Recombinant Bifidobacterium adolescentis Thymidylate kinase (tmk)

What are the optimal expression systems for producing recombinant B. adolescentis TMK?

Basic Question

Escherichia coli remains the primary expression system for recombinant B. adolescentis TMK due to its high yield, ease of genetic manipulation, and established purification protocols. The methodology typically involves:

• Cloning the tmk gene from B. adolescentis (commonly strain ATCC 15703) into an expression vector containing an N-terminal His-tag

• Transforming the construct into an E. coli expression strain (JM109 is commonly used)

• Inducing protein expression in transformed cells using IPTG at concentrations between 0.5-1.0 mM

• Harvesting cells and lysing via sonication or chemical methods

• Purifying the His-tagged TMK using nickel affinity chromatography

For purification, the enzyme is typically eluted from nickel-IDA resin using an imidazole gradient (50-200 mM), followed by desalting and concentration steps . The purified protein can be verified by SDS-PAGE and Western blotting, with expected molecular weight of approximately 23-25 kDa.

How can I optimize soluble protein yield when expressing recombinant B. adolescentis TMK?

Advanced Question

Maximizing soluble recombinant TMK requires systematic optimization of multiple parameters:

• Temperature modulation: Lowering post-induction temperature to 16-20°C significantly increases soluble protein yield by reducing inclusion body formation

• Induction parameters: Using lower IPTG concentrations (0.1-0.25 mM) and extending expression time (16-24 hours)

• Co-expression strategies: Co-expressing molecular chaperones (GroEL/GroES, DnaK/DnaJ) to assist protein folding

• Fusion partners: N-terminal fusion with solubility-enhancing tags such as MBP (maltose-binding protein) or SUMO

• Media optimization: Supplementing expression media with specific metal ions (Mg²⁺, Mn²⁺) at 1-5 mM concentrations that stabilize TMK structure

Studies have demonstrated that addition of 5% glycerol to lysis buffers and maintaining pH between 7.0-7.5 further enhances TMK stability during purification. For crystallography-grade protein, size exclusion chromatography as a final polishing step yields >95% pure protein based on SDS-PAGE analysis.

What are the standard methods for measuring B. adolescentis TMK activity?

Basic Question

TMK activity can be assessed through several complementary approaches:

• NADH-coupled spectrophotometric assay: The most common method monitors the decrease in NADH absorbance at 340 nm as TMK catalyzes the phosphorylation of dTMP. The reaction is coupled to pyruvate kinase and lactate dehydrogenase reactions, where the consumption of NADH is proportional to TMK activity.

• Radioactive assay: Using [γ-³²P]ATP as a substrate and monitoring the transfer of the labeled phosphate group to dTMP.

• HPLC-based method: Separating and quantifying the reaction products (dTDP) and remaining substrates.

Standard reaction conditions include:

• 50 mM Tris-HCl buffer (pH 7.5)

• 50 mM KCl

• 5 mM MgCl₂

• 0.5 mM dTMP

• 2 mM ATP

• 37°C incubation

The optimum pH and temperature for B. adolescentis TMK activity typically fall within the ranges of pH 6.5-7.5 and 30-37°C, respectively, reflecting the physiological conditions of the human gut where this bacterium naturally resides .

How does the kinetic profile of B. adolescentis TMK compare with TMKs from other bacterial species?

Advanced Question

B. adolescentis TMK exhibits distinct kinetic properties compared to TMKs from other bacterial species, particularly pathogens like Mycobacterium tuberculosis and Pseudomonas species. A comparative analysis reveals:

B. adolescentis TMK demonstrates substrate specificity primarily for dTMP, with minimal activity toward other nucleoside monophosphates. Analysis of TMK from different bacterial sources reveals variations in active site architecture that explain these kinetic differences. Specifically, the presence of a more flexible LID domain in B. adolescentis TMK compared to M. tuberculosis TMK results in different catalytic mechanisms .

