Recombinant Enterococcus faecalis Formamidopyrimidine-DNA glycosylase (mutM)

What is Formamidopyrimidine-DNA glycosylase (MutM) and what is its primary function in bacterial cells?

Formamidopyrimidine-DNA glycosylase, also known as MutM or Fpg protein, is a bifunctional DNA glycosylase that initiates the base excision repair pathway in bacteria. Its primary function is to recognize and remove oxidatively damaged DNA bases, particularly 8-oxo-7,8-dihydroguanine (8-oxoG) and formamidopyrimidine (Fapy) lesions. After removing the damaged base, MutM possesses an AP lyase activity that cleaves the DNA backbone at the resulting abasic site. This repair mechanism is critical for maintaining genomic integrity by preventing G:C→T:A transversion mutations that would otherwise occur when 8-oxoG mispairs with adenine during DNA replication .

How does the structure of E. faecalis MutM compare to well-characterized MutM proteins from other bacteria like E. coli?

While the search results don't provide specific structural comparisons between E. faecalis MutM and other bacterial MutM proteins, we can infer that they share considerable structural and functional conservation. The E. coli Fpg/MutM protein contains a zinc finger domain that is essential for its DNA binding and catalytic functions . The crystal structure of MutM from Thermus thermophilus has also been determined, providing insights into the enzyme's mechanism of action . E. faecalis MutM likely shares these conserved structural features, given that Fpg/MutM is described as a "functionally conserved repair enzyme" across bacterial species . The specific structural differences would require detailed structural analysis through crystallography or computational modeling.

What are the substrate specificities of recombinant E. faecalis MutM?

E. faecalis MutM, like other bacterial Fpg/MutM proteins, primarily recognizes and excises oxidatively damaged DNA bases. Its main substrates include:

• 8-oxo-7,8-dihydroguanine (8-oxoG), a common product of oxidative damage to guanine

• Formamidopyrimidine (Fapy) lesions derived from purine bases

• Other oxidized purine derivatives
  The enzyme exhibits bifunctional activity: it first removes the damaged base through N-glycosylase activity, then cleaves the phosphodiester backbone at the resulting abasic site through AP lyase activity . This dual functionality enables efficient processing of oxidative lesions in bacterial DNA. The precise kinetic parameters and relative efficiencies for different substrates specifically for E. faecalis MutM would require experimental determination through biochemical assays.

How does E. faecalis MutM contribute to bacterial oxidative stress resistance?

E. faecalis MutM plays a critical role in protecting bacterial cells against oxidative stress by initiating the removal of oxidative DNA lesions. Reactive oxygen species (ROS) produced during cellular metabolism or from environmental sources cause various types of DNA damage, with 8-oxoG being one of the most prevalent and mutagenic. By efficiently recognizing and removing these damaged bases, MutM prevents the accumulation of mutations that would otherwise compromise genomic integrity and cellular function .
In mycobacteria with G+C rich genomes (which may have parallels in E. faecalis), MutM has been shown to play "a distinct role in down-regulation of accumulation of G, C mutations and protection against oxidative stress" . Given that E. faecalis encounters oxidative stress during host-pathogen interactions and antibiotic exposure, MutM likely contributes significantly to its survival and persistence in these challenging environments.

What is the relationship between MutM and other DNA repair pathways in E. faecalis?

MutM functions as part of the "GO system" (guanine oxidation), a specialized DNA repair system that prevents mutations caused by oxidized guanine. While the search results don't specifically describe the interaction between MutM and other repair pathways in E. faecalis, information from studies in other bacteria suggests that MutM works in coordination with:

• MutY - Another glycosylase that removes adenine incorrectly paired with 8-oxoG

• MutT - A nucleotide sanitization enzyme that hydrolyzes 8-oxo-dGTP to prevent its incorporation into DNA
  Together, these enzymes form a comprehensive defense against the mutagenic effects of oxidized guanine. In Pseudomonas aeruginosa, inactivation of mutY and mutM (both involved in the GO system) led to "elevated mutation rates that correlated to increased development" of antibiotic resistance . This suggests that in E. faecalis, MutM likely works in concert with other DNA repair pathways to maintain genomic stability.

What phenotypes are associated with E. faecalis mutM deletion mutants?

