Recombinant Salmonella enteritidis PT4 Cobalt transport protein CbiN (cbiN)

What is the structural composition of CbiN protein in Salmonella enteritidis PT4?

CbiN in Salmonella enteritidis PT4 is a membrane protein consisting of 93 amino acids with a molecular structure comprising two transmembrane helices connected by an extracytoplasmic loop of 37 amino acid residues . The complete amino acid sequence is: MKKTLMLLAMVVALVILPFFINHGGEYGGSDGEAESQIQAIAPQYKPWFQPLYEPASGEIESLLFTLQGSLGAAVIFYILGYCKGKQRRDDRA . The protein can be expressed recombinantly with an N-terminal histidine tag to facilitate purification and experimental manipulation . This structural arrangement is critical for its function in cobalt transport and interactions with other components of the transport system.

What is the functional role of CbiN in bacterial cobalt transport?

CbiN functions as an auxiliary component in the CbiMQO₂ Co²⁺ transporter system, which belongs to the energy-coupling factor (ECF) transporter family responsible for the uptake of vitamins and transition-metal ions in prokaryotic cells . Notably, CbiN can induce significant Co²⁺ transport activity even in the absence of CbiQO₂ when co-expressed with the substrate-specific component CbiM or as a Cbi(MN) fusion protein . Functionally, CbiN-CbiM loop-loop interactions facilitate metal insertion into the binding pocket, enabling efficient cobalt transport across the bacterial membrane . This transport function is essential for various metabolic processes in Salmonella enteritidis PT4, particularly those requiring cobalt as a cofactor.

What expression systems are recommended for recombinant CbiN production?

For optimal expression of recombinant Salmonella enteritidis PT4 CbiN protein, E. coli expression systems have been successfully employed . The recommended approach involves:

• Cloning the full-length cbiN gene (encoding amino acids 1-93) with an N-terminal His-tag for purification purposes

• Optimizing expression conditions (temperature, induction time, media composition)

• Employing specialized membrane protein expression strains if needed

How should recombinant CbiN protein be handled and stored for maximum stability?

Optimal handling and storage of recombinant CbiN protein requires specific conditions to maintain structural integrity and functional activity:

Repeated freeze-thaw cycles should be strictly avoided as they significantly reduce protein activity and stability . For reconstitution, it is advisable to briefly centrifuge the vial before opening to bring contents to the bottom, then reconstitute in deionized sterile water to the recommended concentration .

How do mutations in the extracytoplasmic loop of CbiN affect cobalt transport functionality?

Research demonstrates that the extracytoplasmic loop of CbiN plays a crucial role in cobalt transport functionality. Any deletion within this 37-amino acid loop region abolishes transport activity completely . This finding indicates the structural integrity of this loop is essential for proper function.

Experimental approaches to study these effects include:

• Cysteine-scanning mutagenesis combined with crosslinking experiments to identify critical residues

• Electron paramagnetic resonance analysis following site-directed spin labeling to monitor structural changes

• Solid-state nuclear magnetic resonance of isotope-labeled protein in proteoliposomes to detect dynamic changes

Results from these methodologies have revealed that in wild-type Cbi(MN), the N-terminal loop of CbiM containing three of the four metal ligands is partially immobilized, while in inactive variants with CbiN loop deletions, this loop becomes completely immobile . This decreased dynamics in the inactive form suggests that proper flexibility of the interaction interface is essential for metal transport function.

What techniques can be employed to study CbiM-CbiN protein-protein interactions?

Understanding the CbiM-CbiN interaction interface is critical for elucidating the mechanism of cobalt transport. Several complementary techniques have proven effective:

• In silico prediction methods: Computational approaches can identify potential protein-protein contacts between segments of the CbiN loop and loops in CbiM .

• Cysteine-scanning mutagenesis and crosslinking: This experimental approach validates predicted interaction sites by introducing cysteine residues at specific positions and analyzing their ability to form crosslinks .

• Electron paramagnetic resonance (EPR) analysis with site-directed spin labeling: This technique reveals ordered structures within the CbiN loop and monitors conformational changes upon interaction with CbiM .

• Solid-state nuclear magnetic resonance (NMR): Using isotope-labeled proteins in proteoliposomes, researchers can detect changes in protein dynamics that occur during the interaction process .

• Fusion protein approaches: Creating Cbi(MN) fusion proteins allows for analysis of intramolecular interactions that would normally occur intermolecularly .

The integration of these techniques has confirmed that specific loop-loop interactions between CbiM and CbiN are essential for facilitating metal insertion into the binding pocket and subsequent transport .

How does CbiN function within the broader context of Salmonella enteritidis PT4 pathogenicity?

While CbiN primarily functions in cobalt transport, its role extends to supporting Salmonella enteritidis PT4 pathogenicity through several mechanisms:

When designing experiments to study this relationship, researchers should consider:

• Creating cbiN knockout mutants to assess changes in virulence

• Monitoring expression of cbiN under infection-relevant conditions

• Analyzing cobalt levels in various cellular compartments during infection processes

What methodologies are recommended for analyzing CbiN-mediated cobalt transport kinetics?

