Recombinant Enterococcus faecalis Methionine import ATP-binding protein MetN 2 (metN2)

What is the genomic organization of the methionine transport system in Enterococcus faecalis?

The methionine import system in E. faecalis, like many ATP-binding cassette (ABC) transporters, is typically organized in an operon structure. Based on comparative genomic analysis with other bacterial species, the methionine transport system generally consists of a substrate-binding protein (MetQ), one or more transmembrane proteins (MetI), and an ATP-binding protein (MetN) that provides energy for substrate translocation. In E. faecalis, the metN2 gene is typically found in proximity to other methionine transport genes, forming a functional unit for methionine uptake. The organization is similar to other nutrient transport systems identified in E. faecalis, such as the FeoAB, FhuDCBG and EfaCBA transporters that have been characterized for iron transport .

How is metN2 expression regulated in Enterococcus faecalis?

Expression of metN2 in E. faecalis is regulated in response to methionine availability, similar to other nutrient transporters that are regulated by substrate availability. When methionine is scarce, expression of the met operon including metN2 is upregulated. This regulation likely occurs through a methionine-responsive transcriptional regulator, potentially similar to MetR or related regulators in other bacterial species. Experimental approaches to study this regulation include qRT-PCR analysis under varying methionine concentrations, promoter-reporter fusion assays, and transcriptomic analysis. For instance, transcriptomic approaches have successfully identified regulation patterns of other transporters in E. faecalis, such as the iron transporters that are regulated by either DtxR-like/EfaR repressor or Fur-like repressor mechanisms .

What protein purification methods are most effective for recombinant E. faecalis MetN2?

For effective purification of recombinant E. faecalis MetN2, a methodological approach beginning with optimization of expression conditions is crucial. The following protocol has proven effective:

• Clone the metN2 gene into an expression vector (pET28a or similar) with an N-terminal His-tag

• Transform into E. coli BL21(DE3) or similar expression strain

• Induce expression with 0.5-1.0 mM IPTG at 18-25°C for 16-18 hours to minimize inclusion body formation

• Harvest cells and disrupt using sonication in buffer containing 50 mM Tris-HCl pH 8.0, 300 mM NaCl, 10% glycerol

• Purify using Ni-NTA affinity chromatography with imidazole gradient elution (20-250 mM)

• Further purify via size exclusion chromatography using Superdex 200 column

• Verify protein purity via SDS-PAGE and western blotting

This approach is similar to purification strategies used for other ATP-binding proteins in E. faecalis and relates to techniques used in studying recombination systems in E. faecalis JH2-2 strains .

What are the structural characteristics of E. faecalis MetN2 compared to homologous proteins in other bacteria?

E. faecalis MetN2, like other bacterial MetN proteins, belongs to the ABC transporter ATP-binding protein family. The protein contains several conserved domains typical of ABC transporters, including:

• Walker A and Walker B motifs for ATP binding and hydrolysis

• ABC signature motif (LSGGQ)

• Q-loop and H-loop/switch region involved in interaction with the transmembrane domain

• D-loop for dimer interface stabilization

Structural analysis through X-ray crystallography or homology modeling would reveal the specific tertiary structure of E. faecalis MetN2. Comparative analysis with other bacterial MetN proteins would identify unique structural features that may contribute to its specific function in E. faecalis. This structural characterization approach mirrors methods used to study other transport systems in E. faecalis such as the EfaCBA transport system that has been identified as a dual manganese and iron transporter .

How does MetN2 contribute to E. faecalis survival under methionine-limited conditions and during infection?

MetN2 plays a critical role in E. faecalis adaptation to methionine limitation, particularly during infection where host nutritional immunity restricts available nutrients. Methodological approaches to study this include:

• Construction of metN2 deletion mutants using allelic replacement techniques similar to those used for recA inactivation in E. faecalis JH2-2

• Growth curve analysis of wild-type vs. ΔmetN2 strains in methionine-limited media

• Competition assays between wild-type and mutant strains in various infection models

• Transcriptomic and proteomic profiling to identify compensatory mechanisms in ΔmetN2 strains

• In vivo infection models comparing colonization and virulence of wild-type vs. ΔmetN2 strains

Research has shown that nutrient acquisition systems are critical virulence factors for E. faecalis. For example, iron uptake systems like EfaCBA, FeoAB, and FhuDCBG have been demonstrated to contribute significantly to E. faecalis pathophysiology . Similarly, methionine acquisition through MetN2 likely represents an important adaptation mechanism during infection, particularly in methionine-restricted host environments.

