Recombinant Bacillus weihenstephanensis Large-conductance mechanosensitive channel (mscL)

What is Bacillus weihenstephanensis and how is it characterized taxonomically?

Bacillus weihenstephanensis is a member of the Bacillus cereus group, which comprises seven bacterial species: B. cereus, B. anthracis, B. thuringiensis, B. mycoides, B. pseudomycoides, B. cytotoxicus, and B. weihenstephanensis. This species is distinctively characterized by its psychrotolerant properties - specifically, its ability to grow at 7°C but not at 43°C. The taxonomic identification of B. weihenstephanensis relies on the presence of specific signature sequences in the 16S rRNA and cspA genes, as well as in several housekeeping genes including glpF, gmK, purH, and tpi .

Molecular phylogenetic analysis using maximum-likelihood trees constructed from concatenated nucleotide sequences reveals that B. weihenstephanensis clusters within Group II, which includes psychrotolerant B. cereus and B. mycoides strains. This clustering pattern provides strong evidence for the taxonomic relatedness of these psychrotolerant bacteria .

What is the physiological role of MscL in Bacillus weihenstephanensis?

Based on research with related Bacillus species, the MscL protein in B. weihenstephanensis likely plays a critical role in osmotic regulation. Studies with B. subtilis have demonstrated that MscL prevents the selective release of cytoplasmic proteins during hypo-osmotic shock conditions . When bacteria experience a sudden decrease in external osmolarity, water rushes into the cell, creating tension in the cytoplasmic membrane. MscL channels open in response to this membrane tension, allowing the rapid efflux of small solutes and thereby preventing cell lysis.

In B. weihenstephanensis, which is adapted to growth at lower temperatures, the MscL channel likely has evolved specific adaptations that maintain its functionality in cold environments. The psychrotolerant nature of this bacterium suggests that its membrane proteins, including MscL, must function efficiently at temperatures as low as 7°C .

How can researchers express and purify recombinant B. weihenstephanensis MscL?

For researchers working with recombinant B. weihenstephanensis MscL, the following methodology provides a framework for protein expression and purification:

Expression System:

• Clone the mscL gene (BcerKBAB4_4501) into an appropriate expression vector with a tag for purification.

• Transform the construct into a suitable expression host (typically E. coli BL21(DE3) or similar strains).

• Induce protein expression under controlled temperature conditions (20-25°C is often optimal for membrane proteins to ensure proper folding).

Purification Protocol:

• Harvest cells and disrupt using sonication or French press in a buffer containing 50mM Tris-HCl pH 7.5, 150mM NaCl.

• Solubilize membrane fraction using a detergent such as n-dodecyl-β-D-maltopyranoside (DDM) at 1% concentration.

• Purify using affinity chromatography based on the chosen tag.

• For storage, maintain in a Tris-based buffer with 50% glycerol at -20°C for short-term or -80°C for extended storage .

Note: Repeated freezing and thawing should be avoided to maintain protein integrity. Working aliquots can be stored at 4°C for up to one week .

What experimental approaches can be used to study MscL channel activity?

Several experimental approaches are available to researchers studying MscL channel activity:

Patch Clamp Electrophysiology:

• Allows direct measurement of channel opening and closing in response to membrane tension.

• Can be performed using reconstituted channels in liposomes or in bacterial spheroplasts.

Fluorescence-Based Assays:

• Using fluorescent dyes that are released through the channel upon activation.

• Can be performed in high-throughput format for screening modulators.

Osmotic Shock Survival Assays:

• Compare survival rates of wild-type and mscL mutant strains under hypo-osmotic shock conditions.

• Specifically useful for B. weihenstephanensis, researchers can test growth and survival at various temperatures (e.g., 7°C, 25°C) combined with osmotic challenges.

Protein Release Assays:

• Monitor the release of specific cytoplasmic proteins during osmotic shock in wild-type versus mscL mutant strains.

• Research with B. subtilis has shown that MscL prevents selective release of cytoplasmic proteins during hypo-osmotic shock .

