Recombinant Enterococcus faecalis Phosphoribosylformylglycinamidine cyclo-ligase (purM)

What is the role of Phosphoribosylformylglycinamidine cyclo-ligase (purM) in E. faecalis metabolism?

Phosphoribosylformylglycinamidine cyclo-ligase (purM) is a critical enzyme in the de novo purine biosynthesis pathway of Enterococcus faecalis. It catalyzes the ATP-dependent conversion of formylglycinamide ribonucleotide (FGAR) to formylglycinamidine ribonucleotide (FGAM), representing the fourth step in the pathway. This enzymatic activity is particularly important for E. faecalis survival in nutrient-limited environments where purine salvage pathways become insufficient. Research has demonstrated that enzymes involved in de novo purine biosynthesis are specifically enriched in E. faecalis biofilms exposed to certain environmental conditions, suggesting their importance in biofilm formation and bacterial persistence . In experimental approaches, researchers should consider measuring purine nucleotide levels using HPLC analysis before and after purM inhibition to quantify its metabolic contribution. Additionally, growth rate analyses in media with and without purine supplementation can help establish the dependency of E. faecalis on de novo purine synthesis in different environmental conditions.

How does E. faecalis purM compare structurally and functionally to purM from other bacterial species?

While specific structural details of E. faecalis purM are not widely reported in the current literature, comparative analyses with purM from other bacterial species reveal important insights. The enzyme belongs to the AIR synthetase family, typically exhibiting a homodimeric structure with ATP-binding domains. When designing experiments to study E. faecalis purM, researchers should consider performing multiple sequence alignments with purM from better-characterized organisms like E. coli or B. subtilis to identify conserved catalytic residues. Homology modeling based on crystal structures of purM from other species can provide insights into substrate binding sites and potential inhibitor development. Functional complementation assays, where E. faecalis purM is expressed in purM-deficient strains of other bacteria, can elucidate the functional conservation across species. Researchers should also note that proteomics studies have identified purM among proteins enriched in E. faecalis biofilms, particularly in response to certain environmental conditions such as exposure to epigallocatechin gallate (EGCG) .

How is purM gene expression regulated in E. faecalis under different growth conditions?

To properly investigate purM regulation, researchers should consider the following methodological approaches:

• qRT-PCR analysis comparing purM expression levels in:

  • Planktonic vs. biofilm growth

  • Purine-rich vs. purine-limited media

  • Stress vs. non-stress conditions

• Promoter fusion assays using reporter genes (e.g., GFP, luciferase) to monitor real-time expression changes

• Chromatin immunoprecipitation (ChIP) analyses to identify transcription factors that bind to purM regulatory regions

The differential expression pattern observed in biofilms suggests that purM may play roles beyond basic metabolism in E. faecalis adaptation and persistence mechanisms.

What are the optimal expression systems for producing recombinant E. faecalis purM?

The selection of an appropriate expression system for E. faecalis purM requires careful consideration of protein solubility, activity preservation, and yield requirements. Based on established protocols for similar proteins, the following methodological approach is recommended:

Table 1: Comparison of Expression Systems for E. faecalis purM

When designing expression constructs, researchers should incorporate a His6-tag or similar affinity tag for purification purposes. The expression of E. faecalis proteins in heterologous hosts like E. coli often requires optimization of codon usage and induction conditions. For the best results, expression at lower temperatures (16-25°C) tends to improve protein solubility. For E. faecalis-based expression systems, nisin-inducible promoters have been successfully employed for controlled expression, as demonstrated in similar genetic modification studies .

What purification strategy yields the highest purity and activity for recombinant E. faecalis purM?

