Recombinant Xanthomonas axonopodis pv. citri Large-conductance mechanosensitive channel (mscL)

What is the Xanthomonas axonopodis pv. citri large-conductance mechanosensitive channel?

The large-conductance mechanosensitive channel (mscL) in Xanthomonas axonopodis pv. citri is a membrane protein that functions as a pressure-release valve during osmotic downshock. The protein consists of 143 amino acids with a molecular structure that forms a pentameric complex embedded in the bacterial membrane. According to the available sequence data, the protein contains multiple transmembrane domains with conserved regions critical for channel gating and function. The full amino acid sequence includes "MGMVSEFKQFAMRGNVIDLAVGVVIGAAFGKIVTALVEKIIMPPIGWAIGNVDFSRLAWVLKPAGVDATGKEIPAVAIGYGDFINTVVQFLIIAFAIFLVVKLINRVTHRKPDAPKGPSEEVLLLREIRDALKNDTLKPPGAL" . This channel plays a crucial role in bacterial survival under changing osmotic conditions, particularly during plant infection processes.

How does the Xac mscL differ from mechanosensitive channels in other bacterial species?

Mechanosensitive channels are widely conserved across bacterial species, but Xac mscL exhibits specific structural and functional adaptations related to its phytopathogenic lifestyle. While the core mechanosensitive function remains similar to other bacterial mscL proteins, the Xac variant contains unique amino acid substitutions that may influence its sensitivity to membrane tension and interaction with other virulence factors. Unlike mechanosensitive channels in some other species, Xac mscL appears to be integrated into specialized pathogenicity mechanisms. This is evidenced by studies of various Xanthomonas virulence factors that show coordinated expression during plant infection . The channel likely works in concert with other membrane proteins and secretion systems that are essential for the bacterium's ability to colonize plant tissues and cause disease symptoms.

What role does mscL play in Xanthomonas axonopodis pv. citri pathogenicity?

While not directly identified in the mutant screens focused on virulence factors , the mscL channel likely contributes to Xac pathogenicity through multiple mechanisms. During plant infection, bacteria experience significant osmotic stress as they transition from the plant surface to intercellular spaces. The mscL channel helps maintain cellular integrity during these transitions by releasing osmotic pressure. Additionally, proper osmotic regulation is crucial for the function of various secretion systems that deliver virulence factors into host cells. Research on other Xanthomonas virulence determinants has shown that membrane proteins play critical roles in adhesion, biofilm formation, and host colonization . The mscL channel may therefore be part of a larger network of proteins that collectively enable successful host infection and disease progression.

What are the optimal expression systems for recombinant Xac mscL production?

The optimal expression system for recombinant Xac mscL production depends on research objectives. For structural studies requiring high protein yields, E. coli-based expression systems (particularly BL21(DE3) or C41(DE3) strains) are preferred due to their high expression levels and compatibility with membrane proteins. Expression vectors containing T7 promoters coupled with appropriate fusion tags (His6, MBP, or GST) enhance solubility and facilitate purification. For functional studies preserving native properties, expression in closely related Xanthomonas species might provide a more native-like membrane environment. When using E. coli, expression should be conducted at lower temperatures (16-25°C) following induction to prevent inclusion body formation. The expression protocol should include optimization of induction conditions (IPTG concentration: 0.1-0.5 mM) and membrane fraction isolation through differential centrifugation. Commercial recombinant Xac mscL is typically supplied in Tris-based buffer with 50% glycerol for stability .

What purification challenges are specific to Xac mscL and how can they be overcome?

Purification of Xac mscL presents several membrane protein-specific challenges. The primary difficulties include maintaining protein stability, preventing aggregation, and preserving native conformation during extraction from membranes. A methodological approach to overcome these challenges includes:

• Membrane solubilization: Use mild detergents such as n-dodecyl-β-D-maltoside (DDM) or LDAO at concentrations slightly above critical micelle concentration.

• Two-step affinity chromatography: Employ immobilized metal affinity chromatography (IMAC) followed by size exclusion chromatography.

• Detergent exchange: Transition to amphipols or nanodiscs for improved stability.

• Buffer optimization: Include glycerol (10-50%) and reducing agents to prevent oxidation.

Cross-contamination with lipopolysaccharides or other membrane components should be monitored through analytical techniques such as SDS-PAGE and Western blotting. The purification protocol should be validated by functional assays to ensure the channel maintains its native properties. Research shows that storage in Tris-based buffer with 50% glycerol at -20°C or -80°C provides optimal stability for extended periods .

What electrophysiological methods are most effective for studying Xac mscL function?

The gold standard for characterizing mechanosensitive channel function is patch-clamp electrophysiology, which allows direct measurement of channel activity. For Xac mscL, the most effective methodological approach involves:

• Reconstitution of purified mscL into artificial liposomes or planar lipid bilayers.

• Application of membrane tension through either negative pressure (suction) in patch pipettes or osmotic gradients.

• Recording channel currents at different membrane potentials (-100 to +100 mV range).

• Analysis of single-channel conductance, gating kinetics, and tension sensitivity.

Giant bacterial spheroplasts can also be prepared from E. coli expressing recombinant Xac mscL for direct patch-clamping. For higher throughput screening, fluorescence-based liposome assays using calcein release upon channel opening provide a practical alternative. These methods should be combined with site-directed mutagenesis of key residues predicted to affect channel gating, particularly those in transmembrane domains identified in the amino acid sequence . Comparing the electrophysiological properties of wild-type and mutant channels provides crucial insights into structure-function relationships.

How can the osmotic protection function of mscL be quantitatively assessed?

The osmotic protection function of Xac mscL can be quantitatively assessed through survival assays under osmotic downshock conditions. The methodological approach includes:

• Expression of recombinant Xac mscL in an E. coli strain lacking endogenous mechanosensitive channels (ΔmscL, ΔmscS, ΔmscK).

• Growth of bacterial cultures in high osmolarity media (LB + 0.5M NaCl).

• Sudden dilution into low osmolarity media (typically 20-fold dilution).

• Quantification of survival rates through colony forming unit (CFU) counts.

• Calculation of survival percentages relative to non-shocked controls.

Data should be presented as survival curves plotting the relationship between osmotic gradient magnitude and survival percentage. Complementary approaches include fluorescence-based assays measuring cytoplasmic content release during osmotic shock using calcein-loaded bacterial cells or liposomes containing purified mscL. These quantitative assays should be performed with both wild-type and mutant versions of the channel to establish structure-function relationships. The osmotic protection function directly relates to the channel's ability to respond to increased membrane tension by opening and allowing rapid solute efflux, which prevents cell lysis during hypoosmotic stress.

What methods are available for studying the interaction between Xac mscL and the bacterial membrane?

Studying interactions between Xac mscL and bacterial membranes requires specialized biophysical techniques that can probe protein-lipid interfaces. Effective methodological approaches include:

• Fluorescence resonance energy transfer (FRET) between labeled mscL and membrane probes to measure insertion depth and orientation.

• Electron paramagnetic resonance (EPR) spectroscopy with site-directed spin labeling to determine dynamic structural changes during channel gating.

• Molecular dynamics simulations based on the known amino acid sequence to predict lipid-protein interactions.

• Lipid binding assays using native mass spectrometry to identify specific lipid interactions.

• Quartz crystal microbalance with dissipation monitoring (QCM-D) to measure binding kinetics to different membrane compositions.

For more native-like systems, fluorescently labeled mscL can be visualized in bacterial spheroplasts using super-resolution microscopy techniques. The lipid composition significantly affects channel function, so experiments should systematically vary membrane composition, particularly phospholipid head groups and acyl chain length. Crosslinking studies can identify neighboring proteins in the native membrane environment, providing insights into potential functional interactions with other virulence factors identified in Xanthomonas research .

How does mscL expression change during different stages of Xac infection?

While specific data on mscL expression during Xac infection stages is limited in the provided search results, we can infer patterns based on research on other Xanthomonas virulence factors. Like other membrane proteins involved in host-pathogen interactions, mscL expression likely follows distinct temporal patterns coordinated with the infection process. During initial attachment to plant surfaces, expression may increase to help manage osmotic transitions. In the colonization phase, when bacteria multiply in intercellular spaces, continued expression would protect against osmotic fluctuations in the apoplastic environment. During later stages involving canker formation, expression patterns may shift as the bacterium adapts to changing tissue conditions. Research on other Xanthomonas virulence factors has shown that genes like XacFhaB (filamentous hemagglutinin-like protein) are specifically induced in planta during plant-pathogen interaction . Similar regulation may apply to mscL, particularly as the bacterium transitions between different microenvironments within host tissues.

What is the relationship between mscL function and Xac biofilm formation?

Biofilm formation is a critical aspect of Xanthomonas pathogenicity, and mechanosensitive channels likely play supporting roles in this process. While direct evidence for mscL involvement in Xac biofilm formation is not explicitly mentioned in the search results, functional connections can be inferred based on bacterial physiology principles. Proper osmoregulation facilitated by mscL is essential during biofilm development as bacteria transition from planktonic to attached states, experiencing changing osmotic conditions. Research on other Xanthomonas adhesion factors, such as the filamentous hemagglutinin-like protein (XacFhaB), has demonstrated that membrane proteins significantly impact biofilm formation . Mutants lacking adhesion proteins show impaired leaf surface attachment and altered biofilm structures. The methodological approach to study mscL's role in biofilm formation would include:

• Generation of mscL knockout or overexpression strains

• Quantitative biofilm assays using crystal violet staining

• Confocal microscopy to visualize biofilm architecture

• Flow cell systems to study biofilm development under controlled conditions

• Correlation analysis between mscL expression levels and biofilm biomass measurements

How does mscL function coordinate with other virulence mechanisms in Xac?

The coordination between mscL and other virulence mechanisms in Xac involves complex interactions within the bacterium's pathogenicity networks. Mechanosensitive channels function as part of the bacterial stress response system, which intersects with virulence factor regulation. During plant infection, Xac deploys multiple virulence strategies including adhesins for attachment, secretion systems for effector delivery, and exopolysaccharide production for biofilm formation . The mscL channel likely supports these mechanisms by:

• Maintaining cellular homeostasis during osmotic transitions encountered during infection

• Supporting membrane integrity required for functional secretion systems

• Contributing to proper biofilm hydration and matrix organization

• Enabling bacterial adaptation to changing host tissue environments

Research on Xanthomonas mutants has identified numerous genes involved in virulence, including those encoding for xylose repressor-like protein (XACΔxylR), Fe-S oxidoreductase (XACΔaslB), helicase IV (XACΔhelD), and cell division proteins . These diverse factors form an interconnected network in which osmoregulation through mscL likely plays a supporting but essential role. The methodological approach to study these interactions would include comparative phenotypic analysis of single and double mutants, transcriptomic profiling during infection, and protein-protein interaction studies.

How can Xac mscL be utilized as a target for novel antimicrobial development?

Xac mscL represents a promising target for novel antimicrobial development due to its essential role in bacterial survival under osmotic stress. A methodological approach to developing mscL-targeted antimicrobials includes:

• High-throughput screening of compound libraries against purified recombinant mscL

• Virtual screening using molecular docking based on the 143-amino acid sequence structure

• Development of gain-of-function compounds that trigger premature channel opening

• Design of peptide inhibitors targeting the pore region or gating mechanism

• Liposome-based assays to validate candidate compound efficacy

Effective compounds would either lock the channel in an open state (causing cytoplasmic leakage) or prevent channel opening during osmotic downshock (leading to cell lysis). The advantage of targeting mscL is its conservation across Xanthomonas species while differing sufficiently from mammalian mechanosensitive channels to ensure selectivity. Candidate compounds should be validated in planta using controlled infection assays and microscopy to verify their ability to disrupt bacterial colonization. This approach offers an alternative to traditional antibiotics, potentially circumventing existing resistance mechanisms and providing more targeted control of this important plant pathogen.

What are the implications of mscL structural variations across different Xanthomonas strains?

Structural variations in mscL across different Xanthomonas strains have significant implications for bacterial adaptation, host range, and virulence. Comparative genomic and proteomic analyses suggest that while core channel functions are conserved, subtle sequence variations may tune channel sensitivity to specific host environments. Methodologically, researchers should:

• Perform multilocus sequence analysis similar to approaches used for other Xanthomonas genes

• Identify conserved domains versus variable regions through multiple sequence alignment

• Correlate sequence variations with strain-specific virulence phenotypes

• Conduct functional comparisons using electrophysiology on recombinant channels from different strains

• Apply site-directed mutagenesis to validate the functional impact of identified variations

Research on other Xanthomonas virulence factors has shown that even single nucleotide polymorphisms can affect protein function and virulence . For mscL, variations might influence gating threshold, conductance, or interaction with other membrane components. Understanding these variations has practical applications for developing strain-specific diagnostic tools and targeted control strategies. A comprehensive database of mscL sequence variations across Xanthomonas pathovars would be valuable for predicting virulence potential and host range.

How might CRISPR-Cas9 genome editing be applied to study mscL function in Xac?

CRISPR-Cas9 genome editing offers powerful approaches for studying mscL function in Xac through precise genetic manipulation. A comprehensive methodological workflow would include:

• Design of guide RNAs targeting the mscL gene with minimal off-target effects

• Construction of Cas9 expression vectors optimized for Xanthomonas

• Development of homology-directed repair templates for:

  • Gene knockout via premature stop codons

  • Point mutations in key functional residues

  • Insertion of fluorescent protein tags for localization studies

  • Addition of inducible promoters for controlled expression

• Transformation protocols optimized for Xanthomonas using electroporation

• Screening strategies combining antibiotic selection and PCR verification

• Phenotypic characterization under various osmotic stress conditions

• In planta virulence assays using citrus host plants

This approach enables precise dissection of structure-function relationships by creating specific mutations in the native genetic context. For example, researchers could modify the hydrophobic pore constriction of the channel to alter its gating properties or create chimeric channels combining domains from different bacterial species. The resulting mutant library would provide valuable insights into how specific amino acid residues in the 143-amino acid sequence contribute to channel function and bacterial virulence during plant infection.

How should researchers address variability in electrophysiological recordings of recombinant Xac mscL?

Variability in electrophysiological recordings of recombinant Xac mscL presents significant challenges for data interpretation. A methodological approach to address this variability includes:

• Standardization of recording conditions:

  • Consistent membrane composition in reconstitution systems

  • Controlled temperature (21-23°C)

  • Defined buffer composition with precise pH control

  • Standardized protocols for applying membrane tension

• Statistical analysis requirements:

  • Minimum of 30-50 independent channel recordings

  • Application of normality tests before selecting parametric/non-parametric analyses

  • Use of mixed-effects models to account for batch variations

  • Bootstrap resampling for confidence interval determination

• Data presentation standards:

  • Single-channel traces alongside ensemble averages

  • Current-voltage relationships with error bars

  • Pressure-response curves with 95% confidence intervals

  • Tabulation of key parameters (conductance, tension sensitivity, open probability)

Researchers should implement quality control metrics such as seal resistance stability and noise-level monitoring throughout recordings. Channel identification should be confirmed through pharmacological verification using known mechanosensitive channel modulators. Technical replicates should be distinguished from biological replicates, with the latter providing more meaningful insights into natural variability. Standardized reporting of recording parameters facilitates cross-laboratory comparisons and reproducibility.

What statistical approaches are appropriate for analyzing structure-function relationships in mscL mutants?

Analyzing structure-function relationships in mscL mutants requires specialized statistical approaches that can handle multivariate data and account for the complex relationship between sequence, structure, and function. Appropriate methodological frameworks include:

• Multiple sequence alignment coupled with principal component analysis to identify co-evolving residues

• Hierarchical clustering of functional parameters across mutant libraries

• Machine learning approaches (random forests, support vector machines) to predict functional outcomes from sequence variations

• Bayesian network analysis to establish causal relationships between structural features and channel properties

• Statistical coupling analysis to identify energetically linked residue networks

Data visualization should employ heat maps for large-scale mutant comparisons and radar plots for multidimensional parameter representation. When comparing multiple mutants, ANOVA with post-hoc tests should be used, with Bonferroni correction for multiple comparisons. For structure-based analyses, researchers should develop quantitative structure-function relationship (QSFR) models that correlate specific amino acid properties (hydrophobicity, charge, volume) with functional outcomes. These analyses should be performed with careful consideration of the 143-amino acid sequence and its predicted structural elements within the bacterial membrane context.

How can researchers distinguish between direct and indirect effects when studying mscL in Xac pathogenicity?

Distinguishing between direct and indirect effects of mscL on Xac pathogenicity requires a systematic experimental approach combining genetic, biochemical, and in planta studies. A comprehensive methodological framework includes:

• Generation of complementary experimental systems:

  • Clean deletion mutants (ΔmscL)

  • Complemented strains with wild-type or mutant mscL variants

  • Point mutants affecting specific channel functions

  • Conditional expression systems for temporal control

• Multi-level phenotypic characterization:

  • Cellular level: osmotic shock survival, membrane integrity

  • Population level: growth kinetics, biofilm formation

  • Host interaction: attachment efficiency, colonization patterns, symptom development

• Transcriptomic and proteomic analyses:

  • RNA-Seq comparing wild-type and mutant strains during infection

  • Proteome profiling to identify compensatory mechanisms

  • Epistasis analysis with other virulence factors

The direct effects of mscL function should be evident in immediate cellular responses to osmotic challenges, while indirect effects would manifest in altered expression of other virulence factors or adaptive responses. Temporal analysis is crucial, as the timing of phenotypic changes can distinguish primary from secondary effects. Spatial analysis using fluorescently tagged proteins can reveal subcellular localization patterns relevant to function. This approach allows researchers to build a comprehensive model of how mscL integrates into the broader virulence network that includes numerous other factors identified in previous studies .

What are the most promising approaches for structural determination of Xac mscL?

Structural determination of Xac mscL presents significant challenges due to its membrane protein nature, but several promising methodological approaches are emerging:

• Cryo-electron microscopy (cryo-EM):

  • Advantages: Captures protein in near-native lipid environment, can resolve different conformational states

  • Requirements: High-purity protein preparation, optimized detergent or nanodisc reconstitution

  • Expected resolution: 3-4 Å achievable with current technology

• X-ray crystallography with advanced approaches:

  • Lipidic cubic phase crystallization

  • Antibody fragment (Fab) co-crystallization to increase polar surface area

  • Fusion protein strategies to aid crystallization

• Integrative structural biology combining:

  • Hydrogen-deuterium exchange mass spectrometry for dynamics

  • Cross-linking mass spectrometry for distance constraints

  • DEER/EPR spectroscopy for conformational changes

  • Molecular dynamics simulations based on the 143-amino acid sequence

Each approach has specific advantages for understanding different aspects of channel structure and function. Researchers should prioritize capturing the channel in multiple conformational states (closed, intermediate, and open) to understand the gating mechanism. The resulting structural data would provide invaluable insights for structure-based drug design and engineering of channel properties for biotechnological applications.

How might synthetic biology approaches utilize engineered Xac mscL variants?

Synthetic biology offers exciting possibilities for utilizing engineered Xac mscL variants across multiple applications. A methodological framework for this research direction includes:

• Engineering channel sensitivity:

  • Mutation of hydrophobic pore residues to alter tension thresholds

  • Introduction of light-sensitive domains for optogenetic control

  • Addition of ligand-binding domains for chemical regulation

  • pH-sensitive modifications for environmental responsiveness

• Application-specific modifications:

  • Biosensing: Coupling channel opening to reporter systems for detecting membrane-active compounds

  • Controlled release: Engineering bacterial cells for programmed content delivery

  • Biocontainment: Creating conditional survival switches based on environmental sensing

  • Bioremediation: Developing strains that respond to specific environmental contaminants

• Technical implementation:

  • Golden Gate assembly for combinatorial domain fusion

  • Directed evolution with functional selection

  • Computational design based on the known 143-amino acid sequence

  • In vitro testing in liposome systems before cellular implementation

These approaches could lead to novel biotechnological applications including bacteria-based environmental sensors, smart probiotic delivery systems, and programmable cellular factories. The fundamental understanding of channel structure-function relationships derived from the natural Xac mscL provides the foundation for rational design of these synthetic biology applications.

What potential exists for developing Xac mscL-based biosensors for agricultural applications?

The development of Xac mscL-based biosensors represents a promising frontier for agricultural applications, particularly for early detection of plant stress and pathogen presence. A methodological roadmap for this research direction includes:

• Sensor design strategies:

  • Engineering mscL variants with modified tension sensitivity

  • Fusion of fluorescent or colorimetric reporters to channel components

  • Coupling channel activity to transcriptional activation systems

  • Integration with microfluidic or field-deployable detection platforms

• Detection targets:

  • Plant stress hormones (ethylene, abscisic acid)

  • Pathogen-associated molecular patterns

  • Agricultural chemicals (pesticides, fertilizers)

  • Environmental stress indicators (pH changes, osmolytes)

• Implementation approaches:

  • Cell-free systems using purified recombinant channels in synthetic membranes

  • Engineered non-pathogenic bacteria as living sensors

  • Immobilized channel proteins on electrochemical detection platforms

  • Smartphone-compatible colorimetric readout systems

The natural sensitivity of mscL to membrane tension can be repurposed to detect various agriculturally relevant analytes by coupling tension-inducing mechanisms to specific molecular recognition elements. Preliminary validation would require laboratory testing followed by controlled field trials. The potential applications include early disease detection, irrigation optimization, and monitoring of plant-microbe interactions in agricultural settings. This approach leverages the fundamental biological properties of the 143-amino acid mscL protein while repurposing it for novel agricultural technology applications.