How does MscL function differ between psychrotolerant B. weihenstephanensis and mesophilic Bacillus species?

The functional differences of MscL between psychrotolerant B. weihenstephanensis and mesophilic Bacillus species likely relate to adaptations for membrane fluidity and protein flexibility at different temperatures.

Psychrotolerant bacteria like B. weihenstephanensis typically exhibit:

• Modified membrane lipid composition with increased proportions of unsaturated fatty acids to maintain membrane fluidity at low temperatures.

• Proteins with structural adaptations that maintain flexibility and function at lower temperatures.

For MscL specifically, these adaptations may include:

Structural Modifications:

• Amino acid substitutions that enhance protein flexibility at lower temperatures

• Altered hydrophobic gating regions that respond appropriately to membrane tension even when membrane properties change at low temperatures

Functional Differences:

• Potentially different tension thresholds for channel opening

• Modified gating kinetics optimized for lower temperature environments

• Possible interactions with cold shock proteins like CspA, which is a marker gene for psychrotolerance in B. weihenstephanensis

Research comparing the MscL function between B. weihenstephanensis (growth optimal at 7-30°C) and mesophilic Bacillus species (growth optimal at 30-43°C) would provide valuable insights into cold adaptation mechanisms of membrane proteins.

What is the relationship between MscL and bacterial susceptibility to antimicrobial compounds?

Research with Bacillus subtilis has demonstrated an unexpected relationship between MscL and antimicrobial susceptibility. Studies show that MscL affects the susceptibility of bacteria to certain antimicrobial compounds, particularly lantibiotics like sublancin 168 .

Key findings include:

• The susceptibility of B. subtilis and S. aureus cells towards sublancin 168 depends on the presence of the large mechanosensitive channel MscL .

• MscL may either serve as a direct target for lantibiotics or function as a gate of entry to the cytoplasm .

• Environmental conditions, such as NaCl concentration, can influence this susceptibility. Higher NaCl concentrations reduced the susceptibility of cells to sublancin 168 .

For B. weihenstephanensis specifically, researchers might investigate:

• Whether its MscL similarly affects susceptibility to antimicrobial compounds

• If temperature modulates this relationship, given its psychrotolerant nature

• How this might impact food safety practices, as B. weihenstephanensis is found in refrigerated foods

How does the expression and function of MscL relate to cereulide production in B. weihenstephanensis?

Some strains of B. weihenstephanensis have been identified as producers of cereulide, an emetic toxin previously thought to be restricted to B. cereus . The relationship between MscL and cereulide production presents an intriguing research question.

The psychrotolerant B. weihenstephanensis strains MC67 and MC118 have been shown to produce cereulide at temperatures as low as 8°C . These strains display atypical characteristics compared to mesophilic emetic B. cereus strains, including differences in their cereulide peptide synthetase gene (cesB) .

Potential research avenues include:

• Investigating whether MscL expression levels correlate with cereulide production.

• Examining if MscL mutations affect cereulide export or retention within the cell.

• Determining whether osmotic stress, which activates MscL, influences cereulide synthesis or export.

• Studying how temperature affects the relationship between MscL activity and cereulide production, particularly at refrigeration temperatures (4-8°C).

A comparative study between mscL wild-type and mutant strains of cereulide-producing B. weihenstephanensis would help elucidate this relationship.

How conserved is the MscL protein sequence among different B. weihenstephanensis strains and other Bacillus species?

The conservation pattern of MscL across Bacillus species provides insights into both functional constraints and evolutionary adaptations. While specific data comparing multiple B. weihenstephanensis MscL sequences is limited, we can make several observations:

Within B. weihenstephanensis:
The MscL protein sequence from strain KBAB4 provides a reference point (UniProt: A9VKI1) . Complete sequence conservation analysis would require comparing this sequence with MscL proteins from other B. weihenstephanensis strains such as WSBC 10204 (the type strain) and WSBC 10202 .

