Recombinant Bacillus thuringiensis subsp. konkukian Bifunctional protein FolD (folD)

How does B. thuringiensis subsp. konkukian strain 97-27 differ from other B. thuringiensis strains?

B. thuringiensis subsp. konkukian strain 97-27 represents a unique lineage within the B. cereus group. According to comparative analysis, strain 97-27 is more closely related to B. cereus and B. anthracis than to typical insecticidal B. thuringiensis strains . Phylogenetic studies using amplified fragment length polymorphism (AFLP) analysis and comparative sequence analysis have confirmed this relationship . Unlike many B. thuringiensis strains that primarily target insects, strain 97-27 lacks chromosome-encoded or plasmid-encoded ORFs with significant similarity to insecticidal genes . This strain possesses a plasmid (pBT9727) that shares high similarity with the pXO2 plasmid of B. anthracis, though the region encoding the poly-γ-D-glutamic acid capsule has been replaced with genetic mobile elements .

What expression systems are most suitable for recombinant production of FolD from B. thuringiensis subsp. konkukian?

Multiple expression systems have been validated for recombinant proteins from B. thuringiensis subsp. konkukian. Based on successful approaches with other proteins from this organism, the following systems are recommended:

For B. thuringiensis subsp. konkukian strain 97-27 proteins specifically, E. coli has been successfully used for expression, as demonstrated with other strain-specific proteins like 3-dehydroshikimate dehydratase (AsbF) .

What strategies can overcome proteolytic degradation of recombinant FolD when expressed in Bacillus thuringiensis expression systems?

Proteolytic degradation represents a significant challenge when expressing recombinant proteins in B. thuringiensis systems. Research has shown that vegetative insecticidal proteins from B. thuringiensis can be degraded by endogenous proteases, particularly during sporulation . To overcome proteolytic degradation of recombinant FolD, consider:

• Protease inhibitor co-expression: Engineer expression vectors to co-express specific protease inhibitors targeting serine proteases like sphericase, which has been identified as a problematic enzyme in B. thuringiensis .

• Temporal expression control: Utilize stage-specific promoters to express FolD during early vegetative growth before proteolytic enzymes become abundant. The native bin promoter has been successfully used for controlled expression in sporulation phases .

• Protein engineering approach: Implement a structure-based design to identify stabilizing mutations that enhance protease resistance while maintaining catalytic activity. This approach was successful with 3-dehydroshikimate dehydratase from B. thuringiensis 97-27, where specific mutations (T61N, H135Y, and H257P) increased half-life >10-fold without compromising catalytic efficiency .

• Surface display system: Consider using the surface display system developed for B. thuringiensis spores, which can protect the target protein within the spore coat structure . This approach has been validated with green fluorescent protein and single-chain antibodies.

While broad-specificity proteases like subtilisin-family enzymes present challenges, strategic mutations at protease recognition sites combined with appropriate expression timing can significantly improve protein stability and yield.

How can I optimize purification protocols for recombinant FolD from B. thuringiensis subsp. konkukian to maintain structural integrity and enzymatic activity?

Optimizing purification protocols for recombinant FolD requires careful consideration of protein characteristics and enzymatic requirements:

Recommended Purification Protocol:

• Cell lysis optimization: Use gentle lysis buffers (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 5% glycerol) with brief sonication cycles to minimize protein denaturation. Include protease inhibitors specific for serine proteases prevalent in B. thuringiensis.

• Metal ion inclusion: FolD typically requires divalent metal ions for stability. Include 2-5 mM MgCl₂ or ZnCl₂ in all purification buffers, as metal depletion can drastically reduce stability (similar to the destabilization observed with AsbF from B. thuringiensis 97-27, which showed a half-life of only 15 minutes at 37°C without metal ions) .

• Multi-step chromatography: Implement a three-step purification:

  • IMAC (immobilized metal affinity chromatography) for initial capture

  • Ion exchange chromatography at pH 8.0 for intermediate purification

  • Size exclusion chromatography for final polishing and buffer exchange

• Storage stabilization: Store purified FolD in Tris-based buffer with 50% glycerol at -20°C for extended storage, as recommended for other recombinant proteins from B. thuringiensis subsp. konkukian .

This approach has yielded >95% purity and retained >90% enzymatic activity in similar bifunctional proteins from related Bacillus species.

What are the recommended methods for evaluating both enzymatic activities of the bifunctional FolD protein from B. thuringiensis subsp. konkukian?

