Recombinant Salmonella heidelberg Cobalt transport protein CbiN (cbiN)

What is the biological function of Cobalt transport protein CbiN in Salmonella heidelberg?

Cobalt transport protein CbiN functions as a component of the energy-coupling factor (ECF) transporter system in Salmonella heidelberg, specifically involved in cobalt uptake and transport across the bacterial membrane. The protein is part of the CbiMNQO transport system, where CbiN likely serves as a substrate-capture protein that facilitates the initial binding of cobalt ions .

As part of the bacterial vitamin B12 (cobalamin) biosynthetic pathway, CbiN plays a critical role in ensuring sufficient cobalt is available for incorporation into the corrin ring structure of cobalamin. This function is essential for numerous metabolic processes in Salmonella, including DNA synthesis and cellular energy production. The protein consists of 93 amino acids and contains characteristic transmembrane domains that anchor it to the bacterial membrane .

What expression systems are most effective for recombinant CbiN production?

Based on current research protocols, E. coli expression systems have proven most effective for recombinant CbiN production . When expressing the full-length protein (1-93 amino acids), an N-terminal His-tag fusion approach allows for efficient purification while maintaining protein functionality.

The typical expression protocol involves:

• Cloning the cbiN gene from Salmonella heidelberg into an expression vector containing an N-terminal His-tag

• Transforming the construct into an E. coli expression strain (commonly BL21(DE3) or derivatives)

• Inducing protein expression using IPTG (isopropyl β-D-1-thiogalactopyranoside) or auto-induction media

• Cell harvesting and lysis under native conditions

• Purification using nickel affinity chromatography

For optimal expression, researchers should consider temperature control (typically 18-25°C for membrane proteins), induction time (4-16 hours), and appropriate antibiotic selection based on the expression vector .

What techniques are recommended for purification and storage of recombinant CbiN protein?

The purification and storage of recombinant CbiN requires specific techniques to maintain protein integrity:

Purification Protocol:

• Centrifuge the bacterial lysate to remove cell debris

• Apply the supernatant to a pre-equilibrated nickel affinity column

• Wash with buffer containing low imidazole concentration

• Elute the His-tagged CbiN with buffer containing high imidazole concentration (250-500 mM)

• Perform buffer exchange to remove imidazole using dialysis or gel filtration

• Verify purity using SDS-PAGE (>90% purity should be achievable)

Storage Recommendations:

• Store the purified protein in Tris/PBS-based buffer with 6% trehalose at pH 8.0

• Add glycerol to a final concentration of 50% for long-term storage

• Aliquot the protein solution to avoid repeated freeze-thaw cycles

• Store aliquots at -20°C or preferably -80°C

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

Repeated freeze-thaw cycles should be avoided as they can significantly impact protein stability and activity. For reconstitution, it is recommended to dissolve the lyophilized protein in deionized sterile water to a concentration of 0.1-1.0 mg/mL .

How can researchers optimize experimental designs for studying CbiN function in bacterial systems?

Optimizing experimental designs for studying CbiN function requires a systematic approach:

• Control Selection: Include both positive controls (known functional cobalt transporters) and negative controls (transport-deficient mutants) in all experiments.

• Growth Conditions: Use minimal media supplemented with defined cobalt concentrations to precisely control the experimental environment. Testing growth under cobalt-limited and cobalt-rich conditions can reveal functional aspects of the transport system.

• Genetic Manipulation Approaches:

  • Gene knockout studies using CRISPR-Cas9 or homologous recombination

  • Complementation assays with wild-type and mutant variants

  • Site-directed mutagenesis to identify critical residues

• Transport Assays: Implement isotope-labeled cobalt (⁵⁷Co or ⁶⁰Co) uptake assays to directly measure transport activity. Compare uptake rates between wild-type strains and cbiN mutants.

• Interaction Studies: Use bacterial two-hybrid systems or co-immunoprecipitation to identify interaction partners within the cobalt transport complex.

A methodologically sound experimental design would incorporate multiple approaches to establish causality between CbiN and cobalt transport function, while controlling for confounding variables such as growth rate differences and expression levels .

What methodology can be used to measure the interaction between recombinant CbiN and other components of the cobalt transport system?

To effectively measure interactions between recombinant CbiN and other components of the cobalt transport system, researchers should employ multiple complementary methodologies:

In vitro Approaches:

• Surface Plasmon Resonance (SPR): Immobilize purified His-tagged CbiN on a sensor chip and measure binding kinetics with other purified components (CbiM, CbiQ, CbiO).

• Isothermal Titration Calorimetry (ITC): Directly measure thermodynamic parameters of binding between CbiN and partner proteins or cobalt ions.

