Recombinant Salmonella paratyphi C Cobalt transport protein CbiN (cbiN)

What is the basic structure of Recombinant Salmonella paratyphi C Cobalt transport protein CbiN?

Recombinant Salmonella paratyphi C Cobalt transport protein CbiN is a relatively small membrane protein with an amino acid sequence of MKKTLmLLAMVVALVILPFFINHGGEYGGSDGEAESQIQALAPQYKPWFQPLYEPASGEIESLLFTLQGSLGAAVIFYILGYCKGKQRRDDRA . The protein is encoded by the cbiN gene (locus tag SPC_1693 in S. paratyphi C strain RKS4594) with an expression region spanning positions 1-93 . Structurally, CbiN functions as a component of the Energy-coupling factor transporter system, specifically serving as the substrate-capture protein (S-component) that facilitates cobalt ion recognition and initial binding during the transport process. The hydrophobic regions in its sequence suggest multiple transmembrane domains characteristic of transport proteins involved in metal acquisition systems.

How does CbiN function in cobalt transport and what is its physiological role?

CbiN functions as a critical component of the cobalt transport system in Salmonella paratyphi C, acting as the substrate-specific component that recognizes and binds cobalt ions with high specificity. Physiologically, CbiN works in concert with other ECF transporter components to facilitate the uptake of cobalt, an essential micronutrient required for multiple biological processes, particularly vitamin B12 biosynthesis. In S. paratyphi C, efficient cobalt acquisition through CbiN and related transport systems likely contributes to bacterial survival in cobalt-limited host environments during infection. The protein's transmembrane topology enables it to capture extracellular cobalt ions and transfer them to other components of the transport machinery for subsequent translocation across the bacterial membrane, making it crucial for maintaining proper cellular cobalt homeostasis.

What are the optimal storage conditions for Recombinant Salmonella paratyphi C CbiN protein?

For optimal stability and activity maintenance, Recombinant Salmonella paratyphi C CbiN protein should be stored at -20°C, and for extended storage, it is recommended to conserve the protein at -20°C or -80°C . The commercially available recombinant protein is typically supplied in a Tris-based buffer containing 50% glycerol, which has been optimized for this specific protein . For working solutions, aliquots can be stored at 4°C for up to one week, but repeated freezing and thawing cycles should be avoided as they can compromise protein integrity and function . When reconstituting lyophilized forms of the protein, it is advisable to use deionized sterile water to achieve a final concentration of 0.1-1.0 mg/mL, with a recommended final glycerol concentration of 50% for long-term storage aliquots .

What is the genetic context of the cbiN gene in S. paratyphi C?

The cbiN gene in Salmonella paratyphi C (strain RKS4594) is identified by the locus tag SPC_1693 and encodes the Cobalt transport protein CbiN . This gene is part of the cobalt uptake and vitamin B12 biosynthesis gene cluster, which is conserved across various Salmonella species but may show sequence variations reflecting evolutionary adaptations. Genomic analyses of S. paratyphi C RKS4594 reveal that the strain contains a chromosome of 4,833,080 bp and a plasmid of 55,414 bp, with 4,640 intact coding sequences (4,578 in the chromosome and 62 in the plasmid) . The cbiN gene is one of the chromosomally encoded genes essential for the bacterium's metabolic functions. Interestingly, S. paratyphi C shares 4,346 genes with S. choleraesuis (a primarily swine pathogen) but only 4,008 genes with S. typhi (another human-adapted typhoid agent), indicating the closer evolutionary relationship between S. paratyphi C and S. choleraesuis .

How can recombinant CbiN protein be used in structural biology studies?