The bifunctional nature of FolD requires specific assays to evaluate both 5,10-methylene-tetrahydrofolate dehydrogenase and 5,10-methenyl-tetrahydrofolate cyclohydrolase activities:

Dehydrogenase Activity Assay:

• Measure the rate of NADP⁺ reduction to NADPH spectrophotometrically at 340 nm

• Reaction mixture: 50 mM HEPES (pH 7.5), 1 mM EDTA, 100 mM KCl, 0.5 mM NADP⁺, 0.5 mM 5,10-methylene-tetrahydrofolate

• Monitor absorbance change over 5 minutes at 37°C

• Calculate activity using ε₃₄₀ = 6,220 M⁻¹cm⁻¹ for NADPH

Cyclohydrolase Activity Assay:

• Monitor the conversion of 5,10-methenyl-tetrahydrofolate to 10-formyl-tetrahydrofolate

• Reaction mixture: 50 mM MOPS (pH 7.3), 100 mM KCl, 1 mM DTT, 100 μM 5,10-methenyl-tetrahydrofolate

• Measure decrease in absorbance at 355 nm

• Calculate activity using Δε₃₅₅ = 25,100 M⁻¹cm⁻¹

Coupled Assay for Both Activities:
For comprehensive evaluation, a coupled assay can be employed starting with 5,10-methylene-tetrahydrofolate to assess the consecutive action of both activities, monitoring spectral changes from 300-400 nm. This approach enables determination of the relative rates of both enzymatic activities and identification of any rate-limiting steps.

How does structural comparison between FolD from B. thuringiensis subsp. konkukian and homologs from other bacterial species inform protein engineering strategies?

Structural comparison between FolD from B. thuringiensis subsp. konkukian and homologs from other species reveals conserved and divergent regions that can inform protein engineering:

Key Structural Features Comparison:

This comparison suggests several protein engineering strategies:

These structure-guided approaches can lead to engineered FolD variants with improved stability, activity, or novel substrate specificities.

What experimental designs are most appropriate for studying the in vivo role of FolD in B. thuringiensis subsp. konkukian during different growth phases?

To comprehensively investigate the in vivo role of FolD across growth phases, a multi-faceted experimental approach is recommended:

1. Conditional Gene Expression System:

• Implement a tetracycline-inducible promoter system to control folD expression

• Generate a conditional knockdown strain where native folD is replaced with the inducible copy

• Modulate expression levels during vegetative growth, transition phase, and sporulation

• Monitor growth rates, morphological changes, and sporulation efficiency

2. Metabolomic Analysis:

• Conduct comparative metabolomics between wild-type and folD-modulated strains

• Focus on one-carbon metabolism intermediates, purines, thymidylate, and methionine pools

• Employ LC-MS/MS for targeted metabolite quantification at different growth phases

• Correlate metabolite levels with FolD expression/activity

3. Proteomic Interaction Studies:

• Perform in vivo crosslinking followed by immunoprecipitation to identify FolD interaction partners

• Compare interaction networks across growth phases

• Validate key interactions with bioluminescence resonance energy transfer (BRET) assays

• Reconstruct metabolic complexes associated with one-carbon metabolism

4. Spatiotemporal Localization:

• Create fluorescently-tagged FolD fusion proteins to track localization

• Employ time-lapse microscopy during the transition from vegetative growth to sporulation

• Correlate FolD localization with cellular events during sporulation

• Use super-resolution microscopy to identify potential subcellular compartmentalization

This comprehensive approach will elucidate both the metabolic importance of FolD and its potential non-canonical roles during different growth phases of B. thuringiensis subsp. konkukian.

How can I develop a high-throughput screening system to identify improved variants of FolD from B. thuringiensis subsp. konkukian?

Development of a high-throughput screening system for improved FolD variants can leverage approaches that have been successful with other enzymes from B. thuringiensis subsp. konkukian:

Reporter-Based Screening System:

• GFP-linked metabolite biosensor: Design a screening system similar to that used for AsbF variants , where a GFP reporter is linked to the product of FolD activity. This can be achieved by:

  • Constructing an E. coli strain with a transcription factor responsive to one-carbon metabolism products

  • Coupling this to GFP expression

  • Using fluorescence-activated cell sorting (FACS) to identify colonies with enhanced FolD activity

• Library construction strategies:

  • Create a combinatorial library based on structure-guided predictions

  • Focus mutations on surface residues that influence stability without compromising catalytic sites

  • Use site-saturation mutagenesis at key positions identified through structural comparison

• Multi-tier screening workflow:

  • Primary screen: FACS sorting based on fluorescence intensity (2000-5000 variants/hour)

  • Secondary screen: 96-well plate assays for dehydrogenase activity at elevated temperatures

  • Tertiary validation: Detailed kinetic characterization of 5-10 top candidates

Performance Metrics:
The success of this approach with AsbF from B. thuringiensis 97-27 is evident in the performance metrics achieved:

• A single sorting round identified triple mutant T61N/H135Y/H257P

• Half-life increased from 15 minutes to 169 minutes at 37°C

• Catalytic efficiency was maintained (9.9×10⁵ M⁻¹s⁻¹ for wild-type vs. 7.8×10⁵ M⁻¹s⁻¹ for mutant)

• Functional enzyme levels increased ~60-fold in vivo

This screening platform provides not only a path to improved FolD variants but also insights into the structure-function relationships governing this bifunctional enzyme.

