Recombinant Putative yfeABCD regulator yfeE (yfeE)

What is the yfeABCD-yfeE system and what is its biological significance?

The yfeABCD-yfeE system represents a siderophore-independent iron acquisition mechanism originally identified in Yersinia pestis. This system consists of two main components: the yfeABCD operon and the yfeE coding region . The biological significance of this system lies in its central role in bacterial iron acquisition, which is critical for pathogen survival within hosts where iron availability is restricted.

The yfeE protein functions as a regulator of the yfeABCD system, which belongs to the ABC transporter family involved in metal transport . Unlike siderophore-dependent iron acquisition systems, the yfeABCD-yfeE system enables bacteria to obtain iron directly without the production of siderophores, providing an alternative iron acquisition pathway that may be particularly important under specific environmental conditions.

How does the structure of yfeE relate to its function in iron acquisition?

The yfeE protein is a full-length protein (1-184 amino acids) that functions as a regulator of the yfeABCD system . While the detailed three-dimensional structure has not been fully elucidated, the protein appears to contain structural domains typical of bacterial regulatory proteins. Its function is closely associated with the yfeABCD components, which form an ABC transporter system.

The yfeABCD system components have specific roles: YfeA functions as a periplasmic binding protein, YfeB as an ATP-binding protein, and YfeC and YfeD as membrane permease components . Together with YfeE, these proteins facilitate the transport of iron across bacterial membranes. The regulatory function of YfeE likely involves sensing iron availability and modulating the expression or activity of the transport components accordingly.

What homologous systems exist in other bacterial species?

Several homologous systems have been identified in other bacterial species. A notable example is the AfeABCD system in Actinobacillus actinomycetemcomitans . This system, while functionally and organizationally related to the YfeABCD system, represents an independent pathway with its own distinctive characteristics.

The A. actinomycetemcomitans afeABCD iron transport system has been identified in multiple isolates, and its expression is controlled by iron and the ferric uptake regulator (Fur) . The protein products of this locus are related to bacterial ABC transporters involved in metal transport. Experimental evidence shows that transformation of Escherichia coli 1017 iron acquisition mutant with a plasmid harboring afeABCD promoted cell growth under iron-chelated conditions .

Other bacterial species may contain similar systems, although they may be named differently based on their specific characteristics and the organisms in which they are found.

What are the optimal conditions for expressing recombinant yfeE protein?

For optimal expression of recombinant yfeE protein, E. coli expression systems have proven effective. The following considerations are critical for successful expression:

• Expression Vector Selection: Vectors with strong promoters like T7 or tac are recommended. His-tag fusion constructs have been successfully used for purification .

• Host Strain: BL21(DE3) and its derivatives are commonly used due to their reduced protease activity and compatibility with T7 expression systems.

• Induction Conditions:

  • Temperature: Lower temperatures (16-25°C) often yield higher amounts of soluble protein

  • IPTG concentration: 0.1-0.5 mM typically sufficient

  • Induction duration: 4-16 hours depending on temperature

• Media Composition: Rich media like LB or TB are suitable for initial trials, while defined media may be necessary for specific applications or isotope labeling.

• Iron Availability: Since yfeE is involved in iron regulation, controlling iron availability in the growth medium may affect expression levels. Consider using iron-depleted media to potentially enhance expression.

What purification strategies yield the highest purity and functional activity of recombinant yfeE?

A multi-step purification strategy is recommended to obtain high-purity, functionally active recombinant yfeE:

• Initial Capture: Immobilized metal affinity chromatography (IMAC) using Ni-NTA resin is effective for His-tagged yfeE protein .

• Intermediate Purification: Ion exchange chromatography (IEX) based on yfeE's theoretical isoelectric point can remove remaining contaminants.

• Polishing Step: Size exclusion chromatography (SEC) to separate monomeric yfeE from aggregates and eliminate remaining impurities.