Based on studies in other bacterial species, E. faecalis mutM deletion mutants would likely exhibit:

• Increased spontaneous mutation rates, particularly G:C→T:A transversions

• Enhanced sensitivity to oxidative stress

• Potential hyperrecombination phenotypes (as indicated by E. coli studies showing that "mutM suppresses illegitimate recombination induced by oxidative stress" )

• Altered virulence or persistence in infection models
  In Pseudomonas aeruginosa, inactivation of mutM led to elevated mutation rates that correlated with increased development of antibiotic resistance . Similarly, in mycobacteria, MutM has been shown to be important for protection against oxidative stress and limiting G,C mutations . E. faecalis mutM mutants would likely show comparable phenotypes, though the exact manifestations might differ based on the specific ecological niche and genomic context of this organism.

What are the optimal conditions for expressing and purifying recombinant E. faecalis MutM?

While the search results don't provide specific protocols for E. faecalis MutM expression and purification, general approaches for recombinant bacterial DNA repair enzymes can be adapted:
Expression System Options:

• E. coli BL21(DE3) or derivatives with a pET vector system

• Cold-induction expression to improve protein folding

• Fusion tags such as His6, GST, or MBP to facilitate purification
  Purification Strategy:

• Affinity chromatography using the fusion tag

• Ion exchange chromatography (typically on a heparin column due to DNA-binding properties)

• Size exclusion chromatography for final polishing
  Buffer Considerations:

• Include reducing agents (DTT or β-mercaptoethanol) to maintain cysteine residues in the zinc finger domain

• Add zinc or other divalent cations if required for structural integrity

• Consider glycerol (10-20%) for stability during storage
  Commercial recombinant Fpg protein from E. coli is available as "buffered aqueous glycerol solution" with activity >20,000 units/mg protein , suggesting similar conditions might be appropriate for E. faecalis MutM.

What assays can be used to measure the enzymatic activity of recombinant E. faecalis MutM?

Several established assays can be adapted to measure E. faecalis MutM activity:
1. Oligonucleotide Cleavage Assay:

• Synthetic DNA substrates containing 8-oxoG or other damaged bases

• Fluorescent or radioactive labeling for detection

• Analysis by denaturing PAGE to visualize cleavage products
  2. Circular Plasmid Nicking Assay:

• Plasmid DNA treated with oxidizing agents to create substrate lesions

• Incubation with MutM converts supercoiled DNA to nicked form

• Agarose gel electrophoresis to separate and quantify different topological forms
  3. Enzyme-Coupled Spectrophotometric Assays:

• Monitoring release of modified bases through coupled enzymatic reactions

• Real-time measurement of activity using absorbance or fluorescence changes
  4. DNA Glycosylase Activity Measurement by HPLC:

• Quantification of released damaged bases by HPLC analysis

• Allows precise determination of substrate specificity and kinetic parameters
  Activity measurements should include appropriate controls (heat-inactivated enzyme, no-enzyme controls) and can be compared against commercially available E. coli Fpg/MutM as a standard .

How stable is recombinant E. faecalis MutM and what are the best storage conditions?

Based on information available for similar recombinant DNA repair enzymes:
Storage Conditions:

• Short-term (weeks): -20°C in buffer containing 50% glycerol

• Long-term (months to years): -80°C, preferably in small single-use aliquots
  Stability Considerations:

• Include reducing agents (1-5 mM DTT) to protect cysteine residues

• Add stabilizing agents such as BSA (0.1 mg/ml) or glycerol (10-50%)

• Avoid repeated freeze-thaw cycles
  Commercial recombinant Fpg protein is supplied as a "buffered aqueous glycerol solution" , suggesting that glycerol is an important component for stability. The enzyme should be kept on ice during experiments and returned to storage promptly after use to maximize activity retention.

What are the challenges in expressing recombinant E. faecalis MutM in heterologous systems?