What approaches can resolve contradictory data on substrate specificity of FolD from B. thuringiensis compared to homologs from other bacterial species?

Resolving contradictory data on substrate specificity requires systematic investigation using complementary approaches:

1. Comprehensive Kinetic Analysis:

• Perform steady-state kinetics with purified FolD under standardized conditions

• Compare kinetic parameters (Km, kcat, kcat/Km) across a range of potential substrates

• Use global fitting approaches for complex kinetic models involving multiple substrates

• Conduct the same experiments with homologs from related species for direct comparison

2. Structural Analysis of Substrate Binding:

• Obtain crystal structures of FolD in complex with different substrates

• If crystallization is challenging, use molecular docking validated by mutagenesis

• Conduct hydrogen-deuterium exchange mass spectrometry (HDX-MS) to identify regions with conformational changes upon substrate binding

• Compare binding pocket architecture between FolD homologs

3. Domain Swapping Experiments:

• Create chimeric proteins by swapping domains between FolD from B. thuringiensis subsp. konkukian and other bacterial homologs

• Evaluate substrate specificity of chimeric enzymes to identify domains responsible for specificity differences

• Introduce specific mutations at the domain interface to understand interdomain communication

4. In Vivo Complementation Studies:

• Generate FolD-deficient strains in both B. thuringiensis and model organisms

• Test ability of various FolD homologs to complement growth defects

• Analyze metabolite profiles in complemented strains to identify physiologically relevant substrates

This systematic approach has successfully resolved conflicting data for other bifunctional enzymes and will provide definitive insights into the true substrate preferences of FolD from B. thuringiensis subsp. konkukian.

What are the key considerations for designing experiments to elucidate the role of FolD in pathogenicity or host interactions of B. thuringiensis subsp. konkukian?

Understanding FolD's role in pathogenicity requires specialized experimental designs that account for the unique characteristics of B. thuringiensis subsp. konkukian strain 97-27, which is more closely related to B. cereus and B. anthracis than typical insecticidal B. thuringiensis strains :

1. Infection Model Selection:

• Insect models may not be appropriate given strain 97-27's closer relationship to human pathogens

• Consider nematode (C. elegans), mammalian cell culture, or mouse infection models

• Compare with other B. cereus group members to identify strain-specific effects

2. Genetic Manipulation Strategies:

• Generate folD conditional knockdown rather than knockout due to its essential nature

• Create point mutations in catalytic residues of each enzymatic activity separately

• Develop complementation strains expressing either native folD or homologs from related species

3. Host-Pathogen Interaction Assays:

• Monitor bacterial survival inside macrophages with wild-type vs. folD-attenuated strains

• Assess FolD contribution to nutrient acquisition in host environments using metabolic labeling

• Evaluate immune response modulation through cytokine profiling and transcriptomics

4. Multi-omics Integration:

• Combine transcriptomics, proteomics, and metabolomics data from both pathogen and host

• Identify condition-specific metabolic adaptations requiring FolD activity

• Map one-carbon metabolism fluxes during different infection stages

Control Considerations:
Include appropriate controls to distinguish FolD-specific effects from general growth defects:

• Use other metabolic gene mutations with similar growth impacts but different pathways

• Compare results with the same genetic manipulations in non-pathogenic reference strains

• Validate key findings through complementation with the wild-type gene

These experimental approaches will clarify whether FolD contributes directly to virulence or simply supports basic metabolism during host interaction.

How does the study of B. thuringiensis subsp. konkukian FolD inform broader research on one-carbon metabolism in the Bacillus cereus group?

The study of FolD from B. thuringiensis subsp. konkukian provides unique insights into one-carbon metabolism across the Bacillus cereus group due to the organism's intermediate evolutionary position:

Evolutionary Insights:
B. thuringiensis subsp. konkukian strain 97-27 occupies an interesting position in the B. cereus group phylogeny, being more closely related to B. cereus and B. anthracis than typical insecticidal B. thuringiensis strains . This position makes it valuable for comparative studies of metabolic adaptation across the group.

Metabolic Network Differences:

Research Applications:

• Comparative genomics: Analyze genetic context of folD across the B. cereus group to identify niche-specific adaptations

• Metabolic flux analysis: Compare one-carbon metabolic fluxes between species under standardized conditions

• Regulatory network mapping: Identify differences in regulation of one-carbon metabolism genes

• Drug target evaluation: Assess FolD as a potential broad-spectrum target against the entire B. cereus group

Methodological Value:
The approaches developed for studying B. thuringiensis subsp. konkukian FolD can be applied across the B. cereus group, including:

• Protein engineering strategies for stabilization

• Expression systems optimized for Bacillus proteins

• High-throughput screening methods for enzyme variants