Recombinant Burkholderia cepacia Large-conductance mechanosensitive channel (mscL)

What is Burkholderia cepacia complex and what role does the mechanosensitive channel play in these bacteria?

The Burkholderia cepacia complex (Bcc) comprises at least nine closely related bacterial species that function as opportunistic pathogens in various hosts, including plants, invertebrates, animals, and humans. The complex is particularly concerning as a pathogen in cystic fibrosis patients and shows high intrinsic resistance to antibiotics .

The Large-conductance mechanosensitive channel (mscL) is a membrane protein that responds to mechanical tension in the bacterial cell membrane. It functions as a pressure release valve during osmotic downshock, preventing cell lysis by allowing the rapid efflux of solutes when bacteria transition from high to low osmolarity environments. In Burkholderia cepacia, this channel likely plays a critical role in environmental adaptation and survival under varying osmotic conditions.

How does mscL function differ between Burkholderia cepacia and other bacterial species?

While the basic function of mscL as an emergency release valve during hypoosmotic shock is conserved across bacterial species, there are significant structural and functional variations in mscL proteins from different bacteria that affect their gating properties, ion selectivity, and conductance.

In Burkholderia cepacia, the mscL protein operates within a complex genomic context influenced by the bacteria's unique methylation patterns. DNA methylation in B. cepacia affects numerous cellular functions including biofilm formation, cell shape, motility, siderophore production, and membrane vesicle production . These methylation patterns may indirectly influence mscL expression and function through complex regulatory networks.

What expression systems are most effective for producing recombinant Burkholderia cepacia mscL?

For expression of recombinant bacterial membrane proteins like mscL, several systems have proven effective with varying advantages:

E. coli-based expression systems:

• BL21(DE3) strain with pET-based vectors provides high yield but may lead to inclusion body formation

• C41(DE3) and C43(DE3) strains, derived from BL21(DE3), are engineered specifically for membrane protein expression

• Tuner™ strains allow fine control of protein expression levels through variable IPTG concentration

Expression methodology recommendations:

• Use lower induction temperatures (16-25°C) to slow production and improve folding

• Consider fusion tags that enhance solubility (MBP, SUMO) or aid purification (His6, Strep-tag)

• Supplement growth media with glycerol (0.5-1%) to enhance membrane protein production

When designing expression constructs, researchers should account for the complex genomic context of B. cepacia genes, which may include specific methylation patterns that influence gene expression .

What are the recommended protocols for isolating and purifying recombinant Burkholderia cepacia mscL protein?

Recommended purification protocol:

• Cell lysis and membrane preparation:

  • Harvest cells by centrifugation (6,000 × g, 15 min, 4°C)

  • Resuspend in buffer containing 50 mM Tris-HCl pH 7.5, 200 mM NaCl, 1 mM EDTA, protease inhibitors

  • Disrupt cells using sonication or French press

  • Remove unbroken cells and debris by centrifugation (10,000 × g, 20 min, 4°C)

  • Isolate membranes by ultracentrifugation (150,000 × g, 1 h, 4°C)

• Membrane protein solubilization:

  • Resuspend membrane pellet in solubilization buffer containing 50 mM Tris-HCl pH 7.5, 200 mM NaCl, and detergent

  • For mscL, detergents such as n-dodecyl-β-D-maltopyranoside (DDM, 1-2%) or n-octyl-β-D-glucopyranoside (OG, 2-3%) are effective

  • Incubate with gentle rotation (2-4 h or overnight at 4°C)

  • Remove insoluble material by ultracentrifugation (150,000 × g, 30 min, 4°C)

• Affinity chromatography:

  • If using His-tagged constructs, apply solubilized protein to Ni-NTA or TALON resin

  • Wash with increasing imidazole concentrations (10-40 mM) to remove non-specific binding

  • Elute with higher imidazole (250-500 mM)

  • Pool protein-containing fractions and concentrate using 30-50 kDa cutoff concentrators

• Size exclusion chromatography:

  • Further purify by gel filtration using Superdex 200 or similar matrix

  • Use buffer containing reduced detergent concentration (0.03-0.05% DDM)

  • Collect fractions containing tetrameric/pentameric mscL

The key to successful purification is maintaining the protein in a native-like membrane environment, which can be achieved using appropriate detergents or reconstitution into lipid nanodiscs or liposomes for downstream applications.

