Recombinant Shewanella pealeana Protein CrcB homolog (crcB)

How is recombinant S. pealeana CrcB homolog typically expressed and purified?

The recombinant CrcB homolog protein from S. pealeana is typically expressed in Escherichia coli expression systems with an N-terminal histidine tag to facilitate purification . The gene encoding the full-length protein (amino acids 1-124) is cloned into an appropriate expression vector and transformed into competent E. coli cells. Following are the key methodological steps:

• Expression: After transformation, bacteria are grown to an appropriate optical density before induction with IPTG or other inducers, depending on the expression system.

• Cell harvesting and lysis: Cells are harvested by centrifugation and lysed using mechanical disruption or chemical methods.

• Purification: The His-tagged protein is purified using immobilized metal affinity chromatography (IMAC), typically with Ni-NTA resin. Additional purification steps may include size exclusion chromatography or ion exchange chromatography.

• Quality control: SDS-PAGE analysis is performed to confirm purity, which should be greater than 90% .

This expression and purification approach is similar to methods used for other recombinant Shewanella proteins, such as shewasin A from S. amazonensis, which was successfully expressed in E. coli and purified to homogeneity for subsequent functional studies .

What are the optimal storage conditions for recombinant S. pealeana CrcB homolog?

For optimal stability and activity maintenance, the following storage guidelines are recommended:

• Long-term storage: Store the lyophilized powder at -20°C/-80°C upon receipt . For extended storage, -80°C is preferable.

• Working solution: After reconstitution, it is advisable to add glycerol to a final concentration of 5-50% (with 50% being typical) and aliquot the protein for long-term storage at -20°C/-80°C .

• Short-term usage: Working aliquots can be stored at 4°C for up to one week .

• Reconstitution procedure: Prior to opening, briefly centrifuge the vial to bring contents to the bottom. Reconstitute in deionized sterile water to a concentration of 0.1-1.0 mg/mL .

• Freeze-thaw cycles: Repeated freezing and thawing is not recommended as it can lead to protein denaturation and loss of activity .

The protein is typically provided in Tris/PBS-based buffer with 6% Trehalose at pH 8.0, which helps maintain stability during lyophilization and reconstitution .

What functional assays can be used to characterize the fluoride ion transport activity of CrcB homolog?

Characterizing the fluoride ion transport activity of the CrcB homolog requires specialized assays that measure ion movement across membranes. Methodological approaches include:

• Liposome-based transport assays: Reconstitute purified CrcB protein into liposomes loaded with a pH-sensitive or fluoride-sensitive fluorescent dye. Measure fluorescence changes upon addition of fluoride ions to the external medium.

• Electrophysiological techniques: Use patch-clamp techniques with cells overexpressing CrcB to measure ion currents across membranes in response to fluoride gradients.

• Fluoride electrode measurements: Monitor fluoride concentration changes in suspension containing proteoliposomes with reconstituted CrcB protein.

• Bacterial survival assays: Compare growth of bacteria expressing wild-type versus mutated CrcB in media containing varying fluoride concentrations, as fluoride resistance is a functional readout for CrcB activity.

• Isotopic flux assays: Use radioactive fluoride isotopes to directly track fluoride movement in CrcB-containing systems.

When designing these assays, it's critical to include appropriate controls such as liposomes without protein, inactive protein mutants, and known fluoride channel blockers to validate specific transport activity.

How does the S. pealeana CrcB homolog compare to similar proteins in other Shewanella species?

Comparative analysis of CrcB homologs across Shewanella species reveals important insights into evolutionary relationships and functional conservation:

• Sequence conservation: Phylogenomic analysis based on conserved single-copy genes (CSCGs) can be used to determine the evolutionary relationships among Shewanella species . For CrcB specifically, multiple sequence alignment tools like MUSCLE can identify conserved motifs critical for function.

• Structural comparison: While specific structural data for S. pealeana CrcB is limited, comparative modeling using templates from related proteins can predict structural similarities and differences across species.

• Genomic context analysis: Examining the genomic neighborhood of crcB genes across different Shewanella species can provide insights into functional associations and potential co-regulated pathways.

The genus Shewanella has expanded rapidly in the past decade, with more than 70 species now identified . Comparative genomic analyses have revealed that while core functions are often conserved, specific adaptations exist that reflect the ecological niches of different species. For example, S. algae has been associated with carbapenem resistance and contains genes for β-lactams, trimethoprim, tetracycline, colistin, and quinolone resistance, as well as multiple efflux pump genes .

