Recombinant Escherichia coli Uncharacterized protein ytfB (ytfB)

What is YtfB and where is it located in Escherichia coli cells?

YtfB is a protein of approximately 25 kDa that localizes primarily to the outer membrane of E. coli cells. Experimental evidence using FLAG-fusion constructs with expression driven by the native promoter has confirmed its predominant association with the outer membrane fraction, despite lacking a traditional prokaryotic signal sequence . This unusual localization pattern is similar to its homolog OapA in Haemophilus influenzae, which is present in both inner and outer membranes despite also lacking a computationally predicted signal sequence .

What domains are present in the YtfB protein structure?

YtfB contains several key structural elements:

• A hydrophobic transmembrane domain between residues 33 and 49

• A LysM-like domain at the extreme C-terminus

• A short sequence spanning the transmembrane domain proximal to the N-terminus that shares homology with OapA from H. influenzae

The LysM-like domain is particularly significant as these domains are involved in binding to polysaccharides found on bacterial, plant, and eukaryotic cell surfaces, and are present in proteins with various functions including adherence and virulence .

What is the evolutionary distribution of YtfB across bacterial species?

Phylogenetic analysis using BLAST and JackHMMER has revealed that YtfB is found primarily in Gamma-proteobacteria, with sequence identity ranging between 23% and 100% to identified homologues. Single homologues have also been identified in Epsilon-proteobacteria, Firmicutes, and Actinobacteria . The Pasteurellaceae family (which includes H. influenzae) is considered the probable ancestor based on identification of ancestral sequences. Within the Enterobacteriaceae family, Escherichia and Shigella are the dominant species containing highly conserved YtfB homologues .

How does YtfB interact with other cell division proteins, and what experimental techniques can validate these interactions?

YtfB has been shown to interact with several cell division proteins, most notably DamX. These interactions have been identified through multiple methodologies:

• Protein-protein interaction screening: Analysis using STRING predicts a moderate interaction between YtfB and the cell division protein DamX .

• Phenotypic characterization: YtfB exhibits similar phenotypic characteristics to DamX when studying cell division defects .

• Genetic interaction studies: The substantial effect of YtfB on cell division becomes evident when ΔytfB is combined with ΔdedD, suggesting functional interaction between these proteins .

To validate these interactions, researchers should employ:

• Bacterial two-hybrid assays

• Co-immunoprecipitation followed by mass spectrometry

• Fluorescence resonance energy transfer (FRET) with tagged proteins

• In vitro binding assays with purified components

What experimental design approaches are recommended for studying YtfB's localization during cell division?

When designing experiments to study YtfB localization during cell division, researchers should consider:

• Fluorescent protein fusions: Create N- and C-terminal fusions to fluorescent proteins (ensuring the fusion doesn't disrupt protein function)

• Time-lapse microscopy: Monitor localization throughout the cell cycle using:

  • Confocal microscopy

  • Super-resolution techniques (STORM, PALM)

  • Correlative light and electron microscopy

• Co-localization studies: Include markers for:

  • FtsZ ring (primary division marker)

  • Other divisome components (FtsI, FtsQ)

  • Membrane markers

• Control experiments:

  • Validate fusion protein functionality through complementation studies

  • Ensure expression levels match physiological conditions

  • Use multiple tag types to rule out tag-specific artifacts

• Experimental design considerations:

  • Use between-subjects designs when comparing wild-type and mutant strains

  • Apply within-subjects designs when tracking localization over time

  • Control environmental variables that may affect cell division

What glycan structures does YtfB bind to and what experimental methods revealed these interactions?

YtfB demonstrates high-affinity binding to specific glycan structures, particularly N-acetylglucosamine (GlcNAc) and mannobiose glycans. These interactions were characterized through multiple complementary techniques:

• Glycan microarray analysis: Purified recombinant His-tagged BW25113 YtfB (C-terminal region, residues 52-212) was incubated with a glycan array of 415 distinct glycan structures, revealing binding to 10 unique moieties .

• Surface plasmon resonance (SPR): This technique determined specific binding affinities, with hexaacetylchitohexaose showing the highest affinity to YtfB with a KD of 24.8 nM ± 5.3 nM .

