Recombinant Escherichia coli O17:K52:H18 UPF0060 membrane protein ynfA (ynfA)

What is YnfA and which protein family does it belong to?

YnfA is a membrane protein classified as an efflux transporter belonging to the Small Multidrug Resistance (SMR) family. This protein is approximately 11.9 kDa in size and contains multiple transmembrane helices that form a structure capable of transporting various compounds across the bacterial cell membrane . The SMR family consists of small proteins that function as efflux pumps, extruding various antimicrobial compounds from the bacterial cell, thereby contributing to antimicrobial resistance mechanisms .

To characterize YnfA:

• Conduct sequence analysis to confirm conserved SMR family motifs

• Perform hydropathy analysis to identify transmembrane domains

• Use prediction algorithms to determine membrane topology

• Compare with other characterized SMR family members like EmrE

How does YnfA contribute to bacterial survival under antimicrobial stress?

YnfA contributes to bacterial survival primarily through its function as an efflux transporter. When bacteria are exposed to antimicrobial compounds, YnfA pumps these substances out of the cell, reducing their intracellular concentration to sub-lethal levels . Studies comparing wild-type strains with YnfA knockout (KO) mutants demonstrate that:

• KO mutants show increased sensitivity to various antimicrobial agents

• Complementation with functional YnfA restores resistance

• Growth curve analysis reveals that while YnfA doesn't significantly affect growth in the absence of antimicrobials, it provides a survival advantage when bacteria are exposed to these compounds

• YnfA-mediated efflux reduces the effective intracellular concentration of toxic compounds, allowing bacteria to survive otherwise lethal exposures

What substrates are transported by YnfA?

YnfA primarily transports cationic compounds, consistent with other SMR family transporters. Experimental studies have confirmed that YnfA can transport several substrates:

These substrates have been verified through comparative studies between wild-type strains, YnfA knockout mutants, and complemented strains, with the knockout mutants showing significantly decreased transport activity . Transport specificity can be determined through:

• Fluorescence-based efflux assays

• MIC90 determination using microtiter plate dilution methods

• Growth assays on plates containing antimicrobial compounds

What methodologies are used for detecting YnfA expression?

YnfA expression can be detected using several molecular biology techniques:

• Western Blotting:

  • YnfA can be tagged with epitopes like Myc or His-tags to facilitate detection

  • Anti-HisA antibodies can detect His-tagged YnfA in Western blot analysis

  • Due to its membrane protein nature, YnfA typically displays anomalous migration patterns on SDS-PAGE gels, running at a slightly higher apparent molecular weight than its calculated mass of 11.9 kDa

• Quantitative RT-PCR:

  • Specific primers targeting YnfA mRNA can quantify expression levels

  • Normalization to housekeeping genes provides relative expression data

• Reporter Gene Fusions:

  • YnfA promoter fused to reporter genes (GFP, lacZ) can monitor expression

  • Enables real-time monitoring of expression under various conditions

• Mass Spectrometry:

  • Targeted proteomics approaches can quantify YnfA protein levels

  • Label-free or isotope-labeled methods provide absolute quantification

What approaches are most effective for generating YnfA knockout mutants?

Creating YnfA knockout mutants requires precise genetic manipulation techniques:

• Gene Targeting Strategy:

  • Design targeting constructs that replace the YnfA coding sequence with a selectable marker

  • Include 40-50bp homology arms flanking the YnfA gene

  • Use antibiotic resistance cassettes (kanamycin, chloramphenicol) as selection markers

• Recombination Methods:

  • Lambda Red recombination system for efficient gene replacement in E. coli and related species

  • CRISPR-Cas9 system for precise genomic editing

  • Homologous recombination with suicide vectors for species lacking efficient recombination systems

• Confirmation Methods:

  • PCR verification of the deleted region

  • Sequencing of the junction regions

  • Western blotting to confirm absence of the protein

  • RT-PCR to verify absence of transcript

• Complementation Construction:

  • Generate a plasmid-based complementation system (such as pBAD-Myc-HisA vector system)

  • Include inducible promoters for controlled expression

  • Include epitope tags for detection and purification

The effectiveness of the knockout can be assessed through functional assays, such as antimicrobial susceptibility testing and transport assays using fluorescent substrates like ethidium bromide and acriflavine .

How can site-directed mutagenesis be optimized for studying YnfA structure-function relationships?

