:

  • Double-reciprocal (Lineweaver-Burk) plots

  • Direct linear plots (more resistant to outliers)

  • Nonlinear regression to the appropriate rate equation

• Secondary plot analysis:

  • Slope and intercept replots to determine kinetic constants

  • Dixon plots for inhibition constants

• Global fitting approaches:

  • Simultaneous fitting of all data sets to candidate mechanisms

  • Model discrimination using AIC (Akaike Information Criterion) or BIC (Bayesian Information Criterion)

Interpreting kinetic parameters:
The following table shows typical kinetic parameters for recombinant B. cereus murA1 and how to interpret variations:

Most B. cereus murA1 enzymes follow an ordered Bi Bi mechanism with UDP-GlcNAc binding first, similar to the substrate binding mechanisms seen in other UDP-GlcNAc-modifying enzymes in B. cereus .

What statistical approaches are most appropriate for analyzing variations in murA1 sequence and function across different Bacillus cereus strains?

When analyzing murA1 sequence and functional variations across B. cereus strains, multiple statistical approaches should be employed:

Sequence analysis methods:

• Multiple sequence alignment (MSA) using MUSCLE or MAFFT algorithms

  • Gap penalties: Gap opening penalty of 10, extension penalty of 0.5

  • Output format: CLUSTAL with conserved residue marking

• Phylogenetic analysis:

  • Maximum likelihood methods with appropriate substitution models (typically LG+G)

  • Bootstrap analysis (≥1000 replicates) for statistical support

  • Correlation with established B. cereus phylogenetic clades

• Population genetics metrics:

  • Nucleotide diversity (π)

  • Tajima's D to detect selection

  • Ka/Ks ratio to identify selective pressure on coding regions

Functional data analysis:

• Principal Component Analysis (PCA) for multivariate kinetic parameters

  • Data transformation: Log transformation for parameters with large ranges

  • Scaling: Standardization to unit variance

• Hierarchical clustering:

  • Distance measure: Euclidean for continuous data

  • Linkage method: Ward's minimum variance

  • Validation: Silhouette coefficient to determine optimal cluster number

• Structure-function correlation:

  • Multiple linear regression models relating sequence variations to functional parameters

  • ANOVA for comparing kinetic parameters between strain groups

  • Non-parametric tests (Kruskal-Wallis) for non-normally distributed data

These analyses help distinguish between variation patterns in B. cereus strains causing anthrax-like disease versus typical foodborne strains, providing insights into the evolutionary adaptation of murA1 across different pathogenic lifestyles .

How can contradictory results in murA1 inhibition studies be reconciled and interpreted?

Contradictory results in murA1 inhibition studies are common due to methodological differences. A systematic approach to reconcile such discrepancies includes:

Sources of experimental variation:

• Protein preparation differences:

  • Tag position (N-terminal vs. C-terminal) can affect enzyme activity

  • Purification method impacts protein folding and active fraction

  • Storage conditions influence stability and activity retention

• Assay methodology variations:

  • Detection method sensitivity and interference profiles

  • Buffer composition effects on enzyme activity

  • Temperature and pH optimization differences

• Data analysis approaches:

  • Different fitting algorithms and constraints

  • Varying definitions of IC50 (especially with tight-binding inhibitors)

  • Interpretation of complex inhibition mechanisms

Reconciliation methodology:

• Meta-analysis approach:

  • Systematically catalog experimental conditions across studies

  • Convert different measures to comparable parameters (e.g., Ki values)

  • Weight results based on methodological rigor

• Standardized validation:

  • Reproduce key experiments using identical protocols

  • Test reference compounds across different assay formats

  • Evaluate time-dependent effects on inhibition

• Mechanistic investigation:

  • Test for time-dependent inhibition with various pre-incubation times

  • Evaluate enzyme stability under assay conditions

  • Investigate potential contaminating activities

Interpretation framework:
When analyzing contradictory results from different B. cereus strains, consider:

• Strain-specific variations in murA1 sequence and structure

• Potential differences in post-translational modifications

• Variable expression of efflux systems affecting inhibitor access in cellular assays

This approach helps distinguish genuine biological variability from methodological artifacts in murA1 inhibition studies.

What are the most promising future research directions for B. cereus murA1 studies?

The most promising research directions for B. cereus murA1 include:

• Structural biology approaches:

  • Cryo-EM studies of murA1 in different conformational states

  • Neutron diffraction to identify precise hydrogen bonding networks

  • Time-resolved crystallography to capture catalytic intermediates

• Systems biology integration:

  • Network analysis of murA1 regulation within the cell wall synthesis pathway

  • Metabolic flux analysis to measure in vivo activity under different conditions

  • Transcriptomic profiling to identify co-regulated genes

• Comparative analysis with related organisms:

  • Functional characterization across the B. cereus sensu lato group

  • Evolutionary analysis of murA1 specialization in different ecological niches

  • Interactions with other UDP-GlcNAc-utilizing pathways

• Therapeutic applications:

  • Development of murA1-specific inhibitors with reduced resistance potential

  • Exploration of synergistic combinations targeting multiple cell wall enzymes

  • Investigation of strain-specific inhibition strategies for anthrax-like B. cereus