Recombinant Shewanella denitrificans Protein CrcB homolog (crcB)

What is the Shewanella denitrificans CrcB homolog protein and what is its primary function?

The CrcB homolog protein in Shewanella denitrificans is a membrane protein that functions as a fluoride ion transporter. It belongs to a family of proteins that are widely distributed across bacteria and archaea, specifically evolved to mitigate fluoride toxicity. These proteins are proposed to function by actively removing fluoride from the cell, thereby preventing the harmful effects associated with elevated intracellular fluoride concentrations .

The full-length protein consists of 124 amino acids with the sequence: MTNLLFVALGGSIGAVLRYLMSIIMIQLFGSSFPFGTLLVNVLGSFFMGIVYALGQVSHVSPELKALVGVGLLGALTTFSTFSNETLLLMQQGYWFKSLINVLLNVSLCIFMVYLGQQLVFSRV . The protein is characterized by its predominantly hydrophobic composition, suggesting multiple transmembrane domains consistent with its role as an ion transporter.

How is the expression of CrcB regulated in Shewanella denitrificans?

The expression of the crcB gene in S. denitrificans, like in many other bacteria, is regulated by a fluoride-responsive riboswitch. This RNA structure, located in the 5' untranslated region of the mRNA, selectively binds fluoride ions while rejecting other anions including chloride . Upon fluoride binding, the riboswitch undergoes a conformational change that increases the expression of downstream genes, including crcB .

This regulatory mechanism represents an elegant example of bacterial adaptation, allowing cells to respond specifically to elevated environmental fluoride concentrations by increasing the production of fluoride resistance proteins. The fluoride riboswitch activates gene expression only when fluoride levels are elevated, ensuring that resources for producing CrcB are allocated efficiently .

What is known about the habitat and physiological characteristics of Shewanella denitrificans?

Shewanella denitrificans was originally isolated from the oxic-anoxic interface of an anoxic basin in the central Baltic Sea. It is a member of the gamma-Proteobacteria and is phylogenetically related to other Shewanella species, showing 95-96% sequence similarity with S. baltica, S. putrefaciens, and S. frigidimarina .

Physiologically, S. denitrificans is characterized as:

• Unpigmented

• Polarly flagellated

• Mesophilic

• Facultatively anaerobic

• Capable of using nitrate, nitrite, and sulfite as electron acceptors

• Able to grow at salinities ranging from 0% to 6%, with optimal growth between 1% and 3%

The bacterium's ability to thrive at the oxic-anoxic interface and its remarkable denitrification capacity suggest it plays a significant role in nitrogen cycling in marine ecosystems.

What methodologies are most effective for expressing and purifying recombinant S. denitrificans CrcB for structural studies?

For effective expression and purification of recombinant S. denitrificans CrcB, researchers should consider the following methodological approach:

• Expression System Selection: Due to CrcB being a membrane protein, specialized expression systems are recommended. E. coli strains C41(DE3) or C43(DE3), specifically engineered for membrane protein expression, often yield better results than standard BL21(DE3).

• Vector Design: The expression vector should include:

  • A strong, inducible promoter (T7 or araBAD)

  • A fusion tag (such as His6, FLAG, or MBP) to facilitate purification

  • A precision protease cleavage site for tag removal

  • Consideration of codon optimization for the expression host

• Purification Protocol:

  • Membrane extraction using detergents such as n-dodecyl-β-D-maltoside (DDM) or lauryl maltose neopentyl glycol (LMNG)

  • Initial purification via affinity chromatography targeting the fusion tag

  • Size exclusion chromatography for further purification and assessment of homogeneity

  • Assessment of protein stability in various buffers and detergents

• Functional Verification: Assay for fluoride transport activity using techniques such as fluoride ion-selective electrodes or fluoride-sensitive probes in proteoliposomes.

When working with this protein, researchers should be aware that its hydrophobic nature (as evident from its amino acid sequence: MTNLLFVALGGSIGAVLRYLMSIIMIQLFGSSFPFGTLLVNVLGSFFMGIVYALGQVSHVSPELKALVGVGLLGALTTFSTFSNETLLLMQQGYWFKSLINVLLNVSLCIFMVYLGQQLVFSRV) may require optimization of solubilization and stabilization conditions .

How do the fluoride resistance mechanisms of S. denitrificans compare to those in other bacterial species?

Fluoride resistance mechanisms in bacteria primarily revolve around two main protein families: CrcB-type fluoride transporters and fluoride-specific channels (EriCF or ClCF). S. denitrificans employs the CrcB system, which shares functional similarities but displays structural differences compared to other bacterial fluoride resistance systems .

