Recombinant Delftia acidovorans Large-conductance mechanosensitive channel (mscL)

What is the taxonomic context of Delftia acidovorans and how does it influence mscL research?

Delftia acidovorans belongs to the family Comamonadaceae, closely related to genera like Comamonas and Variovorax . Originally classified within the Pseudomonas genus, it was reclassified based on 16S rRNA gene sequence analysis. This taxonomic history is relevant for mscL research as it informs comparative genomic approaches. When studying D. acidovorans mscL, researchers should consider examining homologous channels in related species such as D. tsuruhatensis, D. lacustris, and D. litopenaei, which share significant sequence similarity (>97%) . Evolutionary conservation analysis of mscL across these species can provide insights into structural and functional significance of specific domains.

What are the physiological roles of mscL in D. acidovorans in its natural environments?

D. acidovorans has been isolated from diverse environments including soil, rhizosphere, clinical settings, and wastewater treatment facilities . In these varied habitats, the mscL channel likely plays critical roles in osmotic regulation and survival under fluctuating environmental conditions. D. acidovorans inhabits soil environments where it associates with plant roots, as seen in Cistus ladanifer rhizosphere isolation , and also appears in clinical settings as an opportunistic pathogen . In both contexts, mscL would function as a pressure release valve during hypoosmotic shock, preventing cell lysis when environmental osmolarity rapidly decreases. The channel's gating properties may be specifically adapted to the osmotic challenges in D. acidovorans' ecological niches, potentially contributing to its survival in both environmental and clinical settings.

How does D. acidovorans mscL compare structurally to well-characterized mscL channels from model organisms?

While the search results don't provide specific structural information on D. acidovorans mscL, mechanosensitive channels are typically pentameric transmembrane protein complexes. Based on comparative analysis with better-studied mscL proteins from E. coli and M. tuberculosis, the D. acidovorans channel likely shares the conserved transmembrane helices (TM1 and TM2) that form the channel pore, with the N-terminal S1 domain and C-terminal helical bundle contributing to channel gating and stability.

A key consideration for researchers is that D. acidovorans, as a member of Betaproteobacteria with a unique environmental adaptability , may exhibit distinctive features in its mscL structure that reflect its particular ecological niches. This could include modifications in the hydrophobic gate region or in cytoplasmic domains that interact with other cellular components, potentially optimizing channel function for D. acidovorans' specific membrane composition or osmotic stress responses.

What are the optimal strains and conditions for cloning and expressing recombinant D. acidovorans mscL?

For successful expression of recombinant D. acidovorans mscL, researchers should consider several strain options. E. coli BL21(DE3) is often suitable for initial expression attempts, but when membrane protein toxicity becomes problematic, C41(DE3) or C43(DE3) strains may yield better results due to their adapted membrane protein expression machinery.

Expression conditions should be optimized through a factorial design approach:

• Temperature range: Test 16°C, 25°C, and 30°C, with lower temperatures often favoring proper folding

• Induction: Use IPTG at concentrations between 0.1-0.5 mM, with gradual induction potentially improving membrane integration

• Media: M9 minimal medium supplemented with glycerol (0.4%) may reduce inclusion body formation compared to rich media like LB

• Additives: Consider including 5-10% glycerol and 100-500 mM NaCl in growth media to stabilize membrane proteins

The sequence of D. acidovorans mscL should be codon-optimized for the expression host, particularly given that D. acidovorans has a different GC content profile compared to common expression hosts . Including fusion tags (His6, MBP, or SUMO) at either the N- or C-terminus can facilitate purification, though their impact on channel functionality must be assessed experimentally.

What purification strategies are most effective for recombinant D. acidovorans mscL?

Purification of recombinant D. acidovorans mscL requires careful consideration of membrane protein behavior. A systematic purification protocol should include:

• Membrane Isolation:

  • Harvest cells during mid-log phase (OD600 0.6-0.8)

  • Disrupt cells via sonication or French press in buffer containing 50 mM Tris-HCl (pH 7.5), 200 mM NaCl, 10% glycerol, and protease inhibitors

  • Separate membranes by ultracentrifugation (100,000×g for 1 hour)

• Solubilization:

  • Test multiple detergents at concentrations 2-3× their critical micelle concentration:

    • n-Dodecyl-β-D-maltoside (DDM): 1.0%

    • n-Octyl-β-D-glucopyranoside (OG): 1.5%

    • Lauryl maltose neopentyl glycol (LMNG): 0.5%

  • Incubate solubilization mixture at 4°C for 1-2 hours with gentle agitation

• Affinity Chromatography:

  • For His-tagged constructs, use Ni-NTA resin with imidazole gradient elution (20-300 mM)

  • Include 0.05% detergent in all purification buffers

• Size Exclusion Chromatography:

  • Final polishing step using Superdex 200 column

  • Buffer: 20 mM HEPES (pH 7.5), 150 mM NaCl, 0.03% DDM or appropriate detergent

The stability of purified D. acidovorans mscL should be monitored through thermal shift assays and dynamic light scattering to identify optimal buffer conditions. Given D. acidovorans' ability to tolerate various environmental conditions, as demonstrated in biofilm studies , its mscL may exhibit distinct stability profiles compared to other bacterial mscL proteins.

