Recombinant Nitrosomonas europaea Probable inorganic polyphosphate/ATP-NAD kinase (ppnK)

Advanced Research Questions

• What are the most effective methods for heterologous expression and purification of recombinant N. europaea ppnK?

  Based on successful approaches with other N. europaea enzymes, the following protocol is recommended:

  1. Gene amplification: PCR-amplify the ppnK gene (NE1324) from genomic DNA using high-fidelity polymerase

  2. Cloning strategy: Insert the gene into an expression vector (pET-based systems work well for similar enzymes)

  3. Expression system: Transform into E. coli BL21(DE3) or similar strain

  4. Induction conditions: IPTG induction (0.5-1.0 mM) at 16-25°C for 16-20 hours to minimize inclusion body formation

  5. Cell lysis: Sonication or French press in buffer containing 50 mM Tris-HCl pH 8.0, 300 mM NaCl, 10% glycerol

  6. Purification: IMAC purification if using His-tagged construct, followed by ion exchange and size exclusion chromatography

  7. Activity preservation: Include 1-5 mM DTT or β-mercaptoethanol to maintain sulfhydryl groups in reduced state

  For functional characterization, it's crucial to verify that the recombinant enzyme retains both ATP- and polyphosphate-dependent NAD kinase activities.

• How can the kinetic parameters of ppnK be accurately determined using different substrates?

  To determine kinetic parameters of N. europaea ppnK with different substrates, employ the following methodological approach:

  1. Spectrophotometric assay:

     • Primary reaction: NAD+ + ATP (or polyP) → NADP+ + ADP (or polyP n-1)

     • Coupling system: NADP+ + glucose-6-phosphate → 6-phosphogluconate + NADPH (using glucose-6-phosphate dehydrogenase)

     • Monitor NADPH formation at 340 nm (ε = 6220 M-1 cm-1)

  2. Reaction conditions:

     • Buffer: 50 mM Tris-HCl or HEPES, pH 7.5-8.0

     • MgCl2: 5-10 mM (essential cofactor)

     • Temperature: 30°C (physiological for N. europaea)

  3. Substrate variations:

     • For ATP-dependent activity: Vary ATP (0.05-5 mM) with constant NAD+ (1-2 mM)

     • For polyP-dependent activity: Use polyP of defined chain length (polyP15 and polyP3 have shown different kinetic parameters in other bacterial ppnK enzymes)

     • For NAD+ affinity: Vary NAD+ (0.05-5 mM) with saturating ATP or polyP

  4. Data analysis:

     • Use Michaelis-Menten or Hill equations depending on the kinetic pattern observed

     • Calculate Vmax, Km, and Hill coefficients if cooperative behavior is observed

     • Determine kcat by dividing Vmax by enzyme concentration

• How does polyphosphate chain length affect the kinetic properties of N. europaea ppnK?

  Research with other bacterial polyphosphate kinases has shown significant differences in enzyme kinetics depending on polyphosphate chain length. For example, in Rhipicephalus microplus pyrophosphatase with polyphosphatase activity:

  Parameter	polyP3	polyP15
  Km (μM)	177.3 ± 8.9	25.5 ± 1.5
  Vmax (μmol/min/mg)	0.728 ± 0.010	0.655 ± 0.010
  kcat/Km (s-1μM-1)	0.016	0.099
  Hill coefficient	1.40 ± 0.08	1.75 ± 0.14

  These data indicate greater affinity, catalytic efficiency, and cooperativity with longer polyphosphate chains .

  To investigate similar effects with N. europaea ppnK:

  1. Prepare polyphosphate samples of defined chain lengths (commercially available or synthesized enzymatically)

  2. Use identical reaction conditions while varying only the polyphosphate chain length

  3. Analyze binding kinetics using isothermal titration calorimetry (ITC) to determine binding stoichiometry and thermodynamic parameters

  4. Correlate kinetic parameters with structural features using molecular docking simulations

• What role does ppnK play in stress responses of Nitrosomonas europaea?