Comparative Analysis of Bacterial Fluoride Resistance Systems:

While most bacterial species employ similar strategies for fluoride resistance, the structural properties and efficiency of these systems may vary based on their evolutionary history and typical environmental fluoride exposure. The presence of fluoride riboswitches across diverse bacteria, including S. denitrificans, indicates that fluoride toxicity has been a consistent selective pressure throughout bacterial evolution .

Interestingly, even bacteria that do not regularly encounter high fluoride concentrations in human-associated environments (such as S. denitrificans in marine settings) maintain these resistance mechanisms, suggesting either a broader importance of fluoride resistance or additional functions for these proteins .

What experimental approaches can be used to characterize the fluoride transport kinetics of the CrcB homolog?

Several experimental approaches can be employed to characterize the fluoride transport kinetics of the S. denitrificans CrcB homolog:

These methods should be complemented with mutational analyses, where key residues in CrcB are systematically altered to identify amino acids critical for fluoride recognition, channel formation, and transport function.

How does the structural homology of S. denitrificans CrcB compare with crystallographically-characterized fluoride channels?

While the crystal structure of S. denitrificans CrcB has not been specifically reported in the provided search results, the structural homology can be inferred by comparison with other characterized fluoride channels and transporters:

The S. denitrificans CrcB homolog likely shares the core structural features of other CrcB family proteins, which typically contain multiple transmembrane helices that form a pathway for fluoride transport across the membrane. The amino acid sequence (MTNLLFVALGGSIGAVLRYLMSIIMIQLFGSSFPFGTLLVNVLGSFFMGIVYALGQVSHVSPELKALVGVGLLGALTTFSTFSNETLLLMQQGYWFKSLINVLLNVSLCIFMVYLGQQLVFSRV) suggests a predominantly hydrophobic protein with multiple membrane-spanning regions .

The functional similarities between CrcB proteins and the fluoride-specific channels (EriCF/ClCF) suggest some convergent evolutionary features, though they belong to different protein families. The ClCF proteins have been shown to function as fluoride/proton antiporters, and it's possible that CrcB proteins employ a similar mechanism, though this remains to be definitively established .

Homology modeling based on related structures, combined with techniques such as cryo-electron microscopy or X-ray crystallography, would provide valuable insights into the structural basis of fluoride selectivity and transport by S. denitrificans CrcB.

What are the recommended protocols for studying the in vivo expression and localization of CrcB in S. denitrificans?

To study the in vivo expression and localization of CrcB in S. denitrificans, researchers can employ the following protocols:

• Gene Expression Analysis:

  • Quantitative RT-PCR to measure crcB transcript levels under different conditions

  • RNA-seq to understand transcriptional responses to fluoride exposure

  • Reporter gene fusions (e.g., crcB promoter fused to GFP or luciferase) to monitor expression dynamics

• Protein Detection and Quantification:

  • Western blotting using antibodies against CrcB or epitope-tagged versions

  • Mass spectrometry-based proteomic analysis to quantify relative protein abundance

• Subcellular Localization:

  • Fluorescent protein fusions (C- or N-terminal GFP fusions to CrcB)

  • Immunofluorescence microscopy using anti-CrcB antibodies

  • Cell fractionation followed by Western blotting to determine membrane association

• Functional Localization:

  • Fluoride-sensitive fluorescent probes to visualize local changes in fluoride concentration

  • Genetic complementation studies with localization-defective mutants

When studying membrane proteins like CrcB, it's important to ensure that any tagging strategy does not interfere with proper membrane insertion or function. For fluorescent protein fusions, researchers should verify that the fusion protein retains fluoride transport activity.

By drawing parallels with studies of bacterial aspartic proteases like shewasin D from the same organism, which have demonstrated expression of active enzyme in S. denitrificans cells with activity at acidic pH and inhibition by pepstatin, similar methodological approaches could be adapted for CrcB .

How can mutagenesis studies be designed to identify critical residues for fluoride transport in CrcB?

A comprehensive mutagenesis strategy for identifying critical residues in S. denitrificans CrcB should include:

• Targeted Mutagenesis Approaches:

  • Alanine scanning mutagenesis: Systematically replacing charged and polar residues with alanine to identify those crucial for function

  • Conservative substitutions: Replacing residues with chemically similar amino acids to probe specific chemical requirements

  • Radical substitutions: Introducing charge inversions or dramatic size changes to test structural hypotheses

• Selection of Target Residues Based On:

  • Sequence conservation across CrcB homologs from different species

  • Predicted transmembrane topology and potential channel-forming regions

  • Charged or polar residues that might interact with fluoride ions

  • Residues in the MTNLLFVALGGSIGAVLRYLMSIIMIQLFGSSFPFGTLLVNVLGSFFMGIVYALGQVSHVSPELKALVGVGLLGALTTFSTFSNETLLLMQQGYWFKSLINVLLNVSLCIFMVYLGQQLVFSRV sequence that align with functional regions in related transporters

• Functional Assays for Mutant Proteins:

  • In vivo complementation: Testing ability of mutant crcB to restore fluoride resistance in a crcB knockout strain

  • Transport assays: Using fluoride-sensitive indicators or electrodes to measure transport activity

  • Protein expression and stability controls: Ensuring that observed effects are due to functional rather than structural defects

• Structural Interpretation:

  • Mapping of identified critical residues onto structural models

  • Integration with computational docking studies to understand fluoride coordination

This approach would yield a functional map of the protein, identifying residues involved in fluoride recognition, channel formation, and transport mechanics.