How can researchers assess the proper folding and assembly of recombinant D. acidovorans mscL?

Proper folding and assembly of recombinant D. acidovorans mscL can be assessed through multiple complementary techniques:

• Gel Filtration Analysis:

  • Expected elution profile for pentameric assembly (~85-95 kDa for pentamer with detergent micelle)

  • Monitor for aggregation peaks or monomeric species

• Circular Dichroism (CD) Spectroscopy:

  • Characteristic α-helical content (expected ~60-70% for properly folded mscL)

  • Thermal stability assessment (40-90°C scan)

  • CD profile at 222 nm should show cooperative unfolding transition

• Blue-Native PAGE:

  • Allows visualization of native oligomeric state

  • Expected migration pattern for pentameric assembly (~80-90 kDa)

• Negative-Stain Electron Microscopy:

  • Visual confirmation of homogeneous particle size distribution

  • Preliminary structural assessment prior to functional studies

A correctly folded and assembled mscL pentamer is essential for functional studies. Researchers should validate their purification workflow by comparing structural characteristics to well-characterized bacterial mscL channels and confirming that the protein exhibits stability under conditions relevant for functional assays. The environmental adaptability of D. acidovorans suggests its mscL may have evolved unique structural properties that merit careful characterization.

What electrophysiology approaches are most suitable for characterizing D. acidovorans mscL conductance properties?

For rigorous electrophysiological characterization of D. acidovorans mscL, researchers should consider the following methodologies:

• Patch Clamp Electrophysiology (Spheroplast Patch):

  • Prepare E. coli spheroplasts expressing D. acidovorans mscL

  • Utilize excised inside-out patch configuration

  • Apply negative pressure steps (0-300 mmHg) while recording at holding potentials from -60 to +60 mV

  • Key parameters to measure: pressure threshold for activation, conductance (expected ~2.5-3.5 nS in 200 mM KCl), subconductance states, and adaptation behavior

• Planar Lipid Bilayer Recordings:

  • Reconstitute purified mscL into liposomes (POPE:POPG, 7:3 ratio)

  • Transfer protein-containing liposomes to planar lipid bilayer setup

  • Apply hydrostatic or osmotic pressure gradients while recording

  • Advantage: Allows precise control of lipid composition, mimicking D. acidovorans native membrane environment

• Fluorescence-Based Flux Assays:

  • Reconstitute mscL into liposomes containing calcein or other fluorescent dyes

  • Apply osmotic downshift to trigger channel opening

  • Monitor fluorescence dequenching as indicator of channel activity

  • Enables high-throughput screening of channel modulators

D. acidovorans' adaptation to various environmental conditions may be reflected in unique gating properties of its mscL. Researchers should compare conductance, pressure sensitivity, and gating kinetics with channels from other bacterial species, particularly focusing on how membrane tension thresholds might relate to D. acidovorans' ecological niches.

How can researchers investigate the impact of membrane composition on D. acidovorans mscL function?

The function of mechanosensitive channels is intimately linked to membrane properties. To investigate these relationships for D. acidovorans mscL:

• Systematic Membrane Composition Analysis:

  • Reconstitute purified mscL into liposomes with varying lipid compositions:

    • Varying PE:PG:CL ratios to mimic different bacterial membranes

    • Incorporating bacterial-specific lipids (e.g., cyclopropane fatty acids)

    • Testing effects of membrane-active compounds identified in D. acidovorans ecological niches

• Membrane Tension Measurements:

  • Use micropipette aspiration of giant unilamellar vesicles (GUVs) containing D. acidovorans mscL

  • Correlate channel activity with directly measured membrane tension

  • Compare gating threshold tensions across different lipid compositions

• Fluorescence Microscopy Approaches:

  • Employ FRET-based tension sensors to visualize local membrane deformations during channel gating

  • Use fluorescently labeled lipids to examine potential lipid sorting around mscL clusters

Given D. acidovorans' ability to form biofilms with distinct morphochemical characteristics , researchers should investigate how biofilm-specific membrane modifications might influence mscL function. The bacterial strain's tolerance to antimicrobials like chlorhexidine could be partially mediated through membrane adaptations that also affect mechanosensitive channel activity.

What approaches can be used to study D. acidovorans mscL interaction with antimicrobial compounds?

