Recombinant Salmonella newport Cobalt transport protein CbiN (cbiN)

What is the Cobalt transport protein CbiN in Salmonella newport?

Cobalt transport protein CbiN from Salmonella newport (strain SL254) functions as a component of the energy-coupling factor (ECF) transporter system, specifically as a substrate-capture protein. It plays a crucial role in cobalt ion uptake, which is essential for bacterial metabolism and survival. The protein is encoded by the cbiN gene (SNSL254_A2198 locus) and consists of 93 amino acids in its full-length form. CbiN is alternatively referred to as "Energy-coupling factor transporter probable substrate-capture protein CbiN" or "ECF transporter S component CbiN" in scientific literature .

The amino acid sequence of this protein is:
MKKTLMLLAMVVALVILPFFINHGGEYGGSSDGEAERQIQAIAPQYKPWFQPLYEPASGEI ESLLFTLQGSLGAAVIFYILGYCKGKQRRDDRA

This small membrane protein is part of a larger complex involved in nutrient acquisition, which is particularly important for understanding bacterial pathogenicity and developing potential antimicrobial strategies.

How is the recombinant CbiN protein typically produced for research applications?

The recombinant Salmonella newport Cobalt transport protein CbiN is typically produced using heterologous expression systems. While the specific expression system can vary, common approaches include using E. coli strains optimized for recombinant protein expression. The process generally involves:

• Gene synthesis or cloning of the cbiN gene (expression region 1-93) into an appropriate expression vector

• Transformation into a suitable expression host

• Induction of protein expression under controlled conditions

• Cell lysis and protein extraction

• Purification using affinity chromatography (tag-dependent)

• Buffer exchange into a storage-stable formulation

The final purified product is typically provided in a Tris-based buffer containing 50% glycerol for stability . The specific tag used for purification may vary depending on the production process, as indicated in the product specifications. For long-term storage, the protein should be kept at -20°C or -80°C, with working aliquots maintained at 4°C for up to one week to avoid degradation from repeated freeze-thaw cycles .

What are the optimal storage conditions for preserving CbiN protein activity?

To maintain the structural integrity and functional activity of the recombinant Salmonella newport CbiN protein, specific storage conditions must be observed:

• Long-term storage: The protein should be stored at -20°C for regular storage, or -80°C for extended preservation periods

• Working solution: Aliquots intended for immediate use should be kept at 4°C and used within one week

• Buffer composition: The optimal storage formulation consists of a Tris-based buffer with 50% glycerol, which helps prevent protein denaturation

• Freeze-thaw cycles: Repeated freezing and thawing should be avoided as this can lead to protein degradation and loss of activity

• Aliquoting: Upon receipt, the protein should be divided into small working aliquots to minimize the number of freeze-thaw cycles

These storage recommendations ensure maximum stability and activity retention of the recombinant protein for research applications, particularly for functional assays and structural studies that require the protein to maintain its native conformation.

What analytical methods are most effective for confirming the purity and identity of recombinant CbiN protein?

Several complementary analytical methods are recommended for comprehensive characterization of recombinant Salmonella newport CbiN protein:

• SDS-PAGE: For assessing protein purity and approximate molecular weight (expected around 10-11 kDa for the native protein, with variations based on any fusion tags)

• Western blotting: Using anti-CbiN or anti-tag antibodies to confirm protein identity

• Mass spectrometry:

  • MALDI-TOF for accurate molecular weight determination

  • LC-MS/MS for peptide mapping and sequence confirmation of the 93-amino acid sequence

• Circular dichroism (CD) spectroscopy: To verify proper protein folding and secondary structure

• Dynamic light scattering (DLS): To evaluate protein homogeneity and detect potential aggregation

• N-terminal sequencing: To confirm the correct protein sequence beginning and processing

When analyzing results, researchers should compare the experimental data with the expected properties of the full-length protein, including its documented amino acid sequence (MKKTLMLLAMVVALVILPFFINHGGEYGGSSDGEAERQIQAIAPQYKPWFQPLYEPASGEI ESLLFTLQGSLGAAVIFYILGYCKGKQRRDDRA) . Any fusion tags or modifications introduced during recombinant production should be accounted for in the analysis.

How can structural studies of CbiN contribute to understanding cobalt transport mechanisms in Salmonella?

Structural studies of the Salmonella newport CbiN protein can provide crucial insights into the molecular mechanisms of cobalt transport through several research approaches:

• X-ray crystallography and cryo-EM analysis: These techniques can reveal the three-dimensional structure of CbiN, both individually and as part of the ECF transporter complex. Particular attention should be paid to:

  • Potential metal-binding sites

  • Transmembrane helical arrangements (predicted from the hydrophobic regions in the sequence)

  • Interaction interfaces with other components of the ECF transport system

• Molecular dynamics simulations: Using the resolved structure to investigate:

  • Conformational changes during cobalt binding and transport

  • Functional implications of the membrane-spanning regions

  • Energy coupling mechanisms with other ECF components

• Structure-function relationship studies: Comparing CbiN structural elements with homologous proteins from other bacteria can elucidate:

  • Conserved domains essential for cobalt recognition

  • Species-specific adaptations that may relate to pathogenicity

• Mutagenesis approaches: Targeted mutations of key residues identified in structural studies can:

  • Verify the importance of specific amino acids in cobalt binding

  • Elucidate the functional significance of the hydrophobic transmembrane segments

  • Identify critical interaction sites with other ECF transporter components

Understanding the structural basis of CbiN function can provide targets for developing novel antimicrobials that disrupt cobalt acquisition, which is essential for bacterial survival and virulence expression.

What evolutionary insights can be gained from comparative genomic studies of the cbiN gene across Salmonella Newport lineages?

Comparative genomic analysis of the cbiN gene across Salmonella Newport lineages can reveal important evolutionary patterns and functional adaptations:

These evolutionary insights can help researchers understand how nutrient acquisition systems have adapted throughout Salmonella evolution and may contribute to lineage-specific pathogenicity traits.

