Recombinant Escherichia coli O157:H7 Small-conductance mechanosensitive channel (mscS)

What is the mechanosensitive channel MscS and what is its function in E. coli O157:H7?

The small-conductance mechanosensitive channel (MscS) is a critical membrane protein that responds to mechanical forces in bacterial cell membranes. In E. coli O157:H7, as in other E. coli strains, MscS functions as a pressure valve that protects cells against hypoosmotic shock by releasing cytoplasmic osmolytes when membrane tension increases. MscS has a conductance of approximately 1.25 nS and gates in response to increased membrane tension . This channel is part of a family of mechanosensitive channels that includes MscL (large conductance, ~3 nS), MscK (potassium-dependent, ~0.875 nS), and MscM (miniconductance, ~0.375 nS) . In E. coli O157:H7, these channels likely contribute to survival in varied environments, including the bovine gastrointestinal tract and during environmental transitions that occur during transmission to humans .

How do mechanosensitive channels differ between pathogenic E. coli O157:H7 and non-pathogenic E. coli K-12?

While the core structure and function of MscS are conserved between pathogenic and non-pathogenic E. coli strains, genomic analysis reveals that E. coli O157:H7 has undergone both acquisition and loss of DNA during its evolution, which may affect channel regulation and expression . The genomes of E. coli O157:H7 strains contain unique sequences with altered G+C content, indicating horizontal gene transfer from at least 53 different species . These genomic differences may influence membrane composition and properties, potentially affecting MscS function and regulation in response to environmental stresses. Research comparing MscS function between pathogenic and non-pathogenic strains should consider these genomic distinctions and their potential effects on membrane properties.

What techniques are commonly used to express recombinant MscS from E. coli O157:H7?

Recombinant MscS from E. coli O157:H7 can be expressed using standard molecular biology techniques adapted for membrane proteins. The methodological approach typically includes:

• Gene cloning: Amplification of the mscS gene from E. coli O157:H7 genomic DNA using PCR with specific primers, followed by insertion into an appropriate expression vector

• Expression system selection: Often using E. coli BL21(DE3) or similar expression strains with T7 RNA polymerase systems

• Induction protocols: IPTG-inducible systems with careful optimization of induction conditions (temperature, IPTG concentration, and induction time)

• Membrane protein extraction: Using detergents like n-dodecyl-β-D-maltoside (DDM) or n-octyl-β-D-glucopyranoside (OG)

• Purification: Affinity chromatography using histidine or other fusion tags, followed by size exclusion chromatography

How does the electrophysiological characterization of MscS channels from E. coli O157:H7 compare to MscS from non-pathogenic strains?

Electrophysiological characterization of MscS from E. coli O157:H7 requires patch-clamp analysis in reconstituted systems or native membranes. Research comparing E. coli O157:H7 MscS with non-pathogenic counterparts reveals:

Notably, MscS activity must be differentiated from MscM-like activities that may arise from other gene products such as YbdG. To effectively study MscS from E. coli O157:H7, researchers should consider generating giant protoplasts using cephalexin treatment, followed by patch-clamp analysis with appropriate pressure protocols .

What are the genetic and structural differences in MscS homologs between E. coli O157:H7 and other E. coli strains?

E. coli possesses multiple MscS homologs with distinct structures and potentially different functions. Comparative genomic analysis reveals:

• E. coli (including O157:H7) contains at least six MscS homologs: MscS (YggB), MscK (KefA), YjeP, YbiO, YbdG, and YnaI

• YbdG (415 aa) differs from MscS (286 aa) in three key aspects:

  • Larger membrane domain with five transmembrane spans (versus three in MscS)

  • Contains a ~50-amino-acid insertion at the junction between the upper β and αβ domains

  • Different pore sequence conservation pattern, particularly around G101

• The pore-lining helix (TM3a in MscS) contains a conserved pattern of alanine and glycine residues that affects channel gating properties

• Mutations that alter this Ala-Gly packing can modify channel gating characteristics

These structural differences may reflect adaptations to different environmental niches, including those encountered by pathogenic E. coli O157:H7 during infection or survival in cattle .

How can you distinguish between MscS activity and MscM-like conductances in E. coli O157:H7 electrophysiological studies?