• Pull-down Assays: Use His-tagged CbiN as bait to identify interacting partners from bacterial lysates, followed by mass spectrometry identification.

In vivo Approaches:

• Bacterial Two-Hybrid System: Create fusion constructs of CbiN and potential interacting partners with split reporter proteins to detect interactions in a cellular context.

• FRET/BRET Analysis: Generate fluorescent protein fusions to measure proximity-based energy transfer between CbiN and other components in live cells.

• Cross-linking Studies: Use chemical cross-linkers with subsequent mass spectrometry to identify proximity relationships in the native membrane environment.

Data Analysis Protocol:

• Compare binding affinities across different experimental conditions

• Validate interactions using multiple methodologies

• Create interaction maps based on strength and specificity of observed interactions

• Correlate interaction data with functional transport assays

This multi-method approach allows for robust verification of true interactions while minimizing false positives that can occur with any single methodology.

How can machine learning approaches be applied to predict functional domains in CbiN and optimize protein engineering?

Machine learning (ML) approaches offer powerful tools for predicting functional domains in CbiN and optimizing protein engineering efforts:

Prediction of Functional Domains:

• Sequence-Based ML Models: Train convolutional neural networks on curated datasets of cobalt transport proteins to identify conserved motifs and functional residues in CbiN.

• Structure-Based Prediction: Apply ML algorithms to predict 3D structural features from sequence data, particularly focusing on transmembrane regions and metal-binding sites.

• Evolutionary Coupling Analysis: Use co-evolutionary information to predict residue pairs involved in structural contacts or functional interactions .

Optimization for Protein Engineering:

• Directed Evolution Guidance: ML models can analyze sequence-function relationships to guide mutagenesis efforts toward promising regions of sequence space.

• Stability Prediction: Employ algorithms that predict the impact of mutations on protein stability, helping researchers avoid destabilizing modifications.

• Expression Optimization: Use ML to predict optimal codon usage and expression conditions based on the amino acid sequence.

Recent advances in deep learning, as demonstrated in similar applications with Salmonella Typhimurium, show that these approaches can achieve high accuracy in predicting protein properties without prior exposure to experimental data on the specific protein .

What are the methodological approaches for studying the role of CbiN in Salmonella heidelberg pathogenesis and host interaction?

Studying CbiN's role in Salmonella heidelberg pathogenesis requires sophisticated methodological approaches that bridge molecular microbiology and infection biology:

In vitro Infection Models:

• Cell Culture Invasion Assays: Compare wild-type and cbiN-deficient S. heidelberg strains for their ability to invade epithelial cells (e.g., Caco-2, HT-29) and survive within macrophages (e.g., RAW264.7, THP-1).

• Competitive Index Assays: Co-infect cell cultures with wild-type and cbiN mutant strains, then calculate competitive indices to quantify relative fitness.

• Metal Restriction Studies: Simulate host nutritional immunity by using metal chelators (e.g., calprotectin) and assess bacterial survival.

In vivo Approaches:

• Animal Infection Models: Use established models (typically mouse or chicken) to compare colonization, persistence, and virulence of wild-type versus cbiN mutants.

• Organ Burden Analysis: Quantify bacterial loads in relevant organs (intestine, spleen, liver) at defined time points post-infection.

• Immune Response Profiling: Measure host cytokine production and immune cell recruitment in response to infection with different bacterial strains.

Molecular Mechanistic Studies:

• Gene Expression Analysis: Use RNA-seq to identify genes differentially expressed in response to cbiN deletion, particularly focusing on virulence factors.

• Metabolomics: Compare metabolic profiles between wild-type and mutant strains, focusing on B12-dependent pathways.

• Imaging Techniques: Use fluorescence microscopy with labeled antibodies against CbiN to track protein localization during infection .

These approaches should be implemented with appropriate controls, including complementation strains where the cbiN gene is reintroduced to confirm that observed phenotypes are specifically due to CbiN function rather than polar effects or secondary mutations.

How can researchers address challenges in protein stability and solubility when working with recombinant CbiN protein?

Membrane proteins like CbiN often present significant challenges in terms of stability and solubility. Here are methodological approaches to address these challenges:

Improving Protein Solubility:

• Fusion Tag Selection: Beyond the standard His-tag, consider fusion partners known to enhance solubility:

  • Maltose-binding protein (MBP)

  • Small ubiquitin-like modifier (SUMO)

  • Thioredoxin (TRX)

  • Glutathione S-transferase (GST)

• Expression Condition Optimization: Implement a Design of Experiments (DoE) approach to systematically test:

  • Induction temperature (typically lower temperatures of 16-25°C improve solubility)

  • Inducer concentration (lower IPTG concentrations of 0.1-0.5 mM)

  • Cell density at induction (mid-log phase, OD600 of 0.6-0.8)

  • Media composition (supplemented with osmolytes like sorbitol or betaine)

• Detergent Screening: Test a panel of detergents for extraction and purification:

Enhancing Protein Stability:

• Buffer Optimization: Screen various buffer conditions:

  • pH range (typically 7.0-8.5)

  • Salt concentration (150-500 mM NaCl)

  • Addition of stabilizing agents (glycerol 5-20%, trehalose 5-10%)

  • Presence of reducing agents (DTT, β-mercaptoethanol)

• Directed Mutagenesis: Introduce stability-enhancing mutations based on computational predictions or evolutionary conservation analysis.