• Buffer Optimization:

  • For IMAC: 50 mM Tris-HCl pH 8.0, 300 mM NaCl, 10% glycerol

  • For storage: 25 mM Tris-HCl pH 7.5, 150 mM NaCl, 5% glycerol, 1 mM DTT

• Activity Preservation: Addition of stabilizing agents (glycerol, reducing agents) and maintaining cold temperatures throughout purification are crucial for preserving functional activity.

Protein purity should be assessed by SDS-PAGE and Western blotting, while functional activity can be evaluated through DNA binding assays or iron regulation reporter systems.

How can the ARRIVE guidelines be applied to in vivo studies of yfeE function?

When designing in vivo studies to investigate yfeE function, adherence to the ARRIVE guidelines ensures robust experimental design and reporting. Key considerations include:

• Study Plan Development: Create a detailed ARRIVE study plan before commencing experiments, documenting procedural plans and experimental design strategies that minimize bias .

• Sample Size Calculation: Determine appropriate sample sizes through power analysis based on expected effect sizes from preliminary data to ensure adequate statistical power.

• Randomization and Blinding: Implement proper randomization for treatment assignment and blinding procedures for outcome assessment to minimize bias .

• Control Selection: Include appropriate positive and negative controls, including wild-type strains, yfeE deletion mutants, and complemented strains.

• Refinement of Procedures: Clearly document all procedures to be performed on animals, ensuring compliance with ethical guidelines and focusing on the 3Rs (Replacement, Reduction, Refinement) .

• Experimental Design Assistant: Utilize the NC3Rs Experimental Design Assistant (EDA) to receive tailored advice on study design and create visual representations of the experimental workflow .

• Alternative Models Consideration: Before proceeding with animal experiments, evaluate whether cell culture or other in vitro systems could answer the research question .

For studying yfeE specifically, infection models examining bacterial survival and virulence under iron-limited conditions would be particularly relevant, with careful monitoring of both bacterial and host responses.

How can eQTL analysis be applied to study yfeE regulation networks in bacterial systems?

Expression Quantitative Trait Loci (eQTL) analysis, while more commonly applied in eukaryotic systems, can be adapted to study yfeE regulation networks in bacteria with the following approach:

• Population Design: Create a diverse bacterial population through natural variation or mutagenesis, ensuring sufficient genetic diversity to detect associations.

• Expression Profiling: Measure gene expression across the population under various iron concentration conditions, with particular focus on yfeE and related genes.

• Genotyping: Perform whole-genome sequencing or SNP analysis to identify genetic variations across the population.

• eQTL Mapping: Apply statistical methods to identify genomic regions (eQTLs) associated with expression variation of yfeE and its target genes:

  • cis-eQTLs would be located near the yfeE gene itself

  • trans-eQTLs would represent distant regulatory elements affecting yfeE expression

• Regulatory Network Construction: Use eQTL-guided function-related co-expression analysis to identify putative regulators and construct a gene regulatory network for yfeE .

• Hotspot Analysis: Identify trans-eQTL hotspots that may represent master regulators controlling multiple genes including yfeE .

This approach can reveal the complex regulatory architecture governing yfeE expression and its role in iron acquisition networks, potentially identifying novel regulators and environmental response mechanisms.

What are the current challenges in understanding yfeE-mediated iron transport and how might they be addressed?

Several significant challenges exist in understanding yfeE-mediated iron transport, with corresponding strategies to address them:

• Structural Characterization Challenge: Limited structural information on yfeE.

  • Solution: Apply X-ray crystallography or cryo-EM to determine the three-dimensional structure of yfeE alone and in complex with DNA or other components of the yfeABCD system.

• Regulatory Mechanism Challenge: Unclear how yfeE senses iron and regulates yfeABCD.

  • Solution: Employ chromatin immunoprecipitation (ChIP-seq) to identify yfeE binding sites, and use reporter assays with mutated binding sites to characterize the exact regulatory mechanisms.

• Protein-Protein Interaction Challenge: Limited understanding of how yfeE interacts with other components.

