Recombinant Escherichia coli O45:K1 Nucleoside diphosphate kinase (ndk)

What is Nucleoside Diphosphate Kinase (NDK) in E. coli and what are its primary functions?

Nucleoside diphosphate kinase (NDK) in E. coli is a multifunctional protein that plays several critical roles in bacterial metabolism and cellular processes. The primary functions of NDK include:

• Catalyzing the phosphorylation of nucleoside diphosphates to nucleoside triphosphates, playing a central role in nucleotide metabolism and maintaining balanced nucleotide pools

• Contributing to both ribo- and deoxyribonucleoside triphosphate biosynthesis pathways

• Acting as a DNA repair nuclease, specifically processing uracil misincorporated into DNA

• Participating in genetic and metabolic regulation pathways

• Involvement in signal transduction mechanisms

The enzyme displays broad specificity, accepting various nucleoside diphosphates as substrates in phosphotransfer reactions, with the reaction proceeding via a phosphoenzyme intermediate .

How does the E. coli O45:K1 strain differ from other E. coli strains?

E. coli O45:K1 represents a highly pathogenic clone that has emerged as a significant cause of meningitis, particularly in France. It differs from other E. coli strains in several important aspects:

• The O45:K1:H7 strain possesses a unique O-antigen gene cluster that differs significantly from the O45 reference strain (96-3285), suggesting that while they share epitopes, they represent two different antigens

• The strain combines the O45 somatic antigen with the K1 capsular antigen and H7 flagellar antigen, creating a virulent combination

• This strain is closely related to the archetypal O18:K1:H7 meningitis-causing clone but has different somatic antigen characteristics

• The O45 antigen in this strain plays a crucial role in virulence, particularly in neonatal meningitis models

• The O-antigen gene cluster in strain S88 (representative of O45:K1:H7) contains nine open reading frames with low G+C content compared to the E. coli core genome

• Phylogenetic evidence suggests that the O45 antigen gene cluster in these strains may have been acquired through horizontal gene transfer, potentially from another member of the Enterobacteriaceae

Unlike the commonly used laboratory K-12 strains of E. coli that are generally considered safe and are exempt from certain NIH guidelines, O45:K1 strains are pathogenic and require appropriate biosafety measures .

What expression systems are most effective for producing recombinant E. coli NDK?

For the production of recombinant NDK from E. coli O45:K1, several expression systems have proven effective, with key considerations for researchers including:

• E. coli-based expression systems remain the most common and efficient approach, particularly when using strains optimized for recombinant protein production such as BL21(DE3), Rosetta, or HMS174

• Expression vectors containing strong inducible promoters (T7, tac, or pBAD) allow controlled expression

• Fusion tags such as hexahistidine (His6) facilitate purification and can be incorporated at the N-terminus of the protein without significantly affecting enzyme activity

• Codon optimization may be necessary when expressing the gene in a heterologous host to ensure efficient translation

• Expression conditions typically involve induction at mid-log phase (OD600 of 0.6-0.8) with IPTG (0.1-1 mM) followed by expression at lower temperatures (16-25°C) to enhance proper folding

• Solubility can be improved through co-expression with molecular chaperones or by using fusion partners such as GST, MBP, or SUMO

When purifying recombinant NDK, researchers typically employ a combination of immobilized metal affinity chromatography (IMAC) for His-tagged proteins, followed by size exclusion chromatography to achieve >90% purity suitable for enzymatic and structural studies .

What methods are available for measuring NDK enzymatic activity?