Distinguishing between MscS and MscM-like activities in E. coli O157:H7 requires careful experimental design:

• Conductance measurements: MscS has a conductance of ~1.25 nS, while MscM-like conductances are approximately 0.375 nS

• Genetic approaches: Generate deletion mutants lacking specific MscS homologs (e.g., ΔyggB, ΔkefA) to isolate and characterize individual channel activities

• Gating kinetics analysis: MscS typically shows short bursts of activity lasting seconds, while some MscM-like activities (such as from KefA) remain active for extended periods (>30 seconds) without desensitization

• Pressure threshold determination: Different channels activate at distinct pressure thresholds relative to MscL

Research indicates that YbdG may contribute to MscM-like activity, but deletion mutants still exhibit occasional MscM-like conductances, suggesting multiple gene products may produce similar electrophysiological signatures .

What are the optimal conditions for functional recombinant expression of MscS from E. coli O157:H7?

Functional expression of recombinant MscS from E. coli O157:H7 requires careful optimization:

For functional verification, researchers should conduct either electrophysiological analysis or osmotic downshock assays to confirm channel activity. Expression levels can be verified by Western blotting using antibodies against affinity tags or the MscS protein itself .

What methods can be used to study the role of MscS in E. coli O157:H7 survival during osmotic stress?

To investigate MscS contribution to E. coli O157:H7 survival during osmotic stress:

• Genetic manipulation approaches:

  • Generate clean deletion mutants (ΔmscS) using λ-Red recombination

  • Create complemented strains with wild-type or mutant mscS variants

  • Develop double or triple knockout strains (e.g., ΔmscS ΔmscL ΔybdG) to assess channel redundancy

• Osmotic shock survival assays:

  • Culture cells in high osmolarity media (LB + 0.5M NaCl)

  • Subject cells to rapid dilution in low osmolarity media

  • Quantify survival by plating and colony counting

• Microscopic analysis:

  • Phase contrast or fluorescence microscopy to visualize cell lysis during osmotic downshock

  • Time-lapse imaging to track morphological changes

• Transcriptional and translational analysis:

  • qRT-PCR to measure mscS expression under different osmotic conditions

  • Western blotting to quantify protein levels

  • Reporter gene fusions to monitor real-time expression changes

Research indicates that YbdG expression is enhanced by osmotic stress but inhibited by RpoS, in contrast to MscS, which is RpoS-dependent. These regulatory differences may influence channel availability during osmotic challenges faced by E. coli O157:H7 in various environments .

How can you design effective gene knockout experiments to study MscS function in E. coli O157:H7?

Effective gene knockout design for studying MscS function in E. coli O157:H7 requires:

• Selection of appropriate knockout strategy:

  • λ-Red recombination system (Datsenko and Wanner method) for precise gene deletion

  • Selection of antibiotic resistance markers suitable for pathogenic strains

  • Consideration of polar effects on downstream genes

• Verification procedures:

  • PCR confirmation with primers flanking the deletion region

  • Sequencing to confirm precise deletion

  • RT-PCR to verify absence of transcript

  • Western blotting to confirm protein absence

• Control strain development:

  • Complementation strains with wild-type gene reintroduction

  • Point mutation variants to assess specific functional domains

  • Multiple knockout combinations to address functional redundancy

• Functional characterization:

  • Osmotic shock survival assays comparing wild-type, knockout, and complemented strains

  • Patch-clamp analysis to confirm absence of specific channel activities

  • Growth curves under various stress conditions to assess fitness consequences

When working with pathogenic E. coli O157:H7, researchers must follow appropriate biosafety protocols and consider using attenuated strains when possible for initial characterization before moving to fully virulent isolates .

What are the best approaches for purifying recombinant MscS from E. coli O157:H7 for structural studies?

Purification of recombinant MscS from E. coli O157:H7 for structural studies requires specialized techniques for membrane proteins:

• Expression optimization:

  • Use of specialized vectors with appropriate fusion tags (His8, FLAG, or MBP)

  • Expression in E. coli strains designed for membrane proteins (C41/C43)

  • Growth at reduced temperatures (20-25°C) after induction

• Membrane isolation and solubilization:

  • Cell disruption via French press or sonication

  • Membrane fraction isolation by ultracentrifugation

  • Careful detergent screening (DDM, LMNG, or UDM) for optimal solubilization

• Purification strategy:

  • Immobilized metal affinity chromatography (IMAC) as initial purification

  • Size exclusion chromatography to remove aggregates

  • Optional ion exchange chromatography for further purification

• Quality assessment:

  • SDS-PAGE and Western blotting for purity verification

  • Dynamic light scattering to assess homogeneity

  • Negative stain electron microscopy for initial structural assessment

  • Functional verification via reconstitution into liposomes and patch-clamp analysis

• Structural determination approaches:

  • Crystallization trials in the presence of appropriate detergents

  • Cryo-electron microscopy for high-resolution structure determination

  • Lipid cubic phase crystallization as an alternative approach

How can bacterial two-hybrid systems be utilized to identify protein interactions with MscS in E. coli O157:H7?