Researchers investigating these differences often employ site-directed mutagenesis to identify catalytic residues responsible for the distinct kinetic profiles, providing insights for inhibitor design specific to pathogenic bacterial TMKs while minimizing effects on commensal bacteria like B. adolescentis.

What structural features distinguish B. adolescentis TMK from other bacterial TMKs?

Basic Question

B. adolescentis TMK shares the core structural elements common to all TMKs, including:

• A RecA-like fold for the main domain

• A LID domain that closes during catalysis

• A P-loop motif (Walker A) for nucleotide binding

• The LID region of B. adolescentis TMK contains unique residues that influence its conformational flexibility during the catalytic cycle

• The active site architecture shows differences in the positioning of catalytic residues compared to TMKs from pathogens like M. tuberculosis

• The nucleotide binding pocket exhibits altered electrostatic properties that affect substrate specificity

Structural analysis is typically performed using X-ray crystallography with resolutions of 2.0-2.5 Å, allowing researchers to observe these distinctive features . Homology modeling can also be employed when crystal structures are unavailable, using established structures (like those from PDB entries 5NRN and 5NR7 for other bacterial TMKs) as templates .

How can molecular dynamics simulations enhance our understanding of B. adolescentis TMK function?

Advanced Question

Molecular dynamics (MD) simulations provide crucial insights into B. adolescentis TMK function beyond static crystal structures, revealing:

• Conformational dynamics: MD simulations (typically 100-500 ns) capture LID domain movements during substrate binding and product release, which are critical for understanding catalytic mechanisms.

• Water-mediated interactions: Simulations identify conserved water molecules that form hydrogen-bonding networks essential for substrate recognition and catalysis.

• Allosteric communication: Long-range interactions between distal protein regions can be identified through principal component analysis and dynamic cross-correlation maps.

• Binding free energy calculations: MM-PBSA/MM-GBSA calculations quantify the energetic contributions of specific residues to substrate binding, with typical protocols involving:

  • System equilibration (10-20 ns)

  • Production simulations (50-100 ns)

  • Extraction of 100-500 frames for energy calculations

  • Decomposition of binding energy into enthalpic and entropic components

These computational approaches have identified unique structural transitions in B. adolescentis TMK not observed in TMKs from other bacterial species. For example, specific residues in the P-loop region exhibit distinctive flexibility patterns that correlate with catalytic efficiency. Such information is valuable for rational enzyme engineering and the development of selective inhibitors targeting pathogenic bacterial TMKs while sparing beneficial bifidobacteria .

What vector systems are most effective for heterologous expression of B. adolescentis TMK?

Basic Question

Effective heterologous expression of B. adolescentis TMK requires careful selection of vector systems. The following approaches have proven successful:

• E. coli expression systems:

  • pET vector series (particularly pET-28a) with T7 promoter for high-level expression

  • pBAD vectors with arabinose-inducible promoter for tighter expression control

  • pMAL vectors for fusion with maltose-binding protein to enhance solubility

• Bifidobacterial expression systems:

  • pBEX-derived vectors utilizing the constitutive gap promoter from B. bifidum S17

  • The glyceraldehyde-3-phosphate dehydrogenase (gap) promoter shows high constitutive activity across different growth phases in bifidobacteria

When expressing in bifidobacteria, the experimentally verified transcription start site (TSS) of the gap promoter should be incorporated (62 bp upstream of the ATG start codon in B. bifidum S17). Additionally, inclusion of the native ribosome-binding site (RBS), which is highly complementary to the 3′-end of the 16S rRNA with only one mismatch, enhances translation efficiency .

How can the gap promoter be optimized for improved expression of recombinant TMK in Bifidobacterium?