To effectively analyze CbiN-mediated cobalt transport kinetics, researchers should employ a multi-faceted experimental approach:

• Radioisotope uptake assays: Using ⁶⁰Co or other suitable radioisotopes to directly measure transport rates in cells expressing various combinations of CbiM, CbiN, CbiQ, and CbiO proteins .

• Reconstituted proteoliposome systems: Purified components can be reconstituted into artificial membrane vesicles to study transport in a defined environment, allowing precise control over protein composition and buffer conditions .

• Fluorescent metal indicators: Cobalt-sensitive fluorescent probes can be used to monitor transport in real-time in live cells or proteoliposomes.

• Inductively coupled plasma mass spectrometry (ICP-MS): This technique enables precise quantification of intracellular cobalt levels to complement transport kinetics studies.

Research has demonstrated that CbiN can induce significant Co²⁺ transport activity even in the absence of CbiQO₂ when co-expressed with the S component CbiM or as a Cbi(MN) fusion protein . This finding provides important insights for designing simplified experimental systems to study specific aspects of the transport mechanism.

How can structural changes in CbiN during metal transport be monitored experimentally?

Monitoring structural changes in CbiN during the metal transport cycle requires sophisticated biophysical techniques that can capture protein dynamics:

• Electron paramagnetic resonance (EPR) with site-directed spin labeling: This approach has successfully revealed that the CbiN loop adopts an ordered structure that undergoes conformational changes during transport . By introducing spin labels at strategic positions throughout the protein, researchers can monitor local mobility changes during the transport cycle.

• Solid-state nuclear magnetic resonance (NMR): Using isotope-labeled CbiN in proteoliposomes, this technique has detected decreased dynamics in inactive forms with CbiN loop deletions compared to functional proteins . Time-resolved NMR experiments can potentially capture intermediate states during transport.

• Förster resonance energy transfer (FRET): By introducing fluorescent donor-acceptor pairs at key positions in CbiN and CbiM, conformational changes that alter the distance between these markers can be monitored in real-time.

• Hydrogen-deuterium exchange mass spectrometry (HDX-MS): This technique can identify regions of CbiN that undergo changes in solvent accessibility during the transport cycle, providing insights into structural rearrangements.

• Cryo-electron microscopy (cryo-EM): While challenging for small membrane proteins, advances in cryo-EM technology may enable visualization of CbiN in different conformational states during transport.

Research has shown that the N-terminal loop of CbiM containing three of four metal ligands exhibits different mobility patterns between functional and non-functional CbiN variants, highlighting the importance of monitoring these dynamic changes when studying the transport mechanism .

What are the recommended controls for experiments involving recombinant CbiN protein?

When designing experiments with recombinant Salmonella enteritidis PT4 CbiN protein, the following controls should be implemented:

• Negative controls:

  • Empty vector expression (without cbiN gene)

  • Inactive CbiN mutants with loop deletions that abolish transport activity

  • Heat-denatured CbiN protein for binding and activity assays

• Positive controls:

  • Well-characterized cobalt transport proteins from other systems

  • Full CbiMQO₂ complex when studying partial systems

  • Cbi(MN) fusion proteins that show reliable activity

• Specificity controls:

  • Non-substrate metal ions to verify transport selectivity

  • Competitive inhibition assays with known inhibitors

  • Transport assays with varying cobalt concentrations to establish kinetic parameters

• Quality controls:

  • SDS-PAGE to verify protein purity (should exceed 90%)

  • Western blotting using anti-His antibodies to confirm identity of His-tagged recombinant protein

  • Circular dichroism to verify proper protein folding

These controls help ensure experimental rigor and enable proper interpretation of results when studying this complex membrane transport system.

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

An integrated approach combining genetic and biochemical techniques provides the most comprehensive understanding of CbiN function:

• Genetic approaches:

  • CRISPR-Cas9 gene editing to create precise mutations in chromosomal cbiN

  • Construction of cbiN deletion strains complemented with wildtype or mutant alleles

  • Transcriptional reporter fusions to monitor cbiN expression under various conditions

  • Transposon mutagenesis to identify genes that interact functionally with cbiN

• Biochemical approaches:

  • Purification of recombinant CbiN with various affinity tags

  • In vitro reconstitution of transport activity in proteoliposomes

  • Protein-protein interaction assays (pull-downs, crosslinking, SPR)

  • Metal binding assays to characterize the interaction with cobalt

• Integration strategies:

  • Correlate in vivo phenotypes with in vitro biochemical properties

  • Perform structure-function analyses by combining mutagenesis with activity assays

  • Use genetic suppressors to identify functional interaction partners

  • Validate biochemical findings through in vivo complementation experiments

Research has demonstrated the power of this combined approach, as seen in studies where cysteine-scanning mutagenesis was used to create specific CbiN variants, followed by crosslinking experiments to verify protein-protein contacts previously predicted through computational methods .

