Recombinant Escherichia coli Putative lipoprotein YliF (yliF)

What is YliF and what is its known functional classification in E. coli?

YliF is a putative lipoprotein found in Escherichia coli (strain K12) with Uniprot accession number P75801. Current classification places YliF in the GGDEF family of proteins . The protein is encoded by the yliF gene (locus tags: b0834, JW0818) and has a full-length protein expression region of amino acids 24-442 . Despite significant genomic characterization, YliF's precise biological function remains incompletely characterized. GGDEF domain proteins typically function as diguanylate cyclases involved in bacterial signal transduction, often mediating transitions between motile and sessile lifestyles.

How does YliF compare to other GGDEF domain proteins in bacterial systems?

The GGDEF domain is widely conserved across bacterial species where it functions in cyclic-di-GMP (c-di-GMP) signaling pathways. While many GGDEF proteins are involved in biofilm formation regulation, YliF appears to have unique properties. Research has identified YliF as a suppressor of the synthetic inhibition between pta and recBC genes , suggesting potential involvement in metabolic regulation and DNA repair pathways. Unlike well-characterized GGDEF proteins, YliF's precise signaling targets and conditions activating its function remain under investigation. Comparative analysis of YliF's amino acid sequence with other GGDEF proteins reveals both conserved catalytic motifs and unique structural elements that may contribute to its specialized functions.

What is the genomic context of the yliF gene in E. coli strains?

The yliF gene is located at position b0834 in the E. coli K12 genome . Genomic context analysis suggests yliF exists within a region of considerable genomic plasticity. E. coli strains show remarkable genomic diversity with only approximately 20% of genes constituting the core genome shared across all strains . The remaining genes, including many putative lipoproteins like YliF, belong to the flexible genome - genetic elements that can be acquired through horizontal gene transfer and may confer adaptive advantages in specific environmental niches . Understanding yliF's conservation pattern across different E. coli pathotypes and ecological variants provides insight into its potential specialization for particular lifestyles.

What is the relationship between YliF and the pta-ackA pathway in E. coli?

Research has identified YliF as a suppressor of the synthetic inhibition between pta and recBC genes . The pta and ackA genes constitute a two-gene operon responsible for acetate↔acetyl coenzyme A interconversion in E. coli . Mutations in these genes create dependence on recombinational repair enzymes (RecA or RecBCD) at elevated temperatures, suggesting chromosomal lesions requiring repair . The observation that YliF inactivation suppresses this synthetic inhibition suggests that YliF may either:

• Directly interact with acetate metabolism components

• Function in stress response pathways activated by metabolic imbalance

• Participate in DNA damage recognition or signaling

Researchers investigating YliF should consider designing experiments to assess these interactions, particularly focusing on how c-di-GMP signaling potentially interfaces with central metabolism and DNA repair pathways.

What experimental approaches are most effective for determining YliF's role in bacterial stress responses?

Given YliF's connection to synthetic inhibition between metabolic and DNA repair pathways, a multi-faceted experimental approach is recommended:

• Metabolomic profiling: Compare metabolites in wild-type, ΔyliF, Δpta, and ΔyliF Δpta strains under various growth conditions

• Transcriptomic analysis: Perform RNA-seq in these genetic backgrounds with specific focus on:

  • Acetate metabolism genes

  • DNA repair pathways

  • Stress response regulons

• Protein interaction studies: Employ bacterial two-hybrid or co-immunoprecipitation to identify YliF interaction partners

• Cyclic-di-GMP measurements: Quantify intracellular c-di-GMP levels in response to YliF modulation

• Physiological characterization: Assess phenotypes including:

  • Growth rates under various metabolic conditions

  • DNA damage sensitivity

  • Biofilm formation capacity

This integrated approach would help delineate whether YliF functions primarily in metabolism, stress signaling, DNA protection, or integrates these cellular processes.

How might YliF contribute to E. coli adaptation to environmental transitions?

