Recombinant Burkholderia thailandensis Bifunctional protein glk (glk)

What is Burkholderia thailandensis and how does it relate to pathogenic Burkholderia species?

Burkholderia thailandensis is an environmental bacterium closely related to the pathogenic Burkholderia pseudomallei, the causative agent of melioidosis. B. thailandensis is generally considered non-pathogenic compared to B. pseudomallei, though rare cases of human infection have been documented . The organism serves as an important model system for studying Burkholderia biology due to its genetic tractability and lower biosafety requirements.

B. thailandensis E264 is the most commonly studied laboratory strain, with a fully sequenced genome containing two circular chromosomes. Some environmental isolates, including those found in Laos, express a capsular polysaccharide similar to B. pseudomallei, known as B. thailandensis capsular variant (BTCV) .

What are the known functions of the bifunctional glk protein in B. thailandensis?

The bifunctional glk protein in B. thailandensis functions primarily as a glucokinase, catalyzing the phosphorylation of glucose to glucose-6-phosphate in the first committed step of glycolysis. Its bifunctional nature suggests a secondary role, potentially in regulatory processes or metabolic adaptation to varying environmental conditions.

Analysis of gene expression data shows that glk expression in B. thailandensis varies significantly under different environmental stressors, including temperature shifts, pH changes, and nutrient limitation . This suggests its important role in metabolic adaptation.

What genomic features make B. thailandensis suitable for recombinant protein studies?

B. thailandensis possesses several genomic features that make it well-suited for recombinant protein studies:

• Natural competence for DNA transformation

• Well-characterized genome with established genetic tools

• RecA-mediated homologous recombination systems that facilitate genetic manipulation

• Multiple selectable and counter-selectable markers compatible with the organism

• Lower pathogenicity compared to related species, allowing work under BSL-2 conditions

The genome contains IS elements that can facilitate genomic rearrangements, including duplications of large chromosomal regions (up to 208.6 kb) that may affect gene dosage and expression levels .

What expression systems are most effective for producing recombinant proteins in B. thailandensis?

For optimal recombinant protein expression in B. thailandensis, several key systems have demonstrated efficacy:

• Arabinose-inducible promoter systems: The pKaKa1 and pKaKa2 broad-host-range plasmids containing arabinose-inducible promoters have been successfully used for controlled gene expression .

• Rhamnose-inducible systems: These provide an alternative induction mechanism, particularly useful when working with genes involved in arabinose metabolism .

• Chromosomal integration: For stable expression, integration into the genome at specific sites (such as attTn7) has proven effective for long-term studies .

When expressing recombinant glk specifically, consider using inducible promoters to control expression levels, as overexpression of metabolic enzymes can affect growth characteristics.

How do environmental conditions affect glk expression and function in B. thailandensis?

Environmental conditions significantly impact glk expression and function in B. thailandensis as demonstrated by transcriptomic studies:

Studies using the Burkholderia-specific microarray containing probe sets to all intergenic regions have identified regulatory sRNAs that may influence glk expression under specific stress conditions . When designing experiments to study glk function, it is critical to carefully control environmental parameters to ensure reproducible results.

What purification strategies yield highest activity for recombinant glk?

To maintain optimal enzymatic activity of recombinant glk, consider the following purification strategies:

• Affinity purification using N-terminal or C-terminal tags (His6, GST, or MBP tags) with careful evaluation of tag position to avoid interference with the bifunctional nature of the protein.

• Buffer optimization:

  • Phosphate buffers (50-100 mM) at pH 7.2-7.5

  • Addition of glycerol (10-20%) for stability

  • Include reducing agents (1-5 mM DTT or β-mercaptoethanol) to maintain thiol groups

• Temperature considerations:

  • Maintain purification steps at 4°C

  • Consider activity assays at multiple temperatures (30°C, 37°C, 41°C) to determine optimal conditions

• When assessing enzymatic activity, use coupled assays to measure both functions of the bifunctional protein under varying substrate concentrations.

How can I perform precise genetic modifications of glk in B. thailandensis?