Optimizing site-directed mutagenesis for YnfA structure-function studies involves:

• Target Selection Based On:

  • Computational structure predictions

  • Sequence conservation analysis across bacterial species

  • Comparison with homologous proteins like EmrE

  • Functional domains identified through predictive algorithms

• Mutagenesis Strategy:

  • PCR-based site-directed mutagenesis with high-fidelity polymerases

  • Conservative substitutions for transmembrane domains to maintain structural integrity

  • Alanine scanning of key residues (E15, G18, Y60, Y63) to assess functional significance

  • Charge reversals to test electrostatic interactions

• Validation Protocol:

  • Sequence verification of mutations

  • Expression confirmation via Western blotting with anti-HisA antibodies

  • Functional assessment through transport assays

  • Resistance profiling using microtiter plate assays

• Data Analysis Framework:

  • Correlate structural position with functional impact

  • Compare with equivalent mutations in homologous proteins

  • Create comprehensive mutation-function matrices

Based on previous studies, mutations in E15, G18, and Y60 significantly impair transport function and resistance capabilities, identifying these as critical functional residues in YnfA .

What are the optimal conditions for measuring YnfA transport activity?

Measuring YnfA transport activity requires carefully optimized conditions:

For fluorescence-based transport assays:

• Load cells with fluorescent substrate (e.g., ethidium bromide, acriflavine)

• Wash cells to remove extracellular substrate

• Energize cells with glucose to activate transport

• Monitor decreasing fluorescence over time (indicating efflux)

• Compare wild-type, knockout, and complemented strains

• Calculate transport rates from the fluorescence decay curves

This approach provides a reliable measure of YnfA-specific transport activity, particularly when combined with inhibitor studies to confirm specificity.

How can protein-substrate interactions of YnfA be characterized at the molecular level?

Characterizing YnfA-substrate interactions at the molecular level involves:

• Computational Approaches:

  • Molecular docking simulations using predicted 3D structures

  • Molecular dynamics simulations to analyze binding pocket dynamics

  • Quantum mechanics calculations for binding energy estimation

  • Pharmacophore modeling to identify key interaction features

• Experimental Techniques:

  • Isothermal titration calorimetry (ITC) for binding affinities

  • Surface plasmon resonance (SPR) for real-time binding kinetics

  • Fluorescence quenching assays to detect substrate binding

  • Cross-linking studies coupled with mass spectrometry

• Mutagenesis Studies:

  • Systematic mutation of predicted binding pocket residues

  • Evaluation of transport activity using fluorescence-based assays

  • Correlation of mutation effects with computational predictions

  • Competition assays between different substrates

Research has identified key residues (E15, G18, Y60) that likely participate in substrate binding or transport pathway formation, based on functional impairment when mutated to alanine . These residues provide starting points for detailed characterization of the molecular basis of substrate recognition.

What computational methods are most reliable for predicting YnfA structure?

The most reliable computational methods for predicting YnfA structure involve:

• Template-Based Modeling:

  • Homology modeling using EmrE as a template, which has a solved functional structure

  • Threading approaches that align YnfA sequence to multiple structural templates

  • Fragment-based assembly using conserved structural motifs

• Advanced Prediction Algorithms:

  • AlphaFold2 for deep learning-based structure prediction

  • RoseTTAFold for neural network-based modeling

  • MODELLER for comparative modeling with refinement

  • I-TASSER for iterative threading assembly

• Refinement and Validation:

  • Molecular dynamics simulations in explicit membrane environments

  • Energy minimization with appropriate membrane protein force fields

  • Structure validation using PROCHECK, VERIFY3D, and ERRAT

  • Comparison with experimental crosslinking constraints

• Membrane Protein-Specific Approaches:

  • Hydrophobicity analysis to identify transmembrane regions

  • Topology prediction using TMHMM or TOPCONS

  • Membrane insertion energy calculations

  • Lipid-protein interaction modeling

The 3D structure prediction of YnfA has been successfully performed using computational techniques based on the model transporter EmrE, allowing for identification of critical functional residues for mutagenesis studies .

How do mutations in YnfA affect its transport function and resistance profile?

Mutations in YnfA have varying effects on transport function and resistance profile:

Before conducting mutational studies, expression of all YnfA mutants should be confirmed by Western blot with anti-HisA antibody to ensure that reduced function is not due to expression defects . Functional assessment requires:

• MIC90 determination for various antimicrobial compounds

• Transport assays using fluorescent substrates

• Growth assays on solid media containing antimicrobials

• Comparison with wild-type YnfA and empty vector controls

These mutagenesis studies indicate that E15, G18, and Y60 are critical residues for YnfA function, while other mutations had negligible effects on transport activity and resistance conferment capabilities .

How does the topology of YnfA in the bacterial membrane relate to its function?