  While specific data on ppnK in N. europaea stress responses is limited, studies in related bacteria provide important insights that can guide research:

  1. Heavy metal stress: N. europaea exposed to cadmium (1 μM CdCl2), mercury (6 μM HgCl2), or copper (8 μM CuCl2) shows approximately 90% inhibition of ammonia oxidation . Under these conditions, stress response genes are significantly upregulated. To determine if ppnK is involved:

     • Employ RT-qPCR to quantify ppnK expression under heavy metal stress

     • Compare polyphosphate levels between stressed and non-stressed cells

     • Analyze ppnK-knockout mutants for altered sensitivity to heavy metals

  2. Oxidative stress: As NAD kinases influence NADPH production, which is essential for antioxidant defense, investigate ppnK's role by:

     • Exposing cultures to hydrogen peroxide or superoxide generators

     • Monitoring cellular redox status using fluorescent probes

     • Measuring survival rates of wild-type versus ppnK-overexpressing strains

  3. Nutrient limitation: Polyphosphate serves as a phosphate reserve during phosphate limitation. Research approaches include:

     • Culturing N. europaea under phosphate-limited conditions

     • Quantifying polyphosphate mobilization using 31P-NMR spectroscopy

     • Analyzing ppnK expression during different growth phases using microarrays or RNA-seq

• What techniques can be used to monitor polyphosphate dynamics in living N. europaea cells?

  Several complementary techniques can be employed to monitor polyphosphate dynamics in living N. europaea cells:

  1. Fluorescent microscopy with DAPI staining:

     • Prepare cells by fixation with 4% paraformaldehyde

     • Stain with 4',6-diamidino-2-phenylindole (DAPI) at 10-50 μg/mL

     • Observe using fluorescence microscopy (excitation 360-400 nm, emission >490 nm for polyphosphate-DAPI complex)

     • Yellow emission (rather than blue DNA emission) indicates polyphosphate granules

  2. 31P-NMR spectroscopy:

     • Prepare concentrated cell suspensions (OD600 >10)

     • Collect spectra using a high-field NMR spectrometer with 31P probe

     • Identify polyphosphate signals at approximately -20 to -22 ppm

     • Quantify against internal phosphate standards

  3. Biochemical extraction and quantification:

     • Extract polyphosphate using acid extraction protocols

     • Quantify using the malachite green assay after enzymatic hydrolysis

     • Separate polyphosphates of different chain lengths using gel electrophoresis

  4. Transmission electron microscopy:

     • Fix cells with glutaraldehyde and osmium tetroxide

     • Embed in resin and prepare ultrathin sections

     • Electron-dense granules typically represent polyphosphate deposits

     • Confirm identity with energy-dispersive X-ray spectroscopy (EDX)

• How can genetic manipulation techniques be applied to study ppnK function in N. europaea?

  Genetic manipulation of N. europaea to study ppnK function presents unique challenges due to its slow growth and specialized metabolism. A comprehensive approach includes:

  1. Gene knockout strategy:

     • Design a knockout construct with antibiotic resistance marker (e.g., kanamycin) flanked by 1-2 kb homologous regions upstream and downstream of ppnK

     • Introduce DNA via electroporation (2.5 kV, 200 Ω, 25 μF) or conjugation with E. coli donor strain

     • Select transformants on media containing appropriate antibiotics

     • Verify gene deletion by PCR and Southern blotting

     • Phenotypically characterize mutants for growth, ammonia oxidation, and stress responses

  2. Complementation studies:

     • Clone wild-type ppnK into a broad-host-range vector (e.g., pBBR1MCS series)

     • Introduce into knockout strain to verify phenotype restoration

     • Include negative controls with catalytically inactive mutants (e.g., point mutations in predicted active site)

  3. Reporter gene fusion:

     • Create transcriptional or translational fusions of the ppnK promoter with reporter genes (gfp, lacZ)

     • Monitor expression patterns under different growth conditions and stressors

     • Identify regulatory elements in the promoter region through deletion analysis

  4. Overexpression studies:

     • Express ppnK under control of a constitutive promoter

     • Evaluate effects on polyphosphate accumulation, stress resistance, and metabolic parameters

• What molecular approaches can identify potential protein-protein interactions of ppnK in N. europaea?