Bacterial two-hybrid systems offer valuable approaches for identifying MscS protein interactions:

• System selection:

  • BACTH (Bacterial Adenylate Cyclase Two-Hybrid) system, where interaction reconstitutes adenylate cyclase activity

  • LexA-based systems where interactions drive reporter gene expression

  • λ-repressor systems based on transcriptional repression

• Bait and prey construction:

  • Clone full-length mscS and truncated variants into appropriate vectors

  • Create domain-specific constructs to map interaction interfaces

  • Generate genomic libraries from E. coli O157:H7 for unbiased screening

• Screening methodology:

  • Primary screening on indicator plates (X-gal for BACTH)

  • Secondary validation with liquid β-galactosidase assays

  • Plasmid recovery and sequencing of positive clones

• Validation experiments:

  • Co-immunoprecipitation of identified interaction partners

  • Pull-down assays with purified proteins

  • Fluorescence resonance energy transfer (FRET) analysis in live cells

• Functional analysis:

  • Generate knockout strains of identified interaction partners

  • Assess effects on MscS expression, localization, and function

  • Conduct patch-clamp analysis to determine effects on channel properties

This approach can identify proteins that potentially modulate MscS function in E. coli O157:H7, including those involved in channel assembly, trafficking, or regulation in response to environmental stresses .

What methodologies can be employed to study MscS expression and regulation in E. coli O157:H7 under different environmental conditions?

To study MscS expression and regulation under different environmental conditions:

• Transcriptional analysis:

  • qRT-PCR to quantify mscS transcript levels under varying conditions

  • RNA-seq for genome-wide expression profiling in response to stressors

  • 5' RACE to identify transcription start sites and potential alternative promoters

  • Chromatin immunoprecipitation (ChIP) to identify transcription factor binding sites

• Translational analysis:

  • Western blotting with MscS-specific antibodies

  • Translational fusions with reporter proteins (GFP, LacZ)

  • Pulse-chase experiments to determine protein stability

  • Ribosome profiling to assess translation efficiency

• Promoter analysis:

  • Reporter gene fusions to test promoter activity

  • Site-directed mutagenesis to identify key regulatory elements

  • Electrophoretic mobility shift assays (EMSA) to identify protein-DNA interactions

• Environmental stress conditions to test:

  • Osmotic stress (high/low osmolarity transitions)

  • pH variations (acidic conditions mimicking gastric environment)

  • Oxidative stress (H₂O₂, paraquat)

  • Nutrient limitation

  • Temperature shifts

  • Exposure to bile salts

Research indicates that expression of YbdG (an MscS homolog) is enhanced by osmotic stress but inhibited by RpoS, suggesting complex regulatory networks controlling mechanosensitive channel expression. Understanding these regulatory mechanisms could provide insights into how E. coli O157:H7 adapts to environmental challenges during its lifecycle .

How can you troubleshoot poor functional expression of recombinant MscS from E. coli O157:H7?

When encountering poor functional expression of recombinant MscS:

Additionally:

• Verify the coding sequence for mutations

• Test alternative fusion tags (N-terminal vs. C-terminal)

• Consider using fusion partners known to enhance membrane protein expression (MBP, SUMO)

• Experiment with different E. coli expression strains specifically designed for membrane proteins

• Verify proper membrane targeting using subcellular fractionation and Western blotting

How do you address potential safety concerns when working with recombinant E. coli O157:H7 MscS in the laboratory?

Addressing safety concerns when working with recombinant E. coli O157:H7 MscS requires:

• Risk assessment:

  • Determine if full pathogenic strain is necessary or if attenuated strains can be used

  • Consider using an E. coli K-12 background expressing O157:H7 MscS variants

  • Evaluate the presence of virulence factors in recombinant strains

• Biosafety measures:

  • Work at appropriate Biosafety Level (typically BSL-2 for E. coli O157:H7)

  • Use biological safety cabinets for aerosol-generating procedures

  • Implement proper personal protective equipment (gloves, lab coat, eye protection)

• Strain engineering considerations:

  • Remove or inactivate Shiga toxin genes (stx1, stx2) if present

  • Consider using strains lacking other virulence factors (eae, ehxA)

  • Incorporate containment features (auxotrophic markers, suicide systems)

• Laboratory practices:

  • Maintain dedicated equipment for pathogenic strains

  • Implement rigorous decontamination procedures

  • Train personnel specifically on pathogen handling

• Regulatory compliance:

  • Obtain appropriate institutional biosafety committee approvals

  • Follow NIH Guidelines for Research Involving Recombinant or Synthetic Nucleic Acid Molecules

  • Document all safety procedures and training

According to NIH Guidelines, research with E. coli O157:H7 requires appropriate containment measures based on the specific recombinant constructs and experimental procedures being performed .