What approaches can be used to investigate structure-function relationships in the CrcB homolog?

Investigating structure-function relationships in the CrcB homolog requires a multidisciplinary approach:

• Site-directed mutagenesis: Systematically mutate conserved residues, particularly those in the transmembrane domains or predicted ion-binding sites. For example, mutation of catalytic residues in related proteins, such as the Asp residue in the Asp-Thr-Gly motif of shewasin A, resulted in complete loss of enzymatic activity .

• Truncation analysis: Create truncated versions of the protein to identify essential domains for fluoride transport.

• Domain swapping: Exchange domains between CrcB homologs from different species to identify species-specific functional elements.

• Structural determination: Employ X-ray crystallography, cryo-electron microscopy, or NMR spectroscopy to resolve the three-dimensional structure, though membrane proteins present particular challenges for structural studies.

• Molecular dynamics simulations: Use computational approaches to model ion permeation pathways and conformational changes associated with transport.

• Cross-linking studies: Identify potential protein-protein interactions that might regulate function or assembly of transport complexes.

The amino acid sequence analysis indicates that the S. pealeana CrcB homolog contains multiple transmembrane segments, consistent with its role as an ion transporter . The sequence suggests a hydrophobic core with several charged residues that may be involved in ion coordination.

What role might the CrcB homolog play in bacterial stress response and environmental adaptation?

The CrcB protein likely plays important roles in bacterial stress response and adaptation:

• Fluoride toxicity defense: As a putative fluoride ion transporter, CrcB likely helps S. pealeana survive in environments containing toxic levels of fluoride by exporting the ion from the cytoplasm.

• pH adaptation: Ion transporters often contribute to pH homeostasis. The CrcB homolog may assist S. pealeana in adapting to acidic marine environments where fluoride can become more bioavailable.

• Environmental sensing: The protein might function as part of a sensory system that detects environmental changes in ion concentrations.

• Biofilm formation: Ion transport systems can influence bacterial biofilm formation, which is a key survival strategy in marine environments.

Research on other Shewanella species has demonstrated remarkable adaptability to various environmental conditions. For example, S. piezotolerans has adapted to high-pressure environments, with genome-scale models showing how its carbon metabolism and energy conservation systems function under both aerobic and anaerobic conditions . Similarly, the CrcB homolog may contribute to S. pealeana's adaptation to its specific ecological niche.

What are the optimal expression systems for producing functional recombinant CrcB protein?

Several expression systems can be considered for producing functional recombinant CrcB protein, each with specific advantages:

• E. coli expression systems: The most commonly used approach, as evidenced by successful expression of the S. pealeana CrcB homolog with an N-terminal His tag . For membrane proteins like CrcB, specialized strains such as C41(DE3) or C43(DE3) may improve expression of properly folded protein. Consider using vectors with tunable promoters to control expression levels.

• Cell-free expression systems: These can be advantageous for membrane proteins as they allow direct incorporation into lipid environments during synthesis, potentially improving functional yield.

• Yeast expression systems: Pichia pastoris or Saccharomyces cerevisiae may provide a eukaryotic-like membrane environment while maintaining relatively high yields.

• Optimization strategies:

  • Use fusion partners like MBP or SUMO to improve solubility

  • Optimize codon usage for the expression host

  • Test various induction temperatures (typically lower temperatures for membrane proteins)

  • Screen detergents for extraction and purification

The choice of expression system should be guided by the intended experimental applications, with consideration for whether native-like folding, post-translational modifications, or high yield is the priority.

How can researchers effectively analyze the membrane topology of the CrcB homolog?

Determining the membrane topology of the CrcB homolog is crucial for understanding its structure-function relationship. Several complementary approaches can be employed:

• Computational prediction: Use membrane protein topology prediction algorithms such as TMHMM, HMMTOP, or Phobius as a starting point.

• Biochemical methods:

  • Protease protection assays: Treat intact membrane vesicles with proteases; regions accessible to proteolytic cleavage are extramembrane domains.

  • Chemical labeling: Use membrane-impermeable reagents to label exposed residues, followed by mass spectrometry identification.

  • Glycosylation mapping: Introduce glycosylation sites at various positions; only sites in extracellular domains will be glycosylated in eukaryotic expression systems.

• Fluorescence-based approaches:

  • FRET analysis: Measure distances between labeled residues to map protein folding.

