Recombinant Bacillus thuringiensis Urocanate hydratase (hutU), partial

What is urocanate hydratase (HutU) and what is its functional role in Bacillus thuringiensis?

Urocanate hydratase (HutU) is an enzyme involved in histidine catabolism in Bacillus species, including B. thuringiensis. It catalyzes the conversion of urocanate to imidazolonepropionate in the histidine utilization pathway. In B. anthracis and related Bacillus species, hutU expression is negatively regulated by small regulatory RNAs (sRNAs) such as XrrA, suggesting a complex regulatory network for histidine metabolism . This regulation appears to be part of a broader system controlling amino acid metabolism, particularly branched-chain amino acid pathways that are critical for bacterial growth and virulence expression.

How does small RNA regulation affect hutU expression in Bacillus species?

In Bacillus species such as B. anthracis, hutU expression is regulated by small RNAs, particularly XrrA. Transcriptome analysis has revealed that XrrA forms base-pairing interactions with the hutU transcript, resulting in negative regulation of the gene . This regulatory mechanism is part of the AtxA regulon, a master regulator that controls virulence gene expression in B. anthracis. The regulatory impact is substantial, with RNA-seq data showing 4.0- to >100-fold differences in target gene expression between wildtype and sRNA-null mutants . Similar sRNA regulatory mechanisms likely exist in B. thuringiensis, as both species share homologous regulatory systems and belong to the closely related Bacillus cereus sensu lato group.

What expression systems are most effective for producing recombinant HutU in Bacillus thuringiensis?

For recombinant HutU expression in B. thuringiensis, sporulation-specific promoter systems have demonstrated high efficacy. Constructs similar to the pSTAB vectors used for thurincin H and ChiA74 expression can be adapted for hutU . These systems allow for the controlled expression of recombinant proteins during the sporulation phase of B. thuringiensis, even for proteins that are naturally expressed during vegetative growth. For optimal expression:

• Design expression constructs with sporulation-specific promoters (e.g., the cry promoter)

• Transform these constructs into appropriate B. thuringiensis strains (e.g., subsp. kurstaki HD1)

• Culture the transformed strains under conditions that promote sporulation

• Monitor protein expression over time (typically peaking at 72-96 hours)

This approach has been demonstrated to produce significant levels of recombinant proteins (~4000 U mg⁻¹ for antimicrobial proteins) , suggesting it would be suitable for hutU expression.

How can I optimize the expression of recombinant HutU in B. thuringiensis at different growth phases?

Optimizing recombinant HutU expression requires strategic considerations for growth phase-specific expression. The following methodological approach is recommended:

For sporulation-phase expression, which typically yields the highest protein levels, culture the transformed B. thuringiensis strains in nutrient-limiting media and monitor protein production through the 72-96 hour period when crystal formation occurs . This strategy has been successful for other recombinant proteins in B. thuringiensis and can be adapted for hutU expression. The temporal expression pattern must be carefully monitored, as some proteins show maximum expression at different timepoints during the sporulation phase.

What are the advantages of expressing hutU during the sporulation phase rather than vegetative growth?

Expressing recombinant HutU during sporulation offers several significant advantages:

• Higher protein yield: The sporulation-specific expression system typically results in higher recombinant protein yields due to the natural amplification of gene expression during spore formation .

• Increased stability: Proteins expressed during sporulation can become integrated into spore structures, offering protection from degradation and environmental factors. Similar to how Cry proteins form crystals, recombinant proteins expressed during this phase may form stable aggregates or become associated with the spore coat .

• Co-expression capabilities: The sporulation phase allows for the simultaneous expression of multiple recombinant proteins, as demonstrated by the successful co-expression of thurincin H, ChiA74, and Cry proteins . This allows for multifunctional recombinant strains.

• Extended activity period: Proteins expressed during sporulation remain active longer, with studies showing sustained activity up to 72 hours post-inoculation for recombinant proteins in B. thuringiensis .

• Simplified purification: When associated with spore structures, recombinant proteins may be easier to harvest and purify compared to intracellular expression during vegetative growth.

What are the most effective purification methods for recombinant HutU from B. thuringiensis?

