Recombinant Shewanella piezotolerans Protein CrcB homolog (crcB)

What is Shewanella piezotolerans WP3 and why is it significant for research?

Shewanella piezotolerans WP3 is a piezotolerant (pressure-tolerant) and psychrotolerant (cold-tolerant) bacterial species isolated from deep-sea sediment at a depth of 1,914 meters in the west Pacific . This organism is significant for research due to its remarkable ability to grow across a wide range of environmental conditions, including temperatures from 0–35°C and pressures from 0.1–50 MPa . As a member of the group 1 branch of the Shewanella genus, WP3 serves as an excellent model organism for studying adaptation mechanisms to extreme deep-sea environments . Its complete genome has been sequenced, enabling comprehensive genomic and metabolic studies of deep-sea bacterial adaptations .

What are the optimal storage conditions for recombinant Shewanella piezotolerans Protein CrcB homolog?

For optimal stability and preservation of biological activity, the recombinant S. piezotolerans Protein CrcB homolog should be stored at -20°C in a Tris-based buffer containing 50% glycerol that has been optimized for this specific protein . For extended storage periods, maintaining the protein at either -20°C or -80°C is recommended . Working aliquots can be stored at 4°C for up to one week to minimize freeze-thaw cycles . It is important to note that repeated freezing and thawing should be avoided as this can lead to protein denaturation and loss of activity . When handling the protein for experiments, researchers should maintain cold chain conditions and follow standard protein handling protocols to preserve structural integrity.

How does Shewanella piezotolerans fit within the phylogeny of the Shewanella genus?

S. piezotolerans WP3 belongs to the group 1 branch of the Shewanella genus, as confirmed by phylogenomic analysis using 661 conserved single-copy genes from 24 Shewanella species . Within this phylogenetic framework, WP3's closest relatives are Shewanella pealeana and Shewanella halifaxensis . This phylogenetic positioning is significant because most previously modeled Shewanella species belong to group 2, making WP3 the first group 1 species with a genome-scale metabolic model . The phylogenomic reconstruction has demonstrated clear differentiation between group 1 and group 2 Shewanella species, representing distinct evolutionary branches . This positioning helps researchers understand the evolutionary context of WP3's adaptations to deep-sea environments and provides a comparative framework for studying functional divergence across the genus.

How can researchers utilize the genome-scale model of S. piezotolerans for metabolic studies?

The genome-scale metabolic model of S. piezotolerans WP3 (GEM-iWP3) represents a powerful computational platform for systems-level investigations of this deep-sea bacterium . This model contains 806 genes, 653 metabolites, and 922 reactions, providing a comprehensive framework for simulating metabolic behaviors under various environmental conditions . Researchers can use this model to:

• Predict growth phenotypes under varying pressure and temperature conditions

• Simulate metabolic flux distributions during adaptation to deep-sea environments

• Identify essential genes and reactions for survival under extreme conditions

• Study energy conservation mechanisms during anaerobic respiration

• Investigate redox balancing and proton motive force generation

The model is publicly available at https://github.com/zhanglab/GEM-iWP3, allowing researchers to download and customize it for specific research questions . When applying this model, researchers should verify predicted growth profiles with experimental measurements to ensure consistency with the organism's physiology . The model can be particularly valuable for studying WP3's metabolic flexibility in using diverse carbon sources under deep-sea conditions, providing insights into adaptations to fluctuating organic carbon availability in these environments .

What experimental considerations are important when studying CrcB homolog under high hydrostatic pressure?

When investigating the CrcB homolog under high hydrostatic pressure (HHP) conditions, several specialized experimental considerations are essential:

• Pressure cultivation systems: Use stainless-steel high-pressure vessels equipped with a water pump system capable of maintaining stable pressures up to 50 MPa to simulate deep-sea conditions .

• Anaerobic preparation: Prepare cultures in disposable syringes without air and seal with combi stoppers to maintain anaerobic conditions during pressure experiments .

• Media composition: Supplement standard media (such as 2216E) with appropriate electron acceptors (e.g., 4 mM nitrate) and carbon sources (e.g., 20 mM lactate) to support growth under pressure .

• Temperature control: Maintain precise temperature control during pressure experiments, as S. piezotolerans exhibits coupled temperature-pressure responses .

• Comparative controls: Always run parallel experiments at atmospheric pressure (0.1 MPa) as controls to isolate pressure-specific effects .