D. acidovorans demonstrates variable tolerance to antimicrobial compounds, particularly chlorhexidine , making its mscL channel an interesting target for antimicrobial interaction studies:

• Direct Binding Assays:

  • Employ isothermal titration calorimetry (ITC) to measure binding thermodynamics

  • Use surface plasmon resonance (SPR) to determine association/dissociation kinetics

  • Implement microscale thermophoresis (MST) for preliminary screening of compound interactions

• Functional Impact Assessment:

  • Electrophysiology in presence of antimicrobial compounds at sub-MIC concentrations

  • Liposome dye release assays to measure channel activation/inhibition

  • Patch clamp analysis of pressure threshold shifts upon compound application

• Structural Biology Approaches:

  • Use hydrogen-deuterium exchange mass spectrometry (HDX-MS) to identify binding sites

  • Implement cysteine scanning mutagenesis with site-specific labeling to map antimicrobial interaction sites

  • Molecular dynamics simulations to predict compound-channel interactions

Comparative analysis between wild-type and chlorhexidine-tolerant D. acidovorans strains (such as WT15 vs. MT51 ) could reveal how mscL structure and function might contribute to antimicrobial tolerance. The experimental design should account for D. acidovorans' observed variable responses to antimicrobials across different biofilm structures and cell types .

How can D. acidovorans mscL be utilized as a tool for targeted drug delivery systems?

The large pore size of mscL (~30Å when fully open) makes it an attractive candidate for controlled molecular release applications:

• Engineered Drug Release Systems:

  • Design liposomes incorporating modified D. acidovorans mscL with engineered gating properties

  • Develop light-activated or pH-sensitive mscL variants through site-directed mutagenesis

  • Create chimeric channels combining sensing domains from other proteins with D. acidovorans mscL structural framework

• Implementation Strategy:

  • Encapsulate therapeutic compounds in liposomes containing engineered mscL

  • Program channel opening in response to specific physiological conditions

  • Target delivery to environments resembling D. acidovorans' natural habitats (e.g., biofilms)

• Experimental Validation:

  • Measure release kinetics using fluorescent cargo molecules

  • Assess stability in physiological conditions

  • Evaluate specificity of triggering mechanisms

D. acidovorans' natural adaptability to diverse environments suggests its mscL may have evolved unique responsiveness to environmental cues, potentially offering advantages for engineering controlled release systems with specific triggering conditions.

What are the molecular determinants of D. acidovorans mscL's pressure sensitivity and how can they be manipulated?

Understanding and manipulating the pressure sensitivity of D. acidovorans mscL requires detailed structure-function analysis:

• Key Structural Elements for Investigation:

  • Transmembrane domain 1 (TM1): Contains the hydrophobic gate constriction

  • Transmembrane domain 2 (TM2): Contributes to tension sensing

  • N-terminal (S1) domain: Modulates gating behavior

  • C-terminal domain: Influences channel stability and interconnection with cytoskeleton

• Experimental Approaches:

  • Alanine scanning mutagenesis of TM1 and TM2 domains

  • Introduction of charged residues at the hydrophobic gate

  • FRET-based studies to track conformational changes during gating

  • Chimeric channels combining domains from D. acidovorans mscL with those from other bacterial species

• Analytical Framework:

  • Correlate amino acid substitutions with changes in:

    • Pressure threshold for activation

    • Open probability at defined pressures

    • Conductance and subconductance states

    • Adaptation/desensitization kinetics

The evolutionary adaptations of D. acidovorans to various environmental stresses may be reflected in unique pressure-sensing properties of its mscL. Researchers should analyze whether amino acid differences in tension-sensing regions correlate with the ecological niches inhabited by different Delftia species .

How can high-throughput methods be developed to screen for compounds that modulate D. acidovorans mscL activity?

Developing high-throughput screening (HTS) approaches for D. acidovorans mscL modulators should consider:

• Primary Screening Assays:

  • Fluorescence-based liposome assays:

    • Reconstitute mscL into liposomes containing self-quenching fluorescent dyes

    • Monitor fluorescence increase upon channel activation

    • Implement in 384-well format for rapid compound screening

  • Cell-based growth assays:

    • Express D. acidovorans mscL in E. coli lacking endogenous mechanosensitive channels

    • Expose to hypoosmotic shock with/without test compounds

    • Measure survival as indicator of channel function

• Secondary Validation Approaches:

  • Patch clamp electrophysiology of promising hits

  • SPR or ITC to confirm direct binding

  • Mutagenesis to identify binding sites

• Assay Development Considerations:

  • Optimize reconstitution conditions for consistent channel density

  • Determine DMSO tolerance (typically up to 1%)

  • Implement appropriate positive controls (known channel activators like lysophosphatidylcholine)

  • Include inactive channel mutants as negative controls

Given D. acidovorans' interactions with various compounds in its environmental niches , researchers might explore natural product libraries derived from relevant ecosystems as potential sources of specific mscL modulators.