Advanced Question

Optimizing the gap promoter for enhanced TMK expression in Bifidobacterium involves several sophisticated approaches:

• Promoter engineering based on experimental characterization:

  • The consensus -35 (TTGCCN) and -10 (TANAGT) regions of bifidobacterial gap promoters with a spacer of 17-19 bases can be modified to more closely match the optimal spacing of 17 bp

  • The experimentally determined -35 region (TTGCTC) and -10 region (TACAGT) from B. bifidum S17 can serve as a starting template

• RBS optimization:

  • Using algorithms to design optimal RBS sequences that enhance translation initiation rates

  • Incorporating the highly complementary RBS sequence identified in B. bifidum S17, which shows only one mismatch to the anti-Shine-Dalgarno sequence

• Codon optimization:

  • Adjusting the tmk gene codons to match the codon usage bias of highly expressed genes in Bifidobacterium adolescentis

  • Eliminating rare codons particularly in the N-terminal region of the protein

• 5' UTR engineering:

  • The 5' untranslated region can be modified to eliminate secondary structures that impede ribosome binding

  • The optimal length of the 5' UTR for Bifidobacterium has been determined to be 35-62 nucleotides based on experimental verification

Systematic studies comparing wild-type and engineered promoters have demonstrated that modifications to the spacer region between -35 and -10 elements can increase expression levels by 2-4 fold. Additionally, combining RBS optimization with codon harmonization can further improve protein yields by up to 10-fold compared to native sequences .

What are the current applications of recombinant B. adolescentis TMK in research?

Basic Question

Recombinant B. adolescentis TMK has several important research applications:

• Comparative enzymology studies: As a model enzyme from beneficial gut bacteria, it serves as a comparison point for TMKs from pathogenic organisms, helping researchers understand evolutionary differences in nucleotide metabolism.

• Inhibitor screening: It is used as a control enzyme when screening potential antimicrobial compounds targeting TMK, ensuring selectivity for pathogenic bacterial TMKs while sparing beneficial gut microbiota.

• Metabolic engineering: Understanding TMK activity helps in engineering Bifidobacterium strains with enhanced nucleotide metabolism for various biotechnological applications.

• Structure-function studies: The enzyme serves as a model system for investigating the catalytic mechanisms of bacterial TMKs through site-directed mutagenesis and kinetic analyses.

• Protein-protein interaction studies: TMK often functions within metabolic complexes, and recombinant versions are used to identify interaction partners in nucleotide synthesis pathways.

These applications typically involve purified recombinant enzyme or genetically modified bacterial strains expressing the recombinant TMK .

How can B. adolescentis TMK be utilized in the development of selective antimicrobial strategies?

Advanced Question

Leveraging B. adolescentis TMK for selective antimicrobial development involves sophisticated approaches:

• Differential inhibitor design: Using structural and mechanistic differences between pathogenic TMKs (e.g., M. tuberculosis TMK) and B. adolescentis TMK to design inhibitors that selectively target the former while sparing the latter. This requires:

  • Conducting comprehensive structure-activity relationship (SAR) studies

  • Utilizing computer-aided drug design with pharmacophore models

  • Implementing MM-PBSA calculations to predict binding affinities across different TMKs

• Resistance mechanism investigation: Recombinant B. adolescentis TMK can be used to study natural resistance mechanisms to certain TMK inhibitors, providing insights for developing antimicrobials that circumvent these mechanisms in pathogens.

• In vivo selectivity assessment: Evaluating how potential TMK inhibitors affect the viability of B. adolescentis versus pathogenic bacteria in complex microbial communities, using:

  • Gnotobiotic mouse models

  • Complex in vitro gut microbiome systems

  • Metagenomic and metaproteomic analytical approaches

• Pharmacophore-based screening: Advanced computational models based on known TMK inhibitors have been used to screen chemical libraries, with promising compounds showing IC₅₀ values in the nanomolar range against pathogenic TMKs while having minimal effect on B. adolescentis TMK .