E. coli's remarkable adaptability to diverse environments involves complex regulatory networks. YliF may play a role in this adaptability through several potential mechanisms:

• Environmental sensing: As a putative lipoprotein with a GGDEF domain, YliF may sense external conditions and transduce signals via c-di-GMP

• Persistence regulation: E. coli produces persister variants that exhibit metabolic dormancy and increased stress tolerance . YliF could participate in persister formation pathways

• Biofilm-planktonic transitions: GGDEF domain proteins commonly regulate transitions between biofilm and free-living states

• Host-environment adaptation: YliF might facilitate adaptation during transitions between host-associated and environmental phases of E. coli's lifecycle

E. coli has shown capacity to establish in soil, water, and plant-associated communities , and YliF may contribute to this ecological flexibility through its signaling functions.

What are optimal conditions for expressing and purifying recombinant YliF protein?

Based on the protein characteristics and general protocols for similar lipoproteins:

Expression Conditions:

• Expression system: E. coli BL21(DE3) with T7 promoter-based vectors

• Induction parameters: 0.1-0.5 mM IPTG at mid-log phase (OD600 ~0.6)

• Post-induction temperature: 16-18°C for 16-18 hours to maximize soluble protein yield

• Media supplementation: Consider adding 0.5-1% glucose to minimize basal expression

Purification Strategy:

• Lysis buffer optimization: Tris-based buffer (pH 7.5-8.0) with 300-500 mM NaCl and 5-10% glycerol

• Detergent considerations: As a putative lipoprotein, use 0.1% mild detergent (e.g., n-dodecyl-β-D-maltoside)

• Affinity chromatography: His-tag purification with imidazole gradient elution

• Secondary purification: Size exclusion chromatography to achieve >95% purity

• Storage conditions: 50% glycerol in Tris-based buffer at -20°C or -80°C to prevent repeated freeze-thaw cycles

Researchers should validate protein activity through enzymatic assays measuring diguanylate cyclase activity if the GGDEF domain is intact and functional.

What approaches can be used to generate and validate yliF knockout strains?

Generation Methods:

• Lambda Red recombination: The most efficient method for creating clean deletions

  • Design primers with 40-50bp homology arms flanking yliF

  • Replace with antibiotic resistance cassette (e.g., kanamycin)

  • Verify with PCR across junction regions

• P1 transduction: Transfer existing knockout alleles (e.g., from Keio collection JW0818) to experimental strains

• CRISPR-Cas9: Design guide RNAs targeting yliF for precise genome editing

Validation Approaches:

• Genomic PCR: Primers flanking deletion site

• RT-qPCR: Confirm absence of yliF transcript

• Western blotting: Verify protein absence (requires specific antibodies)

• Phenotypic characterization:

  • Test for suppression of pta recBC synthetic inhibition

  • Evaluate c-di-GMP-dependent phenotypes (biofilm formation, motility)

  • Assess growth under conditions that trigger stress responses

Complementation Testing:
Reintroduce wild-type yliF on a plasmid under native or inducible promoter to confirm phenotypes result from yliF deletion rather than polar effects or secondary mutations.

How can researchers investigate potential YliF interaction partners?

Given YliF's putative role in cellular signaling and its connection to both metabolic and DNA repair pathways, multiple approaches should be employed:

In vivo approaches:

• Bacterial two-hybrid screening: Fuse YliF to one domain of a split reporter and screen against a library of E. coli proteins

• Co-immunoprecipitation with mass spectrometry: Pull down YliF and associated proteins under various environmental conditions

• Synthetic genetic arrays: Screen for genetic interactions by introducing yliF deletion into an E. coli knockout library

• Fluorescence resonance energy transfer (FRET): Visualize potential interactions within the cell

In vitro approaches:

• Surface plasmon resonance: Measure direct binding kinetics with candidate partners

• Pull-down assays: Use purified YliF with candidate proteins

• Isothermal titration calorimetry: Determine binding thermodynamics

Focus areas should include components of:

• Acetate metabolism pathway proteins (Pta, AckA)

• RecBC recombination machinery

• c-di-GMP signaling network

• Stress response regulators

How should researchers approach comparative genomic analysis of yliF across E. coli strains?