A multi-step purification approach is recommended to obtain highly pure and active recombinant E. faecalis purM. The following methodological workflow has been optimized based on similar nucleotide-binding enzymes from Gram-positive bacteria:

• Initial Capture: Immobilized Metal Affinity Chromatography (IMAC) using Ni-NTA resin

  • Buffer composition: 50 mM Tris-HCl pH 8.0, 300 mM NaCl, 10 mM imidazole

  • Elution with imidazole gradient (50-250 mM)

  • Expected purity: 70-80%

• Intermediate Purification: Ion Exchange Chromatography

  • Q-Sepharose column for anion exchange at pH 8.0

  • Buffer: 20 mM Tris-HCl pH 8.0, with gradient of 0-500 mM NaCl

  • Expected purity: 85-90%

• Polishing Step: Size Exclusion Chromatography

  • Superdex 200 column

  • Buffer: 25 mM HEPES pH 7.5, 150 mM NaCl, 5 mM MgCl2, 1 mM DTT

  • Expected purity: >95%

Key considerations during purification include maintaining protein stability by including glycerol (10%) and potentially adding ATP (0.1-0.5 mM) to stabilize the enzyme's active site. The purification should be performed at 4°C to minimize proteolytic degradation. For activity preservation, it is essential to include magnesium ions in the final buffer, as they are cofactors for ATP-dependent enzymes like purM. Researchers should verify enzyme purity using SDS-PAGE and confirm activity using spectrophotometric assays measuring ATP consumption or product formation.

How can I assess the kinetic parameters and stability of purified recombinant E. faecalis purM?

Comprehensive characterization of purified recombinant E. faecalis purM requires systematic assessment of its kinetic parameters and stability under various conditions. The following methodological approach is recommended:

Kinetic Parameter Assessment:

• Steady-state kinetics: Measure initial reaction velocities at varying substrate (FGAR) concentrations (0.1-10× Km) and constant ATP concentration

• ATP dependence: Vary ATP concentrations (0.1-10× Km) at fixed FGAR concentration

• Data analysis: Fit data to Michaelis-Menten equation to determine Km, Vmax, and kcat values

Expected Kinetic Parameters (based on related purM enzymes):

Stability Assessment:

• Thermal stability: Differential scanning fluorimetry (DSF) to determine melting temperature (Tm)

• pH stability: Incubate enzyme at various pH values (5.0-9.0) for defined periods, then measure residual activity

• Storage stability: Monitor activity retention at 4°C, -20°C, and -80°C over time

• Freeze-thaw stability: Assess activity loss after multiple freeze-thaw cycles

When conducting these experiments, researchers should consider that E. faecalis is known to adapt to various environmental stresses, particularly in biofilm formation contexts . Consequently, its enzymes might display unique stability profiles compared to homologs from other species. Additionally, the relationship between enzyme activity and the bacterium's ability to form biofilms under stress conditions could provide insights into potential inhibition strategies.

How does purM activity correlate with biofilm formation in E. faecalis?

Recent proteomics studies have revealed a significant correlation between purM expression and biofilm formation in E. faecalis. Notably, enzymes involved in de novo purine biosynthesis were found to be enriched in E. faecalis biofilms, particularly in response to certain environmental conditions . This suggests that purM and related enzymes may play crucial roles beyond basic metabolism in biofilm development and maintenance.

When investigating this correlation, researchers should employ the following methodological approaches:

• Comparative Proteomics Analysis:

  • Compare protein expression profiles between planktonic and biofilm growth states

  • Quantify purM levels using targeted proteomics approaches (MRM-MS)

  • Analyze co-expression patterns with other biofilm-associated proteins

• Gene Knockout/Knockdown Studies:

  • Create purM deletion mutants or employ CRISPR interference for knockdown

  • Assess biofilm formation capacity using crystal violet assays and confocal microscopy

  • Evaluate biofilm matrix composition changes in purM-deficient strains

• Complementation and Overexpression:

  • Restore purM expression in mutants using controlled expression systems

  • Assess whether purM overexpression enhances biofilm formation

  • Use nisin-inducible promoter systems for controlled expression

Research has demonstrated that in response to epigallocatechin gallate (EGCG), E. faecalis significantly upregulates proteins associated with stress responses and purine biosynthesis, while simultaneously enhancing biofilm formation . This contrasts with other microorganisms like S. lugdunensis, where EGCG reduces biofilm formation, highlighting the unique adaptation mechanisms of E. faecalis.

