Recombinant Burkholderia cenocepacia Thymidylate kinase (tmk)

What is Burkholderia cenocepacia and why is it significant in pathogen research?

Burkholderia cenocepacia is a gram-negative opportunistic pathogen belonging to the Burkholderia cepacia complex (Bcc). It causes devastating infections in immunocompromised individuals and cystic fibrosis patients. Recent genomic analyses have revealed that strains registered as B. cenocepacia actually belong to at least two different species, with one clade enriched in clinical isolates containing key virulence factors and another clade predominantly comprising environmental isolates lacking several of these virulence determinants . The organism's ability to survive intracellularly within phagocytic cells makes it particularly challenging to treat, highlighting the importance of understanding its molecular mechanisms for potential therapeutic development .

What is the genomic context of the tmk gene in B. cenocepacia?

The tmk gene in B. cenocepacia encodes thymidylate kinase, an essential enzyme in the thymidine nucleotide biosynthesis pathway. Based on genomic analyses of B. cenocepacia strains, researchers typically use strain J2315 as a reference for genetic studies . To identify and analyze the tmk gene, whole-genome sequencing data can be examined using bioinformatic approaches similar to those used for other B. cenocepacia genes. Phylogenetic analysis can be performed using tools like PhyML with the GTR+GAMMA model, with visualization using platforms such as iTOL v6 . This contextual understanding is crucial for designing recombinant expression strategies.

How does B. cenocepacia clonal complex distribution relate to tmk conservation?

B. cenocepacia clonal complex (CC) 31 represents a predominant lineage causing outbreaks globally, particularly in non-cystic fibrosis patients in regions like India . Whole-genome sequence analysis of 35 CC31 isolates compared with 210 CC31 genomes from the NCBI database has revealed high diversity within this lineage . For tmk research, understanding this genomic diversity is essential as it may impact enzyme structure-function relationships and potential as a drug target. When analyzing tmk sequences from different isolates, phylogenetic approaches similar to those used for whole-genome analyses can help determine conservation patterns and evolutionary relationships.

What expression systems are optimal for recombinant B. cenocepacia tmk production?

Based on successful expression of other B. cenocepacia proteins, E. coli-based expression systems using vectors like pET28a represent a primary choice for tmk expression . A methodological approach would involve:

• PCR amplification of the tmk gene using primers incorporating appropriate restriction sites (e.g., NdeI and XhoI as used for hppD gene expression)

• Ligation into a pre-digested pET28a vector with N-terminal His-tag

• Transformation into E. coli DH5α for plasmid propagation

• Verification by restriction digestion and DNA sequencing

• Transformation into E. coli BL21(DE3) for protein expression

Expression conditions should be optimized by testing various temperatures (18-37°C), IPTG concentrations (0.1-1.0 mM), and induction times (3-16 hours).

What purification strategies yield high-purity B. cenocepacia tmk?

A multi-step purification protocol would typically involve:

• Initial purification: Immobilized metal affinity chromatography (IMAC) using Ni-NTA resin for His-tagged tmk

• Intermediate purification: Ion exchange chromatography (typically Q-Sepharose at pH 8.0)

• Final polishing: Size exclusion chromatography using Superdex 75 or 200

The following table outlines typical buffer conditions for each step:

Quality control should include SDS-PAGE, Western blotting with anti-His antibodies, and activity assays to confirm functional protein production.

How can solubility of recombinant B. cenocepacia tmk be enhanced?

To improve soluble expression, consider implementing these strategies:

• Lower induction temperatures (16-20°C) to slow folding and prevent aggregation

• Co-expression with chaperones (GroEL/GroES, DnaK/DnaJ/GrpE)

• Addition of solubility-enhancing tags (MBP, SUMO, TrxA) beyond the His-tag

• Optimization of lysis conditions (buffer pH 7.5-8.5, salt concentration 100-500 mM NaCl)

• Addition of stabilizing agents (5-10% glycerol, 1 mM DTT or TCEP)

For particularly challenging expression, cell-free protein synthesis systems may be considered as an alternative approach.

What are the optimal assay conditions for measuring B. cenocepacia tmk activity?

Thymidylate kinase activity can be determined through several approaches:

• Coupled enzyme assay: Linking ATP consumption to NADH oxidation through pyruvate kinase and lactate dehydrogenase

• Direct product analysis: HPLC-based detection of dTDP formation

• Radioactive assay: Using [γ-32P]ATP and measuring radioactive dTDP formation

Typical assay conditions include:

How does structural characterization inform B. cenocepacia tmk function?