For structural biology studies of recombinant Salmonella paratyphi C CbiN protein, researchers should consider multiple complementary approaches. X-ray crystallography requires production of highly pure (>95%) protein samples through techniques like affinity chromatography followed by size exclusion chromatography, with careful attention to detergent selection for membrane protein stabilization. Crystallization screening should incorporate various precipitants, detergents, and additives at different temperatures. NMR spectroscopy offers an alternative approach, particularly suited for studying CbiN's dynamic interactions with cobalt ions, requiring isotopically labeled protein (typically with 15N and 13C). For both methods, protein engineering strategies may be necessary, such as creating fusion constructs or removing flexible regions to enhance crystallization propensity. Cryo-electron microscopy represents another powerful option, especially for visualizing CbiN in complex with other ECF transporter components, providing insights into the assembled transport machinery. The integration of these structural approaches with computational modeling can provide comprehensive insights into CbiN's three-dimensional architecture and mechanism of action.

What challenges are associated with expression and purification of functional CbiN protein?

Expression and purification of functional CbiN protein present several significant challenges due to its membrane-associated nature. Heterologous expression systems often struggle with proper membrane insertion and folding of CbiN, potentially leading to inclusion body formation or misfolded protein. When selecting an expression system, E. coli-based systems with specialized modifications for membrane protein expression (such as C41/C43 strains) may provide better results than standard strains. Alternatively, yeast-based systems have demonstrated success for certain CbiN preparations . For purification strategies, researchers must carefully optimize detergent selection, as inappropriate detergents can destabilize the protein's native conformation. The choice between harsh (e.g., SDS) and mild (e.g., DDM, CHAPS) detergents requires empirical testing with functional assays to verify that the purified protein retains its cobalt-binding capacity. Additionally, researchers should implement rigorous quality control testing, including SDS-PAGE, Western blotting, and circular dichroism, to confirm the structural integrity of the purified protein.

How can isothermal titration calorimetry be optimized for studying CbiN-cobalt interactions?

Optimizing isothermal titration calorimetry (ITC) for studying CbiN-cobalt interactions requires careful consideration of several experimental parameters. Buffer composition is critical—phosphate buffers should be avoided due to potential cobalt precipitation, while HEPES or Tris buffers at pH 7.4-8.0 generally provide optimal conditions. All buffers must be thoroughly degassed to prevent artifacts from air bubbles during titration. Protein concentration typically needs to be between 10-50 μM in the cell, while cobalt salt (usually CoCl2) concentration in the syringe should be 10-15 times higher to ensure saturation. Temperature selection is crucial, with most CbiN-cobalt binding studies performed at 25°C to balance signal quality with protein stability. Control experiments must include buffer-into-buffer, cobalt-into-buffer, and buffer-into-protein titrations to account for dilution effects and non-specific interactions. Data analysis should employ multiple binding models (e.g., one-site, sequential binding sites) to determine the most appropriate fit for the interaction. Researchers should also consider the potential influence of detergents or lipids if CbiN is studied in a membrane-mimetic environment, as these can affect both the thermodynamic parameters and the baseline stability during measurement.

What are the recommended methods for functional characterization of CbiN transport activity?

Functional characterization of CbiN transport activity requires multiple complementary approaches. Radioactive 57Co uptake assays provide the most direct measurement of transport activity, where bacterial cells expressing CbiN (ideally in a cbiN-knockout background) are incubated with 57CoCl2, followed by washing and gamma counting to quantify internalized cobalt. Membrane vesicle preparations offer an alternative system that maintains the native membrane environment while allowing greater experimental control over buffer conditions. Non-radioactive approaches include inductively coupled plasma mass spectrometry (ICP-MS) to measure intracellular cobalt concentrations with high sensitivity, or fluorescent cobalt-binding dyes that exhibit spectral shifts upon cobalt binding. Competition assays using other divalent metals (Ni2+, Zn2+, Fe2+) can determine transport specificity. For in vivo relevance, growth complementation assays in cobalt-limited media with wild-type, ΔcbiN mutant, and complemented strains can demonstrate the physiological importance of CbiN-mediated cobalt transport. Growth curves should be performed under both standard and cobalt-limited conditions, with vitamin B12 supplementation as an additional control to bypass the cobalt requirement for strains unable to acquire the metal efficiently.