How can researchers effectively reconstitute purified mscL into liposomes for functional studies?

Liposome reconstitution protocol:

• Lipid preparation:

  • Prepare a lipid mixture mimicking bacterial membrane composition (e.g., 70% phosphatidylethanolamine, 15% phosphatidylglycerol, 15% cardiolipin)

  • Dissolve lipids in chloroform:methanol (2:1), dry under nitrogen stream

  • Remove residual solvent by vacuum desiccation (2-3 h)

  • Rehydrate with reconstitution buffer (10 mM HEPES pH 7.4, 150 mM KCl)

  • Subject to 5 freeze-thaw cycles to form multilamellar vesicles

• Liposome sizing:

  • Extrude through polycarbonate filters (400 nm, then 200 nm) to form unilamellar vesicles

  • Alternatively, sonicate lipid suspension to form small unilamellar vesicles

• Protein incorporation:

  • Mix purified mscL protein with preformed liposomes (protein:lipid ratio 1:50 to 1:200)

  • Add detergent (e.g., Triton X-100) to destabilize liposomes (reaching onset of solubilization)

  • Incubate mixture (30 min, room temperature) with gentle agitation

  • Remove detergent using Bio-Beads SM-2 or dialysis

  • For Bio-Beads: add 30 mg/ml of suspension, incubate 3 h, replace beads, continue overnight at 4°C

• Proteoliposome isolation:

  • Separate proteoliposomes from free protein by centrifugation (150,000 × g, 1 h, 4°C)

  • Resuspend pellet in desired buffer for functional assays

This reconstitution method preserves the native-like environment necessary for proper mscL function and allows for subsequent electrophysiological or fluorescence-based activity assays.

What electrophysiological methods are most suitable for characterizing mscL channel activity?

Several electrophysiological techniques have been successfully applied to study mechanosensitive channels:

• Patch clamp of giant proteoliposomes:

  • Form giant unilamellar vesicles (GUVs) by electroformation or gentle hydration

  • Reconstitute mscL into GUVs using the protocol described above

  • Apply patch-clamp techniques in inside-out configuration

  • Apply negative pressure (suction) through the patch pipette to activate the channel

  • Record single-channel currents at different membrane tensions

• Planar lipid bilayer recordings:

  • Form planar bilayers across a small aperture in a Teflon partition

  • Add proteoliposomes near the aperture for fusion

  • Apply membrane tension through hydrostatic pressure difference

  • Record currents using voltage-clamp amplifiers

• Fluorescence-based assays:

  • Reconstitute mscL into liposomes containing self-quenching fluorescent dyes

  • Measure dye release upon osmotic downshock as an indicator of channel activity

  • This method allows for higher-throughput screening of channel variants or modulators

For mechanistic studies of mscL from Burkholderia cepacia, patch-clamp of giant proteoliposomes provides the most detailed information about channel gating properties and kinetics.

What cloning strategies are most effective for genetic manipulation of the mscL gene in Burkholderia cepacia?