Purification of recombinant HutU from B. thuringiensis requires a strategic approach based on the protein's properties and expression pattern. A comprehensive purification protocol would include:

• Cell harvest and lysis: Collect cells at optimal expression time (72-96h post-inoculation for sporulation-phase expression) . Lyse cells using either sonication or French press techniques for vegetative cells, or alkaline extraction methods for spore-associated proteins.

• Initial fractionation: Separate cellular fractions through differential centrifugation. For spore-associated proteins, wash spores with high-salt buffers to release surface-displayed proteins .

• Chromatographic separation: Apply sequential chromatography:

  • Ion exchange chromatography (based on HutU's predicted isoelectric point)

  • Hydrophobic interaction chromatography

  • Size exclusion chromatography for final polishing

• Activity validation: Monitor purification efficiency using enzyme activity assays specific for urocanate hydratase, measuring the conversion of urocanate to imidazolonepropionate spectrophotometrically.

For recombinant HutU expressed with affinity tags, immobilized metal affinity chromatography (IMAC) can significantly streamline the purification process, allowing for single-step purification with higher yields and purity.

How can I validate the enzymatic activity of recombinant HutU after purification?

Validation of recombinant HutU enzymatic activity requires multiple complementary approaches:

• Spectrophotometric assay: The primary method measures urocanate hydratase activity by monitoring the decrease in absorbance at 277 nm as urocanate is converted to imidazolonepropionate. The reaction mixture typically contains:

  • 50 mM potassium phosphate buffer (pH 7.5)

  • 0.1 mM urocanate

  • Purified enzyme (5-20 μg)

  • Monitor absorbance decrease over 5 minutes at 25°C

• Coupled enzyme assays: For more sensitive detection, couple the HutU reaction to secondary enzymatic reactions that produce chromogenic or fluorogenic products.

• Isotope labeling: Utilize 14C-labeled substrates to track the enzymatic conversion through thin-layer chromatography or liquid scintillation counting, similar to isotope array approaches used for tracking metabolic activities in microbial communities .

• Mass spectrometry validation: Confirm product formation through LC-MS/MS analysis of reaction products, providing unambiguous identification of the imidazolonepropionate product.

Activity should be expressed in standardized units (μmol substrate converted per minute per mg protein) and compared to published values for native HutU to assess the functionality of the recombinant enzyme.

What strategies can be used to display recombinant HutU on the surface of B. thuringiensis spores?

Surface display of recombinant HutU on B. thuringiensis spores can be achieved through fusion with spore coat proteins, particularly leveraging the Cry protein display system. The following methodological approach is recommended:

• Fusion construct design: Create a genetic fusion between hutU and the N-terminal portion of a Cry protein. The expression vector should contain:

  • A sporulation-specific promoter

  • The coding sequence for hutU

  • A linker sequence (typically glycine-rich)

  • The N-terminal domain of the Cry protein that facilitates incorporation into the spore coat

• Transformation and expression: Transform the construct into an appropriate B. thuringiensis strain and induce sporulation through nutrient limitation.

• Spore harvest and purification: Collect spores after 72-96 hours of culture and purify them through repeated washing with high-salt buffers followed by gradient centrifugation.

• Surface display validation: Confirm the presence of HutU on the spore surface through:

  • Immunofluorescence microscopy using anti-HutU antibodies

  • Flow cytometry analysis

  • Enzymatic activity assays with intact spores

This approach has been successfully used for displaying various proteins, including green fluorescent protein and single-chain antibodies on B. thuringiensis spores , making it a viable strategy for HutU surface display.

How can recombinant HutU be used to study small RNA regulatory networks in Bacillus species?

Recombinant HutU serves as an excellent reporter system for studying sRNA regulatory networks in Bacillus species through these methodological approaches:

• Reporter fusion constructs: Create translational fusions between the hutU 5' UTR (containing sRNA binding sites) and reporter genes like gfp or luciferase. This allows visualization of sRNA regulation in vivo by monitoring fluorescence or luminescence changes when specific sRNAs are present or absent.

