Recombinant Burkholderia cepacia Elongation factor Tu (tuf)

What is the genomic structure of the tuf gene in Burkholderia cepacia complex species?

The tuf gene in Burkholderia cepacia complex (Bcc) species is part of its complex genomic structure. B. cepacia has an unusually complex genome with two to four chromosomes (usually three) and numerous insertion sequences, which affects the organization and expression of genes like tuf . The complexity of this genome means that genes that might be duplicated on these chromosomes would supply regions of homology allowing for rearrangement and recombination. This genomic complexity must be considered when designing primers and amplification strategies for the tuf gene.

How can the tuf gene be used for molecular identification of Burkholderia cepacia complex species?

The tuf gene, encoding Elongation Factor Tu, can serve as a valuable molecular marker for identifying and distinguishing between members of the Burkholderia cepacia complex. Similar to its application in streptococcal species, where a 761-bp portion of the tuf gene was used to develop genus-specific PCR primers and conduct phylogenetic analysis , researchers can:

• Design specific PCR primers targeting conserved regions of the tuf gene

• Amplify the target region from genomic DNA

• Sequence the amplicons to identify species-specific variations

• Analyze sequence divergence to distinguish between closely related species

The high conservation of tuf combined with species-specific variations makes it particularly useful for distinguishing between the genomovars within the B. cepacia complex.

What are the key differences between tuf genes across the Burkholderia cepacia complex genomovars?

The B. cepacia complex consists of at least five genomovars based on DNA hybridization, with genomovars II and V named B. multivorans and B. vietnamiensis respectively . While the search results don't specifically detail tuf gene variations across these genomovars, differences would likely occur in non-conserved regions. Researchers should expect:

• Core functional regions of tuf to remain highly conserved

• Variability in third-base positions of codons (synonymous mutations)

• Potential genomovar-specific signature sequences that could be used for identification

• Variations reflecting the evolutionary history and adaptation of different genomovars

What are the optimal PCR conditions for amplifying the tuf gene from Burkholderia cepacia complex strains?

When amplifying the tuf gene from B. cepacia complex strains, researchers should consider:

• DNA extraction method: Use specialized protocols designed for Gram-negative bacteria with high GC content

• Primer design:

  • Target conserved regions flanking variable sections

  • Account for the high GC content of Burkholderia genomes

  • Consider using degenerate primers if amplifying from diverse strains

• PCR conditions:

  • Include additives like DMSO (5-10%) to help with GC-rich templates

  • Use a touchdown PCR approach: starting with higher annealing temperatures (65°C) and gradually decreasing

  • Employ longer extension times to account for secondary structures

  • Use polymerases optimized for GC-rich templates

Similar to protocols used for streptococcal species, verification should include gel electrophoresis followed by sequencing of the amplicons .

How should researchers design expression systems for recombinant Burkholderia cepacia EF-Tu?

For optimal expression of recombinant B. cepacia EF-Tu, consider:

• Vector selection:

  • Inducible expression systems (like arabinose-inducible promoters used in Burkholderia genetic tools )

  • Vectors with appropriate selection markers (tetracycline, chloramphenicol, kanamycin, or trimethoprim resistance genes)

  • Fusion tags that enhance solubility and facilitate purification

• Host selection:

  • E. coli strains optimized for expression of genes with high GC content

  • Consider Pseudomonas-based expression systems for a more compatible cellular environment

  • For certain applications, expression in other Burkholderia species using tools like those developed for B. multivorans

• Codon optimization:

  • Adjust codon usage to match the expression host

  • Avoid rare codons while maintaining key structural elements

• Expression conditions:

  • Lower temperatures (16-25°C) often improve folding of complex proteins

  • Optimize induction conditions (concentration and timing)

  • Consider the addition of specific chaperones to aid folding

What purification strategies yield the highest purity and activity for recombinant B. cepacia EF-Tu?

For high-purity, active recombinant B. cepacia EF-Tu, a multi-step purification approach is recommended:

• Initial capture:

  • Affinity chromatography using His-tag or other fusion partners

  • Ensure buffers contain GTP or GDP to stabilize the protein

• Intermediate purification:

  • Ion exchange chromatography to separate based on charge differences

  • Heparin affinity chromatography to remove nucleic acid contaminants (EF-Tu often co-purifies with RNA)

• Polishing:

  • Size exclusion chromatography to obtain homogeneous protein

  • Remove potential aggregates or truncated forms

• Quality control:

  • SDS-PAGE and western blotting for purity assessment

  • Mass spectrometry for identity confirmation

  • GTPase activity assays to confirm functionality

  • Circular dichroism to verify proper folding

How can CRISPR/Cas9 systems be adapted for genomic editing of the tuf gene in Burkholderia cepacia?

