Recombinant Enterococcus faecalis DNA mismatch repair protein MutS (mutS), partial

What is the function of MutS in Enterococcus faecalis?

MutS is a highly conserved protein that serves as the initial recognition component in the DNA mismatch repair (MMR) pathway. In E. faecalis and other bacteria, MutS functions by recognizing mismatched nucleotides in the DNA, forming ATP-bound sliding clamps on the DNA. These sliding clamps subsequently load MutL sliding clamps that coordinate mismatch repair excision. This recognition and repair mechanism is essential for maintaining genomic integrity by correcting replication errors that would otherwise lead to mutations .

How does E. faecalis MutS protein structure compare to MutS in other bacterial species?

E. faecalis MutS shares significant structural homology with MutS proteins from other bacterial species, particularly in the domains responsible for mismatch recognition and ATP binding. Like other MutS homologs, it contains classic Walker A/B ATP binding motifs and functions as a homodimer. The protein's N-terminal domain is involved in mismatch recognition, while the C-terminal domain mediates dimerization and ATP hydrolysis. These structural features are conserved across bacterial species, reflecting the fundamental importance of the MMR pathway in all organisms .

What experimental systems are available for studying recombinant E. faecalis MutS?

Researchers can express recombinant E. faecalis MutS using standard molecular cloning techniques in expression systems such as E. coli. The recombinant protein can be purified using affinity chromatography, typically with a histidine tag to facilitate purification. For functional studies, researchers can use in vitro assays that measure DNA binding, ATP hydrolysis, and mismatch recognition activities. Additionally, immobilized MutS can be used in mutation detection assays, allowing for the identification of mismatches and small insertion/deletion mutations without requiring radioactivity or gel electrophoresis .

How is E. faecalis MutS involved in antibiotic resistance mechanisms?

While MutS itself is not directly responsible for antibiotic resistance, its function in DNA mismatch repair affects mutation rates, which can indirectly influence the development of resistance. E. faecalis is intrinsically resistant to several clinically relevant antibiotics and can transfer resistance to other pathogens . Defects in the MMR system, including MutS dysfunction, can lead to increased mutation rates (hypermutator phenotypes), potentially accelerating the evolution of antibiotic resistance. This relationship between MMR function and resistance acquisition makes MutS an important factor in understanding the adaptability of E. faecalis in clinical settings.

What are the recommended protocols for expressing and purifying functional recombinant E. faecalis MutS?

For optimal expression and purification of functional E. faecalis MutS, researchers should consider the following methodology:

• Clone the E. faecalis mutS gene into a vector containing an inducible promoter (such as T7) and an N-terminal or C-terminal affinity tag (6xHis is commonly used)

• Transform the construct into an E. coli expression strain optimized for protein production (e.g., BL21(DE3))

• Induce expression at lower temperatures (16-25°C) to enhance proper folding

• Lyse cells in a buffer containing:

  • 50 mM Tris-HCl (pH 7.5-8.0)

  • 300 mM NaCl

  • 5-10% glycerol

  • 1 mM DTT

  • Protease inhibitors

• Purify using nickel affinity chromatography followed by size exclusion chromatography

• Verify protein activity through DNA binding and ATPase assays

The critical step is maintaining the native dimeric state of MutS, as the functional unit is a homodimer that undergoes conformational changes upon ATP binding and DNA interaction .

How can researchers assess the DNA binding and mismatch recognition capabilities of recombinant E. faecalis MutS?

Several experimental approaches can be employed to assess the DNA binding and mismatch recognition capabilities of recombinant E. faecalis MutS:

• Electrophoretic Mobility Shift Assays (EMSA): Using synthetic oligonucleotides containing specific mismatches to determine binding specificity and affinity

• Surface Plasmon Resonance (SPR): For quantitative measurement of binding kinetics to different DNA substrates

• Fluorescence Anisotropy: Using fluorescently labeled DNA to measure direct binding in solution

• Immobilized MutS Assays: Where purified MutS is immobilized on a solid support and used to detect and capture mismatched DNA fragments

These methods can be complemented with ATP hydrolysis assays, as ATP binding and hydrolysis are coupled to the mismatch recognition and signaling functions of MutS. Researchers should design DNA substrates containing various types of mismatches to comprehensively characterize the specificity profile of E. faecalis MutS .

What are the current challenges in studying the interaction between E. faecalis MutS and other components of the mismatch repair system?