Expressing E. faecalis proteins in heterologous systems presents several challenges:
Restriction Modification (RM) Barriers:
E. faecalis contains multiple RM systems that can impede DNA transfer and genetic manipulation. These include type I, II, and IV RM systems that recognize and degrade foreign DNA based on methylation patterns . To overcome these barriers when working with E. faecalis genes:

• Use E. coli strains lacking specific methylation activities (like DC10B lacking dcm methylation)

• Passage plasmids through restriction-deficient intermediate hosts

• Consider in vitro methylation of plasmid DNA prior to transformation
  Codon Usage Differences:
  E. faecalis has different codon preferences compared to common expression hosts like E. coli. This may necessitate:

• Codon optimization of the mutM gene sequence

• Use of rare codon tRNA supplementation in expression strains

• Selection of expression hosts with compatible codon usage patterns
  Protein Folding and Activity:
  Ensuring proper folding and activity requires consideration of:

• Expression temperature (often lowered to improve folding)

• Co-expression with chaperones

• Inclusion of zinc or other cofactors in growth media

What vector systems are most appropriate for complementation studies with E. faecalis mutM?

For complementation of E. faecalis mutM mutants, several vector systems can be considered:
Shuttle Vectors:
Shuttle vectors that can replicate in both E. coli and E. faecalis are commonly used for complementation studies. These allow for easy manipulation in E. coli followed by transfer to E. faecalis .
Chromosomal Integration:
For single-copy complementation (which minimizes gene dosage effects):

• Identify a neutral chromosomal site for integration (between convergent genes)

• Use allelic exchange vectors like pCJK47 with counterselection systems

• This approach has the advantage of not requiring antibiotic selection to maintain the gene of interest
  Temperature-Sensitive Plasmids:
  Advanced allelic exchange vectors like pIMAY-Z (which has been successfully used in E. faecium) provide:

• Temperature-sensitive replication

• Blue/white screening for plasmid loss

• Strong antibiotic selection
  When designing complementation experiments, it's important to ensure the gene is under control of its native promoter or an appropriate substitute to maintain physiological expression levels.

What methodologies work best for creating precise mutM deletions or modifications in E. faecalis?

Creating precise genetic modifications in E. faecalis can be challenging due to RM barriers, but several approaches have proven successful:
Allelic Exchange with Counter-selection:

• Use of pCJK47 counterselection system for marker-free deletions

• Incorporation of silent restriction sites to mark complemented strains

• Whole genome sequencing to confirm no secondary mutations occurred
  Transposon Mutagenesis:
  For random mutagenesis approaches:

• High-density transposon mutant libraries have been successfully created in Enterococci

• Transposon sequencing (Tn-seq) can identify insertion sites

• This approach allows identification of essential genes or those with reduced fitness under specific conditions
  CRISPR-Cas Based Methods:
  Although not specifically mentioned in the search results, CRISPR-Cas systems have been adapted for use in Enterococci and could provide efficient gene editing capabilities for mutM modification.
  When making genetic modifications, it's essential to confirm the genotype through sequencing and validate the phenotype through complementation studies to ensure observed effects are due to the intended mutation .

How can recombinant E. faecalis MutM be used to study oxidative DNA damage in vitro?

Recombinant E. faecalis MutM serves as a valuable tool for investigating oxidative DNA damage through several experimental approaches:
1. Detection of Oxidative Lesions in Isolated DNA:

• Purified MutM can be used to detect and quantify 8-oxoG and other oxidative lesions in genomic DNA

• Combined with PCR or sequencing methods, this can map oxidative damage hotspots in genomes

• Comparison with other glycosylases can provide a comprehensive profile of damage types
  2. Mechanistic Studies of DNA Repair:

• Using defined DNA substrates with specific lesions to study repair kinetics

• Investigation of sequence context effects on lesion recognition

• Structure-function analyses through site-directed mutagenesis
  3. Biochemical Analysis of Oxidative Stress:

• Assessment of DNA damage levels under various oxidative stress conditions

• Comparative analysis between wild-type and mutant enzymes

• Evaluation of potential inhibitors or modulators of repair activity
  The high activity of purified recombinant Fpg/MutM (>20,000 units/mg protein ) makes it suitable for sensitive detection of even low levels of oxidative DNA damage.

What role does E. faecalis MutM play in antibiotic resistance development?

