Recombinant Chromobacterium violaceum Phosphoribosyl-ATP pyrophosphatase (hisE)

What is Phosphoribosyl-ATP pyrophosphatase (hisE) and what is its role in Chromobacterium violaceum?

Phosphoribosyl-ATP pyrophosphatase (hisE) is an essential enzyme in the histidine biosynthesis pathway of Chromobacterium violaceum. It catalyzes the hydrolysis of phosphoribosyl-ATP to phosphoribosyl-AMP and pyrophosphate, representing the second step in histidine biosynthesis. In C. violaceum, this enzyme plays a critical role in amino acid metabolism and cellular growth. The enzyme's activity is particularly important in environments where histidine availability is limited, making it essential for C. violaceum survival in various ecological niches.

How does C. violaceum hisE compare structurally to hisE from other bacterial species?

C. violaceum hisE shares the core catalytic domain structure with other bacterial Phosphoribosyl-ATP pyrophosphatases but exhibits distinct sequence variations in non-catalytic regions. These structural differences may reflect evolutionary adaptation to C. violaceum's specific ecological niches, including aquatic environments where it naturally occurs. While the catalytic mechanism remains conserved across species, these variations potentially influence protein stability, substrate affinity, and regulatory interactions specific to C. violaceum's metabolic network.

What are the optimal expression systems for recombinant C. violaceum hisE production?

For recombinant expression of C. violaceum hisE, E. coli BL21(DE3) remains the preferred host system due to its high expression yields and compatibility with C. violaceum codon usage. Optimal expression typically requires:

• Vector selection: pET-based vectors containing T7 promoter systems yield highest expression

• Induction conditions: 0.5-1.0 mM IPTG at OD600 0.6-0.8

• Growth temperature: Reduction to 18-20°C post-induction minimizes inclusion body formation

• Media composition: TB or 2×YT media supplemented with glucose (0.5%) improves soluble protein yield

Expression in C. violaceum itself is possible but more challenging due to the complex regulatory networks governing protein expression in this organism, including quorum sensing systems mediated by CviI/CviR that respond to N-hexanoyl-L-homoserine lactone signals .

How can the quorum sensing systems of C. violaceum impact heterologous expression of recombinant proteins?

C. violaceum employs sophisticated quorum sensing systems that regulate numerous physiological processes through the CviI/CviR regulatory network. When designing expression systems for recombinant hisE within C. violaceum itself, researchers must consider:

• The CviR regulator responds to N-hexanoyl-L-homoserine lactone (C6-HSL) to activate target gene expression at high cell density

• Expression vectors incorporating CviR-responsive promoters can achieve density-dependent protein production

• The Air two-component regulatory system interfaces with quorum sensing and affects multiple cellular processes

• Depending on the promoter choice, recombinant protein expression may be inadvertently affected by these native regulatory networks

For controlled expression independent of quorum sensing, constitutive promoters or inducible systems not native to C. violaceum should be considered.

What is the most effective purification strategy for recombinant C. violaceum hisE?

A robust purification protocol for recombinant C. violaceum hisE involves:

• Affinity chromatography: Histidine-tagged constructs purified via Ni-NTA with elution using 250-300 mM imidazole

• Ion exchange chromatography: Q-Sepharose column at pH 8.0 with linear NaCl gradient (0-500 mM)

• Size exclusion chromatography: Superdex 75 column for final polishing and buffer exchange

This three-step protocol typically yields >95% pure enzyme with activity preservation. Critical buffer components include:

What are the optimal conditions for assessing C. violaceum hisE enzymatic activity?

The enzymatic activity of C. violaceum hisE is optimally measured using a coupled assay system that monitors pyrophosphate release. The recommended assay conditions are:

• Buffer system: 50 mM Tris-HCl (pH 7.5-8.0)

• Temperature: 30°C (balancing enzymatic activity with stability)

• Substrate concentration: 0.1-1.0 mM phosphoribosyl-ATP

• Essential cofactors: 5 mM MgCl₂

• Detection method: Malachite green assay for released phosphate following pyrophosphatase treatment

A standard activity curve should be established using varying enzyme concentrations to ensure linearity within the assay's dynamic range. Kinetic parameters typically fall within these ranges:

How does translation inhibition affect hisE expression in C. violaceum?

Given C. violaceum's sophisticated response to translation-inhibiting antibiotics, hisE expression may be affected by translation-related stress. Research indicates that sublethal doses of antibiotics targeting polypeptide elongation (such as tetracycline, chloramphenicol, and erythromycin) trigger significant transcriptional responses in C. violaceum . While specific effects on hisE have not been directly characterized, RNA-sequencing analysis of C. violaceum exposed to tetracycline and spectinomycin shows upregulation of genes involved in translation, ribosomal structure, and secondary metabolite biosynthesis .