• Expression analysis: Use quantitative RT-PCR or RNA-seq to measure crcB expression under varying pressure conditions to determine pressure-responsive regulation patterns .

• Protein activity assays: Develop high-pressure compatible assays for measuring CrcB function, potentially using fluoride-sensitive probes if the protein functions as a fluoride channel.

These methodological considerations ensure that experimental data accurately reflects the biological responses of the CrcB homolog to high hydrostatic pressure environments.

How does S. piezotolerans adapt to high hydrostatic pressure at the molecular level?

S. piezotolerans WP3 employs several molecular strategies to adapt to high hydrostatic pressure (HHP) environments:

• Redundant respiratory systems: WP3 possesses duplicate respiratory machinery with distinct responses to pressure, such as the two periplasmic nitrate reductase (NAP) systems that exhibit different pressure tolerances . The NAP-α system demonstrates higher tolerance to elevated pressure, while NAP-β plays a dominant role under normal conditions but can be induced by substrate and pressure .

• Pressure-regulated gene expression: The bacterium modulates gene expression in response to pressure changes, with specific pathways being up- or down-regulated at different pressure levels . For example, the NAP systems show pressure-inducible expression patterns when the complementary system is absent .

• Metabolic flexibility: The genome-scale model of WP3 reveals greater flexibility in ATP production under anaerobic conditions compared to group 2 Shewanella species, which may be advantageous for adaptation to fluctuating carbon availability in the deep sea .

• Energy conservation mechanisms: Despite being an obligate respiratory organism, WP3 primarily uses substrate-level phosphorylation for energy conservation under anaerobic conditions, with the directionality of ATP synthase reaction flux correlated with the availability of reducing equivalents in the cell .

• Membrane adaptations: Proteins like CrcB homolog may contribute to maintaining membrane integrity and function under high pressure, potentially by regulating ion movement across the membrane to maintain cellular homeostasis .

Understanding these molecular adaptation mechanisms provides insights into how deep-sea microorganisms thrive in extreme environments and may inspire biotechnological applications for high-pressure processes.

What is the role of CrcB homolog in bacterial stress responses?

While the specific role of the CrcB homolog in S. piezotolerans stress responses has not been fully characterized in the provided search results, we can infer potential functions based on homologous proteins in other bacteria:

• Ion homeostasis: CrcB homologs typically function as fluoride ion channels in many bacteria, protecting cells from fluoride toxicity by exporting this ion from the cytoplasm.

• Membrane integrity: As a membrane protein with multiple transmembrane domains (as suggested by its hydrophobic amino acid sequence), the CrcB homolog likely plays a role in maintaining membrane integrity under stress conditions, potentially including high pressure .

• Pressure adaptation: The presence of this protein in a piezotolerant organism suggests it may contribute to pressure adaptation, possibly by stabilizing membrane functions or regulating ion fluxes that change under pressure.

• Integration with metabolic networks: Given that WP3 has evolved specific adaptations for energy conservation under pressure, the CrcB homolog may interact with these systems to maintain cellular homeostasis during stress .

To definitively establish the role of CrcB in stress responses, researchers should consider gene knockout studies, expression analysis under various stress conditions, and protein-protein interaction studies to identify functional partners within stress response networks.

What approaches can be used to express and purify recombinant CrcB homolog?

Expressing and purifying membrane proteins like the CrcB homolog presents unique challenges. Researchers can employ the following methodological approaches:

Expression Systems:

• E. coli-based expression: Use specialized E. coli strains (C41/C43 or Lemo21) optimized for membrane protein expression.

• Cell-free expression systems: Consider membrane-mimetic cell-free systems that can directly incorporate membrane proteins into liposomes or nanodiscs.

• Homologous expression: Express the protein in a related Shewanella species that grows at ambient pressure to maintain native folding.

Expression Optimization:

• Temperature modulation: Lower expression temperature (16-20°C) to slow protein synthesis and improve folding.

• Induction control: Use titratable induction systems (like rhamnose or tetracycline) to fine-tune expression levels.

• Fusion tags: Incorporate solubility-enhancing tags (MBP, SUMO) that can be later removed by specific proteases.

Purification Strategy:

• Membrane preparation: Isolate membranes using ultracentrifugation following cell lysis.

• Detergent screening: Test multiple detergents (DDM, LMNG, digitonin) for optimal protein extraction and stability.