  • Solution: Use techniques like bacterial two-hybrid systems, co-immunoprecipitation, and surface plasmon resonance to map interaction networks.

• In vivo Relevance Challenge: Difficulty in assessing the contribution of yfeE to virulence.

  • Solution: Generate clean deletion and point mutation variants, complemented strains, and conduct comparative transcriptomics and proteomics under various iron conditions.

• Redundancy Challenge: Multiple iron acquisition systems may mask yfeE contribution.

  • Solution: Create multiple gene deletion mutants to eliminate redundant systems and isolate yfeE-specific effects.

• Host-Pathogen Interface Challenge: Understanding how yfeE function is affected by host iron-sequestration.

  • Solution: Develop cell culture and animal models that accurately mimic host iron restrictions encountered during infection.

Addressing these challenges requires integrative approaches combining structural biology, molecular genetics, systems biology, and infection models.

How does yfeE function differ between Yersinia pestis and other pathogenic bacteria with homologous systems?

The function of yfeE shows both conserved and divergent features across bacterial species with homologous systems:

Comparative genomic and functional analyses across multiple bacterial species can provide insights into the evolution and specialization of yfeE-based iron acquisition systems, potentially revealing species-specific adaptations that could be targeted for antimicrobial development.

What are common pitfalls in yfeE protein expression and how can they be overcome?

Researchers frequently encounter several challenges when expressing recombinant yfeE protein:

• Insolubility Issues:

  • Problem: Formation of inclusion bodies due to misfolding

  • Solutions:

    • Lower induction temperature (16-20°C)

    • Reduce IPTG concentration (0.1-0.2 mM)

    • Co-express with molecular chaperones (GroEL/GroES)

    • Use solubility-enhancing fusion tags (SUMO, MBP)

• Low Expression Levels:

  • Problem: Inadequate protein production

  • Solutions:

    • Optimize codon usage for expression host

    • Test multiple promoter systems

    • Evaluate different E. coli strains (BL21, Rosetta, Arctic Express)

    • Screen for optimal media compositions

• Proteolytic Degradation:

  • Problem: Protein instability leading to degradation

  • Solutions:

    • Add protease inhibitors during purification

    • Use protease-deficient host strains

    • Maintain cold temperatures throughout processing

    • Optimize buffer conditions (pH, salt concentration)

• Loss of Function:

  • Problem: Purified protein lacks regulatory activity

  • Solutions:

    • Verify proper folding using circular dichroism

    • Include metal ions (especially iron) in purification buffers

    • Test different reducing agents to maintain cysteine residues

    • Develop activity assays to monitor function throughout purification

• Batch-to-Batch Variability:

  • Problem: Inconsistent yields and activities

  • Solutions:

    • Standardize growth conditions precisely

    • Develop detailed SOPs for expression and purification

    • Implement quality control checkpoints throughout the process

    • Create master cell banks for consistent starting material

Implementing these strategies systematically can significantly improve the success rate of yfeE protein expression and purification for functional studies.

What analytical methods are most effective for characterizing yfeE-DNA interactions?

Several complementary analytical methods can effectively characterize yfeE-DNA interactions:

• Electrophoretic Mobility Shift Assay (EMSA):

  • Provides qualitative assessment of binding

  • Can determine apparent dissociation constants (Kd)

  • Allows competition studies to assess binding specificity

  • Best performed with purified yfeE protein and labeled DNA fragments containing putative binding sites

• DNase I Footprinting:

  • Identifies precise DNA sequences protected by yfeE binding

  • Reveals binding site architecture and potential cooperative binding

  • Can detect multiple binding sites within regulatory regions

• Surface Plasmon Resonance (SPR):

  • Provides real-time binding kinetics (kon and koff rates)

  • Determines binding affinity under various conditions

  • Assesses how cofactors (iron, other metals) influence binding

• Chromatin Immunoprecipitation (ChIP):

  • Identifies genome-wide binding sites in vivo

  • When coupled with sequencing (ChIP-seq), provides comprehensive binding profiles