Researchers employ several established methodologies to measure NDK activity, each with specific advantages depending on the experimental objectives:

• Spectrophotometric coupled enzyme assays:

  • The most common approach couples NDK activity with pyruvate kinase and lactate dehydrogenase, measuring NADH oxidation at 340 nm

  • This assay monitors the production of ATP from ADP and a phosphate donor (typically GTP)

  • Standard reaction conditions include 50 mM Tris-HCl (pH 7.5), 5 mM MgCl2, 1 mM phosphoenolpyruvate, 0.2 mM NADH, 1 mM ADP, 0.5 mM GTP, and coupling enzymes

• Direct measurement of nucleotide conversion:

  • HPLC-based methods allow direct quantification of substrate depletion and product formation

  • This approach enables determination of substrate specificity profiles for different nucleotides

  • Typical separation employs reversed-phase chromatography with UV detection at 254 nm

• Radiometric assays:

  • Using radiolabeled substrates (typically [γ-32P]ATP) followed by thin-layer chromatography

  • This method offers high sensitivity for detecting low levels of enzyme activity

  • Standard reaction conditions include 50 mM Tris-HCl (pH 7.5), 5 mM MgCl2, 100 μM [γ-32P]ATP, and 100 μM nucleoside diphosphate acceptor

• Real-time assays:

  • Luminescence-based ATP detection systems allow continuous monitoring of NDK activity

  • Particularly useful for high-throughput screening applications

For most accurate results, enzyme activity should be assessed under initial velocity conditions with substrate concentrations at least 5-10 times the Km values, and reactions performed at physiologically relevant pH (7.0-7.5) and temperature (37°C for E. coli enzymes) .

How does NDK contribute to the virulence mechanisms of E. coli O45:K1 strains?

The contribution of NDK to the virulence of E. coli O45:K1 strains involves multiple mechanisms that extend beyond its canonical role in nucleotide metabolism:

• NDK plays a critical role in maintaining balanced nucleotide pools, which is essential for efficient DNA replication and repair during infection

• The DNA repair nuclease activity of NDK, particularly in processing uracil misincorporations, contributes to genomic stability during infection and stress conditions

• In E. coli O45:K1 strains, NDK may interact with the unique O-antigen, which has been demonstrated to be crucial for virulence in neonatal meningitis models

• Mutations in the ndk gene result in a mutator phenotype that can accelerate adaptive evolution during infection through increased mutation rates

• NDK's role in nucleotide metabolism impacts bacterial survival during host-imposed nutrient limitation and oxidative stress conditions

• Potential interactions between NDK and host cellular factors may contribute to immune evasion mechanisms

The relationship between NDK and the O45 antigen is particularly noteworthy. Research has demonstrated that the O45 antigen gene cluster in pathogenic strains like S88 (O45:K1:H7) is critical for virulence, and may have been acquired through horizontal gene transfer. While the direct interaction between NDK and the O-antigen hasn't been fully characterized, their combined functions likely create synergistic effects enhancing bacterial survival and pathogenicity in host environments .

What are the mechanisms underlying NDK's DNA repair functions?

E. coli NDK possesses remarkable DNA repair capabilities, particularly focused on uracil processing, which proceeds through a multi-step catalytic mechanism:

• Recognition of uracil residues in DNA:

  • NDK selectively binds to DNA containing misincorporated uracil residues

  • The recognition process appears distinct from traditional uracil-DNA glycosylases

• Sequential enzymatic activities in uracil repair:

  • Glycosylase activity: NDK cleaves the N-glycosidic bond between uracil and deoxyribose

  • AP lyase activity: Processes the resulting abasic site through β-elimination

  • Endonuclease activity: Makes incisions at specific phosphodiester bonds

• Coordination with downstream repair factors:

  • The processed sites become substrates for DNA polymerase I and DNA ligase

  • This completes the base excision repair pathway for uracil lesions

The DNA repair function of NDK is particularly significant because cells with disrupted ndk genes display a spontaneous mutator phenotype. While this phenotype was initially attributed solely to imbalanced nucleotide pools affecting replicative DNA polymerases, research now indicates that the loss of NDK's direct DNA repair activity also contributes significantly to increased mutation rates.