Current research challenges include:

• Complex formation dynamics: MutS-MutL interactions in E. faecalis are transient and depend on ATP binding states, making them difficult to capture and characterize structurally

• Species-specific differences: Despite conservation, significant differences exist in how MMR is coordinated between gram-positive and gram-negative bacteria

• Strand discrimination mechanisms: Unlike E. coli, which uses dam methylation for strand discrimination, the mechanism in E. faecalis remains poorly understood

• Reconstituting complete MMR in vitro: Establishing a complete in vitro MMR system with all E. faecalis components remains challenging

Recent research suggests that rather than forming static MutS-MutL complexes bound to mismatched DNA via the positively charged cleft (PCC) located on the MutL N-terminal domains, MutS sliding clamps exploit the PCC to position a MutL NTD on the DNA backbone. This enables diffusion-mediated wrapping of the remaining MutL domains around the DNA, forming a MutL sliding clamp that enhances downstream repair activities .

How does MutS function relate to E. faecalis virulence in clinical settings?

While direct evidence linking MutS function to E. faecalis virulence is limited, several indirect connections suggest its importance in pathogenesis:

• Genomic stability: MutS maintains genomic integrity, which is crucial for optimal expression of virulence factors

• Adaptive mutation: Balanced MMR activity allows for sufficient genetic variation without compromising essential cellular functions

• Stress response: Under host-associated stresses, MutS activity may be modulated to allow for increased adaptation

• Biofilm formation: MMR proteins may influence bacterial cell surface properties that affect biofilm formation, a key virulence trait of E. faecalis

E. faecalis has emerged as a major nosocomial pathogen, with intrinsic resistance to several antibiotics . The relationship between MMR function and virulence may be especially important in healthcare settings where selective pressures are strong, potentially affecting the evolution of more virulent or resistant strains.

What methodologies are most effective for studying MutS function in the context of E. faecalis infection models?

To effectively study MutS function in infection contexts, researchers should consider:

• Genetic approaches:

  • Construction of mutS deletion mutants

  • Complementation studies with wild-type and mutant alleles

  • Site-directed mutagenesis of key functional residues

• In vivo infection models:

  • Zebrafish model (has been validated for E. faecalis virulence studies)

  • Mouse urinary tract infection model

  • Caenorhabditis elegans infection model

  • Cell culture infection assays with macrophages and epithelial cells

• Comparative analyses:

  • Competition assays between wild-type and mutS mutant strains in vivo

  • Transcriptomic/proteomic analyses comparing wild-type and mutS mutants during infection

  • Mutation rate analyses under infection-relevant stress conditions

These methodologies can reveal how MutS function influences bacterial fitness, survival, and virulence during host colonization and infection .

How do the biochemical properties of E. faecalis MutS compare to those of MutS homologs in other bacterial pathogens?

Comparative analysis of E. faecalis MutS with homologs from other bacterial pathogens reveals several important distinctions:

What evolutionary adaptations are observed in E. faecalis MutS that might contribute to its pathogenic lifestyle?

Several evolutionary adaptations in E. faecalis MutS may contribute to its success as an opportunistic pathogen:

• Balanced mutation rate: E. faecalis MutS appears optimized to maintain a mutation rate that balances genomic stability with adaptive potential

• Thermal and stress resilience: Structural adaptations that enhance protein stability under the stressful conditions encountered during infection

• Interaction specificity: Optimized interactions with E. faecalis-specific MMR components that may enhance repair efficiency

• Regulatory integration: Potential integration with stress response networks specific to the hospital environment

E. faecalis is highly recombinogenic, with population structure analyses showing significant horizontal gene transfer . The evolution of MutS in this context may reflect a balance between maintaining essential DNA repair functions while allowing sufficient genomic plasticity to adapt to changing environments, including healthcare settings where it has emerged as a significant pathogen .

What are the potential therapeutic applications targeting E. faecalis MutS function?

Targeting MutS function presents several promising therapeutic strategies:

• MMR modulation: Compounds that enhance MutS activity could potentially increase mutation rates beyond viable levels, leading to error catastrophe

• Synthetic lethality: Identifying genes that become essential in MutS-deficient backgrounds could reveal new drug targets

• Biofilm disruption: If MutS influences biofilm formation, targeting this connection could reduce persistent infections

• Resistance prevention: Drugs that maintain MMR function under stress might slow the development of antibiotic resistance

While not directly about MutS, research on E. faecalis has shown that inhibition of septum cleavage during division represents an attractive therapeutic strategy to control infections . Similar approaches targeting fundamental cellular processes like DNA repair could provide novel avenues for combating E. faecalis infections, particularly in hospital settings where this organism poses significant challenges.

What novel experimental techniques could advance our understanding of E. faecalis MutS structure-function relationships?

