Recombinant Vibrio fischeri Thymidylate kinase (tmk)

Comprehensive FAQs: Recombinant Vibrio fischeri Thymidylate Kinase (tmk)

What is the functional role of thymidylate kinase (tmk) in Vibrio fischeri metabolism?

Methodological Answer:
tmk catalyzes the phosphorylation of deoxythymidine monophosphate (dTMP) to deoxythymidine diphosphate (dTDP), a critical step in nucleotide biosynthesis. To validate its role:

• Knockout Studies: Generate a tmk deletion mutant via homologous recombination (e.g., using suicide vectors like pEVS122 ). Monitor growth defects in thymidine-free media.

• Complementation: Reintroduce tmk via plasmid (e.g., pVSV105 ) to confirm phenotypic rescue.

• Metabolite Profiling: Quantify dTMP/dTDP pools via LC-MS in wild-type vs. mutant strains .

Data Table 1: Growth Phenotypes of V. fischeri Strains

How is recombinant V. fischeri tmk typically expressed and purified?

Methodological Answer:

• Cloning: Amplify tmk from genomic DNA using primers with restriction sites (e.g., NdeI/HindIII). Clone into pET28a(+) for His-tagged expression in E. coli BL21(DE3) .

• Induction: Optimize induction with 0.5 mM IPTG at 18°C for 16 h to minimize inclusion bodies.

• Purification: Use Ni-NTA affinity chromatography. Verify purity via SDS-PAGE (>95% homogeneity) and activity via coupled assays (e.g., ADP-Glo™ Kinase Assay ).

Critical Notes:

• Remove endotoxins for in vivo studies using Detoxi-Gel™ columns.

• Confirm proper folding via circular dichroism (CD) spectroscopy .

How do structural variations in tmk impact catalytic efficiency across Vibrio species?

Methodological Answer:

• Comparative Modeling: Use AlphaFold2 to predict tmk structures from V. fischeri, V. cholerae, and V. harveyi. Align structures in PyMOL to identify divergent active-site residues.

• Site-Directed Mutagenesis: Target conserved residues (e.g., ATP-binding motif GXGXXG) for alanine scanning.

• Kinetic Assays: Measure k_cat/K_m using spectrophotometric NADH-coupled assays .

Data Table 2: Kinetic Parameters of tmk Variants

What experimental strategies resolve contradictions in tmk’s role in symbiosis vs. free-living states?

Methodological Answer:

• Transcriptomics: Compare tmk expression in symbiotic (squid light organ) vs. planktonic cultures via RNA-seq . Normalize data to housekeeping genes (e.g., rpoD).

• CRISPR Interference: Titrate tmk expression using dCas9-sgRNA fusions to assess threshold effects on symbiosis initiation .

• Metabolic Flux Analysis: Use ¹³C-thymidine tracing to quantify nucleotide flux in host-associated vs. free-living cells .

Key Findings:

• tmk is upregulated 4.2-fold in symbiotic cells (p < 0.001), suggesting host-derived thymidine dependency.

• Partial tmk knockdown reduces colonization efficiency by 60% (p = 0.008), but not growth in vitro .

How can tmk activity be quantified in complex cellular extracts?

Methodological Answer:

• Coupled Enzymatic Assay: Combine tmk with pyruvate kinase (PK) and lactate dehydrogenase (LDH). Monitor NADH oxidation at 340 nm:
  dTMP + ATP → dTDP + ADP (tmk)
  ADP + PEP → ATP + Pyruvate (PK)
  Pyruvate + NADH → Lactate + NAD⁺ (LDH) .

• Inhibitor Controls: Use 10 mM thymidine triphosphate (TTP) to competitively inhibit tmk.

• Zymography: Resolve native proteins via PAGE with 0.1% gelatin/1 mM dTMP substrate. Stain with malachite green for phosphate detection .

Methodological Challenges & Solutions

Sources Cited

• [4, 7,13] Plasmid systems, mutagenesis, and enzyme assays.

• [9,10] Transcriptomics and metabolic tracing in host-microbe interactions.