Recombinant Syntrophus aciditrophicus Large-conductance mechanosensitive channel (mscL)

What is Syntrophus aciditrophicus and why is it important in microbial research?

Syntrophus aciditrophicus is a model syntrophic bacterium that plays a crucial role in anaerobic environments by degrading fatty and aromatic acids into simpler compounds such as acetate, CO₂, formate, and H₂. These end products are subsequently utilized by methanogens and other hydrogen-consuming microbes in a mutually beneficial relationship . The significance of S. aciditrophicus in research stems from its remarkable metabolic capabilities under extreme energy limitations. The organism operates near thermodynamic equilibrium, requiring syntrophic partnerships to make its metabolism energetically favorable. This makes it an excellent model for studying energy conservation mechanisms, metabolic regulation, and microbial cooperation in anaerobic ecosystems. S. aciditrophicus has become particularly important for understanding how organisms function in environments where energy yields are exceptionally low, providing insights into fundamental aspects of microbial physiology and ecology .

What are mechanosensitive channels and what is their biological significance?

Mechanosensitive channels are membrane proteins that respond to mechanical force by opening a pore in the cell membrane. The large-conductance mechanosensitive channel (MscL) represents one of the primary classes of these channels and functions as a critical biological pressure release valve. MscL channels open in response to increased membrane tension, allowing the passage of water, ions, and small molecules to prevent cell rupture during osmotic shock .

These channels are particularly important for bacterial survival during rapid environmental changes. Beyond their osmoregulatory function, recent research has identified MscL proteins as viable pharmaceutical drug targets, particularly for the development of novel antibiotics . Their highly conserved structure across bacterial species, combined with their absence in mammalian cells, makes them attractive candidates for selective antimicrobial therapy. Understanding the gating mechanism of MscL is essential for rational drug design approaches targeting these channels .

What techniques are used to express recombinant MscL proteins for structural and functional studies?

To express recombinant MscL proteins, researchers typically employ the following methodological approaches:

• Expression Vector Selection: Specialized vectors containing inducible promoters (e.g., T7, tac) are used to control expression levels, often incorporating affinity tags (His6, GST) for purification.

• Host System Optimization: While E. coli is commonly used, expression in cell-free systems or specialized membrane protein expression hosts may yield better results for challenging membrane proteins like MscL.

• Induction Conditions: Careful optimization of temperature (typically lowered to 16-20°C), inducer concentration, and duration is critical for proper membrane protein folding.

• Membrane Extraction: Protocols using mild detergents (DDM, LDAO) that maintain MscL structure and function are employed for protein isolation.

• Purification Strategy: Sequential chromatography steps, often combining affinity purification with size exclusion chromatography, yield highly pure protein.

• Functional Reconstitution: Purified MscL is reconstituted into liposomes or nanodiscs to study channel function in a controlled membrane environment.

For S. aciditrophicus MscL specifically, additional considerations include codon optimization for the expression host and potential modifications to account for the anaerobic native environment of the protein .

How does the metabolic pathway of benzoate degradation function in S. aciditrophicus?

Benzoate degradation in S. aciditrophicus proceeds through a complex, multistep pathway that generates numerous reactive acyl-CoA species (RACS) as intermediates. The pathway begins with the activation of benzoate to benzoyl-CoA, followed by a series of reductive and hydrolytic reactions . This metabolic process is particularly notable for its reversibility, as S. aciditrophicus enzymes can catalyze reactions in either direction depending on substrate availability and environmental conditions .

The key steps in this pathway include:

• Activation of benzoate to benzoyl-CoA

• Reduction of the aromatic ring

• Ring opening

• Sequential β-oxidation-like reactions

• Generation of acetate, CO₂, formate, and H₂ as end products

A significant feature of this pathway is that the intermediates are maintained as CoA derivatives throughout the process, serving as scaffolds for the sequential transformations. This CoA-tethering strategy is critical for maintaining the thermodynamic favorability of the individual reaction steps . The reversibility of this pathway highlights the metabolic versatility of S. aciditrophicus, allowing it to adapt to changing environmental conditions and substrate availability .

What post-translational modifications occur in S. aciditrophicus proteins?

S. aciditrophicus exhibits an extensive and unique profile of post-translational modifications, particularly acylation of lysine residues. Research has identified seven distinct types of acyl modifications, with six corresponding directly to reactive acyl-CoA species (RACS) that function as intermediates in the benzoate degradation pathway .