The unique aspect of NDK's DNA repair function is its integration with nucleotide metabolism, allowing the enzyme to both prevent uracil misincorporation (by maintaining appropriate dUTP/dTTP ratios) and repair uracil when it does appear in DNA, representing an elegant dual protective mechanism .

How does E. coli NDK interact with bacteriophage T4 proteins and what are the implications?

E. coli NDK forms specific interactions with multiple bacteriophage T4 proteins, creating a complex interplay between host and viral metabolism with several important implications:

• Specific protein-protein interactions:

  • NDK directly interacts with T4 thymidylate synthase

  • NDK binds to T4 aerobic ribonucleotide reductase

  • NDK forms complexes with T4 dCTPase-dUTPase

  • NDK interacts with gene 32 single-strand DNA-binding protein

  • NDK binds to deoxycytidylate hydroxymethylase

• Nucleotide modulation effects:

  • Interactions with ribonucleotide reductase and gp32 are enhanced by nucleoside triphosphates

  • This suggests the integrity of the T4 dNTP synthetase complex in vivo is influenced by the composition of the nucleotide pool

• Functional significance:

  • These interactions likely facilitate the coordination of dNTP synthesis during T4 infection

  • The interactions may allow viral hijacking of host nucleotide metabolism

  • Surprisingly, E. coli NDK is dispensable for successful T4 phage infection, suggesting redundant mechanisms

The complex formation between NDK and these T4 proteins has been demonstrated through multiple complementary techniques including optical biosensor analysis, fluorescence spectroscopy, immunoprecipitation, and glutathione S-transferase pull-down assays. These protein-protein interactions represent a fascinating example of how viruses can interface with host metabolism and may provide insights into novel antimicrobial strategies targeting these interfaces .

What methodologies are most effective for studying NDK protein-protein interactions?

For researchers investigating NDK protein-protein interactions, several complementary techniques have proven particularly effective:

• Optical biosensor analysis:

  • Surface plasmon resonance (SPR) allows real-time, label-free detection of protein-protein interactions

  • Typically employs immobilized NDK on a sensor chip with potential binding partners in solution

  • Provides kinetic parameters (kon, koff) and binding affinities (KD)

  • Recommended instrumentation: Biacore systems or equivalent platforms

• Fluorescence-based methods:

  • Fluorescence spectroscopy detects changes in intrinsic tryptophan fluorescence upon binding

  • Förster resonance energy transfer (FRET) using fluorescently labeled proteins can map interaction interfaces

  • Microscale thermophoresis offers solution-based binding measurements with low sample consumption

• Co-immunoprecipitation approaches:

  • Classical co-IP using antibodies against NDK or interacting partners

  • Can be performed under native conditions to preserve physiologically relevant interactions

  • Coupled with Western blotting or mass spectrometry for identification of binding partners

• Pull-down assays:

  • GST pull-down assays using GST-tagged NDK and potential binding partners

  • His-tag pull-downs using metal affinity resins

  • Tandem affinity purification for identifying multi-protein complexes

• Crosslinking mass spectrometry:

  • Chemical crosslinking combined with MS analysis to map interaction interfaces

  • Provides structural information about the binding regions

  • Can capture transient interactions difficult to detect by other methods

• Yeast two-hybrid screening:

  • For systematic identification of novel interaction partners

  • Split-ubiquitin systems may be preferable for bacterial proteins

The integration of multiple approaches is recommended, as each technique has specific strengths and limitations. For example, the interaction between E. coli NDK and T4 proteins was conclusively demonstrated using a combination of optical biosensor analysis, fluorescence spectroscopy, immunoprecipitation, and GST pull-down assays .

How do mutations in the ndk gene affect nucleotide pools and cellular phenotypes?