• Affinity chromatography: Utilize histidine or other affinity tags for initial purification.

• Size exclusion chromatography: Perform final purification and detergent exchange using size exclusion chromatography.

Quality Control:

• Functional validation: Verify protein functionality through fluoride ion transport assays if applicable.

• Structural integrity: Assess protein folding using circular dichroism or thermal stability assays.

• Homogeneity analysis: Confirm protein homogeneity through dynamic light scattering or analytical ultracentrifugation.

This systematic approach maximizes the likelihood of obtaining properly folded, functional CrcB homolog protein for subsequent biophysical and biochemical analyses.

How can researchers design experiments to study the function of CrcB homolog under varying pressure conditions?

To investigate the function of CrcB homolog under different pressure conditions, researchers should implement a multifaceted experimental design:

Equipment and Setup:

• High-pressure bioreactors: Utilize specialized pressure vessels capable of maintaining stable pressure while allowing for sampling and monitoring .

• Pressure-resistant sampling systems: Implement systems that enable sample collection without decompression to prevent artifacts.

• Real-time monitoring: When possible, incorporate sensors for real-time measurement of relevant parameters inside the pressure vessel.

Functional Studies:

• Gene expression analysis: Measure crcB expression using qRT-PCR or RNA-seq across a pressure gradient (0.1-50 MPa) to establish pressure-response patterns .

• Protein localization: Use fluorescent fusion proteins or immunolocalization to track CrcB localization under different pressures.

• Deletion mutants: Create ΔcrcB knockout strains and assess phenotypic changes in growth, survival, and membrane properties under pressure.

• Complementation studies: Reintroduce wild-type or mutated versions of crcB to confirm phenotype restoration.

Biochemical Characterization:

• Membrane fluidity assessment: Measure membrane fluidity changes in wild-type versus ΔcrcB strains under pressure using fluorescent probes.

• Ion flux measurements: If CrcB functions as an ion channel, develop assays to measure ion movement across membranes under pressure.

• Protein-protein interactions: Identify pressure-dependent interaction partners using techniques like bacterial two-hybrid systems or co-immunoprecipitation.

Data Collection and Analysis:

• Pressure gradients: Collect data at multiple pressure points (e.g., 0.1, 10, 20, 30, 40, 50 MPa) rather than just ambient versus high pressure .

• Time-course experiments: Monitor changes over time to capture both immediate and adaptive responses.

• Statistical analysis: Apply appropriate statistical methods to identify significant pressure-dependent effects.

This comprehensive experimental approach will provide insights into the molecular function of CrcB homolog under conditions mimicking the deep-sea environment.

What analytical techniques are most effective for characterizing CrcB homolog structure-function relationships?

Characterizing the structure-function relationships of membrane proteins like CrcB homolog requires specialized analytical techniques:

Structural Analysis:

• Cryo-electron microscopy: Particularly suitable for membrane proteins, cryo-EM can resolve structures without crystallization.

• X-ray crystallography: If crystals can be obtained, this provides high-resolution structural data.

• NMR spectroscopy: Solution NMR or solid-state NMR can provide structural information and dynamics in membrane-mimetic environments.

• Molecular dynamics simulations: Computational modeling of protein behavior under different pressure conditions can predict structural changes.

Functional Characterization:

• Electrophysiology: Patch-clamp recordings in reconstituted systems to measure ion conductance properties if CrcB functions as an ion channel.

• Fluorescence-based transport assays: Using fluoride-sensitive fluorescent probes to measure transport activity in proteoliposomes.

• Isothermal titration calorimetry: Measuring binding affinities for ions or interaction partners.

• Hydrogen-deuterium exchange mass spectrometry: Identifying flexible regions and conformational changes under different conditions.

Mutagenesis Approaches:

• Alanine scanning: Systematic replacement of amino acids with alanine to identify functional residues.

• Domain swapping: Exchange domains with CrcB homologs from non-piezotolerant organisms to identify pressure-adaptive regions.

• Chimeric proteins: Create chimeras with fluorescent proteins to monitor conformational changes in real-time.

High-Pressure Techniques:

• High-pressure spectroscopy: Specialized equipment for spectroscopic measurements under pressure.

• Pressure perturbation calorimetry: Measuring thermodynamic parameters of protein stability under pressure.

• High-pressure stopped-flow: For capturing fast kinetics of conformational changes or substrate binding under pressure.