CRISPR/Cas9 technology can be adapted for B. cepacia based on systems developed for B. multivorans :

• System components:

  • A two-plasmid system consisting of:

    • pCasPA expressing both Cas9 endonuclease and λ-Red system proteins (Exo, Gam, and Bet)

    • pACRISPR expressing sgRNA and carrying the repair template for homology-directed repair

• Modifications for tuf gene editing:

  • Design sgRNAs targeting specific regions of the tuf gene

  • Create repair templates with desired mutations flanked by homology arms

  • Consider conditional approaches since tuf is likely essential

• Implementation process:

  • Introduce plasmids via triparental conjugation from E. coli to Burkholderia

  • Induce expression with L-arabinose

  • Select transformants using appropriate antibiotics

  • Verify edits through sequencing

  • Cure plasmids using counter-selection with the sacB gene or growth at 18-20°C with serial passages

What are the challenges in using CRISPR/Cas9 for studying essential genes like tuf in B. cepacia?

When using CRISPR/Cas9 to study essential genes like tuf in B. cepacia, researchers face several challenges:

• Lethality concerns:

  • Direct knockout is likely lethal

  • Need for conditional expression systems or partial modifications

• Technical challenges:

  • Off-target effects may be more detrimental when modifying essential genes

  • Lower efficiency may occur due to selective pressure against successful edits

  • Multiple chromosomes in B. cepacia may harbor redundant essential genes

• Solution approaches:

  • Create conditional mutants (inducible promoters controlling expression)

  • Engineer point mutations that alter function without eliminating it

  • Use CRISPRi (CRISPR interference) to modulate expression levels rather than knock out the gene

  • Target non-essential domains while preserving core functions

  • Introduce a complementary copy of the gene before editing the native copy

How can researchers verify successful CRISPR/Cas9-mediated modifications of the tuf gene?

Verification of CRISPR/Cas9-mediated modifications to the tuf gene requires a multi-level approach:

• Genomic verification:

  • PCR amplification and sequencing of the targeted region

  • Restriction fragment length polymorphism (RFLP) if the edit introduces or removes a restriction site

  • Next-generation sequencing to detect potential off-target effects

• Transcript analysis:

  • RT-PCR to confirm transcription of the modified gene

  • RNA-Seq to assess potential impacts on global gene expression

• Protein verification:

  • Western blotting to confirm protein expression and size

  • Mass spectrometry to verify amino acid changes

  • Functional assays to assess GTPase activity

• Phenotypic confirmation:

  • Growth rate analysis to detect fitness effects

  • Stress response tests to identify conditional phenotypes

  • Ribosome profiling to assess impact on translation

How does EF-Tu contribute to virulence and pathogenesis in the Burkholderia cepacia complex?

Elongation Factor Tu may contribute to B. cepacia complex pathogenesis through multiple mechanisms:

• Moonlighting functions:

  • Like other bacterial EF-Tu proteins, B. cepacia EF-Tu may have functions beyond protein synthesis

  • Potential roles in adhesion to host cells or extracellular matrix

  • Possible immunomodulatory effects during infection

• Role in stress adaptation:

  • EF-Tu may help bacteria adapt to stressful conditions encountered during infection

  • Contribution to antibiotic tolerance through effects on translation regulation

  • Potential involvement in biofilm formation, a key virulence trait in B. cepacia complex bacteria

• Research approaches:

  • Comparative proteomics of clinical vs. environmental isolates

  • Cell adhesion assays with recombinant EF-Tu

  • Host immune response studies using purified protein

  • Mutational analysis of specific domains

What role does the tuf gene play in taxonomic and evolutionary studies of the Burkholderia cepacia complex?