Mutations in the E. coli ndk gene lead to profound metabolic alterations with cascading effects on nucleotide homeostasis and cellular functions:

• Nucleotide pool imbalances:

  • Decreased capability to phosphorylate nucleoside diphosphates to triphosphates

  • Relative depletion of minor nucleoside triphosphates (CTP, UTP, GTP)

  • Accumulation of corresponding nucleoside diphosphates

  • Compensatory upregulation of substrate-level phosphorylation pathways

• Metabolic adaptations:

  • Elevated CTP synthetase activity observed in ndk mutants as a compensatory mechanism

  • Induction of alternative nucleotide biosynthesis pathways

  • Metabolic shifts rather than direct genetic changes appear responsible for these adaptations

• Mutator phenotypes:

  • ndk-disrupted cells display spontaneous mutator phenotypes

  • Previously attributed solely to imbalanced nucleotide pools affecting replicative DNA polymerase fidelity

  • Now also linked to loss of NDK's direct DNA repair activity (particularly uracil processing)

  • Estimated 20-50 fold increase in mutation rates depending on the genetic background

• Growth characteristics:

  • Slower growth rates under standard laboratory conditions

  • Altered anaerobic growth capabilities, including apparent suppression of growth in pyruvate kinase-negative E. coli mutants

  • Increased sensitivity to certain antibiotics and environmental stressors

• Virulence implications:

  • In pathogenic strains like O45:K1, ndk mutations may affect virulence factor expression

  • Altered interaction with host defense mechanisms

  • Potential impact on biofilm formation and antibiotic tolerance

The regulatory roles initially thought to be genetic in nature have been demonstrated to be primarily metabolic. This highlights the critical importance of NDK in maintaining cellular homeostasis through its multifunctional activities in nucleotide metabolism, DNA repair, and protein-protein interactions .

What are the challenges and solutions in purifying active recombinant E. coli O45:K1 NDK?

Researchers working with recombinant E. coli O45:K1 NDK face several technical challenges that require specific strategies to overcome:

A highly effective purification protocol typically involves:

• Cell lysis in 50 mM Tris-HCl pH 7.5, 300 mM NaCl, 5 mM MgCl₂, 10 mM imidazole with protease inhibitors

• Immobilized metal affinity chromatography using Ni-NTA resin with imidazole gradient elution (10-250 mM)

• Size exclusion chromatography using Superdex 200 in 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 5 mM MgCl₂

• Optional: Ion exchange chromatography for removal of nucleic acid contaminants

This approach typically yields >90% pure protein with specific activity of 1000-2000 units/mg, suitable for both enzymatic and structural studies .

How does O45 antigen expression influence the function of NDK in pathogenic E. coli strains?

The relationship between O45 antigen expression and NDK function in pathogenic E. coli represents a complex interplay with significant implications for bacterial virulence:

• Structural and biochemical interactions:

  • The O45 antigen gene cluster in virulent strains like S88 (O45:K1:H7) contains nine open reading frames with low G+C content

  • While direct physical interaction between O45 LPS and NDK hasn't been definitively established, membrane association of NDK may be influenced by specific O-antigen structures

  • The unique composition of O45 antigen may create a microenvironment that modulates NDK activity or substrate accessibility

• Co-regulation mechanisms:

  • Stress conditions that induce O45 antigen modifications may simultaneously affect NDK expression

  • Both systems respond to environmental cues encountered during infection

  • Potential for coordinated regulation through shared transcriptional factors

• Functional synergy in pathogenesis:

  • O45 antigen has been demonstrated as crucial for virulence in neonatal meningitis models

  • NDK's role in maintaining nucleotide balance supports the high metabolic demands during infection

  • The DNA repair function of NDK may be particularly important in O45-expressing strains facing host-induced oxidative stress

  • Combined effects on serum resistance, immune evasion, and metabolic adaptation

• Evolutionary considerations:

  • The O45 antigen gene cluster in strain S88 appears to have been acquired through horizontal gene transfer

  • This acquisition, combined with the multifunctional capabilities of NDK, may have contributed to the emergence of the highly virulent O45:K1:H7 clone

  • The co-evolution of these systems suggests functional interdependence

The unique O45 antigen in pathogenic strains differs significantly from that in the reference strain E. coli 96-3285, suggesting that while they share some epitopes, they represent different antigens. This distinctive antigen structure, combined with NDK's multiple functions, likely creates a synergistic effect enhancing bacterial survival and pathogenicity in host environments .