• Yeast agglutination assays: Deletion of ytfB in UTI89 resulted in a 10-fold increase in the number of bacteria needed to agglutinate yeast compared to wild type, indicating reduced binding to mannosylated glycoproteins on yeast cell surfaces .

A comprehensive comparison of glycan binding profiles between UTI89 wild-type and ΔytfB mutant cells revealed:

• UTI89ΔytfB cells bound to more glycans (103) compared to UTI89 cells (85)

• 39 glycans were bound by both strains

• Two specific glycans showed binding by both wild type cells and recombinant YtfB protein, but not the UTI89ΔytfB mutant

How does YtfB contribute to uropathogenic E. coli pathogenesis?

YtfB plays a specific role in the pathogenesis of uropathogenic E. coli (UPEC), particularly in kidney cell adhesion and potentially in ascending urinary tract infections:

• Tissue-specific adhesion: Loss of ytfB results in a reduction in the ability of UPEC strain UTI89 to adhere to human kidney cells, but not to bladder cells, indicating a specific role in the initial adherence stage of ascending urinary tract infections .

• Role in dispersal from host cells: TraDIS analysis identified ytfB as strongly predicted to be important during the dispersal stage of UPEC infection cycles .

• Potential cooperative function with DedD: Both YtfB and DedD have extracytoplasmic glycan-binding capacities and are important during dispersal from host cells. They may play roles in stabilizing peptidoglycan and cell division or the cell envelope during envelope stress experienced by UPEC in the latter stages of the infection cycle .

• Connection to motile-sessile lifestyle transition: Results suggest a role for YtfB in the switch from a motile to a sessile lifestyle in the environment of the urinary tract, which may be additional or complementary to its role in cell division .

What are the recommended approaches for expressing and purifying recombinant YtfB for functional studies?

Based on successful approaches in the literature, the following protocol is recommended:

• Expression construct design:

  • Clone the C-terminal region of YtfB (residues 52-212, containing the LysM-like domain) for soluble expression

  • Add an N-terminal His6-tag for purification

  • Consider using pET-based expression systems in E. coli BL21(DE3) or similar strains

• Expression conditions:

  • Induce with IPTG (0.1-0.5 mM) at lower temperatures (16-25°C) to enhance proper folding

  • Extend expression time to 16-20 hours for maximum yield

  • Supplement with glucose to reduce basal expression prior to induction

• Purification strategy:

  • Initial capture using Ni-NTA affinity chromatography

  • Secondary purification using size exclusion chromatography

  • Consider ion exchange chromatography if additional purity is required

• Quality control:

  • Verify purity by SDS-PAGE (expect ~25 kDa band)

  • Confirm identity by Western blot and/or mass spectrometry

  • Assess folding by circular dichroism to ensure native-like secondary structure

• Functional validation:

  • Verify glycan binding activity using surface plasmon resonance with known ligands

  • Compare activity to positive controls (if available)

How should researchers design experiments to study YtfB-glycan interactions?

When designing experiments to study YtfB-glycan interactions, researchers should follow these methodological guidelines:

• Experimental design framework:

  • Define independent variables (glycan structures, concentrations)

  • Define dependent variables (binding affinity, specificity)

  • Control for extraneous variables (temperature, pH, buffer composition)

• Glycan binding analysis techniques:

  Technique	Advantages	Limitations	Best For
  Glycan microarray	High-throughput screening of multiple glycans	Semi-quantitative	Initial screening
  Surface plasmon resonance	Real-time kinetics, quantitative KD values	Requires specialized equipment	Detailed binding kinetics
  Isothermal titration calorimetry	Direct measurement of thermodynamics	Sample intensive	Thermodynamic parameters
  Bio-layer interferometry	Real-time kinetics, lower sample requirements	Lower sensitivity than SPR	Rapid screening
  Fluorescence anisotropy	Solution-based measurements	Requires fluorescent labeling	Competition assays

• Controls to include:

  • Positive control (known glycan binder with similar specificity)

  • Negative control (protein with no glycan binding activity)

  • Competitive inhibition with free sugars

  • Denatured protein control

• Validation approaches:

  • Compare recombinant protein results with whole-cell binding assays

  • Confirm specificity through mutation of predicted binding residues

  • Cross-validate between multiple binding techniques

• Data analysis considerations:

  • Apply appropriate binding models (1:1, cooperative, multiple site)

  • Use statistical approaches to identify significant differences

  • Consider creating comprehensive glycan binding profiles

How should researchers address contradictory data in YtfB functional studies?