The tuf gene serves as a valuable marker for taxonomic and evolutionary studies of the B. cepacia complex:

• Phylogenetic applications:

  • Similar to its use in streptococcal species , tuf sequences can be used to reconstruct evolutionary relationships

  • The conserved nature with sufficient variability makes it ideal for distinguishing closely related species

  • Can help clarify relationships between the five genomovars identified through polyphasic taxonomic evaluation

• Genomic context analysis:

  • Examining the arrangement of genes surrounding tuf across species can provide insights into genome evolution

  • May reveal horizontal gene transfer events or genomic rearrangements

• Comparative analysis approach:

  • Multiple sequence alignment of tuf genes from different isolates

  • Calculation of nucleotide diversity and evolutionary distances

  • Construction of phylogenetic trees to visualize relationships

  • Correlation with whole-genome phylogenies to validate findings

How can recombinant B. cepacia EF-Tu be used to develop diagnostic tools for clinical applications?

Recombinant B. cepacia EF-Tu offers potential for developing novel diagnostic approaches:

• Antibody-based diagnostics:

  • Generate antibodies against unique epitopes of B. cepacia EF-Tu

  • Develop immunoassays for detecting B. cepacia in clinical samples

  • Use in immunofluorescence or immunohistochemistry for direct visualization

• PCR-based detection systems:

  • Design primers targeting distinctive regions of the tuf gene

  • Develop multiplexed assays to differentiate B. cepacia complex genomovars

  • Create real-time PCR assays for quantitative detection

• Considerations for specificity:

  • Cross-reactivity with related species must be addressed

  • Similar to findings with capsular polysaccharide (CPS), where B. cepacia strains were found to express B. pseudomallei-like CPS , overlapping features may complicate diagnostics

  • Comprehensive validation with diverse clinical isolates is essential

• Complementary approaches:

  • Combine tuf-based detection with other specific markers

  • Integrate with mass spectrometry profiling for increased accuracy

What are common troubleshooting approaches for failed PCR amplification of the tuf gene from clinical isolates?

When encountering difficulties amplifying the tuf gene from B. cepacia clinical isolates:

• DNA extraction optimization:

  • Test multiple extraction protocols designed for Gram-negative bacteria

  • Include additional purification steps to remove PCR inhibitors

  • Quantify and assess DNA quality (A260/A280 ratio) before PCR

• PCR condition adjustments:

  • Gradient PCR to identify optimal annealing temperature

  • Increase denaturation temperature or time for GC-rich templates

  • Add PCR enhancers (DMSO, betaine, glycerol)

  • Try different polymerase enzymes with high-GC capabilities

• Primer redesign considerations:

  • Check for potential secondary structures in primers

  • Verify primer specificity against updated sequence databases

  • Consider degenerate primers to accommodate strain variations

  • Design nested PCR approaches for increased specificity

• Control reactions:

  • Include positive controls from type strains

  • Test primers on verified B. cepacia samples

  • Run 16S rRNA gene amplification as a control for DNA quality

How can researchers differentiate between genomic contaminants and genuine results when studying tuf gene expression?

To ensure genuine results when studying tuf gene expression in B. cepacia:

• RNA isolation considerations:

  • Implement stringent DNase treatment protocols

  • Verify absence of genomic DNA by PCR without reverse transcription

  • Use RNA extraction methods optimized for high-GC bacteria

• RT-PCR controls:

  • Include no-RT controls for each sample

  • Use intron-spanning primers when possible

  • Apply normalization with multiple reference genes validated for B. cepacia

• Expression analysis approaches:

  • qRT-PCR with target-specific probes

  • Northern blotting for direct visualization of transcript size

  • RNA-Seq with stringent mapping parameters to distinguish closely related sequences

  • Consider strand-specific RNA-Seq to identify antisense transcription

• Validation strategies:

  • Confirm key findings with alternative methods

  • Sequence amplicons to verify identity

  • Use biological replicates from independent isolations

What are the critical quality control parameters for recombinant B. cepacia EF-Tu in structural and functional studies?

For high-quality structural and functional studies of recombinant B. cepacia EF-Tu, researchers should implement these quality control measures:

• Purity assessment:

  • SDS-PAGE with Coomassie or silver staining (>95% purity)

  • SEC-MALS (Size Exclusion Chromatography-Multi-Angle Light Scattering) for homogeneity analysis

  • Mass spectrometry to confirm intact mass and detect modifications

• Structural integrity verification:

  • Circular dichroism spectroscopy to confirm secondary structure

  • Thermal shift assays to assess stability

  • Dynamic light scattering to detect aggregation

  • Limited proteolysis to confirm proper folding

• Functional validation:

  • GTP binding assays using labeled GTP or fluorescent analogs

  • GTPase activity measurements under various conditions

  • Aminoacyl-tRNA binding assays

  • In vitro translation assays to confirm biological activity