Recombinant Salmonella dublin Cobalt transport protein CbiN (cbiN)

What is the molecular structure and cellular localization of Salmonella dublin Cobalt transport protein CbiN?

Salmonella dublin Cobalt transport protein CbiN (cbiN) is a small membrane protein consisting of 93 amino acids with a molecular weight of approximately 10 kDa. The amino acid sequence (MKKTLmLLAMVVALVILPFFINHGGEYGGSDGEAESQIQAIAPQYKPWFQPLYEPASGEIESLLFTLQGSLGAAVIFYILGYCKGKQRRDDRA) reveals a hydrophobic N-terminal region characteristic of membrane proteins, suggesting integration into the bacterial cell membrane . Structural analysis indicates CbiN contains transmembrane domains that facilitate cobalt ion transport across the membrane. The protein is primarily localized in the cytoplasmic membrane where it functions as part of the Energy-coupling factor (ECF) transporter complex. While high-resolution crystal structures remain limited, computational modeling suggests CbiN adopts a conformation with membrane-spanning regions connected by short loops that are important for substrate binding and transport functions.

How does CbiN function within the ECF transporter complex?

CbiN functions as the substrate-capture component (S-component) within the Energy-coupling factor (ECF) transporter complex . The ECF complex typically consists of four components: the substrate-specific S-component (CbiN), two transmembrane coupling proteins, and an ATPase that provides energy for transport. The mechanism involves:

• Initial binding of cobalt ions to specific residues in CbiN

• Conformational change in CbiN that signals the ECF complex

• ATP hydrolysis by the ATPase component

• Translocation of cobalt ions across the membrane

Research methodologies to study this mechanism include:

• Site-directed mutagenesis of key residues followed by transport assays

• Fluorescence resonance energy transfer (FRET) to monitor protein-protein interactions

• Isothermal titration calorimetry (ITC) to measure binding affinities between CbiN and cobalt ions

What are optimal conditions for expression and purification of recombinant CbiN protein?

Optimal expression and purification of functional recombinant Salmonella dublin CbiN requires careful consideration of expression systems and conditions due to its membrane protein nature. Based on research practices:

After purification, the protein should be stored in Tris-based buffer with 50% glycerol at -20°C for short-term or -80°C for long-term storage . Avoid repeated freeze-thaw cycles as they can denature the protein. For working stocks, maintain aliquots at 4°C for up to one week to preserve functionality.

What methodologies can be employed to assess CbiN-mediated cobalt transport activity?

Several complementary methodologies can be employed to quantitatively assess CbiN-mediated cobalt transport:

• Radioisotope Transport Assays:

  • Incubate bacterial cells or membrane vesicles expressing CbiN with 57Co or 60Co

  • Measure intracellular accumulation of labeled cobalt at various timepoints

  • Compare transport kinetics between wild-type and mutant CbiN variants

• Fluorescent Probes:

  • Utilize cobalt-sensing fluorophores that exhibit spectral changes upon cobalt binding

  • Monitor real-time transport in live cells using confocal microscopy

  • Calculate transport rates from fluorescence intensity changes

• Inductively Coupled Plasma Mass Spectrometry (ICP-MS):

  • Precisely quantify intracellular cobalt concentrations in cells expressing CbiN

  • Compare with control cells lacking CbiN expression

  • Determine absolute transport rates under various conditions

• Electrophysiological Methods:

  • Reconstitute purified CbiN in lipid bilayers

  • Use patch-clamp techniques to measure ion currents

  • Determine ion selectivity by varying ion composition in experimental chambers

For rigorous analysis, researchers should combine at least two methodologies to confirm findings and rule out artifacts from any single approach.

How can protein-protein interactions between CbiN and other ECF transporter components be characterized?