What are the optimal conditions for expressing and purifying enzymatically active recombinant E. coli O45:K1 NDK?

For researchers seeking to optimize expression and purification of enzymatically active E. coli O45:K1 NDK, the following protocol has been established based on experimental data:

• Expression vector design:

  • The complete ndk gene should be cloned into a T7 promoter-based vector (pET-28a recommended)

  • Include an N-terminal hexahistidine tag with a thrombin cleavage site

  • Confirm sequence integrity with particular attention to catalytic residues

• Host strain selection:

  • E. coli BL21(DE3) or Rosetta(DE3) for strains requiring codon optimization

  • Avoid using E. coli strains with ndk mutations that might contaminate purified protein

  • Consider BL21(DE3)pLysS for tighter expression control if toxicity is observed

• Optimal expression conditions:

  • Inoculate expression cultures at 1:100 dilution from overnight cultures

  • Grow cells at 37°C in LB medium supplemented with appropriate antibiotics

  • Induce at OD600 of 0.6-0.8 with 0.2 mM IPTG

  • Shift temperature to 18°C post-induction

  • Continue expression for 16-18 hours

  • Supplement medium with 0.1 mM MgCl₂ to enhance cofactor availability

• Cell lysis and initial clarification:

  • Harvest cells by centrifugation (5,000 × g, 15 min, 4°C)

  • Resuspend in lysis buffer: 50 mM Tris-HCl pH 7.5, 300 mM NaCl, 5 mM MgCl₂, 10 mM imidazole, 1 mM PMSF, 1 mM DTT

  • Lyse cells by sonication or high-pressure homogenization

  • Clear lysate by centrifugation (25,000 × g, 30 min, 4°C)

  • Treat supernatant with DNase I (10 μg/ml) and RNase A (5 μg/ml) for 30 minutes at 4°C

• Purification strategy:

  • IMAC: Apply clarified lysate to Ni-NTA resin, wash with 20-50 mM imidazole, elute with 250 mM imidazole

  • Desalt using dialysis or gel filtration against 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 5 mM MgCl₂, 1 mM DTT

  • Size exclusion chromatography using Superdex 200 in the same buffer

  • Optional: Remove His-tag with thrombin if required for downstream applications

• Quality control parameters:

  • 95% purity assessed by SDS-PAGE

  • A260/A280 ratio <0.7 indicating minimal nucleic acid contamination

  • Specific activity >1000 units/mg protein in standard NDK assay

  • Hexameric quaternary structure confirmed by native PAGE or size exclusion chromatography

This optimized protocol typically yields 15-20 mg of pure, active enzyme per liter of bacterial culture with >90% retention of enzymatic activity .

How can researchers effectively study the dual functions of NDK in nucleotide metabolism and DNA repair?

Investigating the dual functions of NDK requires carefully designed experimental approaches that can distinguish between its nucleotide metabolism and DNA repair activities:

• Separation of functions through site-directed mutagenesis:

  • Identify and mutate key residues specifically involved in:

    • Phosphotransfer activity (e.g., the conserved histidine in the active site)

    • DNA binding (positively charged surface residues)

    • Uracil recognition (residues analogous to those in uracil-DNA glycosylases)

  • Generate single-function variants that retain only one activity

  • Confirm altered activity profiles through biochemical assays

• Complementation studies with mutant strains:

  • Use E. coli strains with ndk gene deletions as experimental platforms

  • Complement with wild-type or mutant NDK variants

  • Assess:

    • Mutation rates (using rifampicin resistance assays)

    • Nucleotide pool balance (by HPLC analysis)

