Recombinant Klebsiella pneumoniae Cobalt transport protein CbiN (cbiN)

What is Cobalt transport protein CbiN in Klebsiella pneumoniae?

Cobalt transport protein CbiN is a relatively small protein (93 amino acids) that functions as a component of the CbiMNQO transport system, one of the most widespread groups of microbial transporters for cobalt ions. In Klebsiella pneumoniae, CbiN is specifically designated as an "Energy-coupling factor transporter probable substrate-capture protein" or "ECF transporter S component CbiN" . This protein is part of an unusual uptake system that contains an ABC protein (CbiO) but lacks an extracytoplasmic solute-binding protein typically seen in other transport systems . The protein is involved in the high-affinity uptake of cobalt ions, which are essential cofactors for various enzymatic processes in bacterial metabolism.

How does CbiN relate to bacterial cobalt homeostasis and vitamin B12 biosynthesis?

CbiN functions within a cobalt transport system that is critical for maintaining appropriate intracellular cobalt concentrations. Cobalt is an essential cofactor for coenzyme B12 (cobalamin) biosynthesis, which is involved in methyl group transfer and rearrangement reactions in bacterial metabolism . Since nickel and cobalt ions are typically present only in trace amounts in natural environments, bacteria have evolved high-affinity uptake systems like the CbiMNQO transporter to efficiently acquire these metals . The genes encoding these transporters are often colocalized with genes involved in nickel-dependent enzymes or coenzyme B12 biosynthesis, highlighting their coordinated function in bacterial metabolism . The riboswitch-based regulation of cobalt transporters further emphasizes the importance of tightly controlling cobalt uptake in response to metabolic needs.

What expression systems are recommended for producing recombinant CbiN protein?

For effective production of recombinant CbiN, researchers should consider several expression systems based on the protein's characteristics:

• E. coli-based expression: The standard approach utilizes E. coli BL21(DE3) with pET vectors for controlled expression. For membrane proteins like CbiN, consider using C41(DE3) or C43(DE3) strains specifically designed for membrane protein expression.

• Tag selection: While the commercial recombinant proteins mentioned in the search results indicate "tag type will be determined during production process" , researchers typically employ affinity tags such as His6, Strep-tag II, or MBP (maltose-binding protein) fusions to facilitate purification. For CbiN, C-terminal tags are often preferable to avoid interfering with the N-terminal signal sequence.

• Induction conditions: Given CbiN's membrane-associated nature, using lower induction temperatures (16-20°C) and reduced IPTG concentrations (0.1-0.5 mM) can improve proper folding and membrane integration.

• Solubilization strategies: Since CbiN contains transmembrane domains, extraction requires careful optimization of detergents. Initial screening with mild detergents like DDM (n-dodecyl-β-D-maltopyranoside) or LMNG (lauryl maltose neopentyl glycol) at concentrations just above their critical micelle concentration is recommended.

The expression region (amino acids 1-93) represents the full-length protein , which should be considered when designing expression constructs.

What are the optimal storage conditions for recombinant CbiN protein?

Based on the information provided for commercial recombinant CbiN preparations, the following storage recommendations apply:

• Buffer composition: Use Tris-based buffers with 50% glycerol, optimized specifically for CbiN stability .

• Storage temperature:

  • Long-term storage: -20°C or -80°C

  • Working aliquots: 4°C for up to one week

• Stability considerations: Avoid repeated freeze-thaw cycles as they can compromise protein integrity . Instead, prepare single-use aliquots when dividing stock solutions.

• Additives for enhanced stability: Consider adding reducing agents (1-5 mM DTT or β-mercaptoethanol) to prevent oxidation of cysteine residues. For membrane proteins like CbiN, maintaining an appropriate detergent concentration above its critical micelle concentration is essential to prevent aggregation.

These conditions are specifically tailored to maintain the structural integrity and functional activity of the recombinant CbiN protein for experimental applications.

How does the CbiMNQO transport system function mechanistically?

The CbiMNQO system represents an unusual type of ABC transporter for cobalt uptake. Unlike conventional ABC transporters, this system lacks an extracytoplasmic solute-binding protein . The transport mechanism involves:

• Component roles:

  • CbiM and CbiN: Form the transmembrane channel component

  • CbiQ: Membrane component that interacts with CbiO

  • CbiO: ABC protein that provides energy through ATP hydrolysis

• Minimal functional unit: Experimental analysis has demonstrated that even a basic module consisting of just CbiM and CbiN components from Salmonella enterica serovar Typhimurium exhibits significant transport activity . This suggests that CbiM and CbiN form the core metal-binding and translocation pathway.

• Energy coupling: Unlike secondary transporters that use ion gradients, the CbiMNQO system utilizes ATP hydrolysis via the CbiO component to drive cobalt uptake against concentration gradients, allowing for high-affinity transport even when environmental cobalt concentrations are extremely low.