What protein-protein interaction methods are suitable for studying CbiN interactions with other ECF transporter components?

Several protein-protein interaction methods are particularly suitable for studying CbiN's interactions with other ECF transporter components. Bacterial two-hybrid systems, especially those optimized for membrane proteins like BACTH (Bacterial Adenylate Cyclase Two-Hybrid), can detect interactions in a cellular environment while maintaining the membrane context. For in vitro approaches, co-immunoprecipitation using antibodies against CbiN or epitope-tagged versions can identify interaction partners, though careful optimization of detergent conditions is crucial to preserve native interactions. Surface plasmon resonance (SPR) offers quantitative binding kinetics when one component is immobilized on a sensor chip, with the advantage of real-time monitoring of association and dissociation phases. Crosslinking mass spectrometry (XL-MS) can identify specific interaction interfaces by capturing transient contacts through chemical crosslinkers, followed by proteolytic digestion and mass spectrometric analysis. Förster resonance energy transfer (FRET) approaches using fluorescently labeled components can monitor interactions in native-like membrane environments, including reconstituted proteoliposomes. For structural characterization of the entire complex, single-particle cryo-electron microscopy has emerged as the method of choice, potentially revealing the assembled architecture of CbiN with its partner proteins in the ECF transporter complex.

How can researchers effectively use site-directed mutagenesis to identify critical residues in CbiN function?

Effective site-directed mutagenesis to identify critical residues in CbiN function requires a systematic approach beginning with computational analysis. Researchers should first use sequence alignments across multiple Salmonella species to identify conserved residues, combined with predictive algorithms for transmembrane topology and secondary structure. Homology modeling, if possible based on related proteins with known structures, can guide the selection of residues likely involved in cobalt coordination (histidine, cysteine, aspartate, glutamate residues) or protein-protein interactions. When designing mutations, conservative substitutions (e.g., aspartate to glutamate) can distinguish between residues important for structural integrity versus those specifically involved in function. Charge reversal mutations (e.g., positive to negative) can be particularly informative for residues involved in electrostatic interactions. Alanine scanning of predicted functional regions provides a systematic approach to identify critical segments. For experimental validation, mutant constructs should be assessed through multiple functional assays, including cobalt transport activity (using radioactive 57Co), protein expression levels (Western blotting), membrane localization (fractionation studies), and protein folding (circular dichroism). Biological relevance should be confirmed by complementation studies in ΔcbiN bacterial strains grown under cobalt-limited conditions. Compilation of mutagenesis results into a comprehensive structure-function map can reveal mechanistic insights into how CbiN recognizes and transports cobalt ions.

What approaches are recommended for analyzing CbiN expression during Salmonella infection models?

Analyzing CbiN expression during Salmonella infection requires integration of multiple technical approaches to overcome the challenges of low-abundance membrane protein detection in complex biological samples. Quantitative PCR (qPCR) targeting the cbiN transcript provides high sensitivity for monitoring gene expression across different infection stages, but should include careful validation of reference genes stable under infection conditions. For direct protein detection, targeted proteomics approaches like selected reaction monitoring (SRM) or parallel reaction monitoring (PRM) offer superior sensitivity compared to standard Western blotting, allowing quantification of CbiN from infected tissue or cell lysates after appropriate extraction. Transcriptional reporter fusions (cbiN promoter driving luciferase or fluorescent protein expression) enable non-invasive monitoring of gene expression dynamics in real-time during infection, particularly valuable for in vivo imaging in animal models. For spatial information, RNAscope in situ hybridization can visualize cbiN mRNA in tissue sections while maintaining anatomical context. Infection models should include multiple time points to capture expression dynamics, and researchers should compare expression across different host niches (intestinal lumen, Peyer's patches, liver, spleen, gallbladder) to understand spatial regulation. Comparison of expression between wild-type S. paratyphi C and strains with mutations in known regulators can help delineate the regulatory networks controlling CbiN expression during infection.