Table 2: Key Proteins Co-expressed with purM in E. faecalis Biofilms

These findings suggest that targeting purM could potentially disrupt E. faecalis biofilm formation, offering a novel approach for controlling infections caused by this opportunistic pathogen .

How can structure-based drug design be applied to develop inhibitors targeting E. faecalis purM?

Structure-based drug design (SBDD) targeting E. faecalis purM represents a promising approach for developing novel antimicrobials against this opportunistic pathogen. The following methodological framework is recommended for researchers pursuing this direction:

• Structural Characterization:

  • Generate homology models based on crystal structures of purM from related organisms

  • If possible, determine the crystal structure of E. faecalis purM using X-ray crystallography

  • Perform molecular dynamics simulations to identify flexible regions and potential allosteric sites

• Virtual Screening Approach:

  • Identify key binding pockets, including the ATP-binding site and substrate-binding region

  • Conduct in silico docking studies using chemical libraries (e.g., ZINC database)

  • Prioritize compounds based on predicted binding energy and interactions with catalytic residues

• Rational Design Strategy:

  • Design ATP-competitive inhibitors with specificity for bacterial purM

  • Exploit structural differences between bacterial and human purine biosynthesis enzymes

  • Develop transition-state mimetics based on the enzymatic reaction mechanism

• Experimental Validation:

  • Synthesize or acquire top candidate compounds

  • Perform enzyme inhibition assays to determine IC50 and Ki values

  • Assess antibacterial activity against E. faecalis strains, particularly in biofilm models

When targeting purM, researchers should consider its upregulation in biofilm formation , which suggests that effective inhibitors might show enhanced efficacy against biofilm-associated infections. Additionally, since E. faecalis is known to develop antibiotic resistance , inhibitors targeting metabolic pathways like purine biosynthesis may offer alternative treatment strategies for resistant strains.

The development pipeline should include assessment of inhibitor specificity by testing against human purine biosynthesis enzymes to minimize potential toxicity. Additionally, researchers should evaluate inhibitor efficacy against both planktonic cells and established biofilms, as the latter are known to be more resistant to conventional antibiotics.

How does purM expression change during E. faecalis adaptation to different environmental stresses?

Environmental stress adaptation in E. faecalis involves complex transcriptional and translational reprogramming, with purM playing a potentially significant role. Recent proteomics studies have revealed distinct expression patterns of purM and related enzymes under various stress conditions . The following methodological approaches are recommended for investigating these adaptations:

• Stress Response Profiling:

  • Subject E. faecalis cultures to relevant stresses (antimicrobial agents, pH extremes, nutrient limitation)

  • Perform time-course transcriptomics and proteomics analyses

  • Use RT-qPCR to validate purM expression changes under different conditions

• Biofilm vs. Planktonic Comparison:

  • Compare purM expression levels between biofilm and planktonic growth states

  • Assess how environmental stressors differentially affect expression in these two growth modes

  • Evaluate correlation between purM expression and biofilm formation capacity

Studies have demonstrated that exposure to epigallocatechin gallate (EGCG) induces significant upregulation of stress-responsive proteins and enzymes involved in gluconeogenesis in E. faecalis biofilms . Notably, enzymes involved in de novo purine biosynthesis, including purM, were among the proteins enriched in EGCG-responsive biofilms, suggesting their role in adaptation to this specific stress.

Table 3: purM Expression Changes Under Different Stress Conditions

These findings suggest that purM expression is dynamically regulated in response to environmental challenges, potentially contributing to E. faecalis persistence and virulence in clinical settings. This knowledge could inform the development of targeted interventions that disrupt adaptive responses by inhibiting purM activity.

What are the most effective genetic tools for manipulating the purM gene in E. faecalis?