  • GFP-fusion analysis: Create fusions with GFP at different positions to determine which side of the membrane each terminus resides.

• Cysteine scanning mutagenesis: Systematically replace residues with cysteine and assess accessibility to membrane-permeable and impermeable sulfhydryl reagents.

Based on sequence analysis, the S. pealeana CrcB homolog likely contains multiple transmembrane segments consistent with its putative function as a fluoride ion transporter .

What techniques are available for studying protein-protein interactions involving the CrcB homolog?

Understanding protein-protein interactions is essential for elucidating the functional context of the CrcB homolog. Several methodologies can be employed:

• Co-immunoprecipitation (Co-IP): Use antibodies against CrcB or its epitope tag to pull down protein complexes, followed by mass spectrometry identification of interacting partners.

• Bacterial two-hybrid (B2H) assays: Adapted for membrane proteins, these can detect interactions in a bacterial host environment.

• Cross-linking coupled with mass spectrometry: Chemical cross-linkers can capture transient interactions, with subsequent MS analysis identifying cross-linked peptides.

• FRET/BRET analysis: Fluorescence or bioluminescence resonance energy transfer can detect interactions between appropriately labeled proteins in real-time and in living cells.

• Split-protein complementation assays: Systems like split-GFP, where fluorescence occurs only when two protein fragments are brought together by interacting partners.

• Surface Plasmon Resonance (SPR): For in vitro studies of purified components, SPR can detect and quantify interactions.

• Proteomics approaches:

  • Proximity labeling: Methods like BioID or APEX can identify proteins in the vicinity of CrcB in its native environment.

  • Quantitative interactomics: SILAC or TMT labeling coupled with co-IP can identify differential interactions under various conditions.

Interacting partners could include other ion transport components, regulatory proteins, or members of stress response pathways, providing insights into the functional context of CrcB in S. pealeana.

How can the S. pealeana CrcB homolog contribute to our understanding of bacterial ion homeostasis?

The study of S. pealeana CrcB homolog offers several avenues for advancing our understanding of bacterial ion homeostasis:

Studying bacterial ion transporters like CrcB also has broader implications for understanding fundamental biological processes and potentially developing new antimicrobial strategies that target ion homeostasis.

What potential biotechnological applications exist for the recombinant CrcB homolog?

The recombinant CrcB homolog presents several potential biotechnological applications:

• Biosensors: Engineering CrcB-based fluoride biosensors for environmental monitoring or industrial process control.

• Bioremediation: Developing engineered bacteria with enhanced fluoride handling capabilities for environmental cleanup of fluoride-contaminated sites.

• Synthetic biology tools: Utilizing CrcB as a component in synthetic circuits that respond to specific ion concentrations.

• Protein engineering platforms: Using CrcB as a scaffold for designing novel ion selectivity filters or transport mechanisms.

• Model system for membrane protein studies: The relatively small size (124 amino acids) of CrcB makes it an attractive model for developing new methodologies in membrane protein research.

• Drug discovery: As ion transporters are important antimicrobial targets, recombinant CrcB could be used in screening platforms for identifying novel antimicrobial compounds that disrupt bacterial ion homeostasis.

The successful expression and purification of functional recombinant CrcB provides a foundation for these applications, though each would require further protein engineering and functional characterization.

How might the study of CrcB homologs contribute to understanding pathogenicity in Shewanella species?

While S. pealeana itself is not a significant human pathogen, studying its CrcB homolog can provide insights relevant to pathogenic Shewanella species:

• Stress adaptation mechanisms: Ion transporters like CrcB contribute to bacterial adaptation to host environments, including survival under the ionic stress conditions encountered during infection.

• Comparative genomics perspective: By comparing CrcB homologs between non-pathogenic species like S. pealeana and emerging pathogens like S. algae , researchers can identify adaptations that contribute to pathogenicity.

• Resistance mechanisms: Some ion transporters contribute to antimicrobial resistance. S. algae isolates have shown resistance to carbapenems and contain multiple resistance genes , and understanding ion transport systems may reveal novel resistance mechanisms.

• Host-pathogen interactions: Ion homeostasis affects bacterial virulence factor expression and function, making transporters like CrcB potential indirect contributors to pathogenicity.

The genus Shewanella contains several species associated with human infections, with S. algae being the most commonly isolated clinical species (35.16% of clinical Shewanella infections) . Understanding core physiological processes like ion transport across both pathogenic and non-pathogenic species provides context for identifying specific pathogenicity factors.