The relationship between MutM function and antibiotic resistance development is complex and significant:
Prevention of Hypermutation:
Functional MutM prevents accumulation of mutations by repairing oxidative DNA damage. In P. aeruginosa, inactivation of mutM and mutY "led to elevated mutation rates that correlated to increased development" of antibiotic resistance . This suggests that MutM normally restrains mutation-driven resistance development.
Connection to Oxidative Stress from Antibiotics:
Many antibiotics induce oxidative stress as part of their killing mechanism. MutM's role in managing oxidative DNA damage likely influences bacterial survival during antibiotic exposure.
Balance with Adaptive Evolution:
While MutM generally reduces mutation rates, its temporary inactivation during stress might theoretically permit accelerated adaptation. This balance between genomic stability and adaptive potential could influence E. faecalis population dynamics during antibiotic treatment.
Experimental Approach:
To study this relationship, researchers could:

• Compare mutation frequencies and antibiotic resistance development rates between wild-type and mutM-deficient strains

• Analyze mutation spectra in resistant isolates to identify signature G:C→T:A transversions

• Measure MutM expression and activity during antibiotic challenge

How can researchers distinguish between the activities of different DNA glycosylases when studying oxidative damage repair in E. faecalis?

Distinguishing between different DNA glycosylases requires careful experimental design:
Substrate Specificity Analysis:
Different glycosylases show preferences for specific lesions:

• MutM/Fpg: primarily recognizes 8-oxoG and Fapy lesions

• Nth (Endonuclease III): targets oxidized pyrimidines

• MutY: removes adenine mispaired with 8-oxoG or G
  By using defined substrates containing specific lesions, researchers can differentiate the activities of these enzymes.
  Biochemical Assays with Specific Inhibitors:

What insights can comparative studies of MutM across different bacterial species provide about E. faecalis DNA repair mechanisms?

Comparative studies of MutM across bacterial species offer valuable insights into E. faecalis DNA repair:
Evolutionary Conservation and Adaptation:

• Core functional domains are highly conserved across diverse bacteria

• Species-specific adaptations may reflect ecological niches and stress exposures

• Variations in substrate specificity or catalytic efficiency could reveal unique aspects of E. faecalis DNA metabolism
  Structural-Functional Relationships:
  Comparing the crystal structure of MutM from thermophilic bacteria (like Thermus thermophilus ) with E. faecalis MutM can reveal:

• Critical residues for substrate recognition

• Thermostability determinants

• Regulatory interfaces with other proteins
  Integration with Other Repair Pathways:
  Different bacteria may show variations in how MutM integrates with other repair systems:

• Backup pathways when MutM is absent

• Regulatory cross-talk between repair systems

• Species-specific repair modulators
  By systematically analyzing these differences, researchers can develop targeted hypotheses about E. faecalis-specific DNA repair mechanisms that could influence its biology in clinical and environmental settings.

How might the function of MutM be affected in E. faecalis biofilms?

The function of MutM in E. faecalis biofilms likely reflects the unique physiological and stress conditions of this growth mode:
Altered Expression and Activity:

• Transcriptomic data from other bacteria suggest DNA repair genes may be differentially regulated in biofilms

• Oxygen gradients within biofilms could create zones with varying oxidative stress levels

• Nutrient limitation may impact protein synthesis and turnover of repair enzymes
  Biofilm-Specific Challenges:

• Increased extracellular DNA (eDNA) from autolysis may compete for repair enzymes

• Slower growth rates could affect the balance between damage accumulation and repair

• Enhanced horizontal gene transfer in biofilms might interact with MutM's role in suppressing recombination
  Experimental Approaches:
  To investigate these effects, researchers could:

• Compare mutM expression and MutM activity between planktonic and biofilm cells

• Assess mutation frequencies and spectra in biofilm-grown wild-type vs. mutM mutants

• Analyze the impact of oxidative stress specifically on biofilm formation and stability
  Understanding MutM function in biofilms has particular relevance for E. faecalis infections, as biofilms contribute significantly to its persistence in clinical settings.

What is the relationship between E. faecalis MutM activity and virulence in infection models?