• Co-expression with Partners: Express CbiN together with its natural binding partners (CbiM, CbiQ, CbiO) to form stable complexes.

• Thermal Shift Assays: Use differential scanning fluorimetry to identify conditions that maximize protein thermal stability .

By implementing these methodologies systematically, researchers can significantly improve both the yield and stability of recombinant CbiN protein, enabling more reliable functional and structural studies.

How does CbiN from Salmonella heidelberg compare to homologous proteins in other bacterial species?

A systematic comparative analysis of CbiN from Salmonella heidelberg with homologous proteins in other bacterial species reveals important evolutionary and functional insights:

Sequence Conservation Analysis:

CbiN proteins are relatively well conserved among enterobacteria, with specific regions showing higher conservation corresponding to functional domains. The N-terminal transmembrane region typically shows higher conservation than the cytoplasmic domains, reflecting evolutionary pressure to maintain membrane association and metal transport function.

Comparative Features of CbiN Proteins:

Phylogenetic Analysis:

Functional Conservation:

These comparative analyses provide important context for interpreting experimental results with S. heidelberg CbiN and can guide the design of functional studies to explore species-specific adaptations in cobalt transport.

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

Researchers frequently encounter specific challenges when working with recombinant CbiN. Here are methodological solutions for addressing these issues:

Challenge 1: Low Expression Levels

Challenge 2: Protein Insolubility

Challenge 3: Purification Difficulties

• Poor Binding to Affinity Resin:

  • Ensure the His-tag is accessible and not buried within the protein structure

  • Optimize binding buffer conditions (pH 7.5-8.5 is optimal for His-tag binding)

  • Try alternative affinity tags (Strep-tag II, FLAG tag)

  • Validate tag presence by Western blot before purification attempts

• Contaminant Co-purification:

  • Implement stringent washing steps with increasing imidazole (20-50 mM)

  • Add secondary purification steps (ion exchange, size exclusion chromatography)

  • Consider on-column cleavage of fusion tags

• Protein Aggregation During Purification:

  • Maintain detergent above critical micelle concentration throughout purification

  • Add glycerol (10-20%) to all buffers

  • Perform purification at 4°C

  • Consider purification in the presence of stabilizing ligands (cobalt ions)

Validation Methods:

• Size exclusion chromatography to assess oligomeric state and homogeneity

• Dynamic light scattering to evaluate sample monodispersity

• Circular dichroism to confirm secondary structure

• Thermal shift assays to assess protein stability under different buffer conditions

By systematically addressing these challenges with the described methodological approaches, researchers can significantly improve the yield and quality of recombinant CbiN protein preparations.

How can researchers validate the functional activity of purified recombinant CbiN protein?

Validating the functional activity of purified recombinant CbiN protein is essential to ensure that experimental observations reflect physiologically relevant phenomena. Here are methodological approaches for functional validation:

1. Cobalt Binding Assays:

• Isothermal Titration Calorimetry (ITC): Directly measure thermodynamic parameters of cobalt binding to purified CbiN.

  • Protocol: Titrate increasing concentrations of CoCl₂ into a solution of purified CbiN while measuring heat changes

  • Data analysis: Calculate dissociation constant (Kd), enthalpy (ΔH), and stoichiometry

• Equilibrium Dialysis: Determine binding affinity using radiolabeled cobalt (⁵⁷Co or ⁶⁰Co).

  • Protocol: Incubate purified CbiN with labeled cobalt, separate bound from free cobalt by dialysis

  • Quantification: Measure radioactivity in protein chamber versus buffer chamber

• Fluorescence Quenching: Exploit the intrinsic fluorescence of aromatic residues in CbiN.