    • Sensitivity to DNA-damaging agents (particularly those causing uracil misincorporation)

  • Determine which function (metabolism or repair) rescues specific phenotypes

• In vitro biochemical dissection:

  • Establish separate assay conditions optimized for each function:

    • Phosphotransfer: Standard coupled enzyme assays with various NDP substrates

    • DNA repair: Uracil-containing oligonucleotide substrates with defined sequences

  • Test the effects of nucleotides on DNA binding/processing

  • Evaluate competitive inhibition between pathways

• Structural biology approaches:

  • Obtain crystal structures of NDK in complex with:

    • Nucleotide substrates

    • DNA containing uracil

    • Both substrates simultaneously (if possible)

  • Use structural information to design more precise functional mutants

• Cellular localization studies:

  • Track NDK localization under different conditions using:

    • Fluorescent protein fusions

    • Immunofluorescence microscopy

  • Correlate localization with nucleotide stress or DNA damage

  • Determine if NDK redistributes between cytoplasm and nucleoid in response to specific stimuli

• Temporal dynamics investigations:

  • Study how NDK activities change during:

    • Cell cycle progression

    • Response to environmental stressors

    • Infection processes (relevant for O45:K1 pathogenic strains)

  • Use inducible expression systems with activity-specific NDK variants

This multifaceted approach enables researchers to determine how these dual functions are coordinated physiologically and how they may contribute to bacterial survival and pathogenesis .

How can recombinant E. coli O45:K1 NDK be utilized in nucleotide analog studies?

Recombinant E. coli O45:K1 NDK offers versatile applications in nucleotide analog research, providing valuable tools for studying modified nucleotides:

• Production of novel nucleotide analogs:

  • NDK's broad substrate specificity allows phosphorylation of various nucleoside diphosphate analogs

  • Typical reaction conditions:

    • 50 mM Tris-HCl pH 7.5

    • 5 mM MgCl₂

    • 1 mM ATP as phosphate donor

    • 0.5 mM nucleoside diphosphate analog

    • 0.1-0.5 μg purified NDK

  • Yields of phosphorylated analogs typically exceed 85%

  • Scalable from analytical (μg) to preparative (mg) quantities

• Structure-activity relationship studies:

  • Systematic testing of modified nucleotides to determine:

    • Modifications tolerated by the enzyme

    • Effects on binding affinity (Km values)

    • Changes in phosphorylation efficiency (kcat/Km)

  • Parameter ranges typically observed:

    • Km: 10-500 μM depending on analog structure

    • kcat: 50-500 s⁻¹

    • Catalytic efficiency (kcat/Km): 10⁵-10⁷ M⁻¹s⁻¹

• Antiviral and anticancer drug development:

  • Phosphorylation of therapeutic nucleoside analogs to active triphosphate forms

  • Assessment of nucleotide analog incorporation into DNA by polymerases

  • Evaluation of analog effects on NDK's DNA repair function

  • Testing nucleotide analogs as potential inhibitors of bacterial NDK

• Biophysical interaction studies:

  • Isothermal titration calorimetry to determine:

    • Binding thermodynamics of nucleotide analogs

    • ΔH, ΔS, and ΔG parameters

    • Binding stoichiometry

  • Fluorescence-based assays for binding kinetics

  • Structural studies using X-ray crystallography or NMR with bound analogs

• Analytical methodology:

  • Development of NDK-based enzymatic assays for nucleotide analog detection

  • Coupling with HPLC or mass spectrometry for enhanced sensitivity

  • Limit of detection typically in the nanomolar range

  • Linear response over 3-4 orders of magnitude

The broad substrate specificity of NDK makes it particularly valuable for studies with nucleotide analogs that are poor substrates for other kinases. When working with the O45:K1 variant specifically, researchers should consider potential differences in substrate specificity compared to NDK from laboratory strains, which may offer unique opportunities for novel analog development .