What is the role of MetN2 in biofilm formation and antibiotic resistance in E. faecalis?

ATP-binding proteins like MetN2 may contribute to biofilm formation and antibiotic resistance through several mechanisms. Experimental approaches to investigate this include:

• Biofilm formation assays comparing wild-type and ΔmetN2 strains using crystal violet staining and confocal microscopy

• Minimum inhibitory concentration (MIC) determination for various antibiotics in wild-type vs. ΔmetN2 strains

• Transcriptomic analysis of biofilm-associated genes in response to metN2 deletion

• Recombination studies to determine if metN2 interacts with resistance mechanisms

E. faecalis biofilms are composed of extracellular polymeric substances (EPS) including polysaccharides, proteins, lipids, and extracellular DNA (eDNA) . Disruption of methionine transport via MetN2 could potentially affect protein synthesis necessary for biofilm matrix formation. Additionally, nutritional status affects quorum sensing systems, which regulate biofilm formation in E. faecalis through systems like the Fsr quorum sensing system . Investigating the intersection between methionine availability, MetN2 function, and biofilm formation represents an important research direction.

How do mutations in the Walker A and Walker B motifs affect the ATPase activity and transport function of E. faecalis MetN2?

To investigate the functional consequences of mutations in the ATP-binding motifs of MetN2, the following methodological approach can be employed:

• Site-directed mutagenesis of conserved residues in Walker A (GxxxxGKT/S) and Walker B (hhhhDE, where h is a hydrophobic residue) motifs

• Expression and purification of wild-type and mutant proteins

• Colorimetric ATPase activity assays measuring inorganic phosphate release

• Determination of ATP binding affinity using techniques such as isothermal titration calorimetry (ITC)

• Complementation studies in ΔmetN2 strains with wild-type or mutant variants

• Bacterial two-hybrid assays to assess interaction with transmembrane components

The table below presents expected results from ATPase activity assays comparing wild-type MetN2 with common Walker motif mutations:

This approach would be analogous to studies of point mutations in the recA gene of E. faecalis that have demonstrated the importance of specific amino acid residues for recombination functions .

How does recombination affect the evolution of metN2 in clinical isolates of E. faecalis?

Homologous recombination can drive genetic diversity in bacterial populations, potentially affecting functional genes like metN2. To investigate this, researchers could:

• Sequence metN2 from diverse clinical isolates of E. faecalis

• Perform phylogenetic analysis to identify recombination events using methods like ClonalFrameML

• Compare metN2 sequences between recombination-proficient and recombination-deficient strains like JH2-2 recA

• Conduct experimental evolution studies under methionine limitation with wild-type and recA mutant strains

• Assess the frequency of variant metN2 alleles in different infection types

Research has shown that recombination plays a significant role in the acquisition of antibiotic resistance in E. faecalis, as demonstrated by studies comparing wild-type and recombination-deficient strains in developing linezolid resistance . Similar mechanisms might influence metN2 evolution, particularly under selective pressure in methionine-limited environments during infection. Tracking metN2 polymorphisms across clinical isolates would provide insights into how recombination shapes the functional diversity of this transporter.

What interacting partners of MetN2 can be identified through protein-protein interaction studies?

To comprehensively map the protein interaction network of MetN2, multiple complementary approaches should be utilized:

• Bacterial two-hybrid or BACTH (Bacterial Adenylate Cyclase Two-Hybrid) system screening

• Co-immunoprecipitation (Co-IP) followed by mass spectrometry analysis

• Cross-linking mass spectrometry (XL-MS) to identify transient interactions

• Surface plasmon resonance (SPR) to quantify binding affinities between MetN2 and identified partners

• Split-GFP complementation assays for in vivo validation of interactions

Expected interaction partners would include:

• MetI (transmembrane component of the methionine transport system)

• MetQ (substrate-binding protein)

• Potential regulatory proteins responding to methionine availability

• Proteins involved in ATP metabolism and energetics

Understanding protein-protein interactions of MetN2 would elucidate its role within larger metabolic networks. Similar approaches have been used to study interactions between components of other transport systems in E. faecalis, such as the EfaCBA transport system .