The acylation profile includes:

• Acetylation

• Propionylation

• Butyrylation

• Crotonylation

• Glutarylation

• Pimeloylation

• 3-hydroxypimeloylation (a modification first identified in this system)

A comprehensive proteomics analysis identified 125 acylation sites across 60 different proteins, with benzoate-degrading enzymes being particularly heavily represented among the acylated proteins . This suggests that acylation may play a regulatory role in the benzoate degradation pathway.

Notably, S. aciditrophicus also possesses functional deacylase enzymes, indicating the presence of a dynamic regulatory system that can modulate protein acylation levels. This reversible acylation system potentially serves as a mechanism for regulating metabolic processes in response to environmental conditions and substrate availability .

How might post-translational acylation affect MscL function in S. aciditrophicus?

The extensive acylation profile observed in S. aciditrophicus proteins suggests a potential regulatory mechanism that could significantly impact MscL channel function. Lysine acylation alters the local charge distribution and can introduce structural changes that may affect channel gating properties. Based on the identified acylation patterns in S. aciditrophicus, several hypothesized effects on MscL function can be proposed:

• Altered Gating Threshold: Acylation of lysine residues in the transmembrane domains or the periplasmic loops could modify the tension required for channel opening. By neutralizing positively charged lysines, acylation might reduce the energy barrier for conformational changes, effectively lowering the gating threshold.

• Modified Conductance Properties: Acylation patterns could alter the electrostatic environment of the channel pore, potentially affecting ion selectivity or conductance rates.

• Regulation of Protein-Protein Interactions: Acylation might modulate interactions between MscL and other proteins in the membrane or cytoplasmic environment, potentially creating selective pressure-sensing complexes.

The observation that benzoate-degrading enzymes are heavily represented among acylated proteins suggests that acylation could serve as a metabolic feedback mechanism. For MscL, this could mean that channel function is dynamically regulated based on the metabolic state of the cell, particularly during benzoate degradation where numerous reactive acyl-CoA species are generated .

The presence of functional deacylase enzymes in S. aciditrophicus further supports the hypothesis that acylation serves as a reversible regulatory mechanism, allowing for dynamic control of MscL function based on environmental and metabolic conditions.

What methodological approaches are most effective for studying MscL gating mechanisms?

Studying MscL gating mechanisms requires a multi-faceted approach combining computational and experimental techniques. Based on current research, the following methodological strategies prove most effective:

• Molecular Dynamics Simulations (MDS):

  • All-atom simulations with explicit membrane and solvent provide detailed insights into conformational changes during gating

  • Coarse-grained simulations enable longer timescale analyses of channel behavior

  • Steered molecular dynamics can simulate membrane tension to trigger gating events

• Structural Analysis:

  • X-ray crystallography for closed-state structures

  • Cryo-EM for capturing intermediate states

  • FRET spectroscopy to monitor distance changes between domains during gating

• Electrophysiological Approaches:

  • Patch-clamp recordings of reconstituted channels in liposomes

  • Spheroplast patch-clamp measurements for native membrane environment studies

  • Planar lipid bilayer recordings for controlled membrane composition experiments

• Site-Directed Mutagenesis:

  • Systematic modification of key residues to assess their contribution to gating mechanics

  • Introduction of reporter groups (fluorescent, spin labels) at strategic positions

• Chemical Biology Approaches:

  • Photocrosslinking to capture transient protein conformations

  • Small molecule screening to identify channel modulators

The combination of these techniques can provide complementary data, as demonstrated in recent studies where molecular dynamics simulations required validation using in vitro and in vivo experiments . For studying S. aciditrophicus MscL specifically, these approaches would need adaptation to account for the anaerobic growth requirements and potential post-translational modifications unique to this organism.

What are the challenges in determining the structure of fully open-channel MscL?

Determining the structure of fully open MscL channels presents several significant challenges that have hindered progress in this area. As noted in the literature, "the fully open-channel MscL structure has not been experimentally determined" , despite considerable research efforts. The primary challenges include:

• Conformational Instability: The open state is energetically unfavorable and transient, making it difficult to stabilize for structural studies.

• Membrane Environment Requirements: The channel requires specific membrane tension to open, which is challenging to replicate in crystallization or cryo-EM preparations.