Cross-species comparison:
When comparing MscL proteins across Bacillus species, functional domains are typically highly conserved, particularly:

• Transmembrane domains that form the channel pore

• Gating regions that respond to membrane tension

• Cytoplasmic domains involved in channel assembly

Evolutionary considerations:
B. weihenstephanensis belongs to Group II in phylogenetic analyses of the B. cereus group, clustering with other psychrotolerant strains . The evolutionary adaptation of MscL to function at lower temperatures likely involves specific amino acid substitutions that would be evident in comparative sequence analyses.

A systematic analysis comparing MscL sequences across the Bacillus cereus group would help identify signature sequences that might correlate with psychrotolerance or other phenotypic characteristics.

What experimental methods can determine how temperature affects MscL function in B. weihenstephanensis?

Given the psychrotolerant nature of B. weihenstephanensis, temperature effects on MscL function represent a key research area. The following experimental approaches can address this question:

Electrophysiological Studies:

• Patch clamp analysis of MscL channels reconstituted in liposomes at various temperatures (4°C, 7°C, 25°C, 37°C)

• Measurement of channel gating parameters (threshold tension, open probability, conductance) as a function of temperature

Cell Survival Assays:

• Compare survival rates of wild-type and mscL mutant B. weihenstephanensis strains exposed to hypo-osmotic shock at different temperatures

• Quantify the relationship between temperature, osmotic shock magnitude, and survival rates

Protein Dynamics Studies:

• Hydrogen-deuterium exchange mass spectrometry to measure protein flexibility at different temperatures

• Molecular dynamics simulations to predict temperature-dependent conformational changes

Comparative Expression Analysis:

• RT-qPCR to measure mscL expression levels at different growth temperatures

• Proteomic analysis to quantify MscL protein abundance and post-translational modifications

Functional Complementation:

• Express B. weihenstephanensis mscL in a mesophilic Bacillus mscL knockout strain

• Test if the psychrotolerant MscL can restore osmotic shock resistance at various temperatures

A comprehensive experimental approach would combine these methods to build a complete picture of how temperature modulates MscL function in this psychrotolerant bacterium.

How can researchers study the role of MscL in protein secretion under stress conditions?

Based on research with B. subtilis, MscL appears to play a role in preventing selective release of cytoplasmic proteins during hypo-osmotic shock . To investigate this phenomenon in B. weihenstephanensis, researchers can employ the following methodological approaches:

Comparative Proteomics Workflow:

• Strain Preparation:

  • Generate B. weihenstephanensis mscL knockout mutants

  • Culture wild-type and mutant strains under standard conditions

• Osmotic Challenge Protocol:

  • Subject cultures to hypo-osmotic shock (e.g., rapid dilution into low-osmolarity medium)

  • Include appropriate controls (isotonic conditions)

  • Perform experiments at different temperatures (7°C, 25°C) to assess temperature dependence

• Protein Release Analysis:

  • Collect extracellular fractions by centrifugation

  • Analyze protein content using mass spectrometry-based proteomics

  • Compare protein profiles between wild-type and mscL mutant strains

• Cell Viability Assessment:

  • Perform live/dead staining and plate counting to distinguish protein release from cell lysis

  • Measure membrane potential to assess membrane integrity

As demonstrated in research with B. subtilis, this approach can reveal whether MscL prevents selective protein release during osmotic stress and identify which specific proteins are affected .

What are the optimal conditions for functional studies of recombinant B. weihenstephanensis MscL?

For functional studies of recombinant B. weihenstephanensis MscL, researchers should consider the following optimal conditions:

Protein Storage and Handling:

• Store purified protein in Tris-based buffer with 50% glycerol

• For extended storage, maintain at -20°C or -80°C

• Avoid repeated freezing and thawing

• Working aliquots can be stored at 4°C for up to one week

Reconstitution Parameters:

• Lipid composition: Consider using lipid mixtures that mimic B. weihenstephanensis membrane composition

• Temperature range: Include experiments at 7°C to reflect the psychrotolerant nature of the source organism

• Buffer conditions: Phosphate or Tris buffers at pH 7.0-7.5 with physiological salt concentrations