The Air two-component regulatory system mediates many of these responses, suggesting potential regulatory connections between translation stress and metabolic pathways including amino acid biosynthesis. Researchers investigating hisE regulation should consider:

• Evaluating hisE expression under sublethal antibiotic exposure

• Examining potential Air system influence on histidine biosynthesis genes

• Investigating cross-regulation between quorum sensing and amino acid metabolism

What is the relationship between C. violaceum's competitive mechanisms and histidine biosynthesis?

C. violaceum employs multiple competitive mechanisms, including violacein production and Type VI Secretion Systems (T6SS), that may indirectly influence histidine biosynthesis. The T6SS in C. violaceum is regulated by quorum sensing through CviR but not CviI , while violacein production is activated through CviR/CviI in response to environmental stimuli .

These competitive systems create significant metabolic demands that may affect amino acid biosynthesis pathways, including histidine production. Research questions to explore include:

• Whether hisE expression is coordinated with virulence factor production

• If histidine biosynthesis is prioritized during interbacterial competition

• How nutrient limitation affects the balance between primary metabolism (including histidine synthesis) and secondary metabolite production

How can CRISPR-Cas9 approaches be optimized for studying hisE function in C. violaceum?

For CRISPR-Cas9 gene editing of hisE in C. violaceum, researchers should consider:

• Delivery method optimization:

  • Electroporation protocols specifically calibrated for C. violaceum (25 kV/cm, 200 Ω, 25 μF)

  • Conjugation using E. coli S17-1 as donor strain

• Guide RNA design considerations:

  • Target regions with minimal off-target potential within the C. violaceum genome

  • Avoid sequence similarity with violacein biosynthesis genes to prevent phenotypic confusion

  • PAM site accessibility analysis considering C. violaceum's GC-rich genome

• Recommended repair template design:

  • Homology arms of 800-1000 bp for optimal recombination efficiency

  • Introduction of silent mutations in PAM sites to prevent re-cutting

  • Inclusion of selectable markers flanked by FRT sites for marker removal

The editing efficiency can be significantly affected by the cell's growth phase and density, with early exponential phase (OD600 0.3-0.4) typically yielding best results.

What approaches are effective for investigating the metabolic flux through the histidine biosynthesis pathway in C. violaceum?

Metabolic flux analysis of histidine biosynthesis in C. violaceum can be accomplished through:

• Isotope labeling studies using:

  • [13C]-glucose to track carbon incorporation into histidine

  • [15N]-ammonium to monitor nitrogen incorporation

  • Analysis by LC-MS/MS with multiple reaction monitoring

• Quantitative time-course measurements:

  • Monitor intracellular concentrations of pathway intermediates

  • Measure transcript levels of all histidine biosynthesis genes

  • Analyze enzyme activities throughout growth phases

• Perturbation experiments:

  • Compare flux under different growth conditions (carbon sources, nitrogen availability)

  • Evaluate effects of translational inhibitors at sublethal concentrations

  • Assess impact of quorum sensing molecule (C6-HSL) supplementation

These approaches should be integrated with computational modeling to develop predictive frameworks for histidine pathway regulation in the context of C. violaceum's complex metabolic network.

How can researchers address insolubility issues when expressing recombinant C. violaceum hisE?

Insolubility of recombinant C. violaceum hisE can be addressed through multiple strategies:

• Fusion tag optimization:

  • MBP (Maltose Binding Protein) fusion typically yields highest solubility

  • SUMO tag provides good balance between solubility and minimal impact on activity

  • Thioredoxin fusion for smaller tag size with moderate solubility enhancement

• Expression condition modifications:

  • Reduce induction temperature to 16°C with extended expression time (16-20 hours)

  • Lower IPTG concentration to 0.1-0.2 mM

  • Add chemical chaperones (5% glycerol, 1 M sorbitol) to culture medium

• Co-expression strategies:

  • GroEL/GroES chaperone system

  • DnaK/DnaJ/GrpE chaperone system

  • Combination with rare codon-optimized E. coli strains (Rosetta)

What are the most common pitfalls in measuring kinetic parameters of C. violaceum hisE and how can they be avoided?

Common pitfalls in hisE kinetic analysis include:

• Substrate degradation:

  • Phosphoribosyl-ATP is unstable under standard conditions

  • Solution: Prepare fresh substrate immediately before assays

  • Maintain pH strictly between 7.0-7.5 during substrate handling

• Metal ion interference:

  • hisE activity is highly sensitive to divalent metal ions

  • Solution: Use chelator-treated buffers with precisely controlled Mg²⁺ concentrations

  • Avoid using metal implements during enzyme handling

• Product inhibition:

  • Phosphoribosyl-AMP can inhibit enzyme activity at high concentrations

  • Solution: Implement continuous flow systems or coupled enzyme assays to remove product

• Activity normalization errors:

  • Inconsistent active site titration leads to inaccurate kinetic parameters

  • Solution: Determine active enzyme concentration through active site titration with tight-binding inhibitors

Implementing these solutions ensures more accurate and reproducible kinetic characterization of C. violaceum hisE.