Effective genetic manipulation of B. cepacia requires consideration of its complex genome and specific molecular biology:

• Vector selection:

  • For expression in B. cepacia: broad-host-range vectors based on pBBR1, pRK290, or pJN105 backbones

  • For heterologous expression: standard E. coli vectors (pET, pBAD) with appropriate promoters

  • Include selectable markers functional in B. cepacia (Trimethoprim, Tetracycline, Gentamicin resistance)

• Promoter considerations:

  • Native promoters may be influenced by B. cepacia-specific regulatory factors

  • For controlled expression in B. cepacia, use inducible promoters (rhamnose-inducible pRha, PBAD)

  • Account for restriction-modification systems when introducing foreign DNA

• Gene delivery methods:

  • Electroporation protocols optimized for B. cepacia (high voltage: 2.5 kV, 25 μF, 200 Ω)

  • Triparental mating using helper strains carrying pRK2013 or pRK2073

  • Pre-treatment of DNA with B. cepacia cell-free extracts to overcome restriction barriers

When working with B. cepacia, researchers should be aware of the complex methylation patterns that may affect gene expression and protein function. DNA methylation in B. cepacia has been shown to influence numerous cellular properties including biofilm formation, cell shape, and motility .

How can site-directed mutagenesis be used to investigate structure-function relationships in Burkholderia cepacia mscL?

Site-directed mutagenesis offers powerful insights into mscL channel mechanics:

• Key residues for targeted mutation:

  • Hydrophobic pore-lining residues: affect gating tension threshold

  • Transmembrane helix-helix interface residues: influence subunit interactions

  • Cytoplasmic and periplasmic loop residues: modulate channel sensitivity

• Recommended mutagenesis approaches:

  • QuikChange PCR-based method for single mutations

  • Gibson Assembly or Golden Gate Assembly for multiple simultaneous mutations

  • CRISPR-Cas9 for chromosomal editing in B. cepacia

• Functional analysis of mutants:

  • Electrophysiological characterization to determine changes in:

    • Activation threshold (tension required for opening)

    • Open probability at given tensions

    • Subconductance states

    • Gating kinetics

  • Complementation studies in mscL-knockout strains to assess function in vivo

• Systematic mutation strategy:

  • Alanine scanning: replace each residue sequentially with alanine

  • Charge substitutions: introduce charged residues at neutral positions

  • Cysteine scanning: introduce cysteines for subsequent chemical modification

By systematically analyzing the effects of specific mutations, researchers can develop a detailed understanding of the molecular mechanisms underlying mscL function in B. cepacia.

What genomic contexts affect mscL gene expression in Burkholderia cepacia, and how can these be experimentally investigated?

The genomic context of mscL in B. cepacia significantly impacts its expression and function:

• DNA methylation effects:

  • Research has shown that DNA methylation patterns in B. cepacia influence multiple cellular properties

  • DNA methylation affects biofilm formation, cell shape, motility, siderophore production, and membrane vesicle production

  • Methylation may directly or indirectly affect mscL expression through regulatory pathways

• Experimental approaches to investigate genomic context:

  • Single-molecule, real-time (SMRT) sequencing:

    • Allows detection and analysis of methylated bases

    • Can identify methylation patterns in proximity to the mscL gene

  • RNA-seq under varying osmotic conditions:

    • Reveals transcriptional networks controlling mscL expression

    • Identifies co-regulated genes in response to osmotic stress

  • Chromatin immunoprecipitation (ChIP):

    • Identifies transcription factors binding near the mscL promoter

    • Reveals regulatory protein interactions with the genomic region

• Methylome analysis techniques:

  • Whole-genome bisulfite sequencing to detect methylation patterns

  • SMRT technology to identify methylated motifs

  • Creation of methyltransferase knockout strains to assess effects on mscL expression

• Integration with taxonomic understanding:

  • The Burkholderia cepacia complex comprises at least nine closely related species

  • Comparative genomics across the complex can reveal conservation of mscL regulatory elements

  • Multilocus sequence typing (MLST) helps identify strain-specific differences in genetic context

Understanding the genomic context of mscL requires integration of methylome analysis, transcriptomic data, and comparative genomics across the Burkholderia cepacia complex.

How can recombinant Burkholderia cepacia mscL be utilized in drug discovery for targeting bacterial membrane proteins?