• MAPS (MS2 Affinity Purification coupled with RNA Sequencing): Adapt this technique using the hutU transcript as bait to capture interacting sRNAs. This involves:

  • Expressing MS2-tagged hutU mRNA

  • Purifying RNA complexes using MS2-binding protein

  • Sequencing associated sRNAs

• Synthetic regulatory circuits: Design synthetic circuits where hutU expression controls downstream genes of interest, creating tunable systems to study sRNA regulation dynamics.

This system is particularly valuable because hutU is regulated by XrrA in B. anthracis , and similar regulatory mechanisms likely exist in B. thuringiensis. Understanding these regulatory networks provides insights into both metabolic regulation and virulence control in Bacillus species.

What are the implications of HutU regulation for understanding virulence mechanisms in Bacillus species?

The regulation of HutU has significant implications for understanding virulence mechanisms in Bacillus species through several interconnected pathways:

• Metabolic-virulence coupling: HutU's involvement in histidine metabolism represents a critical nexus between metabolism and virulence. In B. anthracis, the same sRNA (XrrA) that regulates hutU also influences genes associated with virulence . This suggests a coordinated regulation of metabolic and virulence factors.

• Signal integration: The negative regulation of hutU by XrrA, which itself is controlled by the master virulence regulator AtxA, demonstrates how environmental signals are integrated to simultaneously modulate metabolism and virulence .

• Nutrient acquisition during infection: Histidine utilization may be particularly important during infection when bacteria must adapt to host nutritional environments. The specific regulation of hutU could represent an adaptation for efficient nutrient utilization in vivo.

• Virulence attenuation markers: Experimental evidence shows that xrrA-null mutants exhibit reduced bacterial burden in mouse infection models, with affected organs showing a small but significant reduction in colonization . This connects hutU regulation to in vivo pathogenesis.

Understanding these regulatory connections provides opportunities to develop targeted interventions that could disrupt both metabolic adaptation and virulence expression simultaneously.

How can RNA sequencing and microarray analysis be used to understand the broader impact of HutU and its regulation in bacterial physiology?

RNA sequencing and microarray analysis provide powerful approaches to understand the broader impacts of HutU and its regulation:

• Comparative transcriptomics: Compare transcriptome profiles between wildtype and hutU mutant strains under various conditions using:

  • RNA-seq for unbiased, genome-wide quantification of transcriptional changes

  • Microarrays with specific probes targeting metabolic and virulence genes

• sRNA-target mapping: Identify the complete regulon of sRNAs that affect hutU using techniques such as:

  • CLASH (Crosslinking, Ligation And Sequencing of Hybrids) to identify direct sRNA-mRNA interactions

  • Comparative RNA-seq of wildtype and sRNA deletion mutants, similar to the approach used for XrrA and XrrB in B. anthracis that revealed 4.0- to >100-fold expression differences in target genes

• Isotope array approach: Combine RNA labeling with microarray detection to track metabolic activities in correlation with hutU expression:

  • Use 14C-labeled substrates to follow carbon flux through histidine metabolism

  • Hybridize labeled RNA to microarrays to correlate metabolic activity with gene expression patterns

• Regulatory network reconstruction: Integrate transcriptomic data with metabolomic analyses to build comprehensive models of how HutU and its regulation impact cellular physiology across different growth conditions and developmental stages.

These approaches can reveal unexpected connections between histidine metabolism, stress responses, and virulence factor expression, providing a systems-level understanding of bacterial physiology.

What are the common challenges in expressing recombinant HutU in B. thuringiensis and how can they be addressed?

Researchers commonly encounter several challenges when expressing recombinant HutU in B. thuringiensis:

• Low expression levels:

  • Challenge: Insufficient protein yield despite correct construct design.

  • Solution: Optimize codon usage for B. thuringiensis, strengthen ribosome binding sites, and test multiple promoters. Sporulation-specific promoters have shown high expression levels (up to ~4000 U mg⁻¹) for other recombinant proteins .

• Protein aggregation and inclusion body formation:

  • Challenge: HutU forms insoluble aggregates rather than active enzyme.