Characterizing protein-protein interactions between CbiN and other ECF transporter components requires multiple complementary approaches:

• Co-Immunoprecipitation (Co-IP):

  • Express epitope-tagged CbiN in Salmonella dublin

  • Perform pull-down assays with antibodies against the tag

  • Identify interacting partners by mass spectrometry

  • Validate interactions with reverse Co-IP experiments

• Bacterial Two-Hybrid System:

  • Clone cbiN and potential partner genes into two-hybrid vectors

  • Measure reporter gene activation as indication of interaction

  • Test interaction domains through truncation analysis

• Surface Plasmon Resonance (SPR):

  • Immobilize purified CbiN on sensor chip

  • Flow purified partner proteins over the surface

  • Determine binding kinetics and affinity constants

  • Test effects of mutations on binding parameters

• Crosslinking Mass Spectrometry:

  • Treat intact cells or purified complexes with crosslinking agents

  • Digest and analyze by LC-MS/MS

  • Identify crosslinked peptides to map interaction interfaces

  • Build structural models based on crosslinking constraints

These approaches provide complementary information about the temporal dynamics, specificity, and structural basis of CbiN interactions within the ECF transporter complex.

What structural features of CbiN are critical for cobalt binding and transport?

The structural features critical for CbiN's cobalt binding and transport function can be analyzed through multiple experimental approaches:

• Sequence Conservation Analysis:
  The amino acid sequence of CbiN (MKKTLmLLAMVVALVILPFFINHGGEYGGSDGEAESQIQAIAPQYKPWFQPLYEPASGEIESLLFTLQGSLGAAVIFYILGYCKGKQRRDDRA) contains several conserved regions across Salmonella species . Critical structural features include:

  • N-terminal hydrophobic region (residues 1-20): Membrane anchoring

  • Central hydrophilic domain (residues 21-60): Likely involved in cobalt coordination

  • C-terminal charged region (residues 61-93): Potential interaction with other ECF components

• Site-Directed Mutagenesis Approach:
  Systematic mutation of conserved residues can identify those critical for function:

  Residue Type	Potential Function	Experimental Approach
  Histidine residues	Cobalt coordination	His→Ala mutations followed by binding assays
  Acidic residues (Asp/Glu)	Ionic interactions with Co2+	Charge-reversal mutations
  Conserved glycines	Conformational flexibility	Gly→Pro mutations to restrict flexibility
  Aromatic residues	Substrate recognition	Conservative and non-conservative substitutions

• Structural Modeling Validation:

  • Generate homology models based on related transporter structures

  • Validate models through molecular dynamics simulations

  • Test predictions through targeted mutagenesis

  • Correlate structural predictions with functional data

How does CbiN contribute to Salmonella dublin virulence and host adaptation?

Salmonella dublin is a host-adapted invasive non-typhoidal Salmonella serovar that causes bloodstream infections in humans and demonstrates increasing prevalence of antimicrobial resistance . CbiN's role in virulence can be examined through several research approaches:

• Nutrient Acquisition During Infection:

  • Cobalt is an essential micronutrient required for vitamin B12 synthesis

  • CbiN-mediated cobalt acquisition likely provides metabolic advantages in nutrient-limited host environments

  • Experimental approach: Compare growth of wild-type and ΔcbiN mutants in cobalt-limited media mimicking host conditions

• Host Adaptation Mechanisms:

  • Comparative genomic analysis reveals distinct populations of S. Dublin circulating in different geographical regions

  • CbiN sequence variations may reflect adaptation to different host environments

  • Research methodology: Analyze CbiN sequence conservation across isolates from different hosts and geographical regions

• Virulence Factor Integration:

  • CbiN function may integrate with other virulence mechanisms:

    • Salmonella Pathogenicity Islands (SPIs)

    • The spv operon (spvRABCD genes)

    • Type VI secretion system (T6SS)

  • Experimental approach: Evaluate virulence factor expression in ΔcbiN backgrounds

S. Dublin contains 13 SPIs (including SPIs 1-6, 9, 11, 13, 14, 16, 17, and 19) and numerous virulence genes that may interact functionally with the cobalt transport system . Research correlating CbiN function with these established virulence mechanisms could identify novel therapeutic targets.