• Selectivity mechanism: The precise molecular basis for selective cobalt binding remains under investigation, but structural features of the CbiM and CbiN proteins likely create coordination environments that preferentially accommodate cobalt ions over other divalent metals.

Understanding this mechanism provides insights into potential targets for inhibiting bacterial cobalt acquisition as a novel antimicrobial strategy.

What experimental approaches can be used to measure cobalt transport mediated by CbiN?

Researchers can employ several complementary approaches to characterize CbiN-mediated cobalt transport:

• Radioisotope uptake assays:

  • Use radioactive 57Co or 60Co to directly measure transport into bacterial cells or proteoliposomes

  • Compare uptake between wild-type strains and cbiN deletion mutants

  • Investigate competitive inhibition using other divalent metals

• Metal-dependent growth assays:

  • Culture bacteria in defined media with limited cobalt availability

  • Supplement with varying cobalt concentrations

  • Compare growth rates between wild-type and cbiN mutant strains

  • Use vitamin B12 auxotrophs to link transport function to downstream metabolism

• Fluorescent metal sensors:

  • Employ intracellular fluorescent probes sensitive to cobalt concentration

  • Monitor real-time changes in intracellular cobalt levels during transport experiments

  • Use flow cytometry or fluorescence microscopy for single-cell resolution

• Protein-based approaches:

  • Reconstitute purified CbiMN or complete CbiMNQO complexes in proteoliposomes

  • Measure ATP hydrolysis rates in correlation with transport activity

  • Use isothermal titration calorimetry (ITC) to determine metal binding affinities

• Genetic approaches:

  • Perform site-directed mutagenesis of conserved residues in CbiN

  • Construct chimeric transporters by swapping domains between homologous systems

  • Use complementation assays to restore function in cobalt transport-deficient strains

These methodologies provide comprehensive insights into the kinetics, specificity, and structural requirements of CbiN-mediated cobalt transport.

What structural features characterize CbiN and how do they relate to function?

CbiN exhibits several key structural features that contribute to its function in cobalt transport:

• Transmembrane topology:

  • N-terminal hydrophobic region: The first ~20 amino acids (MKKTLILLAMVAALMILPFF or MKKPLILLAMVVALMILPFF) represent a characteristic transmembrane domain

  • The protein likely adopts a membrane-integrated conformation with specific regions exposed to either the cytoplasm or periplasm

• Conserved motifs:

  • GGEFGGSDG sequence: Located immediately after the transmembrane segment, likely forming a transition to the soluble domain

  • QVVAPDYQPWFQPLYEPASGE: Contains potential metal-coordinating residues

  • C-terminal region (ARGRQRRDDRV): Rich in charged residues that may participate in protein-protein interactions with other components of the transport system

• Functional domains:

  • Metal binding site: Likely involves coordination of cobalt ions through specific amino acid side chains

  • Interaction interfaces: Regions that mediate association with CbiM to form the basic functional transport module

• Structural homology:

  • As an ECF transporter S component, CbiN shares structural features with other substrate-capture proteins in this transporter family

  • The compact size (93 amino acids) suggests a specialized role in the larger transport complex

Understanding these structural elements is crucial for interpreting the results of mutagenesis studies and for designing inhibitors targeting this transport system.

What advanced techniques should be considered for determining the three-dimensional structure of CbiN?

Determining the structure of membrane proteins like CbiN presents unique challenges. Researchers should consider these complementary approaches:

• X-ray crystallography:

  • Challenges: Obtaining well-diffracting crystals of membrane proteins requires extensive detergent screening

  • Solutions: Use of lipidic cubic phase (LCP) crystallization, antibody fragments or nanobodies as crystallization chaperones

  • Considerations: May require fusion partners (T4 lysozyme, BRIL) inserted into loop regions to enhance crystallization propensity

• Cryo-electron microscopy (cryo-EM):

  • Advantages: Can resolve structures of membrane proteins in more native-like environments

  • Approach: Focus on the complete CbiMNQO complex rather than CbiN alone to achieve sufficient particle size

  • Recent advances: Signal subtraction and focused refinement can improve resolution of smaller components like CbiN

• NMR spectroscopy:

  • Solution NMR: Suitable for smaller membrane proteins like CbiN (93 aa)

  • Solid-state NMR: Can determine structures in lipid bilayers

  • Sample preparation: Requires isotopic labeling (15N, 13C) of recombinant CbiN

• Integrative structural biology approaches:

  • Combine lower-resolution methods (SAXS, SANS) with computational modeling

  • Cross-link mass spectrometry to identify interaction interfaces

  • Hydrogen-deuterium exchange mass spectrometry to probe structural dynamics

• Computational prediction:

  • Recent advances in AlphaFold2 and RoseTTAFold show promise for membrane protein structure prediction

  • Molecular dynamics simulations can reveal dynamic aspects of CbiN in lipid bilayers

By integrating multiple structural techniques, researchers can overcome the challenges inherent in membrane protein structural biology and gain insights into the molecular mechanism of CbiN function.