Genetic manipulation of E. faecalis requires specialized approaches due to its unique genetic characteristics and frequent recalcitrance to standard transformation methods. For purM studies, the following techniques have proven most effective:

• Allelic Exchange Mutagenesis:

  • Utilize temperature-sensitive plasmids (e.g., pG+host) for homologous recombination

  • Design constructs with approximately 1 kb homology arms flanking the purM gene

  • Select recombinants using appropriate antibiotic markers

  • Verify gene replacement by PCR and sequencing

• CRISPR-Cas9 System Adaptation:

  • Design sgRNAs targeting purM with minimal off-target effects

  • Utilize Cas9 expressing vectors optimized for Gram-positive bacteria

  • Include homology-directed repair templates for precise modifications

  • Screen transformants by phenotypic assays and sequencing

• Inducible Expression Systems:

  • Implement nisin-inducible promoter systems for controlled gene expression

  • Use the P-nisA promoter with the two-component nisin sensor system (nisR/nisK)

  • Amplify these elements using PCR with appropriate primers (e.g., PNISaF/PNISR)

  • Clone into suitable vectors for E. faecalis transformation

• Phage-Based Delivery Systems:

  • Adapt temperate bacteriophages like φEf11 for genetic delivery

  • Create recombinant phages by replacing lysogeny modules with expression cassettes

  • Transduce E. faecalis with modified phages for gene delivery

When implementing these techniques, researchers should consider the high frequency of lysogeny in E. faecalis strains, which may affect phage-based approaches. For example, the φEf11 prophage is widely disseminated among E. faecalis strains , which could impact the efficiency of phage-based delivery systems. Additionally, modifications to the phage genome, such as deletion of the putative lysogeny gene module (ORFs 31-36) and replacement of the putative cro promoter with a nisin-inducible promoter, have been shown to enhance virus infectivity and extend host range .

How can I develop an E. faecalis strain with regulated purM expression for functional studies?

Developing E. faecalis strains with precisely regulated purM expression requires a strategic approach combining appropriate genetic tools and careful experimental design. The following methodological workflow is recommended:

• Design of Expression Constructs:

  • Create a nisin-inducible purM expression system using the following components:

    • The purM gene with native ribosome binding site

    • The nisin-inducible promoter (P-nisA)

    • The two-component nisin sensor system (nisR/nisK) for activation

    • An appropriate antibiotic selection marker (e.g., erythromycin resistance)

  • Clone these elements into a suitable E. faecalis shuttle vector

• Integration Strategies:

  • Option A: Chromosomal integration at a neutral site

    • Identify a non-essential genomic region for integration

    • Design homology arms (~1 kb) flanking the integration site

    • Use allelic exchange or CRISPR-Cas9 for precise integration

  • Option B: Complementation in purM deletion background

    • First create a purM deletion mutant

    • Introduce the regulated expression construct

    • Select for appropriate antibiotic resistance

• Validation of Expression Control:

  • Verify construct integration by PCR and sequencing

  • Quantify purM expression at different nisin concentrations using RT-qPCR

  • Confirm protein production using Western blotting or targeted proteomics

  • Perform functional assays to assess phenotypic complementation

For optimal results, researchers should consider the insights from genetic modification studies of E. faecalis phages . These studies demonstrate the successful use of nisin-inducible systems and highlight considerations for genetic manipulation in this organism. The PCR amplification of the nisin-inducible expression cassette can be performed using the AccuPrime DNA Taq Polymerase High Fidelity kit with specific primers like PNISaF/PNISR , followed by cloning into appropriate vectors.

Table 4: Troubleshooting Regulated Expression in E. faecalis

What considerations are important when creating purM knockout strains in E. faecalis?

