Recombinant Escherichia fergusonii Thymidylate kinase (tmk)

What is the function of thymidylate kinase in Escherichia fergusonii?

Thymidylate kinase (dTMP kinase; EC 2.7.4.9) in E. fergusonii, like its counterpart in E. coli, catalyzes the phosphorylation of dTMP to form dTDP, representing a critical step in both de novo and salvage pathways of dTTP synthesis. This enzyme plays an essential role in DNA replication and cellular metabolism . The tmk gene encodes this enzyme, and similar to E. coli, its genomic location in E. fergusonii is in proximity to other important genes involved in DNA replication and repair mechanisms.

How does E. fergusonii tmk differ from E. coli tmk at the sequence level?

While both enzymes share significant sequence homology, E. fergusonii tmk shows distinctive nucleotide variations when compared to E. coli tmk. Phylogenetic analysis using adenylate kinase (adk) housekeeping gene (related to nucleotide metabolism) from the E. coli multi-locus sequence typing (MLST) scheme reveals specific nucleotide variations between these species . Although 16S rRNA gene sequences cannot reliably differentiate between E. fergusonii and E. coli, detailed analysis of the tmk gene sequence can potentially provide distinctive markers for species identification . Research suggests four specific loci in the tmk coding region that can serve as reliable markers for differentiating between these closely related species.

What expression systems are most effective for recombinant E. fergusonii tmk production?

For optimal expression of recombinant E. fergusonii tmk, E. coli-based expression systems have proven most effective, particularly when using engineered strains optimized for recombinant protein expression . The methodology should include:

• Host selection: BL21(DE3) or BL21(DE3)pLysS strains are recommended due to their reduced protease activity and ability to maintain stable expression.

• Vector design: pET-based vectors containing T7 promoter systems offer tight regulation and high expression yields for tmk.

• Expression conditions: Induction at OD₆₀₀ of 0.6-0.8 using 0.5-1.0 mM IPTG, followed by expression at 25-30°C for 4-6 hours to balance protein yield with solubility.

Successful expression requires consideration of the physicochemical properties of the tmk protein, including potential toxicity issues when overexpressed, which may necessitate adjustments to induction parameters .

What are the recommended purification strategies for obtaining high-purity recombinant E. fergusonii tmk?

Purification of recombinant E. fergusonii tmk requires a multi-step approach to achieve high purity while maintaining enzymatic activity. Based on protocols established for similar enzymes , the following procedure is recommended:

• Cell lysis: Sonication in buffer containing 50 mM Tris-HCl (pH 7.5), 100 mM NaCl, 10% glycerol, and 0.1% Triton X-100 (critical for stability).

• Initial capture: Affinity chromatography using His-tag or other fusion tags.

• Intermediate purification: Ion exchange chromatography (typically Q-Sepharose).

• Polishing step: Size exclusion chromatography in buffer containing stabilizing agents.

The addition of both 0.1% Triton X-100 and 10% glycerol is crucial for maintaining enzyme stability throughout the purification process and during storage . Without these additives, rapid activity loss occurs, particularly at temperatures above 25°C. Storage at -20°C in buffer containing these stabilizers preserves activity for at least one month.

How can researchers overcome insolubility issues when expressing recombinant E. fergusonii tmk?

Insolubility issues with recombinant E. fergusonii tmk can be addressed through a combination of expression optimization and solubility enhancement strategies:

• Temperature modulation: Lowering expression temperature to 16-20°C significantly improves solubility by slowing folding kinetics.

• Co-expression with chaperones: Systems like GroEL/GroES can aid proper folding.

• Fusion partners: Solubility-enhancing tags such as MBP (maltose-binding protein) or SUMO can improve folding and solubility.

• Buffer optimization during lysis: Inclusion of mild detergents (0.1% Triton X-100), osmolytes (glycerol, sorbitol), or arginine (50-200 mM) in lysis buffers can enhance soluble yields.

For cases of severe insolubility, refolding from inclusion bodies may be necessary using a gradual dialysis approach with decreasing concentrations of urea or guanidine hydrochloride while maintaining the presence of stabilizing agents throughout the refolding process .

What assays are available for measuring E. fergusonii tmk enzymatic activity?

Several complementary approaches can be employed to measure the enzymatic activity of recombinant E. fergusonii tmk:

• Spectrophotometric coupled assay: This method couples ADP production to NADH oxidation through pyruvate kinase and lactate dehydrogenase, measuring the decrease in absorbance at 340 nm. The standard reaction mixture contains:

  • 50 mM Tris-HCl (pH 7.5)

  • 50 mM KCl

  • 5 mM MgCl₂

  • 0.5 mM phosphoenolpyruvate

  • 0.2 mM NADH

  • 10 units/ml pyruvate kinase

  • 10 units/ml lactate dehydrogenase

  • Varying concentrations of dTMP and ATP

• HPLC-based direct product quantification: Separation and quantification of dTDP formation using reverse-phase HPLC.