For precise genetic modifications of glk in B. thailandensis, several advanced techniques have proven effective:

λ Red Recombineering System:
B. thailandensis is amenable to λ Red recombineering, which uses the λ phage proteins (Gam, Bet, and Exo) to mediate homologous recombination with short homology arms . The broad-host-range plasmids pKaKa1 and pKaKa2 containing these λ Red genes under inducible promoters enable efficient recombination .

Implementation protocol:

• Generate PCR products with 40-45 bp homology arms flanking your target modification in the glk gene

• Transform cells expressing λ Red proteins with the PCR product

• Select recombinants using appropriate markers (e.g., glyphosate resistance with the gat gene)

• Verify modifications by PCR and sequencing

Knock-out and Pull-out Strategies:
For functional studies of glk, you can employ either:

• Knock-out (KO): Replace glk with selectable markers like pheS-gat

• Pull-out (PO): Extract the gene using oriT-ColE1ori-gat-ori1600-rep cassettes for downstream manipulation in E. coli

The entire process typically takes approximately 10 days and offers advantages over traditional methods, including smaller PCR products and increased flexibility for downstream processing .

What role does RecA play in genome stability when working with recombinant constructs?

RecA plays a critical role in genome stability when working with recombinant constructs in B. thailandensis:

• Mediating homologous recombination: RecA facilitates the exchange of homologous DNA sequences, which is essential for integrating recombinant constructs into the genome .

• Resolving tandem duplications: RecA-dependent homologous recombination resolves tandem duplications in the genome, making it a key factor in genome plasticity .

• Dynamic regulation of copy number: Research has shown that RecA activity contributes to the dynamic variation in copy number of duplicated regions within B. thailandensis populations .

Experimental evidence demonstrates that in RecA-deficient strains (ΔrecA::nptII), the resolution of tandem duplications is significantly impaired compared to wild-type strains . When engineering recombinant constructs, especially those involving repetitive elements, consider the impact of RecA activity on the stability of your construct.

How can I create conditional knockouts of glk to study its essential functions?

Creating conditional knockouts of glk in B. thailandensis requires sophisticated genetic approaches to study its potentially essential functions:

Inducible promoter replacement strategy:

• Replace the native glk promoter with an inducible promoter (arabinose or rhamnose-responsive)

• Generate the construct using PCR with homology arms targeting the promoter region

• Transform using the λ Red system expressed from pKaKa1 or pKaKa2

• Select transformants on appropriate selection media

• Verify by PCR and sequencing

Complementation-based approach:

• Introduce a second copy of glk under an inducible promoter at a neutral site (e.g., attTn7)

• Delete the native glk gene using counter-selectable markers like sacB or pheS

• Maintain expression of the introduced copy during selection

• Control expression levels to study phenotypic consequences

CRISPR-Cas9 approach:
While not explicitly mentioned in the search results, CRISPR-Cas9 systems have been adapted for Burkholderia species and could be used to create precise modifications in the glk gene while minimizing polar effects on neighboring genes.

What assays best characterize the bifunctional activities of glk protein?

To comprehensively characterize the bifunctional activities of the glk protein from B. thailandensis, employ these complementary assays:

Glucokinase activity assay:

• Spectrophotometric coupled assay with glucose-6-phosphate dehydrogenase

• Measure NADPH formation at 340 nm

• Determine kinetic parameters (Km, Vmax) for glucose and ATP substrates

• Test pH and temperature optima (particularly relevant given B. thailandensis' environmental adaptability)

Secondary function characterization:

• Protein-protein interaction studies using pull-down assays

• Targeted metabolomics to identify alternative substrates

• Structural analysis (X-ray crystallography or cryo-EM) to identify binding domains

In vivo functional characterization:

• Complementation studies in defined knockout strains

• Phenotypic microarrays under varying carbon sources

• Growth analyses under conditions identified in expression studies (temperature, pH, and salt variations)

The bifunctional nature of glk may be related to its role in adaptation to environmental stressors, as suggested by the differential expression patterns observed in transcriptomic studies .

How can I assess the impact of genomic context on glk expression and function?