What strategies can overcome solubility issues when expressing recombinant E. faecalis MetN2?

Solubility challenges are common when expressing recombinant ATP-binding proteins. The following methodological approaches can address these issues:

• Fusion tag optimization: Compare solubility enhancement with different tags including MBP, SUMO, Thioredoxin, and GST alongside the standard His-tag

• Expression condition optimization:

  • Reduce induction temperature to 16-18°C

  • Decrease IPTG concentration to 0.1-0.2 mM

  • Use auto-induction media instead of IPTG induction

• Codon optimization for heterologous expression in E. coli

• Co-expression with chaperones (GroEL/GroES, DnaK/DnaJ, trigger factor)

• Cell-free protein synthesis for difficult-to-express variants

If membrane association occurs, inclusion of detergents such as n-dodecyl-β-D-maltoside (DDM) or lauryl maltose neopentyl glycol (LMNG) at 0.03-0.05% during extraction and purification can improve solubility. These approaches have been successfully employed for other bacterial ABC transporters and could be adapted for MetN2 purification.

How can isotope-labeled MetN2 be efficiently produced for NMR structural studies?

Producing isotope-labeled MetN2 for NMR studies requires specific methodological considerations:

• Expression vector selection: Use T7-based expression systems (pET vectors) for high-level protein production

• Host strain optimization: BL21(DE3) or its derivatives like Rosetta(DE3) for rare codon optimization

• Minimal media preparation:

  • M9 minimal media supplemented with 15NH4Cl for 15N labeling

  • 13C-glucose for 13C labeling

  • D2O-based media for deuteration

• Expression protocol:

  • Grow cells in rich media to OD600 ~0.8

  • Wash cells and transfer to minimal media with isotopes

  • Induce at lower temperatures (18°C) for 18-24 hours

• Purification considerations:

  • Maintain protein stability with appropriate buffers

  • Use deuterated detergents if needed

  • Include protease inhibitors throughout purification

This protocol typically yields 2-5 mg of purified labeled protein per liter of culture, sufficient for most NMR studies. The approach has been used successfully for structural characterization of other bacterial ABC transporters and could be adapted for MetN2.

What are the optimal conditions for measuring the ATPase activity of E. faecalis MetN2?

Accurate measurement of MetN2 ATPase activity requires careful optimization of assay conditions. The following methodology is recommended:

• Buffer optimization:

  • 50 mM Tris-HCl or HEPES pH 7.5-8.0

  • 100-150 mM NaCl

  • 5-10 mM MgCl2 (essential cofactor)

  • 0.5-1 mM DTT to maintain reducing conditions

  • 0.01-0.05% mild detergent if needed for stability

• Assay methods:

  • Malachite green assay: Sensitive colorimetric detection of released phosphate

  • Coupled enzyme assay: ATP hydrolysis coupled to NADH oxidation

  • ADP-Glo assay: Luminescence-based detection of ADP production

• Controls and validations:

  • Include known ATPase inhibitors (vanadate, EDTA) as negative controls

  • Use Walker A motif mutant (K45A) as inactive control

  • Test ATP concentration range (0.1-5 mM) to determine Km values

  • Establish linearity with respect to time and protein concentration

The table below presents optimal conditions for MetN2 ATPase activity:

These conditions should be systematically optimized for specific MetN2 preparations to ensure reproducible activity measurements.

How does the substrate specificity of E. faecalis MetN2 compare to homologous transporters in other bacterial pathogens?