Functional Assay Conditions:

• For patch-clamp studies: Ensure equipment can operate at reduced temperatures when needed

• For fluorescence-based assays: Account for temperature effects on fluorophore properties

• Include appropriate controls with well-characterized mechanosensitive channels (e.g., E. coli MscL)

Expression System Considerations:

• If expressing in E. coli, consider using a strain adapted to lower temperature expression

• Optimize induction conditions (IPTG concentration, temperature, duration) for maximal functional protein yield

• Include appropriate tags that can be removed without affecting protein function

These optimized conditions will help ensure that functional studies reflect the native properties of B. weihenstephanensis MscL, particularly its adaptation to lower temperatures.

How might understanding B. weihenstephanensis MscL contribute to food safety applications?

B. weihenstephanensis is particularly relevant to food safety due to its psychrotolerant nature and the ability of some strains to produce cereulide toxin at refrigeration temperatures . Understanding MscL in this context could contribute to food safety in several ways:

Potential Applications:

• Biocontrol Strategies:

  • If MscL serves as an entry point for antimicrobials like sublancin (as in B. subtilis) , this could be exploited to develop targeted biocontrol strategies for refrigerated foods

  • MscL-targeting compounds might selectively inhibit B. weihenstephanensis without affecting beneficial bacteria

• Stress Response Mechanisms:

  • Understanding how MscL functions in response to food processing techniques (high pressure, osmotic stress) could help optimize processes to control B. weihenstephanensis

  • Temperature-dependent MscL function might explain survival during cold chain distribution

• Toxin Production Control:

  • If MscL function is linked to cereulide production or export , this knowledge could inform strategies to prevent toxin accumulation in foods

  • Modulating MscL activity might potentially inhibit cereulide production at low temperatures

• Detection Methods:

  • MscL-specific antibodies or aptamers could potentially be developed for rapid detection of B. weihenstephanensis in food samples

  • Temperature-dependent MscL characteristics might provide a basis for distinguishing psychrotolerant from mesophilic Bacillus species

Research investigating these applications would require interdisciplinary approaches combining molecular biology, food microbiology, and applied food safety techniques.

What computational approaches can advance our understanding of B. weihenstephanensis MscL structure and function?

Computational approaches offer powerful tools for studying MscL structure and function, especially given the challenges of experimental work with membrane proteins:

Structural Bioinformatics Approaches:

• Homology Modeling:

  • Develop 3D structural models of B. weihenstephanensis MscL based on known structures of MscL from other bacteria

  • Compare models of psychrotolerant and mesophilic MscL proteins to identify key structural differences

• Molecular Dynamics Simulations:

  • Simulate MscL behavior in membrane environments at different temperatures (7°C vs. 37°C)

  • Model channel opening in response to membrane tension at different temperatures

  • Investigate how lipid composition affects channel function

• Sequence-Based Analysis:

  • Perform comprehensive comparative sequence analysis across the Bacillus cereus group

  • Identify sequence motifs correlated with psychrotolerance

  • Apply coevolution analysis to identify functionally coupled residues

• Systems Biology Integration:

  • Model the regulatory networks controlling mscL expression in response to environmental conditions

  • Integrate transcriptomic, proteomic, and phenotypic data to understand MscL in the broader context of cold adaptation

These computational approaches can generate testable hypotheses about structure-function relationships in B. weihenstephanensis MscL, particularly regarding its adaptation to function at lower temperatures compared to mesophilic homologs.

What are common challenges in working with recombinant B. weihenstephanensis MscL and how can they be addressed?

Researchers working with recombinant B. weihenstephanensis MscL may encounter several technical challenges. The following troubleshooting guide addresses these issues:

Additional recommendations specific to B. weihenstephanensis MscL:

• Given its psychrotolerant origin, maintain working temperatures between 4-25°C during handling

• Consider including specific lipids found in psychrotolerant bacterial membranes in reconstitution mixtures

• When designing mutagenesis studies, prioritize residues that differ between psychrotolerant and mesophilic homologs