What approaches can be used to study the interaction between the fluoride riboswitch and CrcB expression in S. denitrificans?

To study the interaction between the fluoride riboswitch and CrcB expression in S. denitrificans, researchers can employ the following methodological approaches:

• Riboswitch Structure and Function Analysis:

  • In-line probing: To detect conformational changes in the riboswitch RNA upon fluoride binding

  • Chemical probing techniques (SHAPE, DMS): To map structural changes at nucleotide resolution

  • Fluorescence-based assays: Using fluorescently labeled RNA to monitor structural transitions

• Reporter Gene Assays:

  • Transcriptional fusions: Coupling the riboswitch and crcB promoter to reporter genes

  • Translational fusions: Including the riboswitch and early coding sequence fused to reporters

  • Dose-response analyses: Measuring reporter activity across a range of fluoride concentrations

• Genetic Approaches:

  • Mutagenesis of riboswitch elements: Identifying nucleotides critical for fluoride sensing

  • Compensatory mutations: Restoring function by making paired changes in interacting regions

  • Chimeric riboswitches: Swapping domains between riboswitches to map functional elements

• Direct Binding Studies:

  • Isothermal titration calorimetry (ITC): Measuring thermodynamic parameters of fluoride binding

  • Surface plasmon resonance (SPR): Assessing binding kinetics

  • Fluorescence polarization: Using fluorescently labeled ligands to detect binding events

These methods would provide comprehensive insights into how the fluoride riboswitch in S. denitrificans responds to fluoride and regulates CrcB expression. Research has shown that fluoride riboswitches selectively respond to fluoride while rejecting other anions, including chloride, and then activate expression of genes encoding fluoride transporters like CrcB .

What potential applications exist for CrcB beyond understanding basic bacterial physiology?

The S. denitrificans CrcB homolog has several potential applications beyond understanding basic bacterial physiology:

• Bioremediation Technologies:

  • Engineered bacteria overexpressing CrcB could be developed for fluoride removal from contaminated water sources

  • CrcB-based biofiltration systems could offer sustainable approaches to treating industrial wastewater with high fluoride content

• Biosensor Development:

  • The fluoride riboswitch-CrcB system could be repurposed to create highly sensitive and specific fluoride biosensors

  • These biosensors could monitor environmental fluoride levels or detect fluoride in industrial processes

• Synthetic Biology Applications:

  • The fluoride-responsive elements could be incorporated into synthetic gene circuits as orthogonal regulatory components

  • CrcB could serve as a selective marker in synthetic biology applications where fluoride resistance is advantageous

• Structural Biology Insights:

  • The unique fluoride selectivity of CrcB could inform the design of novel ion channels or transporters with tailored specificities

  • Understanding CrcB structure could contribute to membrane protein engineering efforts

• Antimicrobial Development:

  • Inhibition of CrcB could potentially sensitize bacteria to fluoride, suggesting a novel antimicrobial strategy

  • This approach might be particularly relevant for oral bacteria like Streptococcus mutans, where fluoride is already used in dental care

These applications highlight how basic research on proteins like CrcB can lead to diverse translational opportunities across environmental science, biotechnology, and medicine.

How might evolution of CrcB proteins in marine bacteria like S. denitrificans differ from those in terrestrial or human-associated bacteria?

The evolution of CrcB proteins in marine bacteria like S. denitrificans likely follows a distinct trajectory compared to terrestrial or human-associated bacteria due to differences in environmental pressures:

• Evolutionary Selection Pressures:

  • Marine environments: Generally lower but more consistent fluoride levels from geological sources

  • Terrestrial environments: Highly variable fluoride exposure depending on soil composition

  • Human-associated niches: Potential exposure to anthropogenic fluoride (e.g., dental products)

• Comparative Adaptation Strategies:

• Genomic Context Differences:

  • Marine bacteria may show more conservation of the genomic neighborhood surrounding crcB

  • Human-associated bacteria might display more evidence of horizontal gene transfer and rapid adaptation

  • The regulatory elements (riboswitches) may show environment-specific tuning of sensitivity thresholds

• Functional Divergence:

  • Marine CrcB proteins might have evolved additional or modified functions beyond fluoride transport

  • The consistent presence of CrcB even in environments not typically associated with high fluoride suggests potential moonlighting functions or importance in addressing periodic exposure events

Interestingly, the search results note that "many organisms that do not encounter fluoride in the human mouth carry fluoride riboswitches or resistance genes," suggesting that fluoride resistance is an ancient and widespread bacterial trait maintained even in environments where exposure might be limited .