The unique properties of mscL make it an attractive target for antimicrobial development:

• Target validation approaches:

  • Determining essentiality of mscL in B. cepacia under various stress conditions

  • Evaluating phenotypic consequences of mscL inhibition or activation

  • Assessing potential for selectivity between bacterial and host cells

• High-throughput screening platforms:

  • Fluorescence-based liposome dye release assays for compound screening

  • Cell-based reporter systems linking mscL activation to fluorescent output

  • Label-free technologies (e.g., impedance measurements) to detect channel modulation

• Rational drug design strategies:

  • Structure-based design targeting unique features of B. cepacia mscL

  • Peptide modulators designed to interact with the channel pore or interfere with gating

  • Small molecules that alter membrane physical properties to indirectly modulate channel activity

• Therapeutic development considerations:

  • Compounds that lock mscL in open state to cause bacterial lysis

  • Molecules that prevent channel opening during osmotic stress

  • Dual-action compounds targeting both mscL and other essential membrane functions

The development of mscL-targeting therapeutics could be particularly valuable for treating B. cepacia infections in cystic fibrosis patients, where these bacteria demonstrate significant antibiotic resistance .

What are the current challenges in structural studies of Burkholderia cepacia mscL, and how can they be addressed?

Structural studies of membrane proteins like mscL face several challenges:

• Key challenges:

  • Obtaining sufficient quantities of stable, properly folded protein

  • Maintaining native conformation during purification and analysis

  • Capturing different conformational states (closed, intermediate, open)

  • Limited structural data specifically for B. cepacia mscL

• Advanced structural biology approaches:

  • Cryo-electron microscopy (cryo-EM):

    • Advantages: Can visualize protein in near-native lipid environment

    • Challenges: Sample preparation, achieving high resolution for smaller membrane proteins

    • Solutions: Use of larger tags, nanodiscs, or amphipols to increase particle size

  • X-ray crystallography:

    • Advantages: Potentially atomic resolution

    • Challenges: Obtaining well-diffracting crystals of membrane proteins

    • Solutions: Lipidic cubic phase crystallization, fusion with crystallization chaperones

  • Nuclear magnetic resonance (NMR) spectroscopy:

    • Advantages: Information about protein dynamics

    • Challenges: Size limitations, complex spectra for large membrane proteins

    • Solutions: Selective isotope labeling, solid-state NMR approaches

• Computational approaches:

  • Homology modeling based on structurally characterized mscL proteins from other bacteria

  • Molecular dynamics simulations to study conformational changes during gating

  • Integration of experimental data with computational predictions

• Innovative techniques for conformational studies:

  • Single-molecule Förster resonance energy transfer (smFRET) to measure distances between domains

  • Hydrogen-deuterium exchange mass spectrometry (HDX-MS) to probe structural dynamics

  • Site-directed spin labeling combined with electron paramagnetic resonance (EPR) spectroscopy

Addressing these challenges requires multidisciplinary approaches combining advanced protein engineering, membrane mimetics, and hybrid structural biology methods.

How does methylation of the Burkholderia cepacia genome influence mscL expression and function?

DNA methylation plays a significant role in B. cepacia biology:

• Methylation patterns in B. cepacia:

  • SMRT sequencing has revealed specific methylation motifs in B. cepacia genomes

  • The B. cepacia complex contains several core restriction-modification systems

  • DNA methylation has been shown to affect numerous cellular properties

• Potential impacts on mscL:

  • Direct regulatory effects:

    • Methylation of promoter regions may directly affect transcription factor binding

    • Changes in DNA structure due to methylation can alter promoter accessibility

  • Indirect regulatory effects:

    • Methylation affects biofilm formation which may alter osmotic stress responses

    • Changes in cell shape and membrane properties due to methylation may influence mscL function

    • Altered membrane vesicle production may affect membrane tension sensing

• Experimental approaches to investigate methylation impacts:

  • Compare mscL expression in wild-type and methyltransferase mutant strains

  • Analyze DNA methylation patterns in the mscL gene and promoter region using bisulfite sequencing

  • Perform electrophysiological studies of mscL from strains with altered methylation patterns

• Integration with systems biology:

  • Methylation affects multiple cellular pathways simultaneously

  • Network analysis can reveal connections between methylation patterns and stress responses

  • Multi-omics approaches (methylome, transcriptome, proteome) provide comprehensive view

The complex relationship between DNA methylation and mscL function represents an area where significant new discoveries await in understanding B. cepacia biology.

How can recombinant Burkholderia cepacia mscL contribute to understanding antibiotic resistance mechanisms?

The study of mscL provides insights into membrane-based resistance mechanisms:

• Membrane permeability and antibiotic entry:

  • mscL channels can potentially serve as entry points for certain antibiotics

  • Understanding mscL gating mechanisms may reveal approaches to increase antibiotic uptake

  • Membrane tension modulators could potentially sensitize resistant bacteria

• Relevance to B. cepacia antibiotic resistance:

  • B. cepacia complex bacteria demonstrate high intrinsic resistance to antibiotics

  • Membrane modifications are key resistance mechanisms in these bacteria

  • mscL may contribute to survival during antibiotic-induced osmotic stress

• Research approaches:

  • Study correlations between mscL expression levels and antibiotic susceptibility

  • Investigate interactions between antibiotics and mscL using electrophysiology

  • Develop mscL-targeting compounds as antibiotic adjuvants

  • Examine mscL function in clinical isolates with varying resistance profiles

• Potential therapeutic applications:

  • Compounds that modulate mscL gating to increase antibiotic influx

  • Dual-targeting approaches affecting both mscL and cell wall synthesis

  • mscL-mediated delivery systems for novel antimicrobials

The high antibiotic resistance of B. cepacia complex bacteria makes them particularly challenging pathogens in clinical settings, especially for cystic fibrosis patients . Targeting mscL could provide new approaches to overcome this resistance.

What role does mscL play in Burkholderia cepacia pathogenesis and host-pathogen interactions?

Understanding mscL's role in pathogenesis involves multiple aspects:

• Osmotic adaptation during infection:

  • Host environments present varying osmotic challenges

  • mscL likely contributes to bacterial survival during osmotic transitions

  • Regulation of mscL may be integrated with virulence factor expression

• Influence on biofilm formation:

  • DNA methylation affects biofilm formation in B. cepacia

  • Biofilms are critical for B. cepacia persistence in host environments

  • mscL-mediated responses to mechanical stress may influence biofilm development

• Experimental approaches to study pathogenesis roles:

  • Comparison of wild-type and mscL mutant strains in infection models

  • Transcriptomic analysis of mscL expression during different infection stages

  • Live cell imaging of mscL-fluorescent protein fusions during host cell interactions

• Connection to clinical outcomes:

  • B. cepacia complex bacteria are particularly concerning as CF pathogens

  • Understanding mscL's role in adaptation to lung environments may explain virulence

  • Strain variation in mscL sequence or expression may correlate with clinical outcomes

Research into mscL's role in pathogenesis should consider the taxonomic complexity of the B. cepacia complex, which comprises at least nine closely related species with different pathogenic potential .

What are the emerging biosensor applications of recombinant Burkholderia cepacia mscL?

Recombinant mscL channels hold promise for advanced biosensing technologies:

• Mechanosensitive biosensor designs:

  • Integration of mscL into artificial membranes for tension sensing

  • Engineering of mscL to respond to specific stimuli beyond membrane tension

  • Coupling channel activity to detectable outputs (electrical, optical, enzymatic)

• Potential applications:

  • Environmental monitoring:

    • Detection of osmotically active contaminants

    • Sensors for membrane-disrupting toxins

    • Mechanical stress measurements in complex matrices

  • Diagnostic platforms:

    • Detection of membrane-active antimicrobial peptides

    • Monitoring of membrane-targeting drug efficacy

    • Screening for compounds affecting membrane properties

• Engineering approaches:

  • Modification of tension sensitivity through targeted mutations

  • Addition of ligand-binding domains for chemical sensing

  • Incorporation of reporter elements that activate upon channel opening

• Technical implementation strategies:

  • Liposome-based sensors with encapsulated reporters

  • Tethered bilayer membranes on electrode surfaces

  • Microfluidic platforms with integrated membrane patches

The unique properties of B. cepacia mscL, particularly its adaptation to specific environmental niches, may make it especially valuable for specialized biosensing applications.

What are the most promising areas for future research on Burkholderia cepacia mscL?

Several research directions offer particular promise:

• Structural biology frontiers:

  • High-resolution structures of B. cepacia mscL in multiple conformational states

  • Comparative structural analysis across the B. cepacia complex species

  • Integration of computational and experimental approaches for complete gating models

• Systems biology integration:

  • Network analysis of mscL regulation within osmotic stress response pathways

  • Multi-omics profiling to connect methylation patterns, transcription, and channel function

  • Machine learning approaches to predict mscL behavior under complex conditions

• Therapeutic development opportunities:

  • Rational design of mscL-targeting antimicrobials

  • Development of combination therapies targeting membrane homeostasis

  • Personalized approaches based on patient-specific B. cepacia strains

• Synthetic biology applications:

  • Engineering mscL variants with novel sensing capabilities

  • Development of mscL-based cellular computation elements

  • Creation of artificial cells with programmable osmotic responses

• Clinical microbiology advancements:

  • Improved molecular diagnostics for B. cepacia complex species

  • Correlation of mscL sequence variants with clinical outcomes

  • Development of rapid antibiotic susceptibility testing based on membrane properties

The broader field will benefit from interdisciplinary approaches combining microbiology, biophysics, structural biology, and clinical research.

How might comparative studies across the Burkholderia cepacia complex advance our understanding of mscL evolution and function?

Comparative studies offer unique insights into mscL biology:

• Evolutionary analysis approaches:

  • Phylogenetic analysis of mscL sequences across the B. cepacia complex

  • Correlation of mscL sequence variation with ecological niches

  • Examination of selection pressures on different mscL domains

• Functional comparative studies:

  • Electrophysiological characterization of mscL from different Bcc species

  • Cross-species complementation studies with mscL variants

  • Correlation of channel properties with species-specific membrane compositions

• Genomic context considerations:

  • Analysis of mscL gene neighborhoods across species

  • Examination of methylation patterns affecting mscL in different species

  • Identification of species-specific regulatory elements

• Methodological framework:

  • Multilocus sequence typing (MLST) for accurate species identification

  • Whole-genome sequencing to place mscL in complete genomic context

  • Standardized functional assays for cross-species comparison

The B. cepacia complex includes at least nine closely related species with both shared and distinct characteristics . Comparing mscL across these species can reveal important adaptive variations in channel structure and function.

What technological advances are needed to overcome current limitations in Burkholderia cepacia mscL research?

Several technological challenges currently limit progress:

• Protein production and purification:

  • Need for improved expression systems yielding greater quantities of functional protein

  • Development of membrane mimetics that better preserve native channel properties

  • High-throughput purification protocols for parallel processing of multiple variants

• Structural biology methods:

  • Higher resolution imaging technologies for membrane protein structures

  • Improved approaches for capturing transient conformational states

  • Methods for structure determination in native-like membrane environments

• Functional characterization:

  • Higher-throughput electrophysiology platforms

  • Improved sensitivity for measuring subtle differences in channel gating

  • Single-molecule techniques applicable to complex membrane proteins

• Genetic manipulation tools:

  • More efficient gene editing systems for B. cepacia

  • Methods to overcome restriction-modification barriers in these bacteria

  • Controllable expression systems specifically optimized for B. cepacia

• Computational resources:

  • More accurate force fields for membrane protein simulations

  • Increased computational power for all-atom simulations of gating transitions

  • Improved algorithms for predicting functional impacts of sequence variations

Addressing these technological limitations will accelerate progress and enable deeper understanding of mscL biology in B. cepacia and related bacteria.