Creating and working with purM knockout strains in E. faecalis requires careful attention to several critical factors due to the essential nature of the purine biosynthesis pathway. The following methodological considerations should guide this process:

• Viability Assessment and Media Supplementation:

  • Evaluate whether purM is essential under your specific experimental conditions

  • Supplement growth media with adenine (50-100 μg/ml) and/or hypoxanthine (50 μg/ml) to bypass the need for de novo purine synthesis

  • Consider using minimal media supplemented with defined purine sources to control pathway complementation

• Knockout Strategy Selection:

  • Complete deletion: Remove the entire purM coding sequence using homologous recombination

  • Partial inactivation: Insert a stop codon or frameshift mutation early in the gene

  • Conditional knockout: Place purM under an inducible promoter and create the knockout in the presence of inducer

• Phenotypic Characterization Protocol:

  • Assess growth rates in both rich and minimal media with and without purine supplementation

  • Evaluate biofilm formation capacity using crystal violet assays and confocal microscopy

  • Determine stress resistance profiles under various environmental challenges

  • Measure competitive fitness in mixed cultures with wild-type strains

• Genetic Stability Concerns:

  • Monitor for suppressor mutations that might arise during propagation

  • Regularly verify the knockout by PCR and sequencing

  • Consider maintaining selective pressure if using a marked deletion

• Complementation Controls:

  • Develop a complementation strain expressing purM from a controlled promoter

  • Include both native promoter and inducible promoter versions for comprehensive analysis

  • Utilize the nisin-inducible system as demonstrated effective in E. faecalis

When creating purM knockout strains, researchers should be aware that E. faecalis can form biofilms as an adaptive response to stress , and gene expression patterns in biofilms differ significantly from planktonic growth. Proteomics studies have shown that enzymes involved in de novo purine biosynthesis are enriched in E. faecalis biofilms under certain conditions , suggesting that purM knockout strains might exhibit altered biofilm formation capacity. This potential phenotype should be carefully evaluated as part of the characterization process.

How can I troubleshoot low activity of recombinant E. faecalis purM?

Low activity of recombinant E. faecalis purM is a common challenge that can arise from multiple factors throughout the expression and purification process. The following systematic troubleshooting approach is recommended:

• Expression System Optimization:

  • Issue: Improper protein folding in heterologous hosts

  • Solution: Test expression in different E. coli strains (BL21, Rosetta, Arctic Express) or consider Bacillus-based systems more similar to E. faecalis

  • Method: Compare protein solubility and activity across expression systems using SDS-PAGE and activity assays

• Buffer Composition Assessment:

  • Issue: Suboptimal buffer conditions affecting enzyme stability or activity

  • Solution: Screen buffer compositions varying pH (6.5-8.5), salt concentration (50-300 mM NaCl), and additives

  • Method: Prepare a buffer matrix and measure enzyme activity after short-term (1 hour) and long-term (24 hour) incubation

Table 5: Buffer Optimization Matrix for E. faecalis purM

• Cofactor Requirements:

  • Issue: Missing or insufficient cofactors

  • Solution: Ensure adequate Mg²⁺ (required for ATP binding) and test other divalent cations (Mn²⁺, Ca²⁺)

  • Method: Activity assays with varying concentrations of different metal ions

• Protein Structural Issues:

  • Issue: Misfolding or aggregation

  • Solution: Add solubilizing agents or molecular chaperones during expression/purification

  • Method: Size exclusion chromatography to assess oligomeric state; circular dichroism to evaluate secondary structure

• Substrate Quality:

  • Issue: Degraded or impure substrates

  • Solution: Use freshly prepared ATP and verify substrate purity

  • Method: HPLC analysis of substrate quality before use in assays

When troubleshooting recombinant purM activity, consider that E. faecalis is known to adapt to various environmental stresses , and its enzymes may have evolved specific requirements for optimal activity. The proteomics analysis of E. faecalis biofilms has shown that stress-responsive proteins and metabolic enzymes can be significantly upregulated under certain conditions , suggesting that purM may also function optimally under specific physiological contexts.

What are the best approaches for analyzing purM structure-function relationships?