By combining these analytical approaches, researchers can develop a comprehensive understanding of how CrcB homolog structure relates to its function in high-pressure environments.

How should researchers interpret gene expression data for crcB under varying environmental conditions?

When analyzing gene expression data for crcB under different environmental conditions, researchers should follow these methodological guidelines:

Data Normalization and Quality Control:

• Reference gene selection: Choose multiple stable reference genes that maintain consistent expression across experimental conditions for accurate normalization.

• Technical replication: Include at least 3 technical replicates to account for measurement variability.

• Biological replication: Analyze at least 3 independent biological replicates to capture natural biological variation.

• Batch effect correction: Apply statistical methods to correct for batch effects if experiments are conducted across different time periods.

Statistical Analysis Framework:

• Differential expression: Use appropriate statistical tests (e.g., ANOVA with post-hoc tests for multiple conditions or t-tests for pairwise comparisons) to identify significant changes.

• Multiple testing correction: Apply corrections (Benjamini-Hochberg or Bonferroni) to control false discovery rates when testing multiple hypotheses.

• Effect size estimation: Report fold changes and confidence intervals in addition to p-values.

Contextual Interpretation:

• Co-expression analysis: Examine whether crcB expression patterns correlate with other genes involved in pressure adaptation or stress responses.

• Pathway enrichment: Determine if crcB expression changes are part of broader pathway responses.

• Temporal dynamics: Consider the kinetics of expression changes—immediate versus adaptive responses—when interpreting data.

• Cross-condition comparison: Compare crcB expression patterns across different stressors (pressure, temperature, pH) to identify condition-specific versus general stress responses .

Validation Approaches:

• Orthogonal methods: Confirm key findings using alternative techniques (e.g., RNA-seq findings validated by qRT-PCR).

• Protein-level validation: Correlate transcript changes with protein abundance changes using proteomics.

• Functional validation: Connect expression changes to phenotypic outcomes through mutant studies.

This systematic approach to data analysis ensures robust interpretation of crcB expression patterns and their biological significance in environmental adaptation.

What approaches can be used to model the effects of pressure on protein structure and function?

Modeling the effects of pressure on the structure and function of proteins like CrcB homolog requires specialized computational approaches:

Molecular Dynamics (MD) Simulations:

• High-pressure MD: Perform simulations under increased pressure conditions (10-500 MPa) to observe conformational changes.

• Long timescale simulations: Extend simulations to microsecond timescales to capture pressure-induced structural transitions.

• Membrane-embedded simulations: For membrane proteins like CrcB, include explicit lipid bilayers in the simulation system.

• Free energy calculations: Compute free energy landscapes under different pressure conditions to identify stable conformational states.

Structural Bioinformatics:

• Comparative modeling: Build homology models based on structurally characterized homologs.

• Cavity analysis: Identify and measure internal cavities that may be compressed under pressure.

• Hydration analysis: Analyze changes in protein hydration shell under pressure.

• Flexibility prediction: Use normal mode analysis to identify flexible regions likely to respond to pressure.

Machine Learning Approaches:

• Sequence-based prediction: Develop models that predict pressure adaptation from sequence features by comparing piezotolerant vs. non-piezotolerant homologs.

• Structure-based classification: Train neural networks to identify structural features associated with pressure resistance.

• Molecular descriptor analysis: Generate and analyze molecular descriptors that correlate with pressure stability.

Integration with Experimental Data:

• Restraint-based modeling: Incorporate experimental data as restraints in computational models.

• Model validation: Compare computational predictions with experimental measurements of pressure effects.

• Iterative refinement: Update models based on experimental feedback.

Specialized Analysis:

• Volume change calculations: Compute changes in partial molar volume under pressure.

• Ionization state prediction: Model pKa shifts under pressure for charged residues.

• Transition state analysis: For enzymes, model how pressure affects catalytic transition states.

These computational approaches provide valuable insights into pressure adaptation mechanisms that may be difficult to observe experimentally, guiding hypothesis generation for further experimental testing.

How can integrated multi-omics approaches enhance our understanding of CrcB homolog function?

Integrating multiple omics approaches provides a comprehensive systems-level understanding of CrcB homolog function in the context of pressure adaptation:

Multi-omics Data Collection:

Integration Strategies:

• Correlation networks: Construct networks connecting genes, proteins, and metabolites that show correlated changes with crcB/CrcB.