Structural characterization should include:

• Secondary structure analysis: Circular dichroism spectroscopy to determine α-helical and β-sheet content

• Thermal stability assessment: Differential scanning fluorimetry (DSF) or calorimetry (DSC)

• Oligomerization state: Size exclusion chromatography with multi-angle light scattering (SEC-MALS)

• Crystallization trials: For high-resolution structure determination

These analyses help identify unique structural features that distinguish B. cenocepacia tmk from human thymidylate kinase, potentially revealing selective inhibitor binding sites.

What substrate specificity does B. cenocepacia tmk exhibit?

A comprehensive substrate specificity analysis should include:

• Testing various nucleoside monophosphates (dTMP, dUMP, dCMP, dGMP, dAMP)

• Evaluating different phosphate donors (ATP, GTP, CTP, UTP)

• Determining kinetic parameters (Km, kcat, kcat/Km) for each substrate combination

Results are typically presented in table format:

This data helps determine the enzyme's preference for natural substrates and reveals potential for substrate analogs as inhibitors.

How can gene disruption approaches be applied to study tmk in B. cenocepacia?

Since tmk is likely essential, conditional approaches are required:

• Insertional inactivation: Using a protocol similar to that described for hppD disruption , where a 300-bp internal fragment of the target gene is amplified by PCR with primers containing appropriate restriction sites, then ligated into a suicide vector like pGPΩTp .

• Conditional expression: Placing tmk under the control of an inducible promoter to create a depletion strain.

• Complementation testing: For validation, chemical complementation with downstream metabolites or genetic complementation using plasmids expressing wild-type tmk should be performed .

What methods effectively validate tmk essentiality in B. cenocepacia?

To establish the essential nature of tmk, researchers should:

• Attempt direct gene knockout and assess viability

• Create a conditional mutant where tmk expression is controlled by an inducible promoter

• Perform growth studies under inducing and non-inducing conditions

• Analyze morphological changes during tmk depletion using microscopy

• Conduct rescue experiments with plasmid-encoded tmk

Essential genes typically show complete growth arrest upon depletion, with potential cell morphology changes indicating disrupted cell division.

How do genomic variant analyses inform tmk function across B. cenocepacia clades?

Recent studies have shown that strains registered as B. cenocepacia belong to at least two different species . Comparative genomic analysis of tmk sequences from these different clades can reveal:

• Conservation patterns of catalytic residues

• Clade-specific sequence variations that might impact enzyme function

• Correlation between tmk sequence variations and virulence profiles

• Potential horizontal gene transfer events affecting tmk

Analysis methods should include multiple sequence alignment, phylogenetic tree construction, and selection pressure analysis (dN/dS ratios) similar to those used for whole-genome comparisons in studies of B. cenocepacia diversity .

What screening approaches effectively identify B. cenocepacia tmk inhibitors?

Effective inhibitor screening approaches include:

• Enzymatic assays: High-throughput screening using the coupled enzyme assay measuring ATP consumption

• Fragment-based screening: Using thermal shift assays (DSF) to identify fragment hits

• Virtual screening: Computational docking of compound libraries against tmk structure

• Phenotypic screening: Testing growth inhibition of B. cenocepacia followed by target validation

The table below compares these approaches:

How can inhibitor selectivity for B. cenocepacia tmk over human homologs be assessed?

Selectivity assessment requires:

• Parallel testing of inhibitors against purified B. cenocepacia tmk and human thymidylate kinase

• Calculation of selectivity indices (SI = IC₅₀ human / IC₅₀ bacterial)

• Structural analysis of binding modes to identify exploitable differences

• Cell-based testing in bacterial versus mammalian cells

• Counter-screening against related kinases to establish specificity profile

What strategies can overcome B. cenocepacia's intrinsic antibiotic resistance when targeting tmk?

B. cenocepacia exhibits high antimicrobial resistance due to multiple mechanisms . Strategies to overcome these barriers include:

• Efflux pump inhibitor combination: Co-administration with efflux pump inhibitors

• Prodrug approaches: Designing tmk inhibitors as prodrugs that are activated inside bacterial cells

• Nanoparticle delivery: Encapsulation in nanoparticles that can penetrate bacterial biofilms

• Permeability enhancement: Structural modifications to increase compound penetration through bacterial outer membrane

• Polypharmacology: Designing dual-target inhibitors affecting tmk and another essential pathway

How can transcriptomics and proteomics reveal tmk's role in B. cenocepacia pathogenesis?