Investigating structure-function relationships in E. faecalis purM requires an integrated approach combining computational predictions, targeted mutagenesis, and functional assays. The following methodological framework is recommended:

• Computational Structure Analysis:

  • Generate homology models based on crystal structures of purM from related organisms

  • Perform molecular dynamics simulations to identify flexible regions and conformational changes

  • Use in silico docking to predict substrate binding modes and catalytic mechanisms

  • Identify conserved residues through multiple sequence alignment of purM from diverse bacterial species

• Site-Directed Mutagenesis Strategy:

  • Target catalytic residues predicted to be involved in substrate binding or catalysis

  • Create conservative mutations (e.g., D→E, K→R) to assess the importance of specific chemical properties

  • Generate alanine-scanning mutants across predicted functional regions

  • Design mutations that might enhance catalytic efficiency based on computational predictions

Table 6: Priority Residues for Site-Directed Mutagenesis in E. faecalis purM

• Functional Characterization Workflow:

  • Express and purify wild-type and mutant proteins using identical protocols

  • Perform thermal stability assays to ensure mutations don't simply destabilize the protein

  • Conduct detailed kinetic analyses to determine changes in Km, kcat, and substrate specificity

  • Use circular dichroism or other structural techniques to confirm integrity of protein fold

• Structure-Based Inhibitor Design:

  • Utilize structural insights to design potential inhibitors

  • Perform in silico screening followed by experimental validation

  • Test inhibitors against wild-type and key mutant proteins to confirm binding mode

When analyzing structure-function relationships in E. faecalis purM, consider the environmental adaptations of this organism, particularly in biofilm contexts . The upregulation of purine biosynthesis enzymes in biofilms suggests that purM may have evolved specific structural features to optimize function under these conditions. Additionally, the genetic manipulation techniques developed for E. faecalis, including the nisin-inducible expression systems , can be adapted for expressing and testing mutant versions of purM in vivo.

How do I interpret contradictory results between in vitro purM activity and in vivo growth phenotypes?

Discrepancies between in vitro enzyme activity measurements and in vivo phenotypic observations are common challenges in enzyme research, particularly with metabolic enzymes like purM. The following methodological framework helps reconcile and interpret such contradictions:

• Systematic Analysis of Discrepancies:

  • Document specific contradictions between in vitro and in vivo results

  • Evaluate experimental conditions for both types of experiments

  • Consider whether the contradictions follow any patterns related to growth conditions or enzyme properties

• In Vivo Factors to Consider:

  • Metabolic context: Alternative pathways may compensate for purM deficiency

  • Regulatory mechanisms: Expression level may not correlate with enzyme amount used in vitro

  • Substrate availability: Intracellular concentrations of substrates may differ from in vitro conditions

  • Protein-protein interactions: purM may interact with other cellular components not present in vitro

• Methodological Approaches to Resolve Contradictions:

  • Metabolomics analysis: Measure purine intermediate levels in vivo to assess pathway flux

  • In-cell enzyme activity: Develop assays to measure purM activity in cell lysates or permeabilized cells

  • Protein localization studies: Determine if purM localization affects its function in vivo

  • Interactome analysis: Identify potential protein partners that may modulate purM activity

• Experimental Design for Resolution:

  • Create a comprehensive experimental matrix varying both in vitro conditions and in vivo growth parameters

  • Test enzyme activity across a range of physiologically relevant conditions

  • Use genetic approaches to modulate purM expression levels and correlate with phenotypic outcomes

These contradictions may be particularly relevant in the context of E. faecalis biofilm formation, where proteomics studies have shown significant reprogramming of metabolic pathways . The enrichment of purine biosynthesis enzymes in biofilms suggests that their function may be optimized for this growth mode, potentially explaining discrepancies when compared to planktonic growth conditions.

Table 7: Common Contradictions and Resolution Strategies in purM Research

When interpreting contradictory results, researchers should consider E. faecalis' ability to adapt to different environments, particularly in healthcare settings where it has emerged as a significant opportunistic pathogen . The bacterium's capacity to form biofilms and develop antibiotic resistance may involve complex metabolic adaptations that affect purM function in ways not easily replicated in vitro.