  • Reveals how binding patterns change under different iron conditions

• Isothermal Titration Calorimetry (ITC):

  • Measures thermodynamic parameters of binding

  • Determines stoichiometry of the interaction

  • Assesses enthalpy and entropy contributions to binding

• Microscale Thermophoresis (MST):

  • Requires minimal sample amounts

  • Works in complex solutions

  • Provides accurate Kd values with native protein and DNA

• Reporter Gene Assays:

  • Functional validation of binding sites

  • Assessment of regulatory outcomes of binding

  • Evaluation of how mutations in binding sites affect regulation

A comprehensive characterization should combine several of these methods to build a complete picture of how yfeE interacts with DNA to regulate the yfeABCD system under various iron conditions.

How can researchers effectively design experiments to elucidate the iron-dependent regulatory mechanisms of yfeE?

Designing effective experiments to understand yfeE's iron-dependent regulatory mechanisms requires a systematic approach:

• Iron Responsiveness Characterization:

  • Culture bacteria in defined media with precisely controlled iron concentrations

  • Use iron chelators (2,2'-dipyridyl, EDDA) and iron sources (FeCl3, heme, transferrin)

  • Monitor yfeE expression using qRT-PCR or reporter constructs across iron concentrations

  • Establish dose-response curves for iron-dependent regulation

• Regulatory Element Identification:

  • Perform 5' deletion analysis of the yfeE promoter region

  • Use site-directed mutagenesis to modify putative regulatory elements

  • Combine with reporter assays to identify essential regulatory sequences

  • Confirm direct interactions using in vitro binding assays

• Protein-Protein Interaction Analysis:

  • Investigate interactions between yfeE and other regulatory proteins (Fur, RyhB)

  • Apply bacterial two-hybrid systems or co-immunoprecipitation

  • Use fluorescence resonance energy transfer (FRET) to detect interactions in vivo

  • Map interaction domains through truncation mutants

• Iron Sensing Mechanism:

  • Generate point mutations in potential iron-binding residues

  • Assess the impact on regulation and iron responsiveness

  • Use spectroscopic methods to detect iron binding to purified yfeE

  • Perform structural analysis with and without bound iron

• Integration with Global Iron Regulatory Networks:

  • Conduct RNA-seq under iron-replete and iron-depleted conditions

  • Compare transcriptomes of wild-type and yfeE mutant strains

  • Apply network analysis to position yfeE within the global iron regulon

  • Validate key nodes in the network through directed mutations

• Temporal Dynamics Analysis:

  • Implement time-course experiments following iron availability shifts

  • Use microfluidics combined with fluorescent reporters for single-cell analysis

  • Apply mathematical modeling to describe the dynamics of the system

  • Test model predictions with targeted experiments

A well-designed experimental approach combining these strategies will provide comprehensive insights into how yfeE senses and responds to iron availability to regulate bacterial iron acquisition systems.

What potential exists for targeting yfeE-mediated iron acquisition systems for antimicrobial development?

The essential nature of iron acquisition for bacterial pathogenesis makes yfeE and related systems attractive targets for novel antimicrobial strategies:

• Inhibitor Development Strategies:

  • Structure-based design of small molecules that bind to yfeE and disrupt its regulatory function

  • Peptide inhibitors that interfere with yfeE-DNA or yfeE-protein interactions

  • Allosteric modulators that lock yfeE in an inactive conformation

  • Decoy DNA sequences that sequester yfeE away from its natural targets

• Potential Advantages:

  • Novel mechanism of action distinct from conventional antibiotics

  • Potentially reduced selection for resistance due to targeting virulence rather than growth

  • Possible narrow-spectrum activity targeting specific pathogens while sparing beneficial microbiota

  • Potential for combination therapy with conventional antibiotics for synergistic effects

• Challenges and Considerations:

  • Ensuring sufficient cellular uptake of inhibitors

  • Addressing potential redundancy in bacterial iron acquisition systems

  • Developing appropriate in vitro and in vivo models to evaluate efficacy

  • Optimizing selectivity to avoid targeting human iron-binding proteins

• Therapeutic Applications Beyond Direct Inhibition:

  • Vaccine development using yfeE or related transport components as antigens

  • Diagnostic applications based on detecting yfeE expression as a marker of active infection

  • Iron restriction therapies that enhance host iron sequestration mechanisms

The development of yfeE-targeted therapeutics represents a promising approach that aligns with the current emphasis on antivirulence strategies to combat bacterial infections while potentially reducing selective pressure for resistance.