To evaluate how genomic context influences glk expression and function in B. thailandensis:

Genomic duplication analysis:
B. thailandensis exhibits RecA-dependent duplication of large genomic regions (208.6 kb) that can contain multiple genes . To determine if glk resides within such a region:

• Design PCR primers for junction analysis (similar to the Junc1/Junc2 approach described for detecting duplicated regions)

• Screen individual colonies from wild-type populations to assess copy number variation

• Quantify gene dosage using qPCR

Transcriptional context assessment:

• Identify potential operonic structures using RNA-seq data

• Map transcription start sites using 5' RACE

• Characterize promoter elements through reporter gene fusions

Phase variation impact:
B. thailandensis undergoes phase variation, generating genotypically and phenotypically heterogeneous populations . To assess whether this affects glk:

• Isolate and characterize phase variants using PCR-based methods

• Compare glk expression levels across variants

• Correlate expression with specific phenotypes

A junction PCR approach can be used to detect multiple copies of genomic regions, which may influence glk expression through gene dosage effects .

What techniques can detect protein-protein interactions involving glk in B. thailandensis?

To identify and characterize protein-protein interactions involving glk in B. thailandensis:

In vivo approaches:

• Bacterial two-hybrid system: Adapt the bacterial two-hybrid system for use in B. thailandensis to detect direct protein interactions

• Cross-linking coupled with mass spectrometry (XL-MS): Perform in vivo cross-linking followed by affinity purification and mass spectrometry identification of interaction partners

• Fluorescence resonance energy transfer (FRET): Express glk and potential partners as fluorescent protein fusions to detect interactions in living cells

In vitro approaches:

• Co-immunoprecipitation (Co-IP): Use antibodies against glk or epitope-tagged versions to pull down interaction partners

• Surface plasmon resonance (SPR): Measure binding kinetics between purified glk and potential partners

• Isothermal titration calorimetry (ITC): Quantify thermodynamic parameters of interactions

Bioinformatic prediction:
Integrate data from small RNA studies in B. thailandensis with protein interaction networks to identify potential regulatory relationships affecting glk function.

How does glk contribute to B. thailandensis adaptation to environmental stressors?

The bifunctional glk protein likely plays a central role in B. thailandensis adaptation to environmental stressors through several mechanisms:

Metabolic adaptation:

• As a glucokinase, glk controls the entry of glucose into glycolysis

• Expression patterns vary significantly under different environmental conditions (temperature, pH, nutrient limitation)

• May facilitate metabolic shifting between carbon sources during environmental transitions

Stress response coordination:
Based on the comprehensive study of small RNA expression across 54 distinct growth conditions, glk expression appears coordinated with stress response pathways . This suggests its involvement in:

• Temperature adaptation responses (37°C to 41°C shift)

• pH homeostasis mechanisms (especially at pH 9)

• Phosphate limitation responses

• Salt stress adaptation

Phase variation mechanisms:
B. thailandensis employs phase variation as a bet-hedging strategy to adapt to fluctuating environmental conditions . The regulation of metabolic genes like glk may be part of this adaptation strategy, creating subpopulations optimized for different environmental niches.

What regulatory elements control glk expression under different conditions?

The expression of glk in B. thailandensis is controlled by multiple regulatory elements that respond to environmental conditions:

Transcriptional regulation:

• Promoter architecture may include binding sites for global regulators

• Environmental sensors like two-component systems likely influence expression

Post-transcriptional regulation:
Small RNAs (sRNAs) play a significant role in regulating gene expression in B. thailandensis. The comprehensive profiling of sRNA expression under 54 distinct growth conditions identified 38 novel sRNAs . These sRNAs can:

• Base-pair with mRNAs to attenuate translation

• Modulate gene expression in response to specific environmental changes

• Enable adaptation of cellular physiology to stressors

How does genomic duplication affect glk expression and metabolic adaptation?