The topology of YnfA in the bacterial membrane directly relates to its function:

The topology can be experimentally determined using:

• Cysteine accessibility methods

• Epitope insertion mapping

• Fusion protein approaches with reporter genes

• Protease protection assays

How does YnfA compare structurally and functionally with other members of the SMR family?

Comparison of YnfA with other SMR family members:

Methodologies for comparative analysis include:

• Sequence alignment and conservation analysis

• Structural superposition of predicted or experimental models

• Cross-complementation studies in knockout strains

• Comparative substrate specificity profiling

• Evolutionary analysis to determine relatedness

YnfA shares key structural and functional characteristics with EmrE, including similar size, predicted transmembrane topology, and transport of cationic compounds. The conservation of critical residues (like the glutamate in TM1) suggests a common evolutionary origin and transport mechanism among SMR family proteins .

What is the spectrum of antimicrobial resistance conferred by YnfA expression?

YnfA confers resistance to a spectrum of antimicrobial compounds:

The resistance profile is determined through:

• MIC90 (Minimum Inhibitory Concentration) assays using microtiter plate dilution methods

• Drug sensitivity assays on plates containing various concentrations of antimicrobials

• Growth curve analysis in the presence of sub-inhibitory concentrations of compounds

• Transport assays using fluorescent or radiolabeled substrates

YnfA knockout mutants show increased susceptibility to these compounds, while complementation restores the resistance phenotype, confirming YnfA's direct role in conferring antimicrobial resistance .

How does YnfA expression respond to antimicrobial stress conditions?

The regulation of YnfA expression under antimicrobial stress involves multiple levels of control:

• Transcriptional Regulation:

  • YnfA expression is likely upregulated in response to certain antimicrobial compounds

  • May be controlled by stress response regulators like SoxS, MarA, or RamA

  • Promoter analysis can identify potential binding sites for stress-responsive transcription factors

  • ChIP-seq can confirm transcription factor binding under stress conditions

• Post-Transcriptional Control:

  • mRNA stability may be enhanced under stress conditions

  • Small RNAs might regulate translation efficiency

  • RNA-seq and qRT-PCR can quantify transcript levels under various stress conditions

  • Ribosome profiling can assess translation efficiency

• Post-Translational Regulation:

  • Protein stability and membrane insertion may be enhanced during stress

  • Possible interaction with chaperones or other membrane proteins

  • Western blotting can track protein levels under stress conditions

  • Pulse-chase experiments can measure protein turnover rates

To study YnfA expression under stress:

• Expose bacteria to sub-inhibitory concentrations of antimicrobials

• Measure mRNA and protein levels at various time points

• Use reporter gene fusions to monitor promoter activity

• Compare expression patterns in regulatory mutants

How does YnfA interact with other bacterial resistance mechanisms?

YnfA operates within a network of resistance mechanisms:

• Synergy with Other Efflux Systems:

  • YnfA may work in concert with larger RND-family pumps (like AcrAB-TolC)

  • Sequential action where YnfA removes compounds from the cytoplasm while other pumps export from the periplasm

  • Multiple pump knockouts can reveal synergistic relationships

  • Overexpression studies can identify compensatory mechanisms

• Interaction with Membrane Permeability Barriers:

  • YnfA's effectiveness is enhanced by reduced porin expression

  • Complementary relationship with LPS modifications that reduce membrane permeability

  • Combined knockout studies can reveal additive or synergistic effects

  • Permeability assays can measure combined barrier function

• Relationship with Target Modification Mechanisms:

  • YnfA provides time for target-based resistance mechanisms to emerge

  • Reduces effective intracellular concentration, allowing survival with partially modified targets

  • Evolution experiments can track development of additional resistance mechanisms

  • Time-kill curves can demonstrate protection during target modification

• Role in Biofilm Formation and Persistence:

  • Potential contribution to antimicrobial tolerance in biofilms

  • May facilitate survival during initial exposure, allowing biofilm formation

  • Biofilm formation assays can compare wild-type and YnfA mutants

  • Persister formation frequency can be assessed in various genetic backgrounds

Research approaches to study these interactions include:

• Construction of multiple deletion mutants

• Combination therapy testing

• Transcriptomic and proteomic profiling

• Evolution experiments under antimicrobial selection

What strategies could potentially inhibit YnfA to restore antimicrobial sensitivity?