What role might CbiN play in Klebsiella pneumoniae pathogenesis?

While direct evidence linking CbiN to K. pneumoniae virulence is limited in the search results, we can infer its potential role in pathogenesis based on the importance of metal acquisition systems in bacterial infections:

• Nutritional immunity: During infection, host organisms sequester essential metals like iron and cobalt as a defense mechanism. Efficient metal acquisition systems, including CbiMNQO, may help K. pneumoniae overcome this host-imposed metal limitation .

• Vitamin B12-dependent metabolism: By facilitating cobalt uptake, CbiN indirectly supports cobalamin biosynthesis, which is required for several metabolic processes that may contribute to bacterial survival during infection .

• Strain-specific variations: The search results indicate slight sequence differences in CbiN between K. pneumoniae strains (ATCC 700721/MGH 78578 vs. strain 342) . These variations might correlate with differences in virulence potential between classical and hypervirulent K. pneumoniae strains, though this would require experimental verification.

• Comparison to other metal acquisition systems: K. pneumoniae employs multiple metal acquisition systems, including siderophores like aerobactin, enterobactin, salmochelin, and yersiniabactin for iron acquisition . The relative importance of cobalt acquisition via CbiN compared to these iron uptake systems remains to be established in the context of infection.

• Potential as a therapeutic target: If cobalt acquisition via CbiN proves important for K. pneumoniae virulence, it could represent a novel target for antimicrobial development, especially against multidrug-resistant strains.

Future studies directly examining the contribution of CbiN to K. pneumoniae virulence in animal models would be valuable for establishing its role in pathogenesis.

How do classical and hypervirulent strains of K. pneumoniae differ in their cobalt transport systems?

• Sequence variations: The CbiN sequences from K. pneumoniae subsp. pneumoniae (strain ATCC 700721/MGH 78578) and K. pneumoniae (strain 342) show high conservation with minor differences in the N-terminal region . Whether these differences affect transport efficiency remains to be determined.

• Context in strain diversity: K. pneumoniae can be broadly categorized into classical (cKp) and hypervirulent (hvKp) pathotypes, which differ in their clinical presentation, epidemiology, and genetic makeup . Classical strains frequently cause hospital-acquired infections and often carry antimicrobial resistance genes, while hypervirulent strains cause community-acquired invasive infections characterized by liver abscesses and metastatic infections .

• Genomic context considerations: The cobalt transport genes may be differently regulated or have distinct genomic contexts in classical versus hypervirulent strains. In some bacteria, metal transport genes are colocalized with virulence factors or are regulated by environmental signals relevant during infection .

• Research priorities:

  • Comparative genomic analysis of cbiMNQO operons across diverse K. pneumoniae strains

  • Experimental measurement of cobalt transport efficiency in representative cKp and hvKp isolates

  • Investigation of cobalt-dependent enzyme expression and activity in different strain backgrounds

  • Assessment of cbiN mutant phenotypes in infection models using both pathotypes

Understanding these strain-specific differences could provide insights into the metabolic adaptations that contribute to the distinct virulence characteristics of classical and hypervirulent K. pneumoniae.

What are the key unanswered questions regarding CbiN function and regulation?

Despite advances in understanding cobalt transport systems, several critical questions about CbiN remain unanswered:

• Structural dynamics during transport:

  • How does CbiN change conformation during the transport cycle?

  • What residues directly coordinate cobalt ions?

  • How do CbiM and CbiN interact to form a functional transport channel?

• Regulatory mechanisms:

  • How is cbiN expression regulated in response to environmental cobalt availability?

  • Do riboswitches control cbiN expression in K. pneumoniae as they do for other cobalt transporters in bacteria ?

  • What transcription factors influence cbiN expression during infection?

• Functional redundancy:

  • Are there alternative cobalt transport systems in K. pneumoniae that can compensate for CbiN deficiency?

  • What is the hierarchy of metal transporters under different environmental conditions?

• Host-pathogen interactions:

  • Does the host specifically target cobalt acquisition during K. pneumoniae infection?

  • Can antibodies or innate immune factors inhibit CbiN function?

• Therapeutic potential:

  • Is it possible to develop small molecule inhibitors specific to CbiN?

  • Would targeting cobalt transport be an effective strategy against multidrug-resistant K. pneumoniae?