• Size and Conformational Changes: The dramatic expansion of the pore during opening (from ~2 Å to ~30 Å) represents one of the most substantial conformational changes known in membrane proteins, complicating structural determination.

• Heterogeneity of Open States: Evidence suggests multiple intermediate states exist between closed and fully open conformations, creating heterogeneous samples.

• Technical Limitations:

  • Crystallization favors the more stable closed state

  • Detergents used for protein isolation may not properly mimic membrane tension

  • Cryo-EM sample preparation can alter membrane properties

Recent approaches combining site-directed spin labeling, electron paramagnetic resonance, and FRET measurements with computational modeling offer promising avenues to overcome these challenges and better characterize the open state structure.

How does membrane composition affect MscL function in bacterial systems?

• Membrane Thickness Effects:

  • Hydrophobic mismatch between the channel's hydrophobic region and membrane thickness can pre-stress the channel

  • Thinner membranes typically lower the tension threshold required for channel opening

  • Thicker membranes may stabilize the closed conformation

• Lipid Composition Influence:

  • Phospholipid headgroup composition affects local electrostatic environment

  • Negatively charged lipids (e.g., phosphatidylglycerol) can interact with positively charged residues in MscL, altering gating tension

  • Lipids with different acyl chain saturation modify membrane fluidity and MscL sensitivity

• Membrane Curvature:

  • Local curvature stress can redistribute tension forces acting on the channel

  • Conical lipids that induce negative curvature may stabilize the closed state

  • Inverted cone-shaped lipids can facilitate channel opening

• Specific Lipid-Protein Interactions:

  • Evidence suggests specific binding sites for certain lipids on MscL

  • These interactions can stabilize particular conformational states

Lane and Pliotas reported that "alteration of membrane properties and components" serves as one of the three major triggers for MscL channel opening . This finding emphasizes the importance of membrane environment in modulating channel function and suggests potential approaches for channel manipulation through membrane engineering.

In the context of S. aciditrophicus, which operates in an anaerobic environment with limited energy availability, membrane composition likely plays a particularly important role in regulating MscL activity to conserve energy while maintaining cellular integrity.

What experimental approaches can validate computational models of MscL gating?

Validating computational models of MscL gating requires a systematic combination of experimental techniques that can directly measure or infer conformational changes. The following approaches provide robust validation methods:

• Cysteine Scanning Mutagenesis and Accessibility:

  • Sequential replacement of residues with cysteine throughout the channel

  • Measuring accessibility changes to thiol-reactive compounds during gating

  • Comparison with accessibility predictions from simulations

• FRET-Based Measurements:

  • Strategic placement of fluorophore pairs to monitor distance changes

  • Real-time monitoring of conformational changes during gating

  • Direct comparison with distance predictions from simulations

• Electrophysiological Validation:

  • Single-channel conductance measurements compared to predicted pore sizes

  • Kinetic analysis of channel opening/closing rates

  • Ion selectivity measurements versus computational predictions

• Crosslinking Studies:

  • Introduction of disulfide bridges at positions predicted to move during gating

  • Assessment of channel function with constrained movement

  • Verification of simulation-predicted critical motion paths

• Site-Directed Spin Labeling and EPR Spectroscopy:

  • Measurement of residue mobility and environmental changes

  • Detection of conformational changes at specific channel regions

  • Comparison with simulation-predicted dynamics

This multi-method approach is essential because "the findings by MDS need further validation using in vitro/in vivo experiments" . The combination of these techniques provides complementary data points that can corroborate or challenge computational models, leading to iterative refinement of simulations.

For S. aciditrophicus MscL specifically, validation would require additional considerations for the anaerobic environment and potential effects of post-translational modifications identified in this organism .

What expression systems are most appropriate for recombinant production of S. aciditrophicus MscL?

Selecting an optimal expression system for recombinant production of S. aciditrophicus MscL requires careful consideration of the protein's anaerobic origin and specific characteristics. Based on research experience with similar membrane proteins, the following expression systems offer distinct advantages:

Table 1: Comparison of Expression Systems for Recombinant S. aciditrophicus MscL Production

For optimal expression of functional S. aciditrophicus MscL, a modified E. coli BL21(DE3) expression system with controlled induction parameters represents the most practical starting point. Critical optimizations include:

• Addition of a cleavable N-terminal tag (His10-SUMO) for purification and stability

• Use of rhamnose-inducible promoters for fine-tuned expression control

• Growth under microaerobic conditions with gradual adaptation to anaerobic growth

• Supplementation with specific lipids to mimic native membrane environment

• Induction at low temperature (16°C) for extended periods (16-24 hours)

This approach balances practical laboratory considerations with the specialized requirements of this anaerobic bacterial membrane protein .