How does the function of CbiN relate to antimicrobial resistance mechanisms in Salmonella Newport strains?

The relationship between CbiN function and antimicrobial resistance in Salmonella Newport involves several interconnected pathways and mechanisms:

• Metabolic dependence: Cobalt is an essential cofactor for various enzymes in Salmonella metabolism:

  • Disruption of cobalt uptake through CbiN could potentially sensitize bacteria to certain antimicrobials by compromising metabolic resilience

  • Conversely, enhanced cobalt acquisition might support metabolic adaptations that contribute to resistance phenotypes

• Co-selection of resistance determinants: In MDR-AmpC Salmonella Newport strains (resistant to ≥9 antimicrobials including extended-spectrum cephalosporins), resistance genes are often co-located on mobile genetic elements:

  • Research should investigate whether cobalt transporter genes like cbiN are genetically linked to resistance determinants such as blaCMY genes or class 1 integrons

  • Analysis of horizontal gene transfer patterns may reveal if cbiN variants are co-transferred with resistance elements

• Stress response coordination: Cobalt homeostasis and antimicrobial resistance mechanisms may share regulatory pathways:

  • CbiN expression patterns should be analyzed in the context of antimicrobial exposure

  • Potential regulatory crossover between metal transport and resistance mechanisms warrants investigation

• Structural analysis for drug development: CbiN protein structure can be leveraged for antimicrobial development:

  • As cobalt acquisition is essential for bacterial survival, CbiN inhibitors could represent novel antimicrobial agents

  • Such inhibitors might be particularly effective against MDR strains where traditional antibiotic targets are compromised

• Experimental design for testing CbiN-resistance relationships:

  • Generate cbiN knockout mutants in both susceptible and MDR-AmpC backgrounds

  • Compare antimicrobial susceptibility profiles between wild-type and ΔcbiN strains

  • Evaluate the impact of exogenous cobalt supplementation on antimicrobial efficacy

This research direction could potentially identify novel therapeutic strategies against antibiotic-resistant Salmonella Newport strains, which have been identified in both human and animal hosts .

What methodological approaches best characterize the interaction between CbiN and other components of the ECF transporter complex?

To effectively characterize the interactions between CbiN and other components of the Energy-Coupling Factor (ECF) transporter complex, researchers should employ a multi-faceted approach:

• Protein-Protein Interaction (PPI) Analysis:

  • Co-immunoprecipitation (Co-IP): Using antibodies against CbiN or other ECF components to pull down protein complexes

  • Bacterial two-hybrid (B2H) assays: For identifying direct protein-protein interactions in a bacterial cellular context

  • Surface plasmon resonance (SPR): For measuring binding kinetics and affinity constants between purified components

  • Isothermal titration calorimetry (ITC): To determine thermodynamic parameters of binding interactions

• Structural Biology Approaches:

  • Crosslinking mass spectrometry: To identify proximity relationships between proteins within the complex

  • Cryo-electron microscopy: For visualization of the entire ECF complex architecture

  • X-ray crystallography: For atomic-level resolution of interaction interfaces

  • NMR spectroscopy: Particularly useful for mapping dynamic interactions

• Functional Reconstitution:

  • Liposome reconstitution assays: To measure transport activity of purified components in artificial membrane systems

  • Complementation studies: Using various combinations of ECF components to restore function in knockout strains

• Genetic and Mutagenesis Approaches:

  • Site-directed mutagenesis: To identify critical residues at interaction interfaces

  • Suppressor mutation analysis: To identify compensatory mutations that restore function

  • Domain swapping experiments: To determine the specificity determinants in CbiN

• In silico Methods:

  • Molecular docking: To predict interaction modes between CbiN and other ECF components

  • Molecular dynamics simulations: To study the dynamic behavior of the complex

  • Evolutionary coupling analysis: To identify co-evolving residues that may participate in protein-protein interfaces

This comprehensive methodological toolkit allows researchers to build a detailed understanding of how CbiN functions within the larger ECF transporter machinery, providing insights into both the structural organization and the mechanistic details of cobalt transport in Salmonella newport.

What are the key considerations for designing experiments to evaluate CbiN function in cobalt transport?

When designing experiments to assess the function of Salmonella newport CbiN in cobalt transport, researchers should consider several critical elements:

• Genetic System Development:

  • Create precise cbiN deletion mutants using methods like lambda Red recombination

  • Develop complementation systems with wild-type and mutant variants of cbiN

  • Consider conditional expression systems to control CbiN levels

  • Establish fluorescent/luminescent reporter systems linked to cobalt-responsive promoters

• Transport Assay Design:

  • Direct measurement approaches:

    • Use radioactive 60Co or ICP-MS to quantify intracellular cobalt accumulation

    • Develop cobalt-specific fluorescent probes for real-time imaging

    • Compare transport kinetics in wild-type vs. ΔcbiN strains

  • Indirect functional assessments:

    • Measure growth in cobalt-limited media

    • Assess activity of cobalt-dependent enzymes

• Experimental Controls:

  • Include positive controls with known cobalt transporters from other systems

  • Establish negative controls using structurally similar but non-transported metals

  • Use appropriate metal chelators to create defined metal-limited conditions

  • Control for potential compensatory mechanisms by analyzing expression of other transporters

• Environmental Variables:

  • Test function across relevant pH ranges

  • Evaluate temperature effects on transport efficiency

  • Assess the impact of competing divalent cations

  • Consider the influence of oxygen availability on transport function

• Data Collection Parameters:

  • Establish appropriate time points for transport kinetics

  • Determine optimal cell density for assays

  • Select appropriate cobalt concentrations spanning physiological ranges

  • Develop methods to distinguish membrane-bound vs. internalized cobalt

• Analysis Framework:

  • Calculate transport kinetic parameters (Km, Vmax)

  • Use appropriate statistical methods to evaluate significance

  • Develop mathematical models to account for complex transport dynamics

  • Consider systems biology approaches to understand the broader metabolic context

These experimental design considerations will help ensure robust and interpretable data regarding CbiN's specific role in cobalt transport within Salmonella newport.