Addressing these questions would significantly advance our understanding of bacterial metal homeostasis and potentially reveal new antimicrobial strategies.

What emerging technologies show promise for advancing CbiN research?

Several cutting-edge technologies are particularly well-suited for investigating the remaining questions about CbiN:

• Single-molecule techniques:

  • Single-molecule FRET to observe conformational changes during transport

  • Nanodiscs combined with atomic force microscopy to study CbiN in a lipid environment

  • Single-molecule force spectroscopy to measure metal binding affinities

• Advanced imaging approaches:

  • Super-resolution microscopy to visualize CbiN distribution and dynamics in bacterial membranes

  • Correlative light and electron microscopy to link function with ultrastructure

  • Live-cell metal imaging with genetically encoded sensors

• High-throughput functional studies:

  • CRISPR interference screens to identify genetic interactions with cbiN

  • Deep mutational scanning to comprehensively map structure-function relationships

  • Synthetic biology approaches to create minimal cobalt transport systems

• Systems biology integration:

  • Multi-omics approaches combining transcriptomics, proteomics, and metabolomics to understand the broader impact of cobalt transport

  • Machine learning to identify patterns in metal homeostasis across diverse bacterial species

  • Computational modeling of cobalt flux in the context of whole-cell metabolism

• Translational applications:

  • Fragment-based drug discovery targeting CbiN

  • Development of CbiN-specific antibodies as research tools and potential therapeutics

  • Engineering of probiotics with modified cobalt utilization to compete with pathogens

These emerging technologies promise to overcome current limitations in studying membrane transport proteins and provide unprecedented insights into the function and regulation of CbiN in bacterial physiology and pathogenesis.

What is the recommended protocol for functional reconstitution of CbiN in liposomes?

For researchers aiming to study CbiN function in a controlled membrane environment, the following protocol outline is recommended:

• Protein purification:

  • Express recombinant CbiN with a removable affinity tag

  • Extract from membranes using mild detergents (DDM, LMNG, or UDM)

  • Purify using affinity chromatography followed by size exclusion

  • Verify purity by SDS-PAGE and Western blotting

• Liposome preparation:

  • Prepare lipid mixture (typically E. coli polar lipid extract with 10-15% cholesterol)

  • Form large unilamellar vesicles by extrusion through 400 nm filters

  • Pre-load vesicles with cobalt chelators if performing transport assays

• Protein reconstitution:

  • Mix purified CbiN with liposomes at protein:lipid ratio of 1:100 to 1:500 (w/w)

  • Remove detergent gradually using Bio-Beads or dialysis

  • Verify reconstitution by freeze-fracture electron microscopy or density gradient centrifugation

• Functional validation:

  • Measure cobalt uptake using radioisotopes or fluorescent indicators

  • Compare activity with reconstituted CbiMN complex vs. CbiN alone

  • Assess the effect of pH, temperature, and competing metals on transport

• Data analysis:

  • Calculate transport kinetics (Km, Vmax) for cobalt uptake

  • Determine specificity by comparing transport rates for different metal ions

  • Model the energetics of transport using appropriate equations for facilitated diffusion or active transport

This protocol provides a controlled system for studying CbiN function outside the complexities of the cellular environment, allowing for precise manipulation of experimental variables.

How can researchers effectively study CbiN interactions with other components of the transport system?

To investigate protein-protein interactions involving CbiN:

• Co-immunoprecipitation approaches:

  • Express epitope-tagged versions of CbiM, CbiN, CbiQ, and CbiO

  • Perform pull-down assays under native conditions

  • Identify interaction partners by Western blotting or mass spectrometry

  • Use crosslinking to stabilize transient interactions

• Genetic interaction studies:

  • Construct strains with combinations of deletions/mutations in cbiM, cbiN, cbiQ, and cbiO

  • Assess epistatic relationships through phenotypic analysis

  • Use bacterial two-hybrid systems to screen for specific interactions

• Advanced biophysical methods:

  • Microscale thermophoresis to measure binding affinities between purified components

  • Surface plasmon resonance to monitor real-time interactions

  • Hydrogen-deuterium exchange mass spectrometry to map interaction interfaces

  • FRET-based assays using fluorescently labeled components

• Structural biology approaches:

  • Cryo-EM of the assembled CbiMNQO complex

  • X-ray crystallography of subcomplexes (e.g., CbiMN)

  • Cross-linking mass spectrometry to identify proximity relationships

• Computational prediction and validation:

  • Molecular docking to predict interaction interfaces

  • Molecular dynamics simulations to assess stability of predicted complexes

  • Mutagenesis of predicted interface residues followed by functional assays

By combining these complementary approaches, researchers can build a comprehensive understanding of how CbiN functions within the larger CbiMNQO transport system, identifying key residues and conformational changes involved in the transport mechanism.