What strategies can be employed to investigate the interaction between E. coli O45 antigen and NDK function?

Investigating the potential interplay between E. coli O45 antigen and NDK function requires sophisticated experimental approaches spanning multiple disciplines:

• Co-expression and co-purification studies:

  • Engineer strains expressing both the O45 antigen gene cluster and tagged NDK

  • Perform membrane fractionation followed by affinity purification

  • Analyze co-purifying components by mass spectrometry

  • Control experiments must include O-antigen negative mutants

• Localization and trafficking analysis:

  • Fluorescent protein fusions to track NDK localization

  • Immunogold electron microscopy to visualize NDK relative to LPS

  • Compare localization patterns in O45-positive and O45-negative backgrounds

  • Live-cell imaging during infection processes

• Functional activity assays in different membrane contexts:

  • Prepare membrane fractions from:

    • O45-positive wild-type strains

    • O45-negative mutants

    • Strains with modified O45 antigen structures

  • Measure NDK activity in these different membrane environments

  • Assess both phosphotransfer and DNA repair functions

• Physicochemical interaction studies:

  • Surface plasmon resonance using purified components

  • Isothermal titration calorimetry to measure binding parameters

  • Identify specific O45 polysaccharide regions involved in any interactions

  • Study the effects of divalent cations on interactions

• Genetic and phenotypic correlation analysis:

  • Create a panel of mutations in both the O45 gene cluster and ndk

  • Analyze epistatic relationships between these mutations

  • Measure virulence parameters including:

    • Serum resistance

    • Invasion efficiency

    • Survival in phagocytes

    • Biofilm formation capabilities

• Structural biology approaches:

  • Membrane protein crystallography or cryo-EM to visualize potential complexes

  • NMR studies of labeled components

  • Molecular dynamics simulations of NDK interactions with membrane models

  • Hydrogen-deuterium exchange mass spectrometry to map interaction interfaces

• In vivo infection models:

  • Compare virulence of wild-type and mutant strains in:

    • Cell culture infection models

    • Invertebrate infection models (Caenorhabditis elegans, Galleria mellonella)

    • Vertebrate models where ethically approved

  • Correlate O45 antigen structure and NDK function with pathogenicity

The unique O45 antigen in strain S88 (O45:K1:H7) differs significantly from the O45 reference strain, suggesting that while they share epitopes, they represent different antigens. This distinctiveness may be relevant to any functional interactions with NDK and should be considered when designing experimental controls .

What are the most promising research avenues for developing inhibitors targeting E. coli O45:K1 NDK?

The development of inhibitors targeting E. coli O45:K1 NDK represents a promising research direction, with several strategic approaches:

• Structure-based design strategies:

  • Target the highly conserved nucleotide-binding pocket

  • Focus on the unique hexameric quaternary structure of bacterial NDK

  • Design compounds that can:

    • Compete with nucleotide substrates

    • Disrupt oligomerization

    • Trap the phosphohistidine intermediate state

  • Utilize molecular docking and virtual screening of compound libraries

• Exploiting unique features of bacterial NDK:

  • Target structural elements distinct from human NDK homologs

  • Focus on the DNA-binding interface involved in repair functions

  • Develop compounds that selectively inhibit bacterial enzymes while sparing mammalian counterparts

  • Screen for inhibitors that specifically disrupt interactions with bacteriophage proteins

• Allosteric inhibition approaches:

  • Identify regulatory sites outside the catalytic center

  • Design molecules that lock the enzyme in inactive conformations

  • Target protein dynamics rather than static structures

  • Develop compounds that prevent conformational changes required for catalysis

• Dual-target inhibitor development:

  • Design compounds that simultaneously inhibit NDK and interact with O45 antigen

  • Create hybrid molecules targeting both nucleotide metabolism and membrane integrity

  • Develop inhibitors that disrupt the potential functional relationship between NDK and O45 LPS