• Radioactive assay: Using [γ-³²P]ATP as substrate and measuring the transfer of radioactive phosphate to dTMP.

The spectrophotometric coupled assay offers the advantage of continuous monitoring but requires control reactions to account for potential interference from sample components .

What are the kinetic parameters of recombinant E. fergusonii tmk?

The kinetic parameters of recombinant E. fergusonii tmk exhibit distinct pH dependence and substrate specificity profiles. While specific values for E. fergusonii tmk are not directly reported in the search results, related thymidylate kinases display the following characteristics :

The enzyme's half-life (t_1/2) at 37°C varies significantly with pH, showing values of approximately 6.9, 15.6, and 3.0 minutes at pH 5.5, 7.0, and 8.5, respectively. The presence of saturating levels of dTMP can increase thermal stability, extending the half-life to approximately 38 minutes at pH 8.5 .

How does the structure of E. fergusonii tmk relate to its function?

The structure-function relationship of E. fergusonii tmk can be inferred from homologous thymidylate kinases. Key structural features include:

• Core structure: A central five-stranded parallel β-sheet surrounded by α-helices, following the typical fold of nucleoside monophosphate kinases.

• Active site: Contains conserved residues involved in:

  • dTMP binding pocket (specificity for thymine base)

  • ATP binding site (P-loop motif)

  • Catalytic residues for phosphoryl transfer

• Conformational changes: The enzyme undergoes significant conformational changes upon substrate binding, transitioning from an "open" to a "closed" state that brings the substrates into optimal orientation for catalysis.

• Conserved motifs: Several sequence motifs are essential for function:

  • DRY/DRH motif: involved in dTMP binding

  • P-loop motif: critical for ATP binding

  • LID region: undergoes major conformational change upon ATP binding

The deduced amino acid sequence of E. fergusonii tmk likely shows significant similarity to thymidylate kinases of vertebrates, yeasts, and viruses, as observed with the E. coli enzyme , suggesting evolutionary conservation of key structural elements essential for function.

How can researchers reliably differentiate E. fergusonii tmk from E. coli tmk in experimental settings?

Reliable differentiation between E. fergusonii tmk and E. coli tmk in experimental settings requires a multi-faceted approach:

• PCR-based discrimination: Design primers targeting the four specific loci in the tmk gene sequence that differ between the species. A duplex PCR approach targeting species-specific regions can provide rapid differentiation .

• Sequence analysis: Full sequencing of the tmk gene followed by alignment with reference sequences can identify species-specific nucleotide variations.

• MLST approach: Using multiple housekeeping genes including adenylate kinase (adk), combined with tmk sequencing, provides more robust differentiation than 16S rRNA gene sequences alone .

• Restriction enzyme analysis: Identify restriction enzymes that cut specifically at sites present in only one of the species' tmk genes, allowing for rapid screening through restriction fragment length polymorphism (RFLP) analysis.

It's important to note that biochemical tests alone, including commercial systems like API 20E, may misidentify E. fergusonii as E. coli , highlighting the necessity of molecular methods for accurate differentiation.

What are the evolutionary implications of comparing tmk genes across Escherichia species?

Comparative analysis of tmk genes across Escherichia species provides valuable insights into bacterial evolution:

• Phylogenetic relationships: The tmk gene, being part of the core genome, serves as a reliable phylogenetic marker that can help reconstruct the evolutionary history of Escherichia species. E. fergusonii represents an early-diverging lineage within the Escherichia genus, as evidenced by comparative genomic studies .

• Selective pressures: Analysis of synonymous versus non-synonymous substitutions in tmk sequences can reveal selective pressures acting on this essential gene. Conservation of catalytic residues across species indicates functional constraints.

• Horizontal gene transfer assessment: Comparing tmk gene trees with species trees can identify potential horizontal gene transfer events, though the essential nature of tmk makes this less likely than for accessory genes.

• Speciation timeframe: Molecular clock analyses using tmk sequences can help estimate the divergence time between E. coli and E. fergusonii, contributing to our understanding of Enterobacteriaceae evolution.

Phylogenetic analysis indicates that E. fergusonii diverged from E. coli approximately 46 million years ago, with the tmk gene showing patterns of sequence conservation consistent with its essential cellular function .

How does E. fergusonii tmk compare to homologous enzymes in other bacterial species regarding substrate specificity?

The substrate specificity of E. fergusonii tmk likely shows similarities and distinct differences compared to homologs in other bacterial species:

• Primary substrate preference: Like other bacterial thymidylate kinases, E. fergusonii tmk preferentially phosphorylates dTMP, but may accommodate other nucleoside monophosphates with varying efficiency.