How can researchers effectively apply FAIR data principles to CbiN research?

To effectively apply FAIR (Findable, Accessible, Interoperable, Reusable) data principles to Salmonella newport CbiN research, investigators should implement the following strategies:

• Findability Enhancements:

  • Register all datasets with persistent identifiers (DOIs)

  • Develop rich metadata specifically tailored to transport protein research

  • Include standardized keywords for CbiN, ECF transporters, and Salmonella newport

  • Deposit data in specialized repositories like UniProt (for protein sequences) and PDB (for structures)

• Accessibility Implementation:

  • Ensure open access to published datasets when possible

  • Provide clear documentation on how to access protected data

  • Include machine-readable metadata alongside human-readable descriptions

  • Maintain standardized access protocols across different datasets

• Interoperability Strategies:

  • Use controlled vocabularies specific to membrane transport proteins

  • Adopt standardized formats for protein characterization data

  • Structure experimental datasets according to established ontologies

  • Implement cross-reference linking between related datasets

• Reusability Best Practices:

  • Provide detailed experimental protocols with each dataset

  • Document all data processing steps transparently

  • Include positive and negative controls in experimental data

  • Specify detailed sample preparation conditions

• Practical Implementation Tools:

  • Design experiment-specific data templates that incorporate FAIR principles

  • Develop standardized formats for reporting CbiN functional assay results

  • Create machine-readable descriptions of experimental workflows

  • Establish quality control metrics specific to transport protein research

By implementing these FAIR data principles throughout the research lifecycle rather than retrospectively, CbiN researchers can enhance collaboration, improve reproducibility, and accelerate scientific progress in understanding this important transport protein .

What are the most significant technical challenges in expressing and purifying functional CbiN protein?

Expressing and purifying functional Salmonella newport CbiN protein presents several significant technical challenges that researchers must address:

• Membrane Protein Expression Barriers:

  • Toxicity issues: Overexpression of membrane proteins like CbiN often leads to cellular toxicity and growth inhibition

  • Inclusion body formation: Hydrophobic regions tend to aggregate during expression

  • Proper insertion: Ensuring correct membrane insertion requires specialized expression systems

  • Expression level optimization: Finding conditions that balance yield with proper folding

• Solubilization and Extraction Challenges:

  • Detergent selection: Identifying detergents that efficiently extract CbiN while maintaining its native structure

  • Lipid requirements: Determining if specific lipids are needed for stability and function

  • Extraction efficiency: Optimizing conditions to maximize recovery from membrane fractions

  • Native state preservation: Ensuring extraction methods don't disrupt critical structural elements

• Purification Complexities:

  • Tag interference: Finding tag positions that don't interfere with membrane topology or function

  • Purification strategy: Developing multi-step protocols that maintain protein stability

  • Contaminant removal: Separating CbiN from other membrane proteins with similar properties

  • Detergent exchange: Optimizing conditions for detergent switching during purification

• Functional Assessment Difficulties:

  • Activity assays: Developing reliable methods to verify that purified CbiN retains cobalt-binding activity

  • Reconstitution requirements: Determining if other ECF components are needed for function

  • Orientation control: Ensuring correct orientation when reconstituting into liposomes

  • Metal contamination: Preventing contamination with trace metals that could affect functional assays

• Stability Challenges:

  • Long-term storage: Establishing conditions that preserve functionality during storage

  • Aggregation prevention: Minimizing protein aggregation during concentration steps

  • Temperature sensitivity: Determining optimal temperature ranges for handling

  • Buffer optimization: Identifying buffer components that enhance stability

Methodological approaches to address these challenges include:

• Using specialized expression systems designed for membrane proteins (e.g., C41/C43 E. coli strains)

• Exploring fusion partners known to enhance membrane protein expression

• Implementing systematic detergent screening

• Developing fluorescence-based assays for rapid functional assessment

• Utilizing nanodiscs or amphipols for stabilization

These technical considerations are critical for obtaining high-quality recombinant CbiN suitable for downstream structural and functional studies.

How can researchers integrate genomic, structural, and functional data to comprehensively understand CbiN's role in Salmonella pathogenesis?

Integrating multiple data types to understand CbiN's role in Salmonella pathogenesis requires a systematic multi-omics approach:

• Genomic Data Integration:

  • Compare cbiN sequences across Salmonella Newport lineages to identify variants associated with enhanced virulence

  • Analyze cbiN genomic context to identify co-evolving genes within the ECF transporter operon

  • Examine synteny with genes involved in virulence and antimicrobial resistance

  • Apply phylogenetic analysis to correlate cbiN variants with pathogenic potential across Salmonella strains

• Transcriptomic Correlation:

  • Profile cbiN expression under infection-relevant conditions

  • Identify co-expressed genes through RNA-seq analysis

  • Determine if cbiN expression correlates with virulence gene expression

  • Map the regulatory networks controlling cbiN expression during infection

• Structural Biology Connections:

  • Relate structural features of CbiN to its functional capacity in cobalt transport

  • Identify potential binding sites for inhibitors through structural analysis

  • Connect structural variations with functional differences between lineages

  • Use structural information to predict protein-protein interactions with host factors

• Functional Validation Framework:

  • Develop infection models to test the impact of cbiN mutations on virulence

  • Assess competitive fitness of wild-type vs. ΔcbiN strains in vivo

  • Measure tissue-specific requirements for cobalt acquisition during infection

  • Evaluate host nutritional immunity responses targeting cobalt availability

• Computational Integration Methods:

  • Apply machine learning to identify patterns across multi-omics datasets

  • Develop predictive models of CbiN contribution to virulence

  • Use network analysis to position CbiN within Salmonella pathogenicity mechanisms

  • Implement systems biology approaches to quantify the impact of cobalt limitation

• Data Visualization Strategies:

  • Create interactive maps connecting genomic variants to structural features and functional outcomes

  • Develop pathway visualizations incorporating CbiN within the context of virulence mechanisms

  • Implement comparative visualization tools to analyze differences across Salmonella lineages

  • Design temporal visualizations showing dynamic changes during infection progression

This integrated approach enables researchers to build a comprehensive understanding of how CbiN-mediated cobalt acquisition contributes to Salmonella Newport pathogenesis, potentially identifying new targets for antimicrobial development and diagnostic biomarkers for epidemiological investigations .