How should researchers interpret differences in cobalt binding affinities between CbiN proteins from different Salmonella strains?

When interpreting differences in cobalt binding affinities between CbiN proteins from different Salmonella strains, researchers must consider multiple factors that influence experimental outcomes and biological significance. Methodological considerations are paramount, as binding parameters derived from different techniques (ITC, SPR, fluorescence spectroscopy) may not be directly comparable due to differences in experimental conditions. Affinity measurements should always be performed under identical buffer conditions (same pH, ionic strength, temperature) across different CbiN variants to enable valid comparisons. For proper interpretation, researchers should correlate binding affinity differences with specific amino acid variations between the proteins, focusing particularly on residues in predicted metal-binding sites. Statistical analysis must be rigorous, with multiple independent protein preparations and technical replicates to establish the significance of observed differences. Beyond technical considerations, interpretation should address the ecological and pathogenic context—differences in cobalt affinity may reflect adaptation to distinct host environments with varying cobalt availability. Researchers should examine whether higher or lower affinity correlates with the pathogen's preferred niche (human-adapted S. paratyphi C versus predominantly animal-adapted strains). The table below summarizes typical ranges of cobalt binding parameters observed for CbiN proteins:

What comparative genomic approaches can reveal the evolution of CbiN and related cobalt transport systems?

Comparative genomic approaches for studying CbiN evolution should integrate multiple analytical strategies. Whole-genome phylogenetic analysis establishes the evolutionary relationships between Salmonella strains, providing context for interpreting CbiN-specific findings . Researchers should perform detailed sequence analysis of the cbiN gene and its flanking regions across multiple Salmonella species to identify conservation patterns, insertions/deletions, and potential recombination events. Synteny analysis examining the organization of cobalt transport genes can reveal operon rearrangements or acquisition of additional components during evolution. Selection pressure analysis using dN/dS ratios can identify regions under purifying or positive selection, potentially correlating with functional importance or adaptive changes. Comparison between S. paratyphi C and S. choleraesuis is particularly informative given their close relationship but different host preferences . Researchers should examine whether genomic islands containing cobalt transport genes show evidence of horizontal gene transfer, suggesting adaptive acquisition of enhanced cobalt uptake capabilities. Integration of these genomic findings with structural models and experimental functional data provides the most comprehensive understanding of how CbiN has evolved across Salmonella lineages in response to varying selective pressures in different host environments.

How can researchers differentiate between direct and indirect effects when studying CbiN knockout phenotypes?

Differentiating between direct and indirect effects when studying CbiN knockout phenotypes requires a comprehensive experimental approach with appropriate controls. Complementation studies represent the gold standard—phenotypes directly caused by CbiN loss should be fully restored by expressing the wild-type protein from a plasmid. Dose-dependent complementation can provide additional evidence for direct effects, where increasing expression levels correlate with progressive phenotype restoration. Time-course experiments examining when phenotypes first appear after CbiN deletion can help distinguish primary (immediate) from secondary (delayed) effects. Metabolomic analysis comparing wild-type, ΔcbiN, and complemented strains can reveal specific metabolic pathways affected by CbiN deletion, with particular attention to cobalt-dependent processes versus broadly dysregulated metabolism. Gene expression profiling can identify compensatory responses to CbiN deletion that may mask or amplify the primary phenotype. For in vivo infection studies, researchers should complement standard virulence assays with targeted experiments addressing specific CbiN functions, such as measuring in vivo cobalt acquisition or vitamin B12-dependent enzyme activities. Chemical complementation through cobalt supplementation or provision of downstream metabolites can determine whether phenotypes result specifically from impaired cobalt uptake. The use of double mutants (ΔcbiN combined with deletion of putative compensatory transporters) can reveal redundant systems that might mask phenotypes in single CbiN knockouts.

What are the key considerations for interpreting structural data of CbiN in different membrane-mimetic environments?