E. coli strains exhibit remarkable genomic plasticity, with the pan-genome containing over 16,000 genes while individual strains typically possess 4,000-5,500 genes . When analyzing yliF across this diversity:

• Conservation assessment:

  • Determine presence/absence patterns across pathogenic and commensal strains

  • Analyze sequence conservation relative to core genome rates

  • Identify lineage-specific variants or paralogs

• Synteny analysis:

  • Map genomic context of yliF across strains

  • Identify co-conserved gene neighborhoods

  • Determine if yliF is associated with mobile genetic elements

• Selection pressure calculation:

  • Calculate dN/dS ratios to assess evolutionary constraints

  • Identify regions under purifying versus diversifying selection

  • Compare evolutionary rates to other GGDEF proteins

• Structural variation analysis:

  • Catalog variations in protein domains and motifs

  • Evaluate impact of polymorphisms on predicted function

  • Assess correlation between structural variations and ecological niches

These analyses should be integrated with phenotypic data to establish structure-function relationships across E. coli's diverse lifestyles.

What are the most informative assays for evaluating YliF's potential role in biofilm formation?

Given that many GGDEF domain proteins regulate biofilm formation and E. coli biofilms are crucial for environmental persistence and host colonization , the following comprehensive assay panel is recommended:

Static biofilm assays:

• Crystal violet staining in 96-well plates under various nutrient conditions

• Confocal microscopy with fluorescent strains to assess biofilm architecture

• Scanning electron microscopy for detailed structural analysis

Flow cell experiments:

• Real-time biofilm development under controlled shear stress

• Competitive biofilm formation between wild-type and ΔyliF strains

• Biofilm dispersion dynamics in response to environmental signals

Molecular characterization:

• Extracellular matrix composition analysis (polysaccharides, proteins, eDNA)

• c-di-GMP measurements in biofilm versus planktonic populations

• Transcriptomic profiling at different biofilm development stages

Environmental condition variations:

• Temperature ranges relevant to host and environmental persistence

• Nutrient limitation reflective of specific niches

• Exposure to stressors (oxidative stress, pH fluctuations, antibiotics)

Researchers should compare ΔyliF mutants with deletion strains of well-characterized biofilm regulators to position YliF within the regulatory hierarchy of biofilm development.

How might systems biology approaches advance understanding of YliF's function?

The complex interconnections between metabolic networks, DNA repair pathways, and environmental adaptation where YliF appears to function necessitate integrated systems approaches:

• Multi-omics integration:

  • Correlate transcriptomic, proteomic, and metabolomic data from wild-type and ΔyliF strains

  • Develop predictive models of YliF-dependent cellular states

  • Identify emergent properties not evident in single-omics approaches

• Network analysis:

  • Map YliF within E. coli's protein-protein interaction network

  • Identify regulatory motifs connecting metabolism and stress responses

  • Quantify information flow through YliF-dependent pathways

• Mathematical modeling:

  • Develop dynamic models of c-di-GMP signaling including YliF

  • Simulate metabolic shifts under various environmental conditions

  • Predict cellular responses to perturbations in YliF-mediated pathways

• Single-cell analysis:

  • Characterize cell-to-cell variability in YliF expression

  • Determine if YliF contributes to phenotypic heterogeneity

  • Assess YliF's role in population-level behaviors like persister formation

These integrative approaches could reveal how YliF functions as an interface between sensing environmental conditions and modulating cellular physiology.

What is the significance of YliF's suppression of pta recBC synthetic inhibition for understanding bacterial genetics?

The observation that YliF inactivation suppresses the synthetic inhibition between pta and recBC genes has fundamental implications for bacterial genetics research:

• Interconnected cellular networks:

  • Demonstrates unexpected links between metabolic pathways and DNA maintenance

  • Suggests second-order genetic interactions may be common in bacterial systems

  • Highlights the value of synthetic genetic approaches for discovering novel gene functions

• Stress response integration:

  • Indicates potential coordination between metabolic stress and DNA repair mechanisms

  • Suggests existence of feedback loops that regulate cellular resource allocation

  • May represent an adaptive strategy for maintaining genomic integrity under stress

• Evolution of genetic robustness:

  • Provides insight into how bacteria maintain fitness despite mutations

  • Suggests genetic buffering mechanisms that prevent lethal accumulation of damage

  • May explain observed patterns of gene essentiality across different conditions

• Therapeutic implications:

  • Identifies potential combination targets for antimicrobial development

  • Suggests strategies for sensitizing bacteria to existing antibiotics

  • Provides framework for understanding how bacteria might develop resistance

This complex genetic interaction exemplifies the sophisticated regulatory networks that enable bacterial adaptation and survival across diverse conditions.