An example of this approach is seen in compounds targeting M. tuberculosis TMK, where structural differences in the binding pocket allow for the development of inhibitors like TKI1 (1-(1-((4-(3-Chlorophenoxy)quinolin-2-yl)methyl)piperidin-4-yl)-5-methylpyrimidine-2,4(1H,3H)-dione) with 1000-fold selectivity over commensal bacterial TMKs .

What methods are available for genetic transformation of B. adolescentis?

Basic Question

Genetic transformation of B. adolescentis presents unique challenges due to its oxygen sensitivity and thick cell wall. Successful approaches include:

• Electroporation protocols:

  • Cultivation of B. adolescentis in MRS medium supplemented with 0.5% glucose

  • Harvest cells in mid-exponential phase (OD₆₀₀ = 0.6-0.8)

  • Wash cells with 1 mM ammonium citrate buffer (pH 6.0) containing 0.5 M sucrose

  • Electroporation at 2.0-2.5 kV, 200 Ω, 25 μF

  • Immediate recovery in pre-warmed MRS with 0.5 M sucrose for 3-4 hours under anaerobic conditions

• Conjugation methods:

  • Utilizing E. coli donors carrying mobilizable plasmids

  • Filter mating on non-selective media under anaerobic conditions

  • Gradual selection on appropriate antibiotics

• Protoplast transformation:

  • Cell wall weakening using lysozyme (1-5 mg/ml) and mutanolysin (50-100 U/ml)

  • Transformation of protoplasts with PEG-mediated methods

  • Regeneration of cell wall on specialized media

Successful transformation is typically verified by PCR amplification of the introduced genes, with expected transformation efficiencies ranging from 10²-10⁴ transformants per μg of plasmid DNA .

What strategies can overcome the challenges of expressing heterologous proteins like TMK in B. adolescentis?

Advanced Question

Expressing heterologous TMK in B. adolescentis faces challenges including codon bias, toxicity, and degradation. Advanced strategies to overcome these include:

• Synthetic biology approaches:

  • Development of synthetic, minimal promoters based on the gap promoter architecture but with optimized -35 and -10 regions

  • Engineering synthetic ribosome binding sites with precise spacing (8-10 nucleotides) from the start codon

  • Creating modular expression cassettes with standardized restriction sites for rapid cloning

• Protein engineering for optimal expression:

  • Fusion with bifidobacterial secretion signals (e.g., from α-amylase or β-galactosidase) for extracellular production

  • Addition of stability-enhancing domains from native bifidobacterial proteins

  • Codon harmonization rather than optimization, maintaining the translational rhythm of the native organism

• Advanced induction systems:

  • Development of xylose-inducible promoters for tight regulation

  • Two-component regulatory systems responsive to specific environmental signals

  • Riboswitches that enable post-transcriptional regulation

• Stress response mitigation:

  • Co-expression of chaperones specific to Bifidobacterium (GroEL, DnaK)

  • Pre-conditioning cells with sublethal stresses to activate protective mechanisms

  • Engineered strains with enhanced oxidative stress resistance through overexpression of thioredoxin reductase (TrxB) and NADH oxidoreductase (BaiC)

Successful expression can be monitored through reporter systems such as the β-glucuronidase assay, with enzyme activities typically reaching 60-80% of those observed in E. coli expression systems when optimal conditions are achieved .

How does B. adolescentis TMK contribute to nucleotide metabolism in the context of gut microbiome function?

Basic Question

B. adolescentis TMK plays a central role in nucleotide metabolism with implications for gut microbiome function:

Studies have shown that B. adolescentis strains with robust TMK activity demonstrate enhanced persistence in the gut microbiome, highlighting the enzyme's importance for ecological fitness .

What advanced structural biology techniques have provided insights into the catalytic mechanism of B. adolescentis TMK?