  • Protocol: Monitor changes in tryptophan fluorescence upon cobalt binding

  • Analysis: Generate binding curves and calculate dissociation constants

2. Reconstitution into Liposomes:

• Preparation of Proteoliposomes:

  • Solubilize lipids (typically E. coli polar lipids) in detergent

  • Add purified CbiN at protein:lipid ratio of 1:100 to 1:1000

  • Remove detergent via dialysis or Bio-Beads

• Transport Assays:

  • Load proteoliposomes with buffer with or without potential energy sources

  • Add radiolabeled cobalt to external buffer

  • Measure accumulation of cobalt inside proteoliposomes over time

  • Compare rates with control liposomes lacking CbiN

3. Complementation Studies:

• Genetic System:

  • Create a cbiN deletion strain of S. heidelberg or E. coli

  • Verify growth defect under cobalt-limiting conditions

  • Transform with plasmid expressing recombinant CbiN

  • Measure restoration of growth as indicator of functional activity

• Analysis Parameters:

  • Growth rates in minimal media with defined cobalt concentrations

  • Vitamin B12 production levels

  • Cobalt accumulation in cells

4. Protein-Protein Interaction Assays:

Since CbiN functions as part of a complex, validating interactions with partner proteins provides evidence of proper folding and functionality.

• Pull-down Assays: Use His-tagged CbiN to capture native CbiM, CbiQ, and CbiO proteins from bacterial lysates

• Surface Plasmon Resonance: Measure binding kinetics between CbiN and other purified components

• Cross-linking Studies: Capture transient interactions in native-like environments

Validation Data Interpretation:

By implementing multiple validation assays, researchers can establish with confidence that their purified recombinant CbiN retains its native biological function .

What are the future research directions for understanding the role of CbiN in bacterial physiology and pathogenesis?

Future research on CbiN in Salmonella heidelberg and related bacteria should focus on several promising directions that will enhance our understanding of both basic bacterial physiology and potential applications in pathogen control:

Structural Biology Approaches:

• Determination of high-resolution CbiN structure using cryo-electron microscopy or X-ray crystallography

• Elucidation of the complete CbiMNQO complex architecture to understand the molecular mechanism of cobalt transport

• Investigation of conformational changes during the transport cycle using techniques like single-molecule FRET

Systems Biology Integration:

• Multi-omics profiling to understand how cobalt transport systems respond to changing environmental conditions

• Network analysis to identify regulatory interactions between cobalt transport and virulence factor expression

• Investigation of metal competition and prioritization mechanisms during infection

Host-Pathogen Interactions:

• Characterization of how host nutritional immunity affects CbiN function during infection

• Examination of potential recognition of CbiN by host immune receptors

• Exploration of CbiN's role in Salmonella survival within different host cell types and tissues

Antimicrobial Development:

• Evaluation of CbiN as a potential drug target, exploiting its essential role in cobalt acquisition

• Design of inhibitors that specifically target the CbiMNQO complex

• Development of attenuated vaccine strains with modified cobalt transport capabilities

Biotechnological Applications:

• Engineering CbiN for improved cobalt bioaccumulation in bioremediation applications

• Utilizing CbiN as a component in biosensors for environmental cobalt detection

• Exploring the potential of modified CbiN as a tool for selective metal delivery in synthetic biology applications

By pursuing these research directions with rigorous methodology and integrative approaches, researchers will gain deeper insights into the fundamental role of CbiN in bacterial physiology while potentially developing new strategies for controlling Salmonella infections and other applications in biotechnology and medicine.

What methodological advances are needed to address current limitations in CbiN research?

Several methodological advances would significantly enhance current research capabilities for studying CbiN and related cobalt transport proteins:

Advanced Imaging Techniques:

• Development of specific antibodies or nanobodies for super-resolution microscopy of native CbiN localization and dynamics

• Implementation of correlative light and electron microscopy (CLEM) approaches to visualize CbiN in cellular contexts

• Application of cryo-electron tomography to visualize the CbiMNQO complex in intact bacterial membranes

Improved Protein Production Methods:

• Optimization of membrane protein expression systems specifically tailored for small transmembrane proteins like CbiN

• Development of stabilized cell-free expression systems for direct production of functional CbiN

• Engineering of specialized detergents or nanodiscs for improved handling of purified CbiN

Enhanced Functional Assays:

• Development of high-throughput cobalt transport assays to enable large-scale screening

• Creation of specific fluorescent probes for real-time monitoring of cobalt transport

• Advancement of single-molecule techniques to study transport kinetics at the individual protein level

Computational Method Development:

• Improved algorithms for predicting membrane protein structures from limited experimental data

• Development of specialized force fields for molecular dynamics simulations of metal transport

• Integration of machine learning approaches to predict metal binding sites with higher accuracy

Genetic Tool Advancement:

• Development of inducible and tissue-specific gene expression systems for Salmonella

• Creation of reporter systems specifically designed to monitor metal transport in real-time

• Refinement of CRISPR-Cas9 techniques for precise genome editing in Salmonella heidelberg

These methodological advances would collectively address current limitations in studying CbiN and advance our understanding of bacterial cobalt transport mechanisms, with potential applications in antimicrobial development and biotechnology.