Potential strategies to inhibit YnfA function:

Development methodology:

• Virtual screening against predicted YnfA structure

• Fragment-based screening approaches

• High-throughput functional assays using fluorescent substrates

• Validation in YnfA-expressing strains vs. knockout controls

• Synergy testing with current antimicrobials

The development of YnfA inhibitors would likely restore sensitivity to various antimicrobial compounds, as demonstrated by the increased susceptibility of YnfA knockout mutants. Targeting the critical residues identified through mutagenesis studies (E15, G18, Y60) offers a promising approach for rational inhibitor design .

How conserved is YnfA across different bacterial species?

YnfA conservation across bacterial species shows specific patterns:

• Distribution Pattern:

  • Highly conserved in Enterobacteriaceae (including E. coli and Shigella)

  • Present in many gram-negative bacteria

  • Homologs with varying sequence identity found in diverse bacterial phyla

  • Phylogenetic analysis can map evolutionary relationships

• Sequence Conservation:

  • Critical functional residues (E15, G18, Y60) show highest conservation

  • Transmembrane domains more conserved than loop regions

  • N and C termini show greater sequence variation

  • Multiple sequence alignment reveals conservation patterns

• Structural Conservation:

  • Four-transmembrane topology conserved across homologs

  • Substrate binding pocket architecture maintained despite sequence variations

  • Dimer interface regions show strong conservation

  • Structural modeling can predict conserved functional elements

• Functional Conservation:

  • Substrate profiles may vary between species

  • Core mechanism of proton-coupled transport preserved

  • Role in antimicrobial resistance consistent across species

  • Heterologous expression studies can assess functional conservation

Methodologies for studying conservation include:

• Genome database mining for YnfA homologs

• Phylogenetic tree construction

• Conservation scoring of individual amino acid positions

• Prediction of selective pressure using dN/dS ratios

How do orthologues of YnfA differ in their substrate specificity across species?

Variation in substrate specificity among YnfA orthologues:

These differences in substrate specificity likely reflect:

• Evolutionary adaptation to different ecological niches

• Selective pressures from exposure to different antimicrobials

• Sequence divergence in substrate binding regions

• Compensatory mutations that maintain function while altering specificity

Research methodologies to compare substrate specificity:

• Heterologous expression of orthologues in a common host

• Standardized transport assays with a panel of potential substrates

• Comparative MIC determination

• Chimeric protein construction to identify specificity determinants

What can phylogenetic analysis reveal about YnfA evolution and function?

Phylogenetic analysis of YnfA reveals:

• Evolutionary History:

  • YnfA likely evolved from an ancient SMR family progenitor

  • Horizontal gene transfer events have shaped its distribution

  • Sequence clustering correlates with bacterial taxonomy

  • Evidence of convergent evolution in some functional residues

• Structure-Function Relationships:

  • Correlation between phylogenetic clustering and substrate preferences

  • Conservation patterns highlight functional constraints

  • Coevolution of specific residue pairs indicates structural interactions

  • Positive selection signatures in substrate binding regions

• Adaptive Significance:

  • Episodes of accelerated evolution correlate with habitat transitions

  • Clinical isolates may show evidence of recent selection

  • Potential correlation with antimicrobial usage patterns

  • Functional divergence following gene duplication events

• Methodological Approaches:

  • Multiple sequence alignment of YnfA homologs

  • Maximum likelihood or Bayesian phylogenetic tree construction

  • Selection analysis using dN/dS ratios

  • Ancestral sequence reconstruction for evolutionary trajectory analysis

How does heterologous expression of YnfA impact antimicrobial resistance in different bacterial hosts?

Heterologous expression of YnfA provides insights into its functional conservation:

• Cross-Species Functionality:

  • E. coli YnfA can function when expressed in other Enterobacteriaceae

  • Expression in more distant species may require optimization

  • Membrane integration efficiency varies by host

  • Functional activity depends on compatibility with host membrane environment

• Resistance Phenotypes:

  • Different hosts show variable levels of resistance enhancement

  • Background resistance mechanisms influence the impact of YnfA

  • Complementation of native efflux knockouts demonstrates functional conservation

  • Host-specific factors may modulate YnfA efficacy

• Expression Optimization:

  • Codon optimization improves expression in divergent hosts

  • Promoter selection affects expression levels

  • Signal sequence modifications may improve membrane targeting

  • Growth conditions influence expression and activity

• Experimental Approaches:

  • Clone YnfA into expression vectors with appropriate promoters

  • Transform into various bacterial species or strains

  • Verify expression using Western blotting

  • Measure resistance profiles using standardized MIC assays

  • Conduct transport assays with fluorescent substrates

Heterologous expression studies demonstrate the conserved functional properties of YnfA across species boundaries, highlighting its fundamental role in antimicrobial resistance and potential as a target for novel inhibitors.