How can post-translational modifications of MscL be characterized in S. aciditrophicus?

Characterizing post-translational modifications (PTMs) of MscL in S. aciditrophicus requires a comprehensive analytical workflow that integrates advanced proteomics with functional studies. Based on the research showing extensive acylation in this organism , the following methodological approach is recommended:

• Sample Preparation:

  • Culture S. aciditrophicus under controlled conditions (varying carbon sources)

  • Membrane fraction isolation using differential centrifugation

  • Affinity purification of MscL using antibodies or tagged recombinant versions

• MS-Based PTM Identification:

  • Multiple protease digestion strategies to maximize sequence coverage

  • High-resolution LC-MS/MS analysis optimized for PTM detection

  • Label-free quantification to determine modification stoichiometry

  • Analysis without antibody enrichment, as "the amounts of modified peptides are sufficient to analyze the post-translational modifications without antibody enrichment"

• Site-Specific Modification Analysis:

  • Targeted MS approaches for identified modification sites

  • Parallel reaction monitoring (PRM) for absolute quantification

  • Correlation of modification levels with metabolic states

• Functional Correlation Studies:

  • Site-directed mutagenesis of modified residues (K→R to prevent modification)

  • Electrophysiological characterization of wild-type vs. mutant channels

  • Patch-clamp analysis to determine effects on gating properties

• Temporal Dynamics Investigation:

  • Time-course experiments during growth on different substrates

  • Correlation of acylation patterns with metabolic intermediates

  • Inhibitor studies targeting deacylase enzymes

This comprehensive approach leverages the finding that S. aciditrophicus contains "functional deacylase enzymes... indicating a potential regulatory system/mechanism by which S. aciditrophicus modulates acylation" . By mapping the dynamic PTM landscape of MscL in this organism, researchers can gain insights into how post-translational regulation might affect channel function in response to metabolic and environmental changes.

What are the most effective approaches for studying protein-protein interactions involving MscL in syntrophic bacteria?

Investigating protein-protein interactions (PPIs) involving MscL in syntrophic bacteria requires specialized approaches that account for membrane localization, anaerobic conditions, and potential effects of post-translational modifications. The following methodological strategies are particularly effective:

• Proximity-Based Labeling Techniques:

  • BioID or TurboID fusion to MscL for in vivo identification of proximal proteins

  • APEX2-based proximity labeling under anaerobic conditions

  • Quantitative MS analysis of labeled proteins under different growth conditions

• Crosslinking Mass Spectrometry (XL-MS):

  • In vivo chemical crosslinking using membrane-permeable reagents

  • MS/MS analysis to identify crosslinked peptides

  • Computational modeling to map interaction interfaces

• Co-Purification Approaches:

  • Tandem affinity purification under gentle solubilization conditions

  • Quantitative comparison of interactors across different metabolic states

  • Validation using reciprocal tagging of identified interaction partners

• Genetic Interaction Screening:

  • Synthetic genetic array analysis with MscL mutants

  • Suppressor screening to identify functional interactions

  • CRISPR interference screens to identify genes affecting MscL function

• Membrane-Specific Techniques:

  • Fluorescence resonance energy transfer (FRET) between MscL and candidate partners

  • Bimolecular fluorescence complementation adapted for anaerobic imaging

  • Native membrane nanodiscs for in vitro reconstitution of complexes

For S. aciditrophicus specifically, these approaches must be adapted to account for anaerobic growth requirements and the potential regulatory role of post-translational modifications. The extensive acylation observed in S. aciditrophicus proteins suggests that interactome studies should compare interaction profiles of native (acylated) MscL with deacylated versions to understand how these modifications might regulate protein-protein interactions.

This systematic approach can reveal how MscL functions within larger protein complexes and signaling networks in syntrophic bacteria, potentially uncovering unique adaptations that enable these organisms to thrive in energy-limited environments.

How can molecular dynamics simulations be optimized for studying S. aciditrophicus MscL gating?