How does antimicrobial resistance affect CbiN expression and function?

The relationship between antimicrobial resistance and CbiN function represents an emerging research area, particularly given the increasing prevalence of antimicrobial resistant (AMR) S. Dublin strains:

• Differential Expression Analysis:

  • Compare cbiN expression levels between antimicrobial-susceptible and resistant strains

  • Methodology: RT-qPCR and RNA-seq under various growth conditions

  • Data interpretation: Correlate expression changes with resistance patterns

• Functional Impact of AMR-Associated Mutations:
  While most German S. Dublin strains show limited antimicrobial resistance, a North American cluster shows multidrug resistance (MDR) patterns . Research questions include:

  • Do AMR-conferring mutations affect metal transport systems?

  • Does acquired resistance alter cellular metal homeostasis?

  • Experimental approach: Transport assays in isogenic strains differing only in resistance determinants

• Metal Transport in Biofilm Formation:

  • AMR strains often display enhanced biofilm formation

  • Cobalt transport may influence biofilm matrix composition

  • Research methodology: Compare biofilm formation between wild-type and ΔcbiN strains under various metal concentrations

• CbiN as Antimicrobial Target:

  • Novel hybrid plasmids encoding both AMR and metal resistance have been identified in S. Dublin

  • These plasmids may enhance survival through coordinated regulation of transport systems

  • Research approach: Screen for small molecule inhibitors of CbiN and test their efficacy against AMR strains

What are the applications of isotope labeling techniques for studying CbiN function?

Isotope labeling provides powerful tools for studying CbiN structure, dynamics, and functional mechanisms:

• Nuclear Magnetic Resonance (NMR) Spectroscopy:

  • 15N and 13C labeling of recombinant CbiN

  • Methodology: Express protein in minimal media with 15NH4Cl and 13C-glucose

  • Applications:

    • Determine solution structure

    • Analyze protein dynamics

    • Monitor conformational changes upon cobalt binding

• Neutron Diffraction:

  • Deuterium (2H) labeling of CbiN

  • Methodology: Express protein in D2O-based media

  • Applications:

    • Locate hydrogen atoms in crystal structures

    • Distinguish between closely related metal ions

    • Determine protonation states of key residues

• Metabolic Flux Analysis:

  • 59Co stable isotope labeling

  • Methodology: Pulse-chase experiments with isotopically labeled cobalt

  • Applications:

    • Track cobalt movement through cellular compartments

    • Determine rate-limiting steps in transport

    • Quantify cobalt utilization in vitamin B12 synthesis

These techniques allow researchers to study CbiN function with minimal perturbation to the native protein structure and provide insights that cannot be obtained through conventional biochemical approaches.

How can structural biology techniques be optimized for membrane proteins like CbiN?

Structural characterization of membrane proteins like CbiN presents unique challenges that require specialized approaches:

• X-ray Crystallography Optimization:

  Challenge	Solution Strategy	Expected Outcome
  Low expression yields	Use specialized expression vectors with strong promoters	Increased protein production
  Protein instability	Screen detergent conditions systematically	Identify stabilizing conditions
  Crystal formation difficulties	Lipidic cubic phase crystallization	Better-ordered crystals
  Phase determination	Heavy atom derivatives (including cobalt soaking)	Improved electron density maps

• Cryo-Electron Microscopy (Cryo-EM) Approaches:

  • Reconstitute CbiN with partner proteins in nanodiscs

  • Use direct electron detectors for high-resolution data collection

  • Apply 3D classification to separate different conformational states

  • Combine with crosslinking data to validate structural models

• Integrative Structural Biology:

  • Combine multiple low-resolution techniques

  • Use small-angle X-ray scattering (SAXS) for solution structure

  • Apply distance constraints from FRET measurements

  • Validate models with biochemical and functional data

These optimized approaches can overcome the inherent challenges of membrane protein structural biology and provide crucial insights into CbiN function at the molecular level.