• Nucleoside analog phosphorylation: Many thymidylate kinases can phosphorylate therapeutic nucleoside analogs, which has implications for activation of prodrugs. The specific activity of E. fergusonii tmk toward analogs like 5-fluorodeoxyuridine monophosphate (FdUMP) would be of particular interest for understanding its potential role in drug metabolism .

• Comparative efficiency table: Based on related thymidylate kinases, the relative efficiency for different substrates might follow this pattern:

• Species-specific variations: Subtle amino acid differences in the active site between E. fergusonii and other species likely impact substrate recognition and catalytic efficiency, potentially providing unique biotechnological applications or drug targeting opportunities.

How can recombinant E. fergusonii tmk be utilized in antimicrobial resistance studies?

Recombinant E. fergusonii tmk offers several valuable applications in antimicrobial resistance research:

• Novel antimicrobial target validation: As an essential enzyme, tmk represents a potential target for antimicrobial development. Recombinant E. fergusonii tmk enables high-throughput screening of inhibitors that could lead to new antimicrobials effective against multidrug-resistant strains .

• Comparative studies with resistant strains: E. fergusonii has emerged as a significant reservoir of antimicrobial resistance genes, including extended-spectrum beta-lactamase genes and the mcr-1 gene conferring colistin resistance . Combining tmk functional studies with resistance gene analysis provides insights into co-evolutionary patterns.

• Inhibitor specificity assessment: Testing inhibitor efficacy against tmk enzymes from different bacterial species, including E. fergusonii, allows for the development of species-specific antimicrobial strategies.

• Resistance mechanism elucidation: When combined with whole-genome analysis of resistant E. fergusonii strains, tmk studies can reveal whether alterations in nucleotide metabolism pathways contribute to antimicrobial resistance phenotypes .

Research has shown E. fergusonii can harbor up to 43 different antimicrobial resistance genes, making it an important subject for studying the reservoir and transfer dynamics of resistance determinants .

What role might E. fergusonii tmk play in understanding bacteriophage resistance mechanisms?

E. fergusonii tmk research provides unique insights into bacteriophage resistance mechanisms through several angles:

• Nucleotide metabolism and phage replication: Phages rely on host nucleotide metabolism for replication, making tmk a critical enzyme in the phage reproductive cycle. Studying how E. fergusonii tmk interacts with phage proteins can reveal mechanisms of phage adaptation to host machinery.

• Phage-encoded tmk variants: Some bacteriophages encode their own thymidylate kinase variants that may compete with or complement host enzymes. Comparing these with E. fergusonii tmk can elucidate evolutionary strategies for phage survival.

• Genetic recombination events: Analysis of tmk sequences across E. fergusonii strains can identify potential horizontal gene transfer events mediated by bacteriophages, contributing to our understanding of bacterial genome evolution.

• Resistance mechanism development: Changes in tmk expression or structure might contribute to phage resistance by altering the nucleotide pool available for phage replication, representing a potential evolutionary strategy for bacterial survival.

Investigating how E. fergusonii tmk functions during phage infection could lead to novel biotechnological applications for phage therapy, particularly relevant in the context of increasing antimicrobial resistance .

How does E. fergusonii tmk contribute to the metabolism of fluoropyrimidine drugs?

E. fergusonii tmk plays a significant role in fluoropyrimidine drug metabolism, which has implications for both antimicrobial and cancer treatment contexts:

• Activation pathway involvement: Thymidylate kinase is a key enzyme in the activation pathway of fluoropyrimidines like 5-fluorouracil (5-FU), converting 5-FdUMP to 5-FdUDP. The efficiency of this conversion contributes to drug efficacy.

• Gut microbiome effects on chemotherapy: Recent research indicates that gut microbiome bacteria, including Escherichia species, can influence fluoropyrimidine efficacy and toxicity in cancer treatment. E. fergusonii tmk may directly metabolize these drugs in the intestinal environment .

• Resistance mechanism contribution: Altered tmk activity or expression can potentially contribute to fluoropyrimidine resistance in both bacterial infections and cancer treatment contexts.

• preTA operon interaction: E. fergusonii, like E. coli, may contain the preTA operon, which has been shown to inactivate 5-FU in the gut environment. The interaction between this pathway and the phosphorylation pathway involving tmk determines the net effect on drug bioavailability .

Research shows that gut colonization by specific bacterial species can affect fluoropyrimidine toxicity patterns, with potential clinical implications for both antimicrobial treatments and cancer chemotherapy regimens .

What computational approaches are most effective for studying E. fergusonii tmk structure and function?