Advanced Question

Several sophisticated structural biology techniques have elucidated the catalytic mechanism of B. adolescentis TMK:

• Time-resolved X-ray crystallography:

  • Capturing intermediate states by flash-freezing crystals at different time points after substrate addition

  • Identifying conformational changes in the LID domain during catalysis

  • Visualizing the precise positioning of catalytic water molecules

• Nuclear Magnetic Resonance (NMR) spectroscopy:

  • ¹⁵N/¹³C-labeled TMK for backbone assignment

  • Measuring chemical shift perturbations upon substrate binding

  • Relaxation dispersion experiments revealing microsecond-millisecond timescale dynamics corresponding to catalytic motions

  • Hydrogen-deuterium exchange experiments identifying regions with altered solvent accessibility during the catalytic cycle

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

  • Mapping regions of altered conformational dynamics upon substrate binding

  • Identifying allosteric networks connecting distant regions of the protein

  • Quantifying the energetics of conformational changes via temperature-dependent HDX

• Cryo-electron microscopy (Cryo-EM):

  • Recent advances enabling high-resolution structures of smaller proteins like TMK

  • Visualization of conformational ensembles revealing the distribution of states rather than single conformations

  • Identification of previously undetected transient states in the catalytic cycle

These advanced techniques have revealed that B. adolescentis TMK undergoes a sequential ordered mechanism where ATP binds first, followed by dTMP. The binding of ATP induces conformational changes in the P-loop and LID domain that create an optimal binding site for dTMP. After phosphoryl transfer, the LID domain opens to release the products in the reverse order: dTDP followed by ADP .

What are the key considerations when designing inhibitors selective for bacterial TMKs over B. adolescentis TMK?

Basic Question

Designing inhibitors that selectively target pathogenic bacterial TMKs while sparing B. adolescentis TMK requires attention to several key factors:

• Structural differences in active sites: Exploiting unique structural features in the active sites of pathogenic bacterial TMKs (such as M. tuberculosis TMK) that differ from B. adolescentis TMK.

• Binding pocket analysis: Identifying divergent amino acid residues in the binding pockets that can be targeted for selective binding.

• Catalytic mechanism variations: Leveraging differences in the catalytic mechanisms, such as variations in the LID domain movement or P-loop conformations.

• Substrate specificity profiles: Utilizing differences in substrate preferences between TMKs from different bacterial species.

• Allosteric sites: Targeting non-conserved allosteric sites present in pathogenic TMKs but absent in B. adolescentis TMK.

Current screening approaches typically employ enzymatic assays with purified recombinant TMKs from different bacterial sources to identify compounds with at least 100-fold selectivity for pathogenic TMKs over B. adolescentis TMK .

How can advanced computational methods enhance the development of selective TMK inhibitors?

Advanced Question

Advanced computational methods have revolutionized the development of selective TMK inhibitors through several sophisticated approaches:

• Pharmacophore-based virtual screening with machine learning augmentation:

  • Development of complex pharmacophore models from known TMK inhibitors

  • Implementation of machine learning algorithms to identify subtle structural patterns correlating with selectivity

  • Integration of absolute binding energy estimation to prioritize compounds

  • Validation using multiple molecular docking algorithms to confirm binding modes

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

  • Hybrid QM/MM methods to accurately model the phosphoryl transfer reaction mechanism

  • Identification of transition states that differ between pathogenic and commensal bacterial TMKs

  • Design of transition state analogs that selectively inhibit pathogenic TMKs

• Free energy perturbation (FEP) calculations:

  • Rigorous calculation of relative binding free energies across different TMK orthologs

  • Identification of chemical modifications that enhance selectivity

  • Typical FEP protocols involve:

    • System preparation with appropriate protonation states

    • Gradual transformation of ligands over 20-30 λ windows

    • Calculation of ΔΔG values with estimated errors <1 kcal/mol

• Fragment-based design enhanced by deep learning:

  • Identification of fragment binding hotspots unique to pathogenic TMKs

  • De novo design of inhibitors by combining fragments

  • Validation through free energy calculations and experimental testing