Emerging techniques that could significantly advance our understanding of E. faecalis MutS include:

• Cryo-electron microscopy: To capture different conformational states of MutS during the mismatch recognition and signaling process

• Single-molecule approaches: Including FRET and optical tweezers to observe real-time dynamics of MutS-DNA interactions

• Hydrogen-deuterium exchange mass spectrometry: To map protein dynamics and conformational changes during mismatch binding and ATP hydrolysis

• Native mass spectrometry: To characterize the composition and stoichiometry of MMR complexes

• In-cell NMR: To study MutS behavior in a near-native environment

• CRISPR-based approaches: For precise genome editing to study MutS variants in their native context

These advanced techniques could help resolve the molecular mechanisms by which MutS recognizes mismatches and initiates repair, particularly focusing on how MutS acts as a clamp loader for MutL, as recent research has revealed this function to be significantly different from previously understood clamp-loading mechanisms .

How might heterologous expression systems be optimized for producing large quantities of functional E. faecalis MutS for structural studies?

Optimizing heterologous expression of E. faecalis MutS requires addressing several challenges:

• Codon optimization: Adjusting the coding sequence to match the codon usage bias of the expression host (typically E. coli)

• Expression constructs:

  • Testing multiple fusion tags (His, MBP, SUMO) for enhanced solubility

  • Creating truncated constructs focusing on specific domains for difficult structural studies

  • Using bicistronic expression of both MutS and MutL for co-expression studies

• Expression conditions:

  • Screening various E. coli strains (BL21(DE3), Rosetta, Arctic Express)

  • Testing induction at different temperatures (16-30°C) and IPTG concentrations

  • Supplementing growth media with ATP or other cofactors

• Purification strategy:

  • Implementing multi-step purification including affinity, ion exchange, and size exclusion chromatography

  • Using ATP-agarose columns for activity-based purification

  • Incorporating on-column refolding protocols if inclusion bodies form

The critical consideration is maintaining the native dimeric state and ensuring the purified protein retains DNA binding and ATP hydrolysis activities. For structural studies, particular attention should be paid to buffer optimization during final concentration steps to prevent aggregation while maintaining protein stability .

What are the most common pitfalls in recombinant E. faecalis MutS expression and purification, and how can they be overcome?

Researchers commonly encounter several challenges when working with recombinant E. faecalis MutS:

• Poor expression levels:

  • Solution: Optimize codon usage for expression host; try different promoters; test expression at lower temperatures

  • Alternative: Use a stronger RBS or consider different E. coli strains like C41/C43 designed for membrane and toxic proteins

• Protein insolubility:

  • Solution: Express as a fusion with solubility-enhancing tags (MBP, SUMO, TrxA); add low concentrations of non-ionic detergents (0.05-0.1% Triton X-100)

  • Alternative: Develop refolding protocols from inclusion bodies using gradual dialysis

• Protein instability after purification:

  • Solution: Include glycerol (10-20%) and reducing agents in storage buffers; avoid freeze-thaw cycles

  • Alternative: Store protein at higher concentrations (>1 mg/ml) with stabilizing additives like arginine

• Loss of functional activity:

  • Solution: Ensure presence of required metal ions (Mg²⁺) and appropriate pH conditions; maintain reducing environment

  • Alternative: Co-purify with short DNA oligonucleotides containing mismatches to stabilize functional conformation

Each of these challenges requires systematic optimization, and conditions successful for MutS from other species may need significant adaptation for E. faecalis MutS .

How can researchers differentiate between specific and non-specific DNA binding activities of recombinant E. faecalis MutS in experimental systems?

Differentiating specific mismatch recognition from non-specific DNA binding requires carefully designed control experiments:

• Competition assays:

  • Perform binding reactions in the presence of excess non-specific competitor DNA

  • Compare binding to perfectly matched versus mismatched DNA substrates of identical sequence

  • Measure displacement of bound MutS from non-specific DNA by adding mismatched DNA

• Mutation analysis:

  • Create point mutations in the mismatch recognition domain of MutS

  • Compare binding profiles of wild-type and mutant proteins

  • Identify mutants with altered specificity but retained DNA binding

• ATP dependence:

  • Analyze binding in the presence and absence of ATP/ADP

  • Specific mismatch binding typically shows distinct ATP-dependent behavior

  • Use non-hydrolyzable ATP analogs to trap specific binding conformations

• Quantitative analysis:

  • Determine binding constants for different DNA substrates

  • Specific binding should show significantly higher affinity for mismatched DNA

  • Analyze binding kinetics, as specific interactions often have different association/dissociation rates

These approaches, particularly when combined, can reliably distinguish the specific mismatch recognition activity of MutS from its general DNA binding properties .