  • Focus on compounds that synergize with existing antimicrobials

• Natural product screening and optimization:

  • Evaluate plant extracts and microbial metabolites for NDK inhibitory activity

  • Isolate active components from traditional medicinal sources

  • Optimize lead compounds through medicinal chemistry approaches

  • Focus on compounds with demonstrated antibacterial activity against E. coli

• Peptide and aptamer-based approaches:

  • Design peptide inhibitors mimicking protein-protein interaction interfaces

  • Develop nucleic acid aptamers that bind specifically to bacterial NDK

  • Create peptide-nucleotide conjugates targeting both substrate binding and regulatory sites

  • Optimize delivery mechanisms to improve cellular uptake

• Therapeutic potential evaluation parameters:

  • Minimal inhibitory concentration (MIC) against E. coli O45:K1 strains

  • Selectivity index (SI) comparing bacterial and human enzyme inhibition

  • In vitro toxicity profiles using mammalian cell lines

  • Pharmacokinetic properties including stability, bioavailability, and half-life

The development of NDK inhibitors specifically targeting E. coli O45:K1 strains could provide valuable tools both for basic research and potentially for therapeutic applications against these emerging pathogens .

How might genomic and proteomic approaches advance our understanding of NDK function in pathogenic E. coli strains?

Modern genomic and proteomic methodologies offer powerful opportunities to elucidate the complex roles of NDK in pathogenic E. coli, particularly O45:K1 strains:

• Comparative genomics approaches:

  • Whole genome sequencing of diverse E. coli strains to:

    • Identify ndk gene variations across pathotypes

    • Detect potential horizontal gene transfer events

    • Map genetic linkages between ndk and virulence factors

  • Analysis of selection pressure on the ndk gene during host adaptation

  • Investigation of regulatory elements controlling ndk expression

  • Comparison of ndk sequences in O45:K1 strains versus non-pathogenic isolates

• Transcriptomics methodologies:

  • RNA-Seq analysis to determine:

    • Differential expression of ndk during infection processes

    • Co-regulated gene networks including ndk

    • Effects of ndk mutation on global transcription patterns

  • Single-cell transcriptomics to assess heterogeneity in ndk expression

  • Ribosome profiling to evaluate translational efficiency of ndk mRNA

  • Analysis of ncRNAs potentially regulating ndk expression

• Proteomics techniques:

  • Quantitative proteomics to measure NDK abundance under various conditions

  • Post-translational modification analysis of NDK including:

    • Phosphorylation states

    • Potential glycosylation

    • Ubiquitination and other regulatory modifications

  • Protein-protein interaction networks using:

    • Affinity purification-mass spectrometry

    • Proximity labeling approaches (BioID, APEX)

    • Crosslinking mass spectrometry

  • Membrane proteomics to investigate NDK association with cell envelope

• Structural biology integration:

  • Cryo-electron microscopy of NDK complexes in native cellular contexts

  • In-cell NMR to study NDK dynamics in living bacteria

  • Integrative structural modeling combining multiple data sources

  • Investigation of NDK within macromolecular assemblies

• Systems biology frameworks:

  • Metabolic flux analysis to track nucleotide metabolism in wild-type and ndk mutants

  • Network modeling of NDK's role in cellular homeostasis

  • Integration of transcriptomic, proteomic, and metabolomic datasets

  • Predictive modeling of NDK's impact on bacterial fitness and virulence

• CRISPR-based functional genomics:

  • CRISPR interference to modulate ndk expression levels

  • CRISPR screening to identify synthetic lethal interactions with ndk

  • Base editing to introduce specific mutations in the ndk gene

  • Regulatory element mapping using CRISPR activation/repression systems

These multi-omics approaches will likely reveal unexpected connections between NDK and cellular processes beyond its canonical functions, potentially identifying new therapeutic targets and virulence mechanisms in pathogenic E. coli O45:K1 strains .