To determine substrate specificity and compare it across bacterial species, the following experimental approach is recommended:

• Heterologous expression systems:

  • Express metN2 along with metI and metQ in an E. coli strain deficient in methionine transport

  • Compare growth complementation using various potential substrates

• Transport assays:

  • Use radiolabeled substrates (35S-methionine, 35S-homocysteine) to measure direct uptake

  • Compare kinetic parameters (Km, Vmax) for different substrates

  • Perform competition assays with unlabeled potential substrates

• Binding studies:

  • Isothermal titration calorimetry with purified MetQ to determine binding affinities for different substrates

  • Surface plasmon resonance to measure binding kinetics

Comparing transport parameters across bacterial species would identify unique features of E. faecalis MetN2. This approach is similar to functional characterization of other transport systems in E. faecalis, such as the FhuDCBG system known to transport ferrichrome .

How can inhibitors of E. faecalis MetN2 be rationally designed and validated?

Structure-based drug design targeting MetN2 requires a methodical approach:

• Target structure determination:

  • X-ray crystallography of MetN2 in different nucleotide-bound states

  • Homology modeling if crystal structure is unavailable

  • Molecular dynamics simulations to identify druggable binding pockets

• Virtual screening:

  • Docking-based screening of chemical libraries against ATP-binding site

  • Fragment-based screening to identify initial scaffolds

  • Pharmacophore-based approaches using known ABC transporter inhibitors

• In vitro validation:

  • ATPase inhibition assays with identified candidates

  • Thermal shift assays to confirm binding

  • Isothermal titration calorimetry to determine binding parameters

• Cellular validation:

  • Growth inhibition assays in methionine-limited media

  • Radiolabeled methionine uptake inhibition

  • Synergy testing with existing antibiotics

• Structural optimization:

  • Structure-activity relationship studies

  • Lead optimization for improved potency and selectivity

  • ADME property optimization

This rational approach to inhibitor development targets a crucial bacterial process while providing important research tools for studying MetN2 function. Similar strategies have been applied to develop inhibitors targeting other bacterial nutrient acquisition systems.

How can CRISPR-Cas9 technologies be optimized for studying metN2 function in E. faecalis?

CRISPR-Cas9 technology offers powerful tools for genetic manipulation of E. faecalis to study metN2 function. A methodological framework includes:

• sgRNA design and optimization:

  • Design multiple sgRNAs targeting different regions of metN2

  • Test efficiency using in vitro cleavage assays

  • Optimize sgRNA expression with suitable promoters for E. faecalis

• Delivery system development:

  • Thermosensitive plasmids similar to pMAD used in recA inactivation

  • Conjugative delivery systems

  • Electroporation protocols optimized for E. faecalis

• Application strategies:

  • Gene knockout via NHEJ or HDR

  • Base editing for point mutations in critical residues

  • CRISPRi for inducible gene repression

  • CRISPRa for overexpression studies

• Validation approaches:

  • Sequencing confirmation of edits

  • Phenotypic characterization

  • Complementation studies

This CRISPR-based toolkit would overcome limitations of traditional genetic manipulation methods in E. faecalis, allowing more precise and efficient genetic studies of metN2 and other genes.

How does methionine transport via MetN2 integrate with other nutrient acquisition systems in E. faecalis?

To understand the integration of methionine transport with other nutrient acquisition pathways, a systems biology approach is recommended:

• Generate a panel of transport system mutants:

  • Single deletion mutants (ΔmetN2, ΔefaC, ΔfeoB, etc.)

  • Double and triple mutants in various combinations

  • Conditional expression strains for essential transporters

• Perform comprehensive phenotypic profiling:

  • Growth under various nutrient limitations

  • Biofilm formation capacity

  • Stress response characterization

  • Virulence in infection models

• Multi-omics integration:

  • Transcriptomics under varying nutrient conditions

  • Proteomics focusing on membrane transporters

  • Metabolomics to track nutrient utilization patterns

  • Network analysis to identify regulatory connections

This approach would reveal how E. faecalis coordinates various transport systems, including MetN2, to optimize nutrient acquisition in different environments. Research has already identified multiple iron transport systems in E. faecalis (EfaCBA, FeoAB, FhuDCBG, FitABCD, and EmtABC) , and similar complexity likely exists for other nutrients including methionine.