• Multi-layer network analysis: Develop integrated networks incorporating regulatory relationships across different omics layers.

• Pathway enrichment: Perform joint pathway enrichment analysis across multiple omics datasets to identify convergent biological processes.

• Causality inference: Apply causal inference methods to determine directional relationships between observed changes.

• Time-course integration: Align temporal data across omics layers to identify sequence of events in pressure response.

Computational Methods:

• Genome-scale metabolic modeling: Incorporate crcB functional data into the existing genome-scale model (GEM-iWP3) to predict systemic effects .

• Bayesian network approaches: Use probabilistic modeling to integrate heterogeneous data types.

• Machine learning integration: Apply supervised or unsupervised learning algorithms to identify patterns across multi-omics datasets.

• Knowledge-based integration: Leverage existing biological knowledge to guide data integration and interpretation.

Visualization and Interpretation:

• Multi-dimensional visualization: Develop visualizations that represent relationships across multiple omics layers.

• Comparative analysis: Compare integrated networks between pressure-adapted and non-adapted states.

• Functional module identification: Identify functional modules that include CrcB and respond coordinately to pressure changes.

This integrated approach moves beyond studying CrcB in isolation to understanding its role within the broader cellular network, providing insights into how this protein contributes to the piezotolerant phenotype of S. piezotolerans.

What are the most promising avenues for future research on CrcB homolog in deep-sea bacteria?

Several high-priority research directions would significantly advance our understanding of CrcB homolog in deep-sea bacteria:

• Structural determination under pressure: Develop methodologies to determine the structure of CrcB homolog under high pressure conditions, potentially revealing pressure-induced conformational changes that explain its function in piezotolerant organisms.

• Comparative genomics across depth gradients: Analyze CrcB sequences from Shewanella species isolated across various ocean depths to identify adaptive mutations correlated with depth/pressure tolerance.

• In vivo visualization: Develop fluorescent protein fusions or antibody-based approaches to visualize CrcB localization and dynamics in living cells under pressure.

• Interactome mapping: Identify pressure-dependent protein-protein interactions of CrcB homolog to place it within cellular response networks.

• Synthetic biology applications: Engineer CrcB variants with enhanced pressure tolerance for potential biotechnological applications in high-pressure bioreactors.

• Integration with genomic model: Incorporate CrcB function into the existing genome-scale model of S. piezotolerans to predict its systemic effects under pressure .

• Cross-species functional analysis: Compare CrcB function between piezotolerant and non-piezotolerant organisms to identify critical adaptations.

• Evolutionary history reconstruction: Trace the evolutionary history of CrcB across bacterial lineages to understand how pressure adaptation evolved.

These research directions would provide comprehensive insights into the role of CrcB in deep-sea adaptation and potentially reveal novel mechanisms of protein function under extreme conditions.

How might findings about CrcB homolog contribute to broader understanding of extremophile adaptation?

Research on the CrcB homolog in S. piezotolerans has potential to advance our understanding of extremophile adaptation through several conceptual frameworks:

• Functional redundancy principles: The discovery that S. piezotolerans employs redundant respiratory systems with distinct pressure responses (as seen with the NAP systems) suggests that similar redundancy might exist for other cellular functions, potentially including CrcB-mediated processes. This represents a broader adaptation strategy where organisms maintain multiple pathways for critical functions, each optimized for different environmental conditions.

• Molecular adaptation mechanisms: Understanding how CrcB structure and function are maintained under high pressure could reveal general principles about protein adaptations to extreme conditions, including the roles of protein flexibility, hydration, and stability.

• Evolutionary trade-offs: Comparing CrcB homologs across species adapted to different environments could illuminate evolutionary trade-offs between adaptation to different stressors (e.g., pressure vs. temperature vs. pH).

• Systems-level adaptation: Integration of CrcB function into genome-scale models could demonstrate how individual protein adaptations contribute to system-wide resilience in extreme environments.

• Convergent evolution: Identifying whether similar adaptations in CrcB have evolved independently in different deep-sea lineages would provide insights into constraints and opportunities in molecular adaptation.

• Biomarkers for environmental adaptation: CrcB variants could potentially serve as biomarkers for predicting an organism's ability to thrive under specific pressure regimes in deep-sea environments.

These broader conceptual advances would extend the significance of CrcB research beyond a single protein to fundamental principles of how life adapts to extreme environments.