What advanced techniques can differentiate the specific role of CbiN from other components in the cobalt transport system?

To precisely delineate the specific function of CbiN from other components in the cobalt transport system, researchers should employ these advanced methodological approaches:

• CRISPR Interference (CRISPRi) and CRISPR Activation (CRISPRa):

  • Use CRISPRi for targeted, tunable repression of cbiN and other ECF components

  • Apply CRISPRa to selectively upregulate individual components

  • Implement multiplexed CRISPR systems to manipulate multiple genes simultaneously

  • Analyze resulting phenotypes to disentangle component-specific roles

• Advanced Proteomic Approaches:

  • Employ proximity-dependent biotinylation (BioID or APEX) to identify proteins that interact with CbiN in vivo

  • Use hydrogen-deuterium exchange mass spectrometry (HDX-MS) to map conformational changes upon cobalt binding

  • Apply cross-linking mass spectrometry (XL-MS) to determine interaction interfaces

  • Implement quantitative proteomics to monitor stoichiometric relationships between transporter components

• Fluorescence-Based Technologies:

  • Utilize Förster resonance energy transfer (FRET) to measure direct interactions between CbiN and other components

  • Apply fluorescence recovery after photobleaching (FRAP) to assess CbiN mobility in membranes

  • Implement super-resolution microscopy to visualize CbiN localization and clustering

  • Develop cobalt-specific fluorescent sensors to track transport in real-time

• Functional Reconstitution Systems:

  • Create defined liposome systems with purified components in different combinations

  • Develop proteoliposome-based transport assays with controlled component composition

  • Use nanodiscs to study CbiN in a native-like membrane environment

  • Implement microfluidic approaches for high-throughput analysis of reconstituted systems

• Single-Molecule Techniques:

  • Apply single-molecule fluorescence to track individual transport events

  • Use atomic force microscopy to visualize conformational changes

  • Implement patch-clamp techniques to measure transport-associated currents

  • Develop high-speed AFM to capture dynamic structural changes during transport

By systematically applying these advanced techniques, researchers can create a comprehensive functional map of the cobalt transport system, clearly defining the specific contribution of CbiN within the larger ECF transporter complex. This level of mechanistic detail is essential for developing targeted interventions that could disrupt cobalt acquisition in pathogenic Salmonella newport.

How can researchers leverage comparative genomics to identify evolutionary patterns in CbiN across Salmonella species?

To effectively leverage comparative genomics for evolutionary analysis of CbiN across Salmonella species, researchers should implement a systematic analytical framework:

• Comprehensive Sequence Collection and Alignment:

  • Gather cbiN sequences from diverse Salmonella serovars, with particular focus on distinct Newport lineages

  • Include evolutionary outgroups from closely related genera

  • Implement progressive multiple sequence alignment algorithms optimized for membrane proteins

  • Generate codon-aware alignments to distinguish synonymous from non-synonymous changes

• Phylogenetic Analysis Methods:

  • Construct maximum likelihood phylogenetic trees specific to cbiN

  • Compare cbiN phylogeny with whole-genome phylogenetic patterns to identify incongruences

  • Apply Bayesian evolutionary analysis to estimate divergence times

  • Implement ancestral sequence reconstruction to infer evolutionary trajectories

• Selection Pressure Analysis:

  • Calculate dN/dS ratios to identify regions under positive or purifying selection

  • Apply site-specific selection analysis to identify key functional residues

  • Implement branch-site models to detect lineage-specific selection patterns

  • Correlate selection patterns with functional domains and predicted structural features

• Horizontal Gene Transfer Detection:

  • Analyze GC content, codon usage bias, and tetranucleotide frequencies

  • Implement phylogenetic network approaches to visualize potential recombination events

  • Apply parametric and non-parametric methods to identify genomic islands

  • Assess the role of mobile genetic elements in cbiN evolution

• Synteny and Genomic Context Analysis:

  • Examine conservation of gene order surrounding cbiN

  • Identify operon structure variations across lineages

  • Analyze promoter regions for regulatory element conservation

  • Assess co-evolution patterns with other ECF transporter components

• Structural Variation Mapping:

  • Project sequence variations onto predicted protein structures

  • Identify lineage-specific structural adaptations

  • Correlate structural changes with environmental adaptations

  • Predict functional consequences of observed variations

This comprehensive comparative genomics approach can reveal how CbiN has evolved across Salmonella lineages, potentially identifying adaptive changes that contribute to host specificity, virulence, or environmental persistence. The analysis may also identify key evolutionary events that could serve as markers for epidemiological tracking of Salmonella Newport lineages .

What are the optimal approaches for studying CbiN's role in metal homeostasis networks in Salmonella?

To comprehensively investigate CbiN's role in Salmonella metal homeostasis networks, researchers should implement these optimal methodological approaches:

• Systems-Level Metallomic Analysis:

  • Employ ICP-MS (Inductively Coupled Plasma Mass Spectrometry) to quantify the metallome under varying conditions

  • Implement metalloproteomics to identify cobalt-binding proteins affected by CbiN function

  • Use synchrotron X-ray fluorescence microscopy for subcellular metal localization

  • Apply stable isotope labeling to track cobalt flux through metabolic networks

• Regulatory Network Mapping:

  • Conduct RNA-seq analysis comparing wild-type and ΔcbiN strains under metal-replete and metal-limited conditions

  • Implement ChIP-seq to identify transcription factors regulating cbiN expression

  • Use proteome-wide approaches to detect post-translational modifications in response to cobalt availability

  • Develop computational models of metal-responsive regulatory networks including CbiN

• Genetic Interaction Screening:

  • Perform synthetic genetic array analysis with cbiN mutations

  • Apply CRISPR interference screening to identify genes that become essential in the absence of CbiN

  • Develop high-throughput phenotypic assays for metal utilization

  • Implement multiplexed growth competition assays under varying metal conditions

• Metabolic Impact Assessment:

  • Profile cobalt-dependent metabolites using LC-MS/MS

  • Monitor activity of cobalt-dependent enzymes (e.g., vitamin B12-dependent pathways) in cbiN mutants

  • Implement metabolic flux analysis using stable isotope labeling

  • Develop computational models predicting metabolic adaptation to cobalt limitation

• Cross-Metal Interaction Studies:

  • Analyze how CbiN function affects the homeostasis of other metals (iron, nickel, zinc)

  • Investigate potential competitive or cooperative interactions between metal transport systems

  • Examine metal-specific stress responses in the context of CbiN function

  • Study the impact of host nutritional immunity on Salmonella metal acquisition systems

• In vivo Relevance Models:

  • Develop animal infection models to assess the importance of CbiN-mediated cobalt acquisition

  • Implement tissue-specific analysis of metal availability during infection

  • Use competition assays between wild-type and cbiN mutants in different host environments

  • Apply single-cell approaches to examine heterogeneity in metal acquisition

These integrated approaches will enable researchers to position CbiN within the broader context of Salmonella metal homeostasis networks, providing insights into how cobalt acquisition interfaces with other essential metal utilization pathways and how these systems collectively contribute to bacterial survival and pathogenesis.

How can researchers develop high-throughput screening methods to identify inhibitors of CbiN function?

Developing effective high-throughput screening (HTS) methods to identify potential inhibitors of Salmonella newport CbiN function requires a multi-faceted approach:

• Assay Development Strategies:

  • Direct binding assays:

    • Develop fluorescence polarization assays using labeled cobalt analogs

    • Implement thermal shift assays to detect compounds that alter CbiN stability

    • Create surface plasmon resonance (SPR) screening platforms with immobilized CbiN

    • Develop microscale thermophoresis (MST) assays for solution-based binding detection

  • Functional transport assays:

    • Engineer reporter strains with cobalt-responsive fluorescent or luminescent outputs

    • Develop liposome-based transport assays with encapsulated cobalt-responsive sensors

    • Create whole-cell assays linking cobalt transport to survival under selective conditions

    • Implement competitive uptake assays with radioactive cobalt isotopes

• Compound Library Selection:

  • Focus on small molecule collections with properties suitable for membrane protein targets

  • Include natural product extracts, particularly from environments with metal competition

  • Incorporate peptidomimetics designed to disrupt protein-protein interactions

  • Select metal-chelating compounds with potential to interfere with cobalt binding

• Screening Platform Optimization:

  • Miniaturize assays to 384- or 1536-well format for true HTS capability

  • Implement automated liquid handling systems for consistent assay performance

  • Develop multiparametric readouts to capture different aspects of inhibition

  • Create counter-screening assays to eliminate false positives early in the process

• Data Analysis Framework:

  • Implement machine learning algorithms to identify structure-activity relationships

  • Develop network pharmacology approaches to predict compound effects on metal homeostasis

  • Create predictive models for selectivity and off-target effects

  • Implement chemoinformatic clustering to prioritize diverse hit structures

• Validation Strategy Pipeline:

  • Secondary assays with orthogonal detection methods

  • Dose-response studies to establish potency metrics

  • Selectivity panels against other metal transporters

  • Cytotoxicity assessment in mammalian cells

  • Stability and solubility profiling

• Structure-Based Optimization Approach:

  • Use computational docking to predict binding modes of hits

  • Implement structure-guided medicinal chemistry for hit optimization

  • Apply fragment-based approaches to develop high-affinity inhibitors

  • Create pharmacophore models based on initial active compounds

This comprehensive HTS workflow would enable the identification of compounds that could serve as both chemical probes to study CbiN function and as starting points for the development of novel antimicrobials targeting cobalt acquisition in Salmonella newport, potentially addressing the challenges posed by multi-drug resistant strains like MDR-AmpC Salmonella Newport .

How might CbiN research contribute to developing novel antimicrobial strategies against Salmonella infections?

CbiN research offers several promising avenues for developing novel antimicrobial strategies against Salmonella infections:

• Direct CbiN Inhibition Approaches:

  • Design small molecule inhibitors that specifically block cobalt binding to CbiN

  • Develop peptidomimetics that disrupt CbiN's interaction with other ECF transporter components

  • Create decoy substrates that competitively bind CbiN without transport functionality

  • Engineer antibody-based therapeutics targeting surface-exposed regions of CbiN

• Cobalt Acquisition Interference Strategies:

  • Design cobalt chelators that selectively reduce available cobalt in infection sites

  • Develop metal-substitution approaches where non-functional metal analogs compete with cobalt

  • Create host-directed therapies that enhance natural metal sequestration mechanisms

  • Implement combination approaches targeting multiple metal acquisition systems simultaneously

• Vaccine Development Applications:

  • Assess CbiN's potential as a vaccine antigen, focusing on conserved epitopes

  • Develop attenuated vaccine strains with modified cobalt acquisition systems

  • Create subunit vaccines incorporating CbiN epitopes with appropriate adjuvants

  • Design DNA vaccines encoding immunogenic CbiN components

• Diagnostic Applications:

  • Develop rapid detection methods for pathogenic Salmonella based on CbiN variants

  • Create diagnostic panels distinguishing between antimicrobial-resistant lineages

  • Implement serological tests targeting anti-CbiN antibodies in infected hosts

  • Design point-of-care diagnostics based on CbiN molecular signatures

• Combination Therapy Approaches:

  • Identify synergistic interactions between cobalt transport inhibition and existing antibiotics

  • Develop dual-targeting approaches affecting both cobalt acquisition and utilization

  • Create therapeutic strategies combining metal starvation with immune stimulation

  • Implement sequential treatment protocols optimized for preventing resistance development

• Resistance Mitigation Strategies:

  • Analyze potential resistance mechanisms to CbiN-targeted therapeutics

  • Design inhibitor cocktails targeting multiple components of the ECF transporter

  • Develop cycling protocols to minimize resistance development

  • Create evolutionary trap approaches where resistance comes with significant fitness costs

This research direction is particularly promising given the rise of MDR-AmpC Salmonella Newport strains that are resistant to multiple conventional antibiotics, including extended-spectrum cephalosporins . By targeting essential nutrient acquisition systems like CbiN, researchers may be able to overcome existing resistance mechanisms and develop therapeutics effective against these challenging pathogens.