What approaches can resolve data inconsistencies in D. acidovorans mscL functional studies?

When facing inconsistent results in mscL functional studies, researchers should implement a systematic troubleshooting approach:

• Common Sources of Variability:

  • Protein Preparation Factors:

    • Detergent lot variability affecting solubilization efficiency

    • Incomplete delipidation altering native lipid content

    • Oxidation of cysteine residues affecting channel structure

  • Reconstitution Parameters:

    • Inconsistent protein-to-lipid ratios

    • Variability in liposome size distribution

    • Incomplete detergent removal

  • Recording Conditions:

    • Patch geometry variations affecting tension distribution

    • Temperature fluctuations altering membrane properties

    • Buffer composition differences impacting channel conductance

• Standardization Protocol:

  • Implement standard quality control metrics for protein preparations:

    • SEC profile consistency check before each experiment

    • Circular dichroism to confirm secondary structure

    • SDS-PAGE and Western blot to verify protein integrity

  • Standardize reconstitution procedures:

    • Use dynamic light scattering to verify liposome size distribution

    • Quantify protein incorporation efficiency for each preparation

    • Implement rigorous detergent removal validation

• Statistical Approach:

  • Collect sufficient biological replicates (n ≥ 5) from independent protein preparations

  • Apply appropriate statistical tests that account for non-normal distributions

  • Use power analysis to determine required sample sizes

D. acidovorans' demonstrated phenotypic heterogeneity in biofilm studies suggests potential protein-level heterogeneity that might contribute to functional variability. Researchers should consider whether observed inconsistencies might reflect biological properties rather than experimental artifacts.

How can researchers overcome expression and purification challenges specific to D. acidovorans mscL?

When facing challenges with recombinant D. acidovorans mscL expression and purification:

• Expression Optimization Strategies:

  • For poor expression levels:

    • Test alternative promoter systems (e.g., pBAD instead of T7)

    • Evaluate expression in specialized membrane protein hosts (C41/C43, LEMO21)

    • Consider cell-free expression systems with supplied lipids or nanodiscs

  • For toxicity issues:

    • Implement tight expression control with glucose repression

    • Use strains with reduced basal expression

    • Consider inducible periplasmic targeting to reduce membrane disruption

• Solubilization Improvements:

  • For inefficient extraction:

    • Test detergent combinations (e.g., DDM with cholesteryl hemisuccinate)

    • Implement temperature-dependent solubilization (gradual increase from 4°C to 25°C)

    • Consider novel amphipathic polymers (SMALPs) for native lipid co-extraction

  • For aggregation issues:

    • Include osmolytes (e.g., glycerol, betaine) in buffers

    • Test ionic strength ranges from 150-500 mM NaCl

    • Add specific lipids (POPE, cardiolipin) during solubilization

• Purification Enhancements:

  • For co-purifying contaminants:

    • Implement ion exchange chromatography steps

    • Use GFP fusion and FSEC to identify optimal detergent conditions

    • Consider on-column detergent exchange during affinity purification

D. acidovorans' adaptability to various growth conditions may be reflected in complex membrane composition. Drawing from biofilm and environmental adaptation studies , researchers might explore how growth media composition affects membrane properties and subsequent mscL extraction efficiency.

What analytical techniques can definitively distinguish between functional and non-functional recombinant D. acidovorans mscL?

To definitively assess the functionality of recombinant D. acidovorans mscL preparations:

• Integrated Analytical Workflow:

  • Structural Integrity Assessment:

    • Size exclusion chromatography-multi-angle light scattering (SEC-MALS) to confirm pentameric assembly

    • Thermal stability analysis via differential scanning fluorimetry

    • Limited proteolysis to verify native-like conformation

  • Functional Validation:

    • Patch clamp analysis of reconstituted channels (gold standard)

    • Stopped-flow fluorescence assays for channel activation

    • Orientation analysis via accessibility of introduced cysteine residues

  • Comparative Analysis:

    • Parallel testing with known functional mscL from model organisms

    • Correlation between structural parameters and functional outcomes

    • Analysis of substrate permeation profiles

• Systematic Controls:

  • Positive Controls:

    • Well-characterized E. coli MscL expressed and purified identically

    • Known channel activators (LPC, membrane thinning agents)

  • Negative Controls:

    • Inactivating mutants (e.g., G22D equivalent in D. acidovorans mscL)

    • Heat-denatured protein preparations

    • Empty liposomes/membranes

• Activity Quantification:

  • Pressure dose-response curves (P50 determination)

  • Single channel conductance measurements

  • Open probability calculations at standardized tension values

The unique environmental adaptations of D. acidovorans may result in distinct functional signatures for its mscL. Researchers should consider developing D. acidovorans-specific functional benchmarks rather than relying solely on comparisons with model organisms.