Optimizing molecular dynamics simulations for S. aciditrophicus MscL gating requires specialized approaches that account for the unique characteristics of this syntrophic bacterium. Based on current research methodologies , the following optimization strategies are recommended:

• System Preparation Considerations:

  • Homology modeling based on multiple MscL templates for improved accuracy

  • Incorporation of experimentally determined post-translational modifications, particularly the seven acyl modifications identified in S. aciditrophicus

  • Membrane composition matching S. aciditrophicus lipid profile

  • Inclusion of key metabolites from benzoate degradation pathway in simulation environment

• Simulation Protocol Optimizations:

  • Extended equilibration phases (>100 ns) to accommodate slow membrane relaxation

  • Stepwise application of membrane tension to prevent artifactual conformational changes

  • Multiple replica simulations with different initial velocities for statistical robustness

  • Adaptive sampling techniques focusing on transition states between conformations

• Advanced Simulation Approaches:

  • Coarse-grained simulations for accessing longer timescales (μs-ms)

  • Subsequent all-atom refinement of key states identified in coarse-grained simulations

  • Constant-tension versus constant-area protocols to better mimic physiological conditions

  • Free energy calculations to determine energetic barriers between conformational states

• Analysis Framework:

  • Principal component analysis to identify dominant motions

  • Correlation analysis to detect allosteric networks

  • Markov state modeling to map conformational landscape

  • Comparison with experimental observables (conductance, accessibility patterns)

• Integration with Experimental Data:

  • Restraint-based simulations incorporating FRET or crosslinking distance constraints

  • Targeted simulations to explore effects of site-directed mutations tested experimentally

  • Iterative refinement based on experimental validation

These optimization strategies address the challenges noted in the literature, where "the fully open-channel MscL structure has not been experimentally determined" and molecular dynamics simulations have become "an indispensable tool to study the gating mechanism" . For S. aciditrophicus MscL specifically, incorporation of acylation patterns observed in this organism is crucial for understanding how post-translational modifications might modulate channel gating in response to metabolic state.

How might understanding S. aciditrophicus MscL contribute to developing new antibiotics?

Understanding S. aciditrophicus MscL structure, function, and regulation offers several promising avenues for novel antibiotic development. Recent research has established mechanosensitive channels as "viable pharmaceutical drug targets for development of precursors or antibiotics" . The unique characteristics of S. aciditrophicus MscL could enhance these approaches in several ways:

• Targeting Syntrophic-Specific Features:

  • Exploiting structural differences in MscL from syntrophic bacteria could enable selective targeting of these organisms in mixed microbial communities

  • Compounds that interfere with syntrophic partnerships could disrupt critical metabolic processes in anaerobic environments

• Leveraging Post-Translational Modifications:

  • The extensive acylation profile observed in S. aciditrophicus offers unique targeting opportunities

  • Inhibitors that mimic or prevent specific acylation patterns could disrupt channel regulation

  • Compounds blocking deacylase activity could lead to dysregulated channel function

• Exploiting Metabolic Vulnerabilities:

  • MscL modulators that respond to metabolic intermediates from the benzoate degradation pathway

  • Compounds that alter MscL gating thresholds, making cells more vulnerable to osmotic stress during specific metabolic states

• Rational Drug Design Approaches:

  • Structure-based design of compounds that lock the channel in open or closed conformations

  • Development of molecules that alter the tension sensitivity of the channel

  • Creation of compounds that disrupt protein-protein interactions specific to syntrophic bacteria

Lane and Pliotas concluded that "there is great potential for new pioneering discoveries through the modulation of bacterial mechanosensitive channels in order to develop understanding of their structures, mechanisms, and functions but also for their use within biotechnology and as targets for antimicrobial therapies" . The unique metabolic context and post-translational modification landscape of S. aciditrophicus MscL provide additional dimensions for exploitation in antibiotic development.

This approach could be particularly valuable for targeting syntrophic communities in settings like anaerobic digesters, wastewater treatment plants, or certain human infections where syntrophic partnerships contribute to pathogenesis.

What are the most promising directions for future research on S. aciditrophicus MscL?

Future research on S. aciditrophicus MscL presents several high-impact directions that could significantly advance our understanding of membrane protein function in syntrophic bacteria and enable novel biotechnological applications:

• Structure-Function Relationships in Extreme Energy Limitation:

  • Determining how MscL structure and gating mechanisms adapt to the near-thermodynamic equilibrium conditions of syntrophic growth

  • Investigating potential energy conservation mechanisms linked to mechanosensitive channel function

  • Comparing MscL properties across syntrophic and non-syntrophic bacteria to identify adaptations

• Regulatory Networks and Signaling:

  • Mapping the interplay between post-translational modifications and MscL function

  • Investigating how the seven identified acyl modifications affect channel properties

  • Elucidating the role of deacylase enzymes in dynamic regulation of channel activity

• Syntrophic Communication Mechanisms:

  • Exploring whether MscL functions in cell-cell communication between syntrophic partners

  • Investigating if channel activity coordinates metabolic activities between organisms

  • Determining if metabolites released through MscL serve as signaling molecules

• Technological Applications:

  • Developing biosensors based on S. aciditrophicus MscL for detecting specific compounds

  • Creating controlled release systems for biotechnology using engineered channels

  • Exploiting the unique properties of syntrophic MscL for nanopore sequencing applications

• Evolutionary Perspectives:

  • Comparative genomic and structural analysis of MscL across syntrophic lineages

  • Investigating the co-evolution of MscL with metabolic pathways in syntrophic bacteria

  • Reconstructing the evolutionary history of mechanosensation in extreme energy environments

These research directions align with observations that "post-translational modifications modulate benzoate degradation in this and potentially other, syntrophic bacteria" and that understanding MscL mechanisms has "great potential for new pioneering discoveries" .

By pursuing these avenues, researchers can gain fundamental insights into membrane protein function under extreme energy limitation while developing novel applications in biotechnology, medicine, and environmental science.

What are common challenges in purifying recombinant MscL from S. aciditrophicus and how can they be addressed?

Purifying recombinant MscL from S. aciditrophicus presents several technical challenges due to the protein's membrane localization and the organism's unique metabolic characteristics. The following table outlines common challenges and effective solutions:

Table 2: Challenges and Solutions for S. aciditrophicus MscL Purification

Additional considerations specific to S. aciditrophicus MscL include preserving the native acylation state if desired, as research has shown that "six [acyl modifications] correspond directly to RACS that are intermediates in the benzoate degradation pathway" . For studies investigating the effects of these modifications, careful monitoring of the acylation profile during purification is essential.

The presence of "functional deacylase enzymes... in the proteome" also suggests that inhibition of these enzymes during purification may be necessary to maintain the native acylation pattern. Alternatively, controlled deacylation could be employed to generate homogeneously unmodified protein for comparative studies.

How can researchers address data interpretation challenges when studying MscL in syntrophic systems?

Data interpretation for MscL studies in syntrophic systems presents unique challenges due to complex metabolic interactions, potential post-translational modifications, and environmental influences. Researchers can address these challenges through the following methodological approaches:

• Establishing Appropriate Controls:

  • Compare MscL behavior in syntrophic versus pure culture conditions

  • Create point mutants resistant to specific acylations (K→R mutations)

  • Develop parallel experiments with well-characterized MscL homologs

• Resolving Confounding Metabolic Effects:

  • Use metabolomic profiling alongside functional MscL studies

  • Implement labeled substrate tracking to correlate metabolic states with channel properties

  • Develop computational models integrating metabolic flux with membrane protein function

• Addressing Heterogeneity in Experimental Samples:

  • Apply single-molecule techniques to capture population diversity

  • Implement sorting methods to separate cells based on metabolic state

  • Use statistical approaches designed for heterogeneous datasets

• Managing Time-Dependent Phenomena:

  • Design time-course experiments that capture dynamics of syntrophic interactions

  • Implement synchronized culture techniques

  • Develop non-destructive monitoring approaches for long-term studies

• Integrating Multi-Omics Data:

  • Combine proteomics, transcriptomics, and metabolomics datasets

  • Develop integrative computational frameworks specific to syntrophic systems

  • Implement machine learning approaches for pattern recognition across diverse datasets

This systematic approach addresses the complexity revealed in studies showing that "N-ε-acyl-lysine RACS are highly abundant in these syntrophic bacteria" and that various triggers can modulate MscL channel opening, including "modification of MscL protein itself via site-directed mutagenesis and post-translational modifications, the small molecular modulators and antimicrobials targeting MscL, and the alteration of membrane properties and components" .

By implementing these approaches, researchers can more confidently interpret experimental data and distinguish between direct effects on MscL and indirect consequences of the complex syntrophic metabolic network.