When encountering contradictory data in YtfB functional studies, researchers should apply a structured approach to resolve inconsistencies:

• Classify contradiction patterns using the (α, β, θ) notation where:

  • α represents the number of interdependent items

  • β represents the number of contradictory dependencies

  • θ represents the minimal number of required Boolean rules to assess these contradictions

• Methodological reconciliation strategies:

  • Examine differences in experimental conditions (growth conditions, strain backgrounds)

  • Consider post-translational modifications that may vary between experimental systems

  • Investigate potential moonlighting functions under different conditions

  • Evaluate whether YtfB has strain-specific functions (K-12 vs. pathogenic strains)

• Resolving contradictions between cellular localization data:

  • YtfB has been reported in different membrane locations

  • Validate using multiple complementary techniques (fractionation, microscopy)

  • Consider dynamic localization depending on growth phase or conditions

• Reconciling dual functions in cell division and adhesion:

  • Design experiments that can simultaneously monitor both functions

  • Investigate whether specific domains mediate different functions

  • Create domain-specific mutations to separate functions

  • Consider environmental triggers that may shift function

• Data integration approaches:

  • Develop weighted scoring systems for evidence quality

  • Apply Boolean minimization techniques to identify minimal rule sets

  • Use probabilistic models to account for condition-dependent functions

What bioinformatic tools and approaches are most useful for YtfB sequence and structure analysis?

Researchers studying YtfB should utilize the following bioinformatic tools and approaches:

• Sequence analysis tools:

  • BLAST and JackHMMER for homology identification

  • MEGA X for phylogenetic tree construction

  • TMHMM and HMMTOP for transmembrane domain prediction

  • SignalP for signal peptide prediction

  • InterProScan for domain annotation

• Structural prediction approaches:

  • AlphaFold2 for tertiary structure prediction

  • PSIPRED for secondary structure prediction

  • ConSurf for evolutionary conservation mapping

  • CASTp for binding pocket prediction

  • Molecular dynamics simulations to explore conformational flexibility

• Functional prediction tools:

  • STRING for protein-protein interaction network analysis

  • Pfam and SMART for domain function prediction

  • COACH for ligand binding site prediction

  • ESPript for structure-based sequence alignment visualization

• Data integration strategies:

  • Combine structural and sequence information to identify critical residues

  • Map conservation data onto structural models

  • Integrate phylogenetic information with functional annotations

  • Cross-reference with experimental data from similar proteins

• Visualization approaches:

  • PyMOL or UCSF Chimera for structural visualization

  • Jalview for multiple sequence alignment visualization

  • Cytoscape for interaction network visualization

By applying these comprehensive bioinformatic approaches, researchers can generate testable hypotheses about YtfB function, identify critical residues for mutagenesis, and guide experimental design for further functional characterization.

What is the recommended methodology for generating and validating ytfB mutants?

To generate and validate ytfB mutants, researchers should follow this comprehensive methodology:

• Mutant generation approaches:

  • λ-Red recombination for precise gene deletion in E. coli

  • CRISPR-Cas9 for scarless mutations or deletions

  • Site-directed mutagenesis for specific amino acid changes

  • Transposon mutagenesis for random insertional inactivation

• Essential validation experiments:

  • PCR verification of gene deletion/mutation

  • Sequencing confirmation of the mutation site and surrounding regions

  • RT-qPCR to confirm absence of transcript

  • Western blot to confirm absence of protein (if antibodies available)

  • Genetic complementation to verify phenotype reversibility

• Phenotypic characterization framework:

  • Growth curves in multiple media conditions (LB, M9-glycerol)

  • Cell morphology analysis via microscopy

  • Division defect quantification (cell length, FtsZ ring formation)

  • Adhesion assays with relevant cell types (kidney, bladder cells)

  • Glycan binding profiles comparison with wild-type

• Controls to include:

  • Wild-type parent strain

  • Complemented mutant strain

  • Known cell division mutants (for comparison)

  • Strains with deletions in related genes (e.g., dedD)

How can TraDIS (Transposon Directed Insertion-site Sequencing) be applied to study YtfB function in different infection models?