The relationship between MutM activity and virulence involves several interconnected aspects:
Oxidative Stress During Host-Pathogen Interaction:

• Phagocytes produce reactive oxygen species as antimicrobial defense

• MutM helps bacteria withstand this oxidative attack by maintaining genomic integrity

• Loss of MutM function may reduce bacterial survival during infection
  Mutation Rate and Adaptation:

• MutM prevents mutagenesis, stabilizing the genome

• During infection, controlled mutation rates might balance between maintaining essential functions and adapting to host defenses

• Studies in other pathogens suggest an optimal mutation rate exists for virulence
  Potential Experimental Approaches:

• Compare virulence of wild-type and mutM-deficient E. faecalis in various infection models

• Analyze mutation accumulation during the course of infection

• Measure expression of mutM in different infection sites and stages

• Test for correlations between MutM activity and known virulence factors
  This research direction could reveal whether targeting DNA repair systems might represent a novel approach to reducing E. faecalis virulence or persistence.

What are common pitfalls when working with recombinant DNA glycosylases and how can they be avoided?

When working with recombinant DNA glycosylases like E. faecalis MutM, researchers should be aware of these common challenges:
Activity Loss During Purification:

• Oxidation of critical cysteine residues in the zinc finger domain

• Loss of zinc or other cofactors

• Proteolytic degradation
  Solutions:

• Include reducing agents (DTT, β-mercaptoethanol) in all buffers

• Add zinc or other required metal ions

• Use protease inhibitors and work at cold temperatures

• Consider fusion tags that enhance stability
  Non-specific DNA Binding:

• Co-purification of bacterial nucleic acids

• High background in activity assays

• Enzyme precipitation when removing bound DNA
  Solutions:

• Include high salt washes during purification

• Treat with nucleases during purification

• Develop specific activity assays with proper controls
  Reproducibility Issues:

• Variation in substrate quality and quantity

• Inconsistent reaction conditions

• Enzyme batch-to-batch variation
  Solutions:

• Standardize substrate preparation

• Use internal standards and positive controls

• Validate each enzyme batch against known standards
  By anticipating these challenges, researchers can design more robust experimental protocols when working with E. faecalis MutM.

How can researchers optimize transformation protocols for genetic manipulation of E. faecalis strains?

Optimizing transformation protocols for E. faecalis requires addressing several barriers:
Overcoming Restriction Barriers:
E. faecalis contains restriction modification systems that protect against foreign DNA . Strategies to overcome these include:

• Using DNA isolated from the same strain or a strain with compatible methylation patterns

• Passing plasmids through an intermediate E. coli host lacking specific methylation activities

• In vitro methylation of DNA prior to transformation

• Using restriction-deficient but methylation-proficient E. faecalis strains as recipients
  Physical Barriers:
  E. faecalis has a thick cell wall that limits DNA uptake . To address this:

• Optimize growth conditions to produce cells in a transformation-competent state

• Use glycine in growth media to weaken the cell wall

• Test different electroporation parameters (field strength, pulse duration)

• Consider alternative methods like protoplast transformation
  Protocol Optimization:

• Carefully control growth phase and density of recipient cells

• Pre-treat cells with cell wall-weakening agents or glycine

• Test different DNA concentrations and quality

• Optimize recovery conditions post-transformation
  Successful transformation protocols often achieve >10^5 CFU transformants, opening the door to sophisticated genetic techniques including "Mu transposon mutagenesis, construction of genomic, surface display and CRISPRi libraries" and other methods .

What controls and validation steps are essential when characterizing a newly purified recombinant E. faecalis MutM?

Rigorous characterization of newly purified recombinant E. faecalis MutM should include these essential controls and validation steps:
Purity Assessment:

• SDS-PAGE analysis (≥90% purity is typical for functional studies )

• Mass spectrometry confirmation of protein identity

• Western blot with specific antibodies if available
  Activity Validation:

• Comparison with commercially available Fpg/MutM from E. coli

• Dose-dependent activity demonstration

• Heat-inactivated enzyme as negative control

• Substrate specificity confirmation using different DNA lesions
  Biochemical Characterization:

• Determination of optimal pH, temperature, and salt conditions

• Metal ion requirements and inhibitor sensitivity

• Kinetic parameters (Km, kcat) for primary substrates

• Analysis of glycosylase and AP lyase activities separately
  Functional Complementation:

• Ability to restore wild-type phenotype in mutM-deficient strains

• Suppression of spontaneous mutation rates

• Restoration of oxidative stress resistance This comprehensive validation ensures that observations made with the recombinant enzyme accurately reflect the biological activity of native E. faecalis MutM.