What emerging technologies could advance our understanding of CbiN structure and function?

Several cutting-edge technologies show particular promise for advancing our understanding of CbiN structure and function:

• Advanced Structural Biology Methods:

  • Cryo-electron tomography: For visualizing CbiN in its native membrane environment at near-atomic resolution

  • Micro-electron diffraction (MicroED): For determining structures from nanocrystals of membrane proteins

  • Serial femtosecond crystallography: Using X-ray free electron lasers (XFELs) for time-resolved structural studies

  • Integrated structural biology approaches: Combining multiple techniques (NMR, cryo-EM, X-ray) for complete structural characterization

• Single-Cell and Single-Molecule Technologies:

  • Single-cell metabolomics: For measuring cobalt uptake heterogeneity in bacterial populations

  • Single-molecule fluorescence microscopy: To track individual transport events

  • Patch-clamp electrophysiology: For real-time measurement of transport-associated currents

  • High-speed atomic force microscopy: To visualize conformational dynamics during transport

• Advanced Genetic Engineering Tools:

  • Base editing and prime editing: For precise modification of specific CbiN residues

  • Optogenetic control systems: To manipulate CbiN function with light

  • Synthetic cellular circuits: Creating artificial regulatory networks to control CbiN expression

  • Cell-free protein synthesis: For rapid production and engineering of CbiN variants

• Novel Biophysical Approaches:

  • Nanopore recording: For direct detection of metal ion translocation

  • Magnetic tweezers: To measure force generation during transport

  • Advanced EPR techniques: For mapping metal coordination environments

  • Time-resolved vibrational spectroscopy: To capture transient transport intermediates

• Computational and AI Developments:

  • AlphaFold and other AI structure prediction methods: For modeling CbiN conformational states

  • Molecular dynamics at exascale computing: For simulating complete transport cycles

  • Quantum mechanical calculations: For accurate modeling of metal coordination

  • Network medicine approaches: To position CbiN in the broader context of cellular systems

• Advanced Imaging Technologies:

  • Correlative light and electron microscopy (CLEM): For connecting function to structure

  • Expansion microscopy: For super-resolution imaging of membrane protein complexes

  • Cryo-correlative light and electron microscopy: To locate specific labeled proteins in frozen cells

  • 4D cellular tomography: For tracking dynamic protein complexes in living cells

These emerging technologies can be strategically combined to create a comprehensive understanding of CbiN structure, dynamics, and function, potentially revealing new mechanisms of cobalt transport and identifying novel therapeutic targets for combating Salmonella newport infections.

How might environmental and host factors influence CbiN expression and function during infection?

The expression and function of Salmonella newport CbiN during infection are likely modulated by complex environmental and host factors:

• Host Nutritional Immunity Mechanisms:

  • Metal sequestration proteins: Host proteins like calprotectin may limit cobalt availability

  • Inflammation-induced metal restriction: Inflammatory responses alter metal distribution in infection sites

  • Cell-type specific microenvironments: Different host cell types provide varying levels of available cobalt

  • Temporal changes during infection: Metal availability likely fluctuates throughout infection progression

• Gastrointestinal Environment Factors:

  • pH gradients: Varying pH conditions throughout the GI tract may affect CbiN function and expression

  • Oxygen availability: Microaerobic and anaerobic conditions influence cobalt requirements

  • Microbiome competition: Commensal bacteria compete for available cobalt

  • Bile acids and digestive enzymes: May affect membrane protein function and stability

• Salmonella Adaptation Mechanisms:

  • Stress response integration: How general stress responses affect cbiN expression

  • Metabolic reprogramming: Shifts in metabolic pathways that alter cobalt requirements

  • Biofilm-specific regulation: Expression patterns in biofilm versus planktonic states

  • Persister cell formation: Role of cobalt transport in persistence phenotypes

• Signal Integration Systems:

  • Two-component regulatory systems: Identification of systems controlling cbiN expression

  • Quorum sensing effects: Population density-dependent regulation

  • Small RNA regulators: Post-transcriptional control mechanisms

  • Sigma factor utilization: Alternative sigma factors for condition-specific expression

• Experimental Approaches to Study Environmental Modulation:

  • In vitro infection models: Recreating relevant host microenvironments

  • Transcriptional reporters: Monitoring cbiN expression under varying conditions

  • Animal infection models: Tissue-specific analysis of cobalt availability and transport

  • Single-cell approaches: Examining heterogeneity in CbiN expression and function

• Clinical Relevance of Environmental Adaptation:

  • Antibiotic treatment effects: How antimicrobial therapy affects cobalt homeostasis

  • Host nutritional status influence: Impact of host cobalt levels on infection dynamics

  • Disease state correlations: Relationship between cobalt acquisition and infection severity

  • Therapeutic targeting opportunities: Environmental conditions that sensitize to cobalt limitation

Understanding these complex interactions between host, environment, and pathogen will provide crucial insights into the role of CbiN during Salmonella newport infection and may reveal specific conditions where targeting cobalt acquisition would be most effective as a therapeutic strategy against Salmonella infections, including multi-drug resistant strains .

What are the potential applications of CbiN in biotechnology beyond understanding bacterial pathogenesis?