Multi-omics approaches provide insights into tmk's broader biological context:

• RNA-Seq analysis: Compare tmk expression levels under different conditions (e.g., planktonic vs. biofilm growth, different infection stages)

• Proteomics: Identify proteins co-regulated with tmk using techniques like LC-MS/MS

• Interactomics: Determine tmk protein interaction partners using pull-down assays coupled with mass spectrometry

• Metabolomics: Analyze changes in nucleotide metabolism upon tmk inhibition

These methods can reveal how tmk expression correlates with virulence factor production and stress responses.

What infection models best evaluate the impact of tmk inhibition on B. cenocepacia virulence?

Appropriate infection models include:

• Cell culture models: Using macrophage cell lines to study intracellular survival following tmk inhibition, similar to studies showing B. cenocepacia's ability to escape to the cytosol

• Galleria mellonella: Invertebrate model for initial in vivo assessment

• Murine models: For evaluating efficacy in acute and chronic infection scenarios

• Biofilm models: Assessing impact on biofilm formation and resistance

Key readouts should include bacterial burden, inflammatory markers, and survival rates.

How can CRISPR-Cas9 technologies advance tmk research in B. cenocepacia?

CRISPR-Cas9 applications for tmk research include:

• Precise genetic modifications: Creating point mutations to study structure-function relationships

• Promoter engineering: Replacing native promoter with inducible systems for expression control

• CRISPRi: Using deactivated Cas9 (dCas9) for transcriptional repression without gene deletion

• Base editing: Introducing specific nucleotide changes without double-strand breaks

• Tagged variant creation: Adding fluorescent or affinity tags for localization and interaction studies

How has tmk evolved across the Burkholderia cepacia complex species?

Evolutionary analysis should include:

• Sequence comparison: Extracting tmk sequences from multiple Burkholderia genomes

• Phylogenetic analysis: Using methods similar to those employed for whole-genome phylogeny

• Selection pressure analysis: Calculating dN/dS ratios to identify conserved functional domains

• Recombination analysis: Using tools like Gubbins to identify potential recombination events

• Correlation with species boundaries: Determining if tmk phylogeny supports the proposed division of B. cenocepacia into multiple species

How does tmk sequence variation correlate with B. cenocepacia's ecological niches?

Recent genomic analyses have shown that B. cenocepacia strains from different ecological niches (clinical vs. environmental) form distinct phylogenetic clades with different virulence factor profiles . Analysis of tmk variation should:

• Compare sequences between clinical and environmental isolates

• Identify any niche-specific tmk variants

• Correlate tmk sequences with the presence/absence of key virulence factors

• Determine if tmk characteristics support the proposed evolutionary trajectory from plant-associated to human pathogen

What insights can tmk provide into B. cenocepacia's host adaptation process?

Genomic analyses suggest B. cenocepacia underwent a host jump from plants/environment to animals . Studying tmk in this context can:

• Identify any selection signatures in tmk associated with host adaptation

• Compare tmk expression regulation between environmental and clinical isolates

• Determine if tmk functional differences contribute to survival in different hosts

• Assess whether tmk could serve as a marker for tracking evolutionary transitions

How does tmk expression change during different phases of B. cenocepacia infection?

This research direction would require:

• Ex vivo studies: Analysis of B. cenocepacia recovered from patient samples at different infection stages

• In vitro modeling: Recreating host conditions (e.g., CF sputum medium, oxygen limitation, antibiotic pressure)

• Transcriptional analysis: Measuring tmk expression using qRT-PCR or RNA-Seq

• Correlation analysis: Linking tmk expression with virulence factor production and antibiotic resistance

What is the potential of tmk as a diagnostic biomarker for B. cenocepacia infections?

Biomarker development approaches include:

• PCR-based detection: Designing tmk-specific primers for sensitive and specific detection

• Antibody development: Generating antibodies against unique B. cenocepacia tmk epitopes

• Mass spectrometry: Identifying tmk-specific peptides in clinical samples

• Validation studies: Testing sensitivity and specificity in patient cohorts

How can tmk inhibitors be effectively integrated into combination therapies for B. cenocepacia?

Given B. cenocepacia's intrinsic resistance to many antibiotics , effective combination strategies would:

• Synergy testing: Evaluate interactions between tmk inhibitors and conventional antibiotics

• Resistance mechanism targeting: Combine tmk inhibitors with agents that compromise resistance mechanisms

• Biofilm penetration assessment: Test combinations that enhance activity against biofilm-embedded bacteria

• Host-directed therapy combinations: Explore combining tmk inhibitors with immunomodulatory agents