TraDIS is a powerful high-throughput technique for identifying gene function in complex environments. For studying YtfB, TraDIS can be applied as follows:

• TraDIS experimental design considerations:

  • Generate saturating transposon library in relevant E. coli strains

  • Select appropriate infection models (cell culture, animal models)

  • Design sampling timepoints to capture different infection stages

  • Include in vitro controls (standard media, stress conditions)

• Specific applications for YtfB research:

  • Identify genetic interactions with ytfB using double mutant analysis

  • Map the complete network of genes required during different stages of infection

  • Compare requirements between bladder and kidney infection models

  • Identify condition-specific functions of YtfB

• Analysis and validation approach:

  • Calculate log fold-change between conditions to identify differential fitness effects

  • Apply statistical thresholds to identify significant hits

  • Validate TraDIS results with targeted mutants (as demonstrated with hisF, neuC, pdhR, yggB, and ykgC in UTI89)

  • Conduct competitive infections with fluorescently labeled strains to confirm fitness effects

• Data interpretation framework:

  • Group genes by functional categories

  • Identify enriched pathways

  • Compare with published datasets

  • Generate network models of genetic interactions

The application of TraDIS has already yielded valuable insights into YtfB function, identifying it as a strongly predicted gene in UPEC dispersal from host cells .

What structural biology techniques are most appropriate for studying YtfB's interaction with glycans and other proteins?

For comprehensive structural characterization of YtfB interactions, researchers should consider these techniques:

• X-ray crystallography:

  • Most appropriate for obtaining high-resolution structures of YtfB alone or in complex with ligands

  • Has been successfully used to determine the structure of a related protein (YhcB) at 2.8 Å resolution, revealing a unique tetrameric α-helical coiled-coil structure

  • Challenges include obtaining diffraction-quality crystals of membrane-associated proteins

• Cryo-electron microscopy (cryo-EM):

  • Valuable for larger complexes (YtfB with interacting division proteins)

  • Does not require crystallization

  • Can capture multiple conformational states

• NMR spectroscopy:

  • Useful for studying dynamics of YtfB-glycan interactions

  • Can determine binding sites through chemical shift perturbation

  • Limited by protein size constraints

• Small-angle X-ray scattering (SAXS):

  • Provides low-resolution envelope of YtfB in solution

  • Useful for confirming oligomeric state

  • Can detect conformational changes upon ligand binding

• Hydrogen-deuterium exchange mass spectrometry (HDX-MS):

  • Maps protein-protein and protein-glycan interaction interfaces

  • Detects conformational changes upon binding

  • Requires less protein than crystallography or NMR

The combination of these complementary techniques would provide a comprehensive understanding of YtfB structure and interactions.

How should researchers interpret contradictory findings between in vitro binding studies and whole-cell functional assays with YtfB?

When faced with contradictions between in vitro binding data and cellular functional assays, researchers should consider:

• Potential sources of discrepancy:

  • Protein conformation differences between recombinant and native forms

  • Missing cofactors or binding partners in reconstituted systems

  • Different environmental conditions (pH, ionic strength)

  • Presence of competing ligands in cellular context

  • Concentration differences between in vitro and in vivo settings

• Reconciliation strategies:

  • Conduct dose-response experiments across a wide concentration range

  • Vary buffer conditions to mimic cellular environments

  • Include relevant binding partners in reconstituted systems

  • Use cell membrane extracts rather than purified components

  • Develop cell-based assays that more closely approximate native conditions

• Specific consideration for YtfB:

  • The glycan microarray analysis revealed that some glycans bound by recombinant YtfB were not detected in whole-cell assays

  • Hexaacetylchitohexaose showed high affinity binding in SPR but was not detected in glycan microarray with recombinant YtfB

  • This suggests YtfB may require native folding on the cell surface for binding to a range of N'acetylglucosamine glycans