CbiN protein offers diverse applications in biotechnology beyond its role in understanding bacterial pathogenesis:

• Biosensor Development:

  • Metal-specific environmental sensors: Engineering CbiN-based biosensors for detecting cobalt contamination in water supplies

  • Metabolic monitoring tools: Creating cellular biosensors that report on intracellular cobalt status

  • High-throughput screening platforms: Developing CbiN-based assays for drug discovery

  • Food safety applications: Biosensors for detecting harmful metal concentrations in food products

• Protein Engineering Applications:

  • Metal binding optimization: Engineering CbiN variants with altered metal specificity or affinity

  • Membrane protein design templates: Using CbiN structural features as scaffolds for designing novel membrane proteins

  • Directed evolution platforms: Creating libraries of CbiN variants for selecting desired properties

  • Fusion protein development: Utilizing CbiN as a membrane anchor for other functional domains

• Bioremediation Technologies:

  • Metal recovery systems: Engineered bacteria with enhanced CbiN expression for environmental cobalt extraction

  • Contamination cleanup: Biological systems for removing toxic metals from contaminated sites

  • Selective metal filtration: Membrane-based systems incorporating CbiN for specific metal binding

  • Metal recycling applications: Biological systems for recovering valuable metals from waste streams

• Synthetic Biology Tools:

  • Cobalt-responsive genetic circuits: Developing regulatory systems controlled by cobalt availability

  • Orthogonal nutrient acquisition systems: Engineering bacteria with novel metal utilization pathways

  • Minimal cell design components: Including optimized metal transport systems in synthetic minimal cells

  • Metabolic engineering tools: Using controlled cobalt transport to regulate cobalt-dependent enzymes

• Therapeutic Delivery Systems:

  • Metal-dependent drug release mechanisms: Creating delivery systems triggered by specific metal concentrations

  • Targeted antimicrobial delivery: Developing phage or nanoparticle systems targeting CbiN in pathogenic bacteria

  • Probiotics with engineered mineral uptake: Optimizing beneficial bacteria for nutrient acquisition

  • Vaccine design platforms: Using CbiN-derived components for antigen presentation

• Industrial Biotechnology Applications:

  • Enzyme cofactor delivery systems: Improving biocatalysis by enhancing cobalt availability

  • Fermentation optimization: Engineering production strains with improved cobalt utilization

  • Biomanufacturing process enhancement: Optimizing metal availability in industrial bioprocesses

  • Biosynthesis of vitamin B12: Improving production by enhancing cobalt acquisition pathways

These diverse applications demonstrate how fundamental research on bacterial transport proteins like CbiN can lead to unexpected biotechnological innovations with potential impacts in fields ranging from environmental science to industrial bioprocessing and medicine.

What are the key controls and validation steps needed in CbiN functional studies?

Rigorous controls and validation steps are essential for ensuring the reliability and reproducibility of Salmonella newport CbiN functional studies:

How can researchers effectively combine biochemical and genetic approaches to study CbiN?

An effective integrated strategy for studying Salmonella newport CbiN should combine complementary biochemical and genetic approaches:

• Sequential Investigation Framework:

  • Begin with genetic manipulations to establish in vivo relevance

  • Follow with biochemical characterization to define mechanisms

  • Return to genetic systems to test mechanistic hypotheses

  • Iterate between approaches to refine understanding

• Genetic Approaches and Their Biochemical Complements:

  • Gene deletion studies:

    • Genetic: Create precise cbiN knockout strains

    • Biochemical: Quantify changes in cellular cobalt content by ICP-MS

    • Integration: Correlate phenotypes with specific biochemical defects

  • Mutagenesis analysis:

    • Genetic: Generate point mutations in conserved CbiN residues

    • Biochemical: Determine effects on protein stability and cobalt binding affinity

    • Integration: Create structure-function maps connecting sequence to activity

  • Suppressor screens:

    • Genetic: Identify mutations that restore function in cbiN mutants

    • Biochemical: Characterize interaction changes in suppressor strains

    • Integration: Define functional networks surrounding CbiN

• Biochemical Approaches and Their Genetic Validations:

  • Protein purification and reconstitution:

    • Biochemical: Purify CbiN and reconstitute in liposomes

    • Genetic: Test if observed in vitro properties match in vivo phenotypes

    • Integration: Refine reconstitution systems to better reflect in vivo conditions

  • Interaction studies:

    • Biochemical: Identify binding partners through pull-down assays

    • Genetic: Confirm biological relevance through genetic interaction studies

    • Integration: Build comprehensive interaction maps with functional validation

  • Structural analysis:

    • Biochemical: Determine CbiN structure through crystallography or cryo-EM

    • Genetic: Test structure-based predictions through targeted mutagenesis

    • Integration: Iteratively refine structural models using genetic data

• Advanced Integrative Approaches:

  • Chemical genetics:

    • Identify small molecule inhibitors of CbiN function

    • Compare chemical inhibition phenotypes with genetic knockouts

    • Use compounds as temporally controlled tools to complement genetic studies

  • In vivo crosslinking:

    • Capture transient interactions in living cells

    • Validate crosslinking results through genetic manipulation of interaction partners

    • Define temporal dynamics of interactions during transport

  • Multi-omics integration:

    • Connect transcriptomic changes in cbiN mutants with proteome and metallome alterations

    • Use genetic backgrounds to validate systems-level models

    • Develop predictive frameworks incorporating both genetic and biochemical data

• Data Integration Strategies:

  • Implement computational models that incorporate both genetic and biochemical data

  • Develop visualization tools that present integrated datasets

  • Establish standardized protocols that bridge genetic and biochemical approaches

  • Create shared repositories for integrated datasets

This systematic integration of genetic and biochemical approaches provides a comprehensive understanding of CbiN function that neither approach could achieve alone, addressing both the in vivo relevance and molecular mechanisms of cobalt transport in Salmonella newport.

What are the most appropriate in vitro systems for studying the transport mechanism of CbiN?