  To identify protein-protein interactions involving ppnK in N. europaea, employ these molecular approaches:

  1. Bacterial two-hybrid system:

     • Clone ppnK into bait and prey vectors

     • Co-transform into reporter E. coli strain

     • Screen for interactions based on reporter gene activation

     • Verify positive interactions with biochemical methods

  2. Co-immunoprecipitation (Co-IP):

     • Express epitope-tagged ppnK in N. europaea or heterologous host

     • Prepare cell lysates under mild conditions to preserve protein complexes

     • Immunoprecipitate with antibodies against the tag

     • Identify co-precipitated proteins by mass spectrometry

  3. Cross-linking mass spectrometry (XL-MS):

     • Treat intact cells or purified complexes with chemical cross-linkers

     • Digest with proteases and enrich cross-linked peptides

     • Analyze by LC-MS/MS to identify interaction interfaces

     • Map interaction sites on structural models

  4. Proximity-dependent biotin identification (BioID):

     • Create fusion protein of ppnK with biotin ligase

     • Express in N. europaea cells

     • Supply biotin to cells to biotinylate proximal proteins

     • Purify biotinylated proteins and identify by mass spectrometry

  5. Pull-down assays with recombinant proteins:

     • Purify recombinant ppnK with affinity tag

     • Incubate with N. europaea cell lysate

     • Analyze bound proteins by SDS-PAGE and mass spectrometry

• How does the structure of N. europaea ppnK compare to characterized homologs from other bacteria?

  While the specific structure of N. europaea ppnK has not been fully characterized, comparative analysis with homologs suggests:

  1. Domain architecture:

     • N. europaea ppnK likely contains regulatory cystathionine β-synthase (CBS) domains common to family II pyrophosphatases

     • May contain the DRTGG domain found between CBS domains in many bacterial ppnK enzymes

     • The DRTGG domain is particularly significant as it has been shown to be important for binding diadenosine polyphosphates (ApnAs) in other bacteria

  2. Structural modeling approach:

     • Generate homology models using related crystal structures as templates

     • Validate models using molecular dynamics simulations

     • Predict substrate binding sites and catalytic residues

     • Design site-directed mutagenesis experiments to test functional predictions

  3. Evolutionary relationships:

     • Phylogenetic analysis places N. europaea ppnK among β-proteobacterial NAD kinases

     • Comparative genomics shows ppnK is often co-located with genes involved in nucleotide metabolism or stress response

     • Structural conservation suggests functional importance across bacterial lineages

• What are the potential biotechnological applications of recombinant N. europaea ppnK?

  Recombinant N. europaea ppnK has several potential biotechnological applications:

  1. Biosensor development:

     • Engineer biosensors for environmental monitoring based on ppnK activity or expression

     • Create whole-cell biosensors with ppnK promoter-reporter fusions to detect nitrification inhibitors

     • Develop immobilized enzyme systems for ATP or polyphosphate detection

  2. NADP+ regeneration systems:

     • Employ recombinant ppnK for efficient NADP+ regeneration in biocatalytic processes

     • Optimize for stability and catalytic efficiency through protein engineering

     • Couple with polyphosphate-dependent reactions for cost-effective cofactor recycling

  3. Wastewater treatment applications:

     • Monitor nitrification activity in biological nitrogen removal processes

     • Detect heavy metal contamination that could inhibit ammonia oxidation

     • Enhance phosphate removal through engineered polyphosphate accumulation

  4. Polyphosphate production:

     • Develop enzymatic systems for controlled production of polyphosphates of defined chain lengths

     • Create polyphosphate-based biomaterials with applications in medicine and industry

     • Engineer metabolic pathways for enhanced polyphosphate accumulation in host organisms

Research Methodology and Resources

• What are the best techniques for measuring ppnK enzyme kinetics with different phosphoryl donors?