  • Solution: Co-express molecular chaperones, lower incubation temperature to 25°C during expression, or use solubility-enhancing fusion tags such as thioredoxin or SUMO.

• Proteolytic degradation:

  • Challenge: Rapid degradation of recombinant HutU by native proteases.

  • Solution: Use protease-deficient B. thuringiensis strains or add protease inhibitors during extraction. Consider fusion with stabilizing domains or co-expression with protease inhibitors.

• Poor enzymatic activity:

  • Challenge: Recombinant enzyme shows lower activity than expected.

  • Solution: Ensure proper folding by optimizing extraction conditions, adding cofactors required for activity, and validating the structural integrity through circular dichroism or limited proteolysis analysis.

By systematically addressing these challenges, researchers can significantly improve the expression and functionality of recombinant HutU in B. thuringiensis expression systems.

How can researchers troubleshoot issues with sporulation-specific expression of recombinant HutU?

Troubleshooting sporulation-specific expression of recombinant HutU requires a systematic approach addressing both sporulation efficiency and protein expression:

• Poor sporulation efficiency:

  • Symptom: Low percentage of cells forming spores.

  • Diagnostic approach: Microscopic examination of cultures at 48-72 hours.

  • Solutions: Optimize media composition (particularly carbon:nitrogen ratio), ensure proper aeration, and gradually adapt cultures to sporulation conditions through sequential passaging in increasingly nutrient-limited media.

• Asynchronous sporulation:

  • Symptom: Wide variation in developmental stages within the culture.

  • Diagnostic approach: Time-course microscopy with phase contrast.

  • Solutions: Start cultures from freshly streaked colonies, standardize inoculum density, and use resuspension methods to synchronize entry into sporulation.

• Delayed or absent protein expression:

  • Symptom: Protein expression not detected at expected timepoints (72-96h) .

  • Diagnostic approach: Western blot analysis at 24h intervals.

  • Solutions: Verify promoter sequence integrity, ensure sigma factor availability by confirming sporulation stage progression, and check for possible toxic effects of the recombinant protein on sporulation.

• Strain stability issues:

  • Symptom: Loss of expression in subsequent generations.

  • Diagnostic approach: PCR verification of construct presence.

  • Solutions: Maintain selection pressure throughout growth, prepare glycerol stocks from verified high-expressing colonies, and consider integrating the expression cassette into the chromosome rather than relying on plasmid-based expression.

Successful expression of other recombinant proteins during sporulation suggests these approaches should be effective for hutU expression as well .

What analytical methods are most suitable for detecting RNA-based regulation of hutU expression in Bacillus species?

For investigating RNA-based regulation of hutU in Bacillus species, several specialized analytical approaches are recommended:

• Northern blotting with specific probes:

  • Methodology: Design oligonucleotide probes targeting hutU mRNA and potential regulatory sRNAs (e.g., XrrA homologs in B. thuringiensis).

  • Applications: Determine if hutU transcript levels change in response to different growth conditions or in sRNA deletion mutants.

  • Validation: Include positive controls by examining known sRNA-regulated genes in parallel.

• RT-qPCR with strand-specific primers:

  • Methodology: Design primers that specifically detect hutU transcript and potential antisense RNAs.

  • Application: Quantify expression levels in different genetic backgrounds (e.g., wildtype vs. sRNA deletion strains).

  • Analysis: Use the comparative CT method with appropriate reference genes for normalization.

• RNA structure probing:

  • Methodology: Use SHAPE (Selective 2'-Hydroxyl Acylation analyzed by Primer Extension) or DMS probing to examine structural changes in hutU mRNA upon sRNA binding.

  • Applications: Determine the specific RNA regions involved in regulatory interactions.

  • Analysis: Compare reactivity profiles with and without potential regulatory sRNAs.

• GFP reporter fusions:

  • Methodology: Create translational fusions between the hutU 5' UTR and gfp to visualize regulation.

  • Applications: Monitor expression in real-time under different conditions.

  • Analysis: Quantify fluorescence using microscopy or flow cytometry.

These methods have been successfully used to characterize sRNA-mediated regulation in Bacillus species, including the XrrA-mediated regulation of multiple targets in B. anthracis , and can be adapted to study hutU regulation in B. thuringiensis.