What are common challenges in expressing and purifying recombinant CbiN protein?

Researchers frequently encounter several challenges when working with recombinant CbiN protein:

• Expression challenges:

  • Low expression levels due to toxicity of membrane proteins

  • Protein misfolding leading to inclusion body formation

  • Proteolytic degradation during expression

• Purification difficulties:

  • Inefficient extraction from membranes

  • Aggregation during purification steps

  • Loss of native conformation in detergent solutions

  • Co-purification of contaminating proteins

• Recommended solutions:

  • Optimize expression temperature (typically lower temperatures improve folding)

  • Screen multiple detergents for optimal extraction and stability

  • Consider fusion partners that enhance solubility and folding

  • Implement stringent washing steps during affinity purification to achieve >90% purity

  • Add stabilizing agents such as trehalose (6%) to storage buffers

  • Prepare single-use aliquots to avoid freeze-thaw cycles

• Reconstitution considerations:

  • Reconstitute in deionized sterile water to a concentration of 0.1-1.0 mg/mL

  • Add glycerol (5-50% final concentration) for long-term storage

  • Brief centrifugation prior to opening vials is recommended

These technical considerations are crucial for obtaining functional protein suitable for downstream applications and experimental analyses.

How can researchers verify the functional activity of purified recombinant CbiN?

Verifying the functional activity of purified recombinant CbiN requires multiple complementary approaches:

• Cobalt transport assays:

  • Radioisotope (⁶⁰Co) uptake measurements in proteoliposomes

  • Fluorescent cobalt indicators to monitor transport in real-time

  • ICP-MS quantification of cobalt accumulation

• Protein-protein interaction verification:

  • Co-purification assays with CbiM

  • Surface plasmon resonance to measure binding kinetics

  • Crosslinking experiments to confirm specific interactions

• Structural integrity assessment:

  • Circular dichroism to verify secondary structure elements

  • Limited proteolysis to confirm proper folding

  • Electron paramagnetic resonance analysis after site-directed spin labeling

• Activity comparison metrics:

  • Compare transport rates to native complexes

  • Establish dose-response relationships with varying protein concentrations

  • Analyze metal specificity profiles to confirm selectivity

Research has shown that functional CbiN exhibits specific interactions with the N-terminal loop of CbiM, and these interactions can be monitored through various biophysical techniques . Loss of this interaction capability correlates with loss of transport activity, providing a useful proxy measure for functional integrity.

What are emerging areas of research regarding CbiN's role in bacterial physiology and pathogenesis?

Several promising research directions are emerging in the study of CbiN:

• Systems biology approaches:

  • Integration of CbiN function within global cobalt homeostasis networks

  • Metabolomic profiling to identify downstream processes dependent on CbiN-mediated cobalt transport

  • Transcriptomic analyses to identify co-regulated genes and regulatory networks

• Host-pathogen interactions:

  • Investigation of CbiN's role during infection and colonization

  • Exploration of host immune responses targeting bacterial metal acquisition systems

  • Nutritional immunity mechanisms that restrict cobalt availability

• Structural biology advances:

  • Cryo-EM structures of the complete CbiMNQO complex

  • Molecular dynamics simulations of transport mechanisms

  • Structure-guided design of inhibitors targeting cobalt transport

• Biotechnological applications:

  • Engineering CbiN for enhanced cobalt accumulation in bioremediation applications

  • Development of biosensors based on CbiN transport activity

  • Vaccine development targeting conserved epitopes in metal transport systems

The study of Salmonella enteritidis PT4 has revealed that 3.66% of its 4506 coding sequences are virulence factors associated with cell invasion, intestinal colonization, and intracellular survival . Understanding how CbiN contributes to these virulence mechanisms represents an important frontier in Salmonella pathogenesis research.

How might comparative analysis of CbiN across different bacterial species advance our understanding?

Comparative analysis of CbiN proteins across diverse bacterial species offers valuable insights:

• Evolutionary perspectives:

  • Tracing the evolutionary history of cobalt transport systems

  • Identifying conserved functional domains versus species-specific adaptations

  • Understanding horizontal gene transfer patterns for metal transport components

• Structure-function relationships:

  • Correlation between sequence variations and transport efficiency

  • Identification of species-specific regulatory mechanisms

  • Discovery of alternative protein-protein interactions in different systems

• Host adaptation mechanisms:

  • Comparing CbiN proteins from host-adapted versus environmental bacteria

  • Identifying signatures of selection in pathogen-specific variants

  • Understanding niche-specific adaptations in metal acquisition strategies

• Methodological approaches:

  • Multiple sequence alignments to identify conserved residues

  • Homology modeling based on known structures

  • Heterologous expression systems to test functional complementation

  • Chimeric protein construction to map functional domains

Salmonella enteritidis PT4 and other Salmonella serotypes show high conservation in critical pathogenicity islands and secretion systems , suggesting that comparative analyses of metal transport systems might reveal similar patterns of conservation in virulence-associated functions.