What are the remaining challenges in understanding the structure-function relationship of bacterial CrcB proteins?

Despite significant advances, several challenges remain in fully understanding the structure-function relationship of bacterial CrcB proteins like that from S. denitrificans:

• Structural Characterization Challenges:

  • Membrane proteins like CrcB present inherent difficulties for structural techniques

  • Limited availability of high-resolution structures hampers detailed mechanistic understanding

  • Crystallization of membrane transporters often requires extensive optimization

• Transport Mechanism Uncertainties:

  • The precise mechanism of fluoride transport remains to be fully elucidated

  • Questions persist about energy coupling (passive transport vs. active pumping)

  • The stoichiometry of transport (how many fluoride ions per transport cycle) is not definitively established

• Physiological Integration Questions:

  • How CrcB function integrates with other cellular processes during fluoride stress

  • Potential interactions with other membrane proteins or cellular components

  • Regulation beyond the riboswitch level, including post-translational modifications

• Evolutionary Considerations:

  • Understanding how structural variations across bacterial species relate to functional differences

  • Determining if CrcB has evolved secondary functions in some organisms

  • Clarifying the evolutionary relationship between CrcB and other fluoride transporters

• Technical Methodology Gaps:

  • Need for improved assays to measure fluoride transport with higher temporal resolution

  • Development of specific inhibitors to probe CrcB function

  • Better tools for studying membrane protein dynamics in native-like environments

Addressing these challenges will require interdisciplinary approaches combining structural biology, biochemistry, genetics, and computational methods. The amino acid sequence (MTNLLFVALGGSIGAVLRYLMSIIMIQLFGSSFPFGTLLVNVLGSFFMGIVYALGQVSHVSPELKALVGVGLLGALTTFSTFSNETLLLMQQGYWFKSLINVLLNVSLCIFMVYLGQQLVFSRV) provides a starting point, but additional experimental data is needed to fully understand this important class of transporters .

What are common challenges in the expression and purification of recombinant CrcB, and how can they be addressed?

Researchers working with recombinant S. denitrificans CrcB homolog protein commonly encounter several challenges during expression and purification. The following table outlines these challenges and provides methodological solutions:

Researchers should be particularly attentive to the detergent selection process, as the hydrophobic nature of the S. denitrificans CrcB (evident from its amino acid sequence: MTNLLFVALGGSIGAVLRYLMSIIMIQLFGSSFPFGTLLVNVLGSFFMGIVYALGQVSHVSPELKALVGVGLLGALTTFSTFSNETLLLMQQGYWFKSLINVLLNVSLCIFMVYLGQQLVFSRV) suggests multiple transmembrane regions that require appropriate solubilization conditions .

A step-wise optimization approach, starting with small-scale expression tests and detergent screens before proceeding to large-scale purification, is recommended for maximizing yield and activity.

How can researchers accurately assess the functional activity of purified recombinant CrcB protein?

Accurate assessment of functional activity for purified recombinant S. denitrificans CrcB protein requires multiple complementary approaches:

• Reconstitution-Based Assays:

  • Proteoliposome Fluoride Transport:

    • Reconstitute CrcB into liposomes loaded with fluoride-sensitive indicators

    • Monitor fluoride movement using fluoride-selective electrodes

    • Quantify transport rates under different conditions (pH, ion gradients)

  • Solid-Supported Membrane Electrophysiology:

    • Adsorb proteoliposomes onto sensor chips

    • Measure electrical signals associated with fluoride transport

    • Allows for rapid screening of transport activity

• Binding Assays:

  • Isothermal Titration Calorimetry (ITC):

    • Directly measure thermodynamic parameters of fluoride binding

    • Distinguish specific from non-specific interactions

  • Microscale Thermophoresis (MST):

    • Detect binding-induced changes in thermophoretic mobility

    • Requires minimal protein amounts and works in detergent solutions

• Structural Integrity Assessments:

  • Circular Dichroism (CD) Spectroscopy:

    • Verify secondary structure content

    • Monitor thermal stability

  • Fluorescence-Based Thermal Shift Assays:

    • Assess protein stability in different conditions

    • Identify stabilizing ligands or conditions

• Functional Complementation:

  • In vivo complementation assays:

    • Express purified CrcB in fluoride-sensitive bacterial strains

    • Measure restoration of fluoride resistance

    • Provides validation of biological activity

When interpreting these assays, researchers should consider that transport proteins like CrcB may require specific lipid environments for optimal activity. Comparing activity measurements across different reconstitution conditions can provide insights into the lipid requirements for CrcB function.