When interpreting structural data of CbiN obtained in different membrane-mimetic environments, researchers must carefully consider how the experimental conditions influence the observed protein conformation and functional properties. Detergent micelles, commonly used for solubilizing membrane proteins, may not accurately replicate the lateral pressure and curvature of native membranes, potentially distorting transmembrane regions or altering oligomeric states. Lipid composition significantly impacts membrane protein structure—data obtained in simplified lipid systems may not capture native interactions with specific lipids that modulate protein function in vivo. When comparing structures determined by different methods (X-ray crystallography, NMR, cryo-EM), researchers should account for method-specific artifacts: crystal packing forces in X-ray structures, averaging effects in NMR, or preferred orientations in cryo-EM. Resolution limitations must be clearly acknowledged, particularly for regions with poor electron density or multiple conformations. Validation metrics appropriate for membrane proteins should be rigorously applied, including Ramachandran statistics, side-chain rotamer analysis, and assessment of transmembrane packing. Functional validation is essential—structural interpretations should be corroborated by functional data showing that the observed conformation is catalytically competent. Molecular dynamics simulations can bridge experimental structures with dynamic behavior, revealing transitions between states not captured in static experimental structures. The integration of multiple structural approaches in different membrane environments provides the most robust picture of CbiN's native conformation and mechanistic details.

How might single-molecule approaches advance our understanding of CbiN transport mechanisms?

Single-molecule approaches offer unprecedented potential to elucidate the dynamic aspects of CbiN transport mechanisms that remain inaccessible to ensemble measurements. Single-molecule FRET (smFRET) can track conformational changes in CbiN during the transport cycle by strategically placing fluorophore pairs at key positions and monitoring distance changes during cobalt binding and release events. This approach could reveal intermediates and conformational dynamics essential for the transport mechanism. Single-molecule force spectroscopy techniques, including atomic force microscopy (AFM) and optical tweezers, can measure the energetics and kinetics of CbiN-cobalt interactions by applying controlled forces to individual protein molecules. These measurements provide insights into the energy landscape governing the transport process. For visualizing CbiN in native-like environments, high-speed atomic force microscopy (HS-AFM) can capture real-time structural dynamics of the protein in lipid bilayers, potentially revealing how CbiN interacts with other ECF transporter components. Complementary to these approaches, nanopore-based single-molecule sensing can detect individual cobalt ion translocation events through reconstituted CbiN channels, providing direct evidence of transport activity at unprecedented temporal resolution. The integration of these single-molecule techniques with computational approaches like molecular dynamics simulations will be particularly powerful for developing a comprehensive mechanistic model of CbiN-mediated cobalt transport.

What potential applications exist for CbiN inhibitors in antimicrobial development?

The development of CbiN inhibitors represents a promising avenue for novel antimicrobial strategies against S. paratyphi C and potentially other pathogenic bacteria reliant on cobalt acquisition. Structure-based drug design approaches can leverage CbiN structural information to identify compounds that competitively bind the cobalt-binding site or allosterically inhibit conformational changes required for transport. High-throughput screening of chemical libraries using cobalt uptake assays can identify lead compounds with CbiN inhibitory activity. Metal chelators modified to target bacterial surfaces represent another strategy, sequestering cobalt before it can interact with CbiN. For antimicrobial development, researchers must carefully assess the therapeutic window between inhibiting bacterial CbiN and affecting host cobalt-dependent processes. CbiN inhibitors could be particularly valuable for combination therapy, potentially sensitizing bacteria to existing antibiotics by restricting nutrient acquisition. Target validation studies should demonstrate that chemical inhibition of CbiN replicates genetic deletion phenotypes in infection models. Importantly, researchers should evaluate the potential for resistance development through mutations in CbiN or upregulation of alternative cobalt acquisition systems. Developing CbiN inhibitors with activity against multiple bacterial species would maximize their therapeutic potential, though this requires addressing structural variations in CbiN homologs. The specificity of CbiN for bacterial cobalt acquisition pathways absent in humans makes it an attractive target for antimicrobial development with potentially minimal off-target effects.