Recombinant Bacillus licheniformis Phosphoribosylformylglycinamidine cyclo-ligase (purM)

What is the function of Phosphoribosylformylglycinamidine cyclo-ligase (purM) in Bacillus licheniformis?

Phosphoribosylformylglycinamidine cyclo-ligase (purM) is an essential enzyme in the purine biosynthesis pathway that catalyzes the ATP-dependent conversion of formylglycinamide ribonucleotide (FGAR) to formylglycinamidine ribonucleotide (FGAM). In B. licheniformis, this enzyme contributes to the de novo purine biosynthesis pathway, which is critical for nucleotide formation and subsequent DNA and RNA synthesis. The enzyme functions as part of a multi-step pathway that ultimately leads to inosine monophosphate (IMP), a precursor for both adenine and guanine nucleotides.

To study purM function in B. licheniformis, researchers typically employ gene deletion or overexpression methodologies similar to those used for other genes in this organism. For deletion studies, temperature-sensitive plasmids with kanamycin resistance markers can be used, with upstream and downstream fragments amplified using specific primers, followed by SOE-PCR fusion and transformation into B. licheniformis . Growth curve analyses in the presence and absence of the gene provide insights into its physiological relevance.

What expression systems are most effective for producing recombinant B. licheniformis purM?

For effective recombinant expression of B. licheniformis purM, several expression systems have demonstrated success, with the choice depending on research objectives:

E. coli-based expression systems:

• BL21(DE3) with pET vector systems offer high protein yields under IPTG induction

• T7 promoter-based systems typically provide strong expression control

• Addition of an N-terminal His-tag facilitates purification via nickel affinity chromatography

Bacillus-based expression systems:

• B. subtilis is often preferred due to its genetic similarity to B. licheniformis

• Strong constitutive promoters like P43UTR12 can be used to replace the native promoter of purM for overexpression

• Temperature-sensitive plasmids facilitate genomic integration, as demonstrated for other B. licheniformis genes

When implementing these systems, cultivation conditions should be optimized (37°C, 220 rpm) and protein expression verified through SDS-PAGE and Western blotting. For B. licheniformis-based expression, electroporation-based transformation methods have shown success for other recombinant proteins in this organism .

How can purM activity be effectively measured in recombinant B. licheniformis strains?

Multiple complementary approaches can be employed to measure purM activity in recombinant B. licheniformis strains:

Spectrophotometric enzyme assays:

• Direct measurement of ADP formation using a coupled enzyme system (pyruvate kinase/lactate dehydrogenase) that monitors NADH oxidation at 340 nm

• Quantification of inorganic phosphate released during ATP hydrolysis using malachite green assay

Growth-based functional assessment:

• Complementation studies using purM-deficient strains on minimal media

• Growth curve analysis under standard conditions (37°C, 230 rpm) comparing recombinant versus wild-type strains

• Survival rate assessment following oxidative stress (similar to methods used in H₂O₂-kill assays for other B. licheniformis proteins)

Table 1. Recommended conditions for purM activity measurement in B. licheniformis:

Growth curves should be generated with OD₆₀₀ measurements taken hourly for at least 12 hours to properly assess the impact of purM expression on bacterial physiology .

How does oxidative stress affect purM expression and function in B. licheniformis?

Oxidative stress significantly influences purM expression and function in B. licheniformis through several mechanisms:

Transcriptional regulation:
Research indicates that oxidative stress-responsive transcription factors may regulate purM expression in B. licheniformis, similar to how YvmB regulates other genes under oxidative conditions . Under H₂O₂ exposure (2 mM), B. licheniformis demonstrates altered gene expression patterns to cope with oxidative damage . While specific purM regulation hasn't been directly documented, the enzyme likely experiences similar regulatory control as other biosynthetic pathways during stress.

Protein modification and stability:
Oxidative stress can induce post-translational modifications of purM through:

• Oxidation of cysteine residues, potentially forming disulfide bonds (similar to YvmB C57 oxidation)

• Carbonylation of susceptible amino acids affecting catalytic function

• Altered protein half-life due to increased proteolysis of damaged proteins

Metabolic impact:
H₂O₂ challenge assays reveal that B. licheniformis adjusts metabolic pathways during oxidative stress . purM function may be diverted from growth-related nucleotide synthesis toward stress-response pathways, as evidenced by survival rate measurements in stress conditions for other B. licheniformis strains .

To investigate these effects experimentally, researchers should employ H₂O₂-kill assays (2 mM H₂O₂, 15 min exposure) followed by CFU counting to determine survival rates , combined with RT-qPCR to measure purM expression changes.

What structural modifications can optimize the thermostability of recombinant B. licheniformis purM?

Enhancing thermostability of recombinant B. licheniformis purM can be achieved through strategic structural modifications based on both computational prediction and experimental validation:

Computational design approaches:

• Homology modeling based on known purM structures from thermophilic Bacillus species

• Molecular dynamics simulations at elevated temperatures (45-60°C) to identify flexible regions

• Identification of destabilizing surface residues for targeted substitution

Experimental stabilization strategies:

• Site-directed mutagenesis:

  • Introduction of additional salt bridges at surface-exposed regions

  • Proline substitutions in loop regions to reduce conformational flexibility

  • Conversion of free cysteines to serines to prevent oxidation-related instability

• Directed evolution:

  • Error-prone PCR to generate variant libraries

  • Screening under thermal challenge conditions

  • Iterative selection of improved variants

Table 2. Potential thermostabilizing mutations based on comparative analysis with thermophilic homologs:

Validation should be performed through differential scanning calorimetry and residual activity measurements after thermal challenge (similar to the 45°C incubation methods used for other B. licheniformis proteins) .

What is the impact of different promoter systems on recombinant purM expression levels and functionality in B. licheniformis?

The choice of promoter significantly influences both the expression level and functionality of recombinant purM in B. licheniformis:

Constitutive promoter systems:
The P43UTR12 promoter from B. subtilis has demonstrated strong expression capability in B. licheniformis for various genes . When applied to purM expression, this promoter provides:

Inducible promoter systems:

• IPTG-inducible systems: Allow titratable expression but may suffer from leakiness

• Xylose-inducible systems: Provide tighter regulation with lower background

• