Recombinant Xanthomonas campestris pv. vesicatoria Large-conductance mechanosensitive channel (mscL)

What is the mscL protein from Xanthomonas campestris pv. vesicatoria?

The mscL (Large-conductance mechanosensitive channel) protein from Xanthomonas campestris pv. vesicatoria is a 143-amino acid membrane protein that functions as a pressure-sensitive channel. It belongs to a family of bacterial mechanosensitive channels that respond to changes in membrane tension by opening pores to release osmolytes, thereby protecting cells from osmotic stress. The full-length recombinant protein can be expressed with a His-tag to facilitate purification and experimental manipulation . The protein plays a crucial role in bacterial osmoregulation, allowing the microorganism to adapt to changing environmental conditions, particularly during infection of plant hosts.

What is the molecular structure of Xanthomonas campestris pv. vesicatoria mscL?

The mscL protein from X. campestris pv. vesicatoria consists of 143 amino acids with the following sequence: MGMVSEFKQFAMRGNVIDLAVGVVIGAAFGKIVTALVEKIIMPPIGWAIGNVDFSRLAWVLKPAGVDATGKEIPAVAIGYGDFINTVVQFLIIAFAIFLVVKLINRVTHRKPDAPKGPSEEVLLLREIRDALKNDTLKPPGAL . Structurally, like other bacterial mscL channels, it is predicted to form a homopentameric complex with two transmembrane domains per subunit, creating a channel that undergoes conformational changes in response to membrane tension. The protein contains hydrophobic regions that anchor it within the lipid bilayer and hydrophilic portions that line the channel pore when opened.

How does mscL function differ between Xanthomonas campestris pathovars?

When comparing mscL functionality between different pathovars such as X. campestris pv. vesicatoria and X. campestris pv. campestris, significant differences exist in protein secretion systems and substrate specificities. While both pathovars utilize type II secretion (T2S) systems, research has shown that several substrates from X. campestris pv. vesicatoria are secreted independently of the T2S systems in X. campestris pv. campestris . This indicates potential variations in the regulatory mechanisms and functional roles of membrane proteins, including mechanosensitive channels, between these closely related pathovars. These differences may contribute to their distinct host specificities and pathogenicity mechanisms.

What experimental approaches can be used to study the gating mechanism of Xanthomonas campestris mscL?

To investigate the gating mechanism of X. campestris mscL, researchers can employ several sophisticated techniques:

• Patch-clamp electrophysiology: This technique allows for direct measurement of channel activity by applying calibrated suction pressures to membrane patches expressing the recombinant mscL. Research has validated the functional expression of engineered MscL in neurons through patch-clamp recordings upon application of defined suction pressures .

• Reconstitution in artificial lipid bilayers: Purified mscL protein can be incorporated into artificial membrane systems to study its behavior under controlled conditions.

• FRET-based tension sensors: By incorporating fluorescent markers at strategic positions within the protein, researchers can monitor conformational changes associated with channel gating.

• Molecular dynamics simulations: Computational approaches can model how the channel responds to membrane deformation at the atomic level.

• Site-directed mutagenesis: Systematic modification of specific amino acid residues can identify critical regions for mechanosensation and channel gating.

How does the heterologous expression of mscL affect mammalian neuronal networks?

The heterologous expression of bacterial mechanosensitive channels like mscL in mammalian neuronal networks introduces novel mechano-sensitivity to these cells. Research has demonstrated that engineered MscL can be successfully expressed in neuronal systems without compromising cell survival, synapse formation, or spontaneous network activity . This mechano-sensitization creates neuronal networks that can be activated by mechanical stimuli, offering potential applications for non-invasive neuromodulation.

Key findings from neuroscience research show that:

• Neurons expressing engineered MscL maintain normal morphology and function

• The number of synaptic puncta remains comparable to control neurons

• Spontaneous network activity continues despite MscL expression

• The pure mechanosensitivity of engineered MscL provides a versatile tool for developing mechano-genetic approaches to neuronal stimulation

These properties make mscL a valuable research tool for developing remote stimulation techniques for neuronal tissues, potentially allowing mechanical stimuli to non-invasively convey information into intact brain tissue.

What is the relationship between mscL and virulence mechanisms in plant-pathogenic bacteria?

The relationship between mscL and virulence in Xanthomonas campestris pv. vesicatoria is complex and involves several interacting systems. While mscL itself has not been directly implicated in virulence, it is part of a sophisticated membrane protein network that contributes to bacterial survival in plant hosts. The Xps-T2S system from X. campestris pv. vesicatoria is known to secrete virulence-associated enzymes, including xylanases, proteases, and lipases that contribute to plant cell wall degradation .

Research has shown that these secreted enzymes:

• Mediate degradation of plant cell wall components during host-pathogen interactions

• Promote bacterial virulence and in planta growth

• Can be transported via both the Xps-T2S system and alternative routes such as outer membrane vesicles (OMVs)

Understanding membrane protein function, including mechanosensitive channels, can provide insights into how these bacteria maintain membrane integrity during the infection process and adapt to changing osmotic conditions within the plant environment.

What are the optimal protocols for purification and reconstitution of recombinant mscL?

For optimal purification and reconstitution of recombinant X. campestris pv. vesicatoria mscL, researchers should follow these methodological guidelines:

Purification Protocol:

• Express the His-tagged recombinant protein in E. coli expression systems

• Harvest cells and lyse using appropriate buffer systems

• Perform immobilized metal affinity chromatography (IMAC) using Ni-NTA resin

• Include detergents during purification to maintain membrane protein solubility

• Conduct size exclusion chromatography for further purification if needed

Reconstitution Guidelines:

• Reconstitute lyophilized protein in deionized sterile water to a concentration of 0.1-1.0 mg/mL

• Add glycerol to a final concentration of 5-50% (with 50% being optimal) for long-term storage

• For functional studies, incorporate the purified protein into liposomes using detergent-mediated reconstitution

• Ensure proper protein orientation by using controlled dehydration-rehydration cycles

• Verify successful reconstitution through functional assays such as patch-clamp electrophysiology or fluorescence-based flux assays

How should recombinant mscL proteins be stored to maintain functionality?

Proper storage of recombinant mscL is critical for maintaining protein functionality. According to product specifications, researchers should:

• Store the received protein at -20°C to -80°C, with aliquoting necessary for multiple use

• Avoid repeated freeze-thaw cycles which can denature the protein and reduce activity

• For working solutions, store aliquots at 4°C for up to one week

• Use Tris/PBS-based buffer with 6% Trehalose at pH 8.0 as an appropriate storage buffer

• Centrifuge vials briefly before opening to bring contents to the bottom

For long-term storage:

• Add glycerol to a final concentration of 50%

• Aliquot in small volumes to prevent multiple freeze-thaw cycles

• Store at -80°C in airtight containers

• Document storage date and conditions for quality control

What experimental systems are most effective for studying mscL function?

Several experimental systems have proven effective for studying mscL function, each with specific advantages depending on research objectives:

In vitro Systems:

• Liposome-reconstituted channels: Allow precise control of lipid composition and membrane tension

• Planar lipid bilayers: Enable detailed electrophysiological characterization

• Giant unilamellar vesicles (GUVs): Permit visualization of channel-mediated solute flux

Cellular Systems:

• E. coli expression systems: Provide native-like bacterial membrane environment

• Mammalian cell lines: Allow investigation of heterologous expression effects

• Neuronal cultures: Enable studies of mechanosensitivity in specialized excitable cells

Analytical Techniques:

• Patch-clamp electrophysiology: Directly measures channel conductance and gating properties

• Fluorescence-based assays: Monitor solute flux through reconstituted channels

• Pressure/tension application devices: Apply calibrated mechanical stimuli to activate channels

The choice of system should be guided by specific research questions, with E. coli expression systems being particularly valuable for initial characterization of bacterial mscL variants .

How can mscL be utilized in neuroscience research?

The mscL protein offers unique applications in neuroscience research through mechano-genetic approaches:

• Remote neuronal stimulation: The heterologous expression of engineered mscL in neurons creates mechano-sensitized neuronal networks that can be activated by mechanical stimuli without invasive procedures .

• Cell-type specific stimulation: By expressing mscL under the control of cell-type-specific promoters, researchers can selectively target distinct neuronal populations.

• Neurodevelopmental studies: mscL expression can help investigate how mechanical forces influence neuronal development and network formation.

• Neuroprosthetic applications: The pure mechanosensitivity of engineered mscL provides a versatile tool for developing mechanical interfaces with neural tissue .

Research has validated that neurons expressing mscL maintain normal morphological and functional properties, including:

• Cell survival comparable to control neurons

• Normal synapse formation as evidenced by synaptic puncta counts

• Preserved spontaneous network activity

This makes mscL a promising tool for developing non-invasive neuromodulation techniques.

What are the comparative advantages of using Xanthomonas campestris mscL versus other bacterial mechanosensitive channels?

When comparing X. campestris mscL to other bacterial mechanosensitive channels for research applications, several advantages emerge:

The specific amino acid composition of X. campestris mscL (MGMVSEFKQFAMRGNVIDLAVGVVIGAAFGKIVTALVEKIIMPPIGWAIGNVDFSRLAWVLKPAGVDATGKEIPAVAIGYGDFINTVVQFLIIAFAIFLVVKLINRVTHRKPDAPKGPSEEVLLLREIRDALKNDTLKPPGAL) provides unique structural properties that can be advantageous for certain applications, particularly those involving heterologous expression in non-native systems.

How can researchers address challenges in functional expression of recombinant mscL?

Researchers face several challenges when working with recombinant mscL, including proper folding, membrane insertion, and functional activity. Here are methodological approaches to address these challenges:

• Optimizing expression conditions:

  • Use specialized E. coli strains designed for membrane protein expression

  • Adjust induction conditions (temperature, inducer concentration, duration)

  • Consider codon optimization for the expression system

• Enhancing membrane incorporation:

  • Include appropriate detergents during purification

  • Optimize lipid composition during reconstitution

  • Consider fusion partners that facilitate membrane targeting

• Functional validation methods:

  • Employ patch-clamp electrophysiology with calibrated suction pressures

  • Use fluorescence-based flux assays to monitor channel activity

  • Implement growth-based complementation assays in bacterial systems

• Addressing aggregation issues:

  • Add stabilizing agents such as trehalose (6%) to storage buffers

  • Maintain pH 8.0 for optimal protein stability

  • Avoid repeated freeze-thaw cycles

• Structural integrity verification:

  • Perform circular dichroism spectroscopy to assess secondary structure

  • Use limited proteolysis to evaluate proper folding

  • Employ native gel electrophoresis to assess oligomeric state

By systematically addressing these challenges, researchers can achieve reliable functional expression of recombinant X. campestris mscL for diverse experimental applications.