How might systems biology approaches enhance our understanding of yfeE function in bacterial iron homeostasis?

Systems biology approaches offer powerful tools to comprehensively understand yfeE's role in bacterial iron homeostasis:

• Multi-omics Integration:

  • Combine transcriptomics, proteomics, and metabolomics data from wild-type and yfeE mutant strains

  • Identify direct and indirect effects of yfeE on global gene expression patterns

  • Map metabolic changes resulting from altered iron acquisition

  • Correlate protein abundance changes with transcriptional responses

• Network Analysis and Modeling:

  • Construct gene regulatory networks centered on yfeE using eQTL-guided approaches

  • Identify hub genes and master regulators that interact with yfeE

  • Apply mathematical modeling to predict system behavior under various conditions

  • Validate model predictions through targeted experiments

• Genome-Scale Analyses:

  • Apply transposon insertion sequencing (Tn-seq) to identify genes synthetic with yfeE

  • Perform genome-wide CRISPR interference screens to map genetic interactions

  • Use ChIP-seq to identify genome-wide binding sites for yfeE

  • Integrate with existing databases of iron-responsive genes

• Temporal and Spatial Resolution:

  • Implement time-resolved omics to track dynamic responses to changing iron availability

  • Use single-cell approaches to characterize population heterogeneity in yfeE expression

  • Apply biosensors to monitor iron levels and yfeE activity simultaneously

  • Develop computational models incorporating spatial aspects of iron distribution

• Host-Pathogen Interface Analysis:

  • Study how host iron restriction affects yfeE-mediated responses during infection

  • Identify cross-talk between bacterial and host iron acquisition systems

  • Characterize the impact of inflammation on yfeE regulation

  • Develop dual RNA-seq approaches to simultaneously monitor host and pathogen responses

Systems biology approaches can reveal emergent properties of iron regulatory networks that would not be apparent from reductionist approaches, potentially identifying unexpected connections and novel regulatory mechanisms involving yfeE.

What comparative genomic insights can be gained from studying yfeE evolution across bacterial species?

Comparative genomic analysis of yfeE across bacterial species offers valuable insights into evolution, adaptation, and function:

• Evolutionary Conservation and Divergence:

  • Phylogenetic analysis of yfeE sequences across bacterial phyla

  • Identification of highly conserved domains suggesting critical functional regions

  • Mapping of variable regions that may reflect species-specific adaptations

  • Assessment of selection pressures (purifying vs. positive selection) on different domains

• Genomic Context Analysis:

  • Examination of synteny and gene neighborhood conservation

  • Identification of co-evolved gene clusters suggesting functional relationships

  • Detection of horizontal gene transfer events that may have spread yfeE systems

  • Analysis of regulatory elements in promoter regions across species

• Structure-Function Relationships:

  • Correlation of sequence variations with known functional differences

  • Prediction of critical residues through evolutionary conservation analysis

  • Identification of species-specific structural features that may reflect niche adaptation

  • Development of homology models for species lacking experimental structures

• Host Adaptation Signatures:

  • Comparison of yfeE systems between generalist pathogens and host-restricted species

  • Identification of modifications associated with specific host environments

  • Assessment of yfeE variation in response to different host iron sequestration strategies

  • Analysis of pseudogenization or gene loss in species with alternative iron acquisition mechanisms

• Applications of Comparative Insights:

  • Development of species-specific diagnostics based on unique yfeE features

  • Design of broad-spectrum inhibitors targeting conserved regions

  • Prediction of functional properties in newly sequenced organisms

  • Reconstruction of the evolutionary history of bacterial iron acquisition strategies

Comparative genomic approaches can reveal the molecular basis for species-specific adaptations in iron acquisition systems, providing insights into bacterial pathogenesis and potential vulnerabilities that could be exploited for therapeutic intervention.