  To comprehensively characterize ppnK enzyme kinetics with different phosphoryl donors:

  1. Continuous spectrophotometric assay:

     • Reaction mixture: 50 mM HEPES (pH 7.5), 5 mM MgCl2, 1 mM NAD+, variable concentrations of phosphoryl donor (ATP or polyphosphate), coupling enzymes (G6PDH/6PGDH), glucose-6-phosphate

     • Monitor NADPH formation at 340 nm

     • Calculate initial velocities from linear portion of progress curves

  2. Radiometric assay for precise measurements:

     • Use [γ-32P]ATP as phosphoryl donor

     • Separate reaction products by thin-layer chromatography

     • Quantify radioactivity in NADP+ band using phosphorimager

  3. Analysis of polyphosphate utilization:

     • Use polyphosphates of defined chain lengths (polyP3, polyP15, etc.)

     • Analyze polyphosphate consumption using polyacrylamide gel electrophoresis

     • Quantify reaction products using HPLC with conductivity detection

  4. Data analysis protocol:

     • Fit initial velocity data to appropriate kinetic models

     • Determine kinetic parameters (Km, kcat, Hill coefficients)

     • Compare catalytic efficiencies (kcat/Km) between different phosphoryl donors

  5. Isothermal titration calorimetry:

     • Measure binding thermodynamics (ΔH, ΔS, Kd)

     • Determine binding stoichiometry

     • Evaluate cooperative binding behavior

• How can transcriptomic and proteomic techniques be used to understand ppnK regulation in N. europaea?

  Integrated omics approaches provide comprehensive insights into ppnK regulation:

  1. Transcriptomic analysis:

     • RNA-Seq or microarray analysis of N. europaea under different conditions

     • Compare expression profiles between wild-type and mutant strains

     • Identify co-regulated genes through cluster analysis

     • Map transcriptional start sites using 5' RACE or RNA-Seq

     A transcriptomic study of N. europaea exposed to heavy metals showed that:

     • 66 genes were upregulated and 50 genes downregulated >2-fold after 1 μM CdCl2 exposure

     • Mercury resistance genes (merTPCADE) showed dramatic upregulation (277-fold average)

     • Similar patterns were observed with mercury and copper exposure

  2. Proteomic approach:

     • Use iTRAQ LC-MS/MS to quantify protein abundance changes

     • Identify post-translational modifications using phosphoproteomics

     • Compare proteome changes in response to stress conditions

     • Analyze protein-protein interactions through immunoprecipitation-mass spectrometry

  3. Integrative analysis:

     • Correlate transcriptomic and proteomic data to identify post-transcriptional regulation

     • Apply network analysis to place ppnK in functional modules

     • Use comparative genomics to identify conserved regulatory elements

     • Develop predictive models of ppnK regulation under different conditions

• What recent advances in understanding polyphosphate metabolism in bacteria could inform research on N. europaea ppnK?

  Recent advances in bacterial polyphosphate metabolism with implications for N. europaea ppnK research include:

  1. Polyphosphate and stress response integration:

     • Polyphosphate has been linked to regulation of ATP-dependent proteolysis of type II toxin-antitoxin systems

     • Polyphosphate interacts with alternative sigma/anti-sigma factors

     • These regulatory connections could explain multiple structural and functional deficiencies in polyP metabolism mutants

  2. Polyphosphate and biofilm formation:

     • Studies in biofilms of N. europaea showed differential gene expression compared to planktonic cells

     • 240 genes were differentially expressed between mature biofilms and exponential batch cells

     • Biofilm cells exhibited increased resistance to inhibitors like phenol and toluene

  3. Methodological advances:

     • Development of fluorescent probes for polyphosphate visualization in living cells

     • Cryo-electron tomography for studying subcellular localization of polyphosphate granules

     • CRISPR-Cas9 gene editing systems adapted for challenging bacterial species

  4. Evolutionary insights:

     • Comparative genomics of polyphosphate metabolism genes across bacterial lineages

     • Identification of novel polyphosphate-utilizing enzymes

     • Understanding the evolutionary history of NAD kinases and their substrate specificity