How can recombinant HutU be incorporated into multi-protein expression systems for enhanced biocontrol applications?

Recombinant HutU can be strategically incorporated into multi-protein expression systems for biocontrol through these methodological approaches:

• Co-expression with complementary proteins: Design expression systems that simultaneously produce HutU alongside proteins with synergistic functions, such as:

  • Chitinases (e.g., ChiA74) that degrade insect exoskeletons

  • Antimicrobial peptides (e.g., thurincin H) that provide antibacterial activity

  • Cry proteins that target specific insect pests

  This approach has been successfully demonstrated with other protein combinations in B. thuringiensis, achieving substantial production levels for multiple proteins simultaneously .

• Promoter engineering for coordinated expression: Create synthetic promoter systems that enable:

  • Synchronized expression of multiple proteins during sporulation (72-96h)

  • Differential expression levels tailored to the desired ratio of each protein

  • Environmental or developmental stage-specific expression

• Spore surface display combinations: Utilize the B. thuringiensis spore display system to create multi-functional spores displaying:

  • HutU for histidine metabolism modulation

  • Binding domains for specific environmental targets

  • Enzymatic cascades that function synergistically

This multi-protein approach creates biocontrol agents with enhanced functionality, targeting multiple biological processes simultaneously and potentially reducing the development of resistance in target organisms.

What are the most promising research directions for understanding the role of HutU in bacterial adaptation to different environmental conditions?

Several promising research directions can advance our understanding of HutU's role in bacterial environmental adaptation:

• Comparative genomics and transcriptomics across diverse Bacillus strains:

  • Analyze hutU sequence conservation, expression patterns, and regulatory elements across Bacillus species from different ecological niches

  • Correlate hutU expression with adaptation to specific environments

  • Identify selective pressures that shape hutU evolution in different ecological contexts

• In situ expression analysis in natural and host environments:

  • Develop reporter systems to monitor hutU expression during plant colonization, insect infection, or soil persistence

  • Apply isotope array approaches to track metabolic activity and hutU expression simultaneously in complex environments

  • Correlate expression patterns with successful environmental adaptation or host infection

• Integration of HutU function with global metabolic networks:

  • Map metabolic flux through histidine utilization pathways under different environmental conditions

  • Determine how hutU regulation connects to broader metabolic reprogramming during stress responses

  • Develop metabolic models that predict how HutU activity influences growth and survival in changing environments

These research directions would significantly advance our understanding of how histidine metabolism contributes to bacterial adaptation and potentially reveal new strategies for enhancing beneficial Bacillus applications or controlling pathogenic species.

How might advanced RNA-sequencing approaches be used to fully characterize the regulatory networks controlling hutU expression?

Advanced RNA-sequencing approaches offer powerful tools to fully characterize the complex regulatory networks controlling hutU expression:

• Dual RNA-seq for host-pathogen interactions:

  • Simultaneously sequence bacterial and host transcriptomes during infection

  • Correlate hutU expression with specific host responses

  • Identify infection-stage specific regulation of histidine metabolism

• Term-seq for transcript termination mapping:

  • Map transcription termination sites genome-wide

  • Identify premature termination events in hutU regulated by riboswitches or attenuation

  • Discover condition-specific termination patterns that affect hutU expression

• CLIP-seq (Cross-Linking Immunoprecipitation-Sequencing):

  • Identify proteins binding to hutU mRNA by crosslinking RNA-protein complexes

  • Immunoprecipitate specific RNA-binding proteins and sequence associated RNAs

  • Map exact binding sites of regulatory proteins on the hutU transcript

• Ribo-seq for translation efficiency analysis:

  • Measure ribosome occupancy on hutU mRNA under different conditions

  • Identify translational regulation mechanisms

  • Correlate with sRNA binding to determine if regulation occurs at transcriptional or translational levels

• Single-cell RNA-seq for population heterogeneity:

  • Analyze hutU expression at single-cell resolution

  • Identify subpopulations with distinct expression patterns

  • Correlate with cell fate decisions (e.g., sporulation) or stress responses