What recombinant yfeE protein products are available for research applications?

Researchers have access to several recombinant yfeE protein products for experimental applications:

These commercially available products provide standardized reagents for investigating yfeE function, though researchers should verify protein quality, purity, and functional activity before use in critical experiments. For specialized applications, custom protein production may be necessary to obtain specific constructs, alternative tags, or mutant variants.

What experimental tools and protocols have been established for studying yfeE function?

Several established experimental tools and protocols have been developed for studying yfeE function:

• Genetic Manipulation Systems:

  • Gene deletion protocols using lambda Red recombination

  • Complementation systems with inducible promoters

  • Site-directed mutagenesis approaches for targeted modifications

  • Reporter fusion constructs (lacZ, gfp) for expression analysis

• Expression and Purification Protocols:

  • Optimized expression conditions in E. coli systems

  • Multi-step purification strategies involving IMAC, IEX, and SEC

  • Quality control methods including SDS-PAGE, Western blotting, and mass spectrometry

  • Activity assays to verify functional integrity

• Functional Assays:

  • Growth assays under iron-restricted conditions

  • Iron uptake measurements using radioactive or fluorescent iron sources

  • DNA binding assays (EMSA, footprinting, SPR)

  • Reporter systems for monitoring iron-responsive gene expression

• Structural Analysis Tools:

  • X-ray crystallography methods for protein structure determination

  • NMR approaches for investigating protein dynamics and interactions

  • Hydrogen-deuterium exchange mass spectrometry for conformational analysis

  • Computational modeling tools for structure prediction and analysis

• In Vivo Models:

  • Cell culture infection models to assess iron acquisition during host interaction

  • Animal infection models following ARRIVE guidelines

  • Ex vivo systems using host tissues or fluids

These established tools provide a foundation for investigating yfeE function, though researchers often need to adapt and optimize protocols for their specific experimental systems and questions.

How can researchers effectively design collaborative studies to advance understanding of yfeE across disciplinary boundaries?

Effective cross-disciplinary collaboration on yfeE research requires strategic planning and communication:

• Interdisciplinary Team Assembly:

  • Include experts from microbiology, structural biology, biochemistry, and systems biology

  • Incorporate clinicians for translational relevance

  • Engage computational biologists for data analysis and modeling

  • Consider collaborating with metallomics specialists for iron-specific expertise

• Shared Research Framework Development:

  • Establish common terminology and conceptual understanding

  • Define clear research questions accessible to multiple approaches

  • Develop integrated experimental plans with defined contributions

  • Create standardized protocols for consistency across laboratories

• Complementary Methodology Application:

  • Combine reductionist biochemical approaches with systems-level analyses

  • Integrate in vitro mechanistic studies with in vivo functional validation

  • Apply both experimental and computational approaches to address hypotheses

  • Leverage specialized equipment and expertise across participating laboratories

• Data Integration Strategies:

  • Implement shared data repositories with standardized formats

  • Develop integrated analysis pipelines combining diverse data types

  • Hold regular data integration meetings to synthesize findings

  • Create visualization tools accessible to collaborators from different backgrounds

• Communication and Coordination Mechanisms:

  • Establish regular virtual meetings with structured agendas

  • Use project management tools to track progress and dependencies

  • Create shared electronic laboratory notebooks for transparent documentation

  • Develop collaborative manuscripts with clear contribution statements

Effective cross-disciplinary collaboration can accelerate understanding of yfeE by bringing diverse perspectives and complementary techniques to bear on complex questions about iron acquisition systems and their regulation.