Several in vitro systems are particularly well-suited for studying the transport mechanism of CbiN, each with specific advantages for different experimental questions:

• Proteoliposome Reconstitution Systems:

  • Simple proteoliposomes: CbiN incorporated into phospholipid vesicles

    • Advantages: Defined composition; control over lipid environment

    • Best for: Basic transport kinetics; substrate specificity determination

    • Technical considerations: Ensuring correct orientation; verifying incorporation

  • Co-reconstituted ECF complexes: Complete transporter complex in liposomes

    • Advantages: Recapitulates native transport system; allows component manipulation

    • Best for: Studying component interactions; energy coupling mechanisms

    • Technical considerations: Complex reconstitution; stoichiometry control

• Nanodiscs and Membrane Scaffolds:

  • Standard nanodiscs: CbiN in disc-shaped lipid bilayers

    • Advantages: Soluble system; defined size; accessible from both sides

    • Best for: Structural studies; binding assays; single-molecule studies

    • Technical considerations: Optimization of scaffold protein; limited size

  • Macro-nanodiscs: Larger diameter nanodiscs

    • Advantages: Accommodate larger complexes; reduce curvature stress

    • Best for: Reconstituting complete ECF transporter complexes

    • Technical considerations: Stability; homogeneity control

• Planar Bilayer Systems:

  • Black lipid membranes (BLM): CbiN in free-standing bilayers

    • Advantages: Electrical measurements possible; large surface area

    • Best for: Electrophysiological studies; flux measurements

    • Technical considerations: Fragility; protein incorporation challenges

  • Supported lipid bilayers: Bilayers on solid supports

    • Advantages: Stability; compatibility with surface techniques

    • Best for: Surface-sensitive measurements; lateral mobility studies

    • Technical considerations: Support interactions; incorporation methods

• Cell-Based Minimal Systems:

  • Spheroplasts: Bacteria with partially removed cell walls

    • Advantages: Near-native environment; accessible interior

    • Best for: Patch-clamp studies; membrane potential effects

    • Technical considerations: Fragility; background transport

  • Inside-out membrane vesicles: Inverted bacterial membranes

    • Advantages: Native membrane composition; high protein density

    • Best for: High-throughput transport assays; native complex studies

    • Technical considerations: Mixed orientation; multiple transporters present

• Advanced Hybrid Systems:

  • Droplet interface bilayers (DIB): Lipid bilayers between aqueous droplets

    • Advantages: Electrical access; dynamic composition changes possible

    • Best for: Real-time transport measurements; gradient studies

    • Technical considerations: Specialized equipment; stability

  • Microfluidic transport systems: Channel-based flow systems

    • Advantages: Controlled gradients; real-time measurements

    • Best for: Kinetic studies; inhibitor screening

    • Technical considerations: System complexity; miniaturization challenges

For optimal results, researchers should select systems based on specific experimental questions and often employ multiple complementary approaches to build a comprehensive understanding of CbiN transport mechanisms.

What bioinformatic tools are most useful for analyzing CbiN sequence, structure, and evolutionary relationships?

For comprehensive bioinformatic analysis of Salmonella newport CbiN, researchers should employ these specialized tools across several analytical domains:

• Sequence Analysis Tools:

  • Multiple Sequence Alignment:

    • MAFFT: Fast, accurate alignment of CbiN homologs

    • T-Coffee: High-accuracy alignment for divergent sequences

    • MUSCLE: Iterative alignment approach for balancing speed and accuracy

    • PRALINE: Alignment optimized for transmembrane proteins

  • Sequence Feature Prediction:

    • TMHMM/TOPCONS: Transmembrane helix prediction

    • SignalP: Signal peptide identification

    • PSIPRED: Secondary structure prediction

    • ConSurf: Conservation analysis for functional residue identification

• Structural Analysis Resources:

  • 3D Structure Prediction:

    • AlphaFold2: State-of-the-art protein structure prediction

    • RoseTTAFold: Alternative AI-based structure prediction

    • SWISS-MODEL: Homology modeling for CbiN based on related structures

    • Robetta: Ab initio and template-based modeling

  • Structural Analysis Tools:

    • PyMOL/UCSF Chimera: Visualization and analysis of predicted structures

    • CASTp: Binding pocket identification

    • HOLE: Channel and pore analysis

    • MDAnalysis: Analysis of molecular dynamics simulations

• Evolutionary Analysis Software:

  • Phylogenetic Tree Construction:

    • RAxML-NG: Maximum likelihood phylogeny for CbiN sequences

    • MrBayes: Bayesian phylogenetic inference

    • IQ-TREE: Fast, model-selection integrated phylogenetic analysis

    • BEAST2: Bayesian evolutionary analysis with time calibration

  • Selection Analysis:

    • PAML: Detection of positive selection at specific sites

    • HyPhy/MEME: Identification of episodic selection

    • SelectionLRT: Likelihood ratio tests for selection

    • RELAX: Tests for relaxed or intensified selection

• Comparative Genomics Platforms:

  • Genomic Context Analysis:

    • MicrobesOnline: Gene neighborhood visualization

    • SyntTax: Synteny analysis across multiple genomes

    • Artemis: Genome browser with comparative capabilities

    • PATRIC: Comprehensive bacterial genomics resource

  • Horizontal Gene Transfer Detection:

    • IslandViewer: Genomic island prediction

    • Alien_Hunter: Horizontal gene transfer prediction

    • HGTector: Detection of horizontally transferred genes

    • T-REX: Phylogenetic network analysis for HGT events

• Protein-Protein Interaction Prediction:

  • STRING: Interaction network analysis

  • PSICQUIC: Standardized access to interaction databases

  • InterPreTS: Structure-based interaction prediction

  • PRISM: Protein interaction prediction based on structural matching

• Integrated Analysis Platforms:

  • Galaxy: Web-based platform for accessible bioinformatics

  • Jalview: Integrated alignment, analysis, and visualization

  • InterProScan: Integrated protein signature recognition

  • UniProt: Comprehensive protein information resource (B4SWZ0 for S. newport CbiN)

This comprehensive toolkit enables researchers to systematically analyze CbiN from sequence to structure to evolutionary context, providing a multi-dimensional understanding of this important transport protein in Salmonella newport.