Advanced computational methodologies offer powerful tools for investigating E. fergusonii tmk:

• Homology modeling and molecular dynamics:

  • Build 3D models based on crystal structures of homologous enzymes

  • Perform molecular dynamics simulations to analyze conformational changes during catalysis

  • Identify water-mediated interactions critical for substrate binding and catalysis

• Quantum mechanics/molecular mechanics (QM/MM) approaches:

  • Study the phosphoryl transfer reaction mechanism at the atomic level

  • Calculate energy barriers for catalysis

  • Investigate the role of specific residues in transition state stabilization

• Machine learning applications:

  • Predict substrate specificity based on sequence features

  • Identify potential inhibitor scaffolds through virtual screening

  • Classify evolutionary relationships between thymidylate kinases across bacterial species

• Network analysis:

  • Map interactions between tmk and other proteins in nucleotide metabolism pathways

  • Identify key regulatory nodes that affect tmk expression in different conditions

  • Predict the systems-level impact of tmk inhibition or modification

These computational approaches, when combined with experimental validation, provide comprehensive insights into the structure-function relationships of E. fergusonii tmk and guide rational design of inhibitors or engineered variants with modified properties .

What are the current limitations in E. fergusonii tmk research and how might they be addressed?

Current research on E. fergusonii tmk faces several significant limitations:

• Limited genomic data: Despite increased sequencing efforts, the number of fully sequenced E. fergusonii genomes remains limited compared to E. coli. Solution: Implement targeted sequencing initiatives focusing on diverse E. fergusonii isolates from various ecological niches to expand the genomic database.

• Experimental challenges:

  • Protein stability issues during purification and storage

  • Potential toxicity when overexpressed in heterologous systems

  • Lack of standardized assays for comparative studies

  Solution: Develop optimized expression systems with tightly regulated promoters and fusion partners specifically designed for E. fergusonii proteins. Standardize activity assays with appropriate controls for inter-laboratory comparisons.

• Functional characterization gaps: Limited understanding of the role of E. fergusonii tmk in various physiological contexts. Solution: Implement systematic functional genomics approaches, including gene knockout studies coupled with metabolomics analysis to elucidate the broader metabolic impact of tmk in E. fergusonii.

• Species misidentification: Frequent misidentification of E. fergusonii as E. coli in clinical and environmental samples . Solution: Develop and validate rapid molecular diagnostic tools specifically targeting unique regions of the tmk gene for accurate species identification.

How might CRISPR-Cas9 technology be applied to study E. fergusonii tmk function in vivo?

CRISPR-Cas9 technology offers revolutionary approaches to investigate E. fergusonii tmk function:

• Precise genetic manipulation:

  • Generate clean knockouts or point mutations in the native tmk gene

  • Create conditional expression systems to study essentiality

  • Introduce tagged versions for protein localization studies

  • Engineer variants with altered substrate specificity or catalytic efficiency

• Regulatory element analysis:

  • Target promoter regions to understand transcriptional regulation

  • Modify ribosome binding sites to control translation efficiency

  • Disrupt potential small RNA binding sites affecting tmk expression

• High-throughput functional screening:

  • Implement CRISPR interference (CRISPRi) to partially repress tmk in a tunable manner

  • Use CRISPR activation (CRISPRa) to upregulate tmk expression and assess metabolic consequences

  • Deploy CRISPR-based screens to identify genetic interactions with tmk

• In vivo tracking:

  • Create fluorescent reporter fusions to monitor tmk expression dynamics

  • Study competitive fitness of tmk variants in mixed populations

  • Analyze the impact of tmk modifications on bacterial survival in host environments

These approaches would provide unprecedented insights into the physiological role of tmk in E. fergusonii and could reveal novel aspects of nucleotide metabolism regulation in diverse environmental conditions .

What potential biotechnological applications exist for recombinant E. fergusonii tmk?

Recombinant E. fergusonii tmk holds promise for diverse biotechnological applications:

• Biocatalysis and enzymatic synthesis:

  • Production of modified nucleotides for research and therapeutic applications

  • Chemoenzymatic synthesis of nucleotide analogs with modified phosphate groups

  • Development of enzyme cascade systems for complex nucleotide derivative synthesis

• Biosensor development:

  • Creating ATP biosensors based on conformational changes in tmk

  • Developing systems to detect thymine derivatives in environmental samples

  • Engineering allosteric switches based on tmk conformational changes

• Drug development platforms:

  • High-throughput screening of antimicrobial compounds targeting bacterial thymidylate kinases

  • Structure-based drug design using E. fergusonii tmk as a template

  • Development of species-selective inhibitors based on structural differences between human and bacterial enzymes

• Nucleotide metabolism engineering:

  • Modifying nucleotide pools in bacterial production strains for enhanced nucleic acid-based product formation

  • Creating strains with altered dTTP metabolism for specialized biotechnological applications

  • Engineering synthetic biology circuits that respond to changes in nucleotide concentrations

Given E. fergusonii's emerging role as a reservoir for antimicrobial resistance genes, studying its tmk also provides opportunities for understanding resistance transfer mechanisms and developing countermeasures against the spread of resistance determinants .