Genomic duplication has significant implications for glk expression and metabolic adaptation in B. thailandensis:

Gene dosage effects:
B. thailandensis E264 populations exhibit heterogeneity in the copy number of a 208.6 kb region that contains 157 coding sequences . If glk is located within or regulated by elements in this region:

• Expression levels would vary among subpopulations

• Create diversity in metabolic capabilities within the population

• Contribute to the bet-hedging strategy for environmental adaptation

RecA-mediated dynamics:
The copy number variation of duplicated regions is maintained through RecA-dependent homologous recombination :

• RecA facilitates both duplication and resolution events

• Creates a dynamic equilibrium in the population

• Generates subpopulations with different metabolic profiles

Experimental evidence:
Studies using reporter constructs have demonstrated that the resolution of tandem duplications is RecA-dependent, with significant differences observed between wild-type and RecA-deficient strains . This dynamic process facilitates rapid adaptation to disparate growth conditions.

The heterogeneity in genomic architecture creates a population with diverse metabolic capabilities, potentially including variable glk expression levels, which may be advantageous when facing fluctuating environmental conditions.

How can I engineer glk for enhanced catalytic efficiency or substrate specificity?

Engineering glk from B. thailandensis for enhanced properties requires detailed understanding of structure-function relationships:

Targeted mutagenesis approach:

• Identify catalytic residues and substrate binding sites through homology modeling

• Generate point mutations using site-directed mutagenesis

• Express and purify variants using the optimized recombinant expression system

• Perform enzyme kinetics to assess changes in catalytic parameters

• Validate in vivo using complementation of knockout strains

Domain shuffling strategy:
For modifying the bifunctional nature of glk:

• Identify distinct functional domains

• Create chimeric proteins with domains from related enzymes

• Screen for novel functionalities or enhanced native activities

Directed evolution:

• Generate a library of glk variants using error-prone PCR

• Transform into an appropriate selection system

• Apply selective pressure relevant to the desired property

• Isolate and characterize improved variants

When designing these experiments, consider the environmental adaptability of B. thailandensis and how engineered changes might affect the protein's function under various stress conditions .

What insights can comparative genomics provide about glk evolution across Burkholderia species?

Comparative genomic analysis of glk across Burkholderia species reveals important evolutionary patterns:

Functional conservation and divergence:

• Compare sequence and structural features of glk between B. thailandensis and pathogenic relatives like B. pseudomallei

• Identify conserved catalytic residues versus variable regions

• Map species-specific adaptations that might relate to environmental niches

Genomic context analysis:
The genomic neighborhood of glk may differ between species:

• In B. thailandensis, the presence of insertion sequence (IS) elements facilitates genomic rearrangements

• These genomic rearrangements may affect the regulation and expression of glk

• Comparing synteny across species can reveal evolutionary events affecting glk function

Evolutionary pressure analysis:
Calculate selection pressures (dN/dS ratios) across different Burkholderia species to identify:

• Regions under purifying selection (conserved function)

• Regions under positive selection (adaptive evolution)

• Correlation with pathogenicity or environmental adaptation

The development of capsular variants in B. thailandensis demonstrates how genomic changes can generate phenotypic diversity across the genus, potentially affecting metabolic functions like those performed by glk.

How can glk be used as a model for studying protein evolution and neofunctionalization?

The bifunctional glk protein from B. thailandensis provides an excellent model for studying protein evolution and neofunctionalization:

Ancestral sequence reconstruction:

• Build a phylogenetic tree of glk homologs across bacterial species

• Infer ancestral sequences at key evolutionary nodes

• Resurrect and characterize these ancestral proteins to understand the evolution of bifunctionality

Experimental evolution studies:
Using the recombineering techniques established for B. thailandensis :

• Create libraries of glk variants

• Subject populations to selection under diverse conditions

• Monitor changes in sequence and function over time

• Identify pathways to neofunctionalization

Structural biology approach:

• Determine the three-dimensional structure of B. thailandensis glk

• Compare with structures of single-function homologs

• Identify structural elements that contribute to bifunctionality

• Design experiments to test the role of these elements

Genomic plasticity context:
The RecA-dependent genomic rearrangements observed in B. thailandensis create a natural laboratory for studying gene duplication and divergence, central processes in protein evolution and neofunctionalization. This genomic plasticity may facilitate the evolution of novel protein functions through duplication and subsequent divergence.