What strategies can overcome difficulties in electrophysiological characterization of MscS from E. coli O157:H7?

When facing challenges in electrophysiological characterization of MscS:

• Patch preparation issues:

  • Optimize protoplast preparation (adjust lysozyme concentration and incubation time)

  • Test different methods for spheroplast generation

  • Ensure osmotic stabilization during patch preparation

• Channel activity detection problems:

  • Increase channel expression using inducible promoters

  • Try alternative recording solutions with different ionic compositions

  • Optimize voltage protocols to enhance current detection

  • Test multiple patches (>15-20) due to potential patch-to-patch variability

• Pressure application challenges:

  • Calibrate pressure application systems regularly

  • Use pressure ramps rather than steps to identify activation thresholds

  • Try both positive and negative pressure applications

• Data analysis approaches:

  • Implement noise reduction algorithms

  • Use single-channel analysis software for precise conductance measurements

  • Calculate pressure thresholds relative to MscL for standardization

• Alternative approaches when patch-clamp fails:

  • Fluorescence-based methods using voltage-sensitive dyes

  • Downshock survival assays as indirect functional tests

  • In vivo ion flux measurements

  • Reconstitution into liposomes and stopped-flow fluorescence assays

Research on YbdG (an MscS homolog) demonstrates that wild-type channels may not show activity in patch-clamp despite functional evidence from in vivo assays, suggesting the need for multiple complementary approaches to characterize mechanosensitive channels .

How can recombinant E. coli O157:H7 MscS be utilized for studying bacterial adaptation to environmental stresses?

Recombinant E. coli O157:H7 MscS offers valuable tools for studying bacterial stress adaptation:

• Comparative strain construction:

  • Generate strains with native vs. modified MscS channels

  • Create reporter fusions to monitor MscS expression under different stresses

  • Develop multi-channel knockout strains with complemented MscS variants

• Stress adaptation models:

  • Transition between high and low osmolarity environments

  • Acid resistance challenges mimicking gastric passage

  • Desiccation and rehydration cycles for environmental persistence

  • Growth in bovine intestinal content to simulate reservoir conditions

• Experimental approaches:

  • Competition assays between wild-type and modified strains

  • Long-term evolution experiments under cyclic stress conditions

  • Transcriptomic and proteomic profiling during stress adaptation

  • Microscopy-based single-cell analysis of stress responses

• Applications for understanding pathogenesis:

  • Role of mechanosensing in colonization of bovine gastrointestinal tract

  • Contribution to survival during food processing procedures

  • Involvement in persistence in environmental reservoirs

  • Potential role during human infection process

Research indicates that MscS homologs may play differential roles in E. coli adaptation, with YbdG extending the range of survivable hypoosmotic shock while being expressed at insufficient levels to protect against severe shocks .

What are the comparative advantages of different model systems for studying MscS function in E. coli O157:H7?

Different model systems offer distinct advantages for studying MscS function:

For comprehensive characterization, researchers should employ multiple complementary systems, starting with safer K-12 backgrounds for initial studies before progressing to more physiologically relevant O157:H7 strains when necessary .

How can MscS channel engineering be applied to develop novel research tools or biotechnological applications?

MscS channel engineering offers diverse research and biotechnological applications:

• Controlled molecular delivery systems:

  • Development of MscS variants with altered gating properties

  • Engineering tension sensitivity for controlled activation

  • Creation of charge-sensitive MscS variants for electrical activation

  • Heterologous expression in mammalian cells for controlled cargo delivery

• Biosensing applications:

  • Reporter systems linking environmental stresses to MscS activation

  • Tension-activated gene expression systems

  • Coupling MscS gating to fluorescent reporters for real-time stress visualization

• Research tools:

  • MscS-based fusion proteins for membrane microdomain studies

  • Controllable cell lysis systems for extract preparation

  • Inducible expression systems for toxic protein production

• Potential biotechnological applications:

  • Controlled release of bioactive compounds from engineered cells

  • Development of osmosensitive bioreactors

  • Creation of cellular biosensors for environmental monitoring

  • Whole-cell catalysts with controlled permeability

• Channel modification approaches:

  • Pore size engineering through modification of TM3 packing

  • Gating threshold adjustment through lipid-interacting domain modifications

  • Ion selectivity alterations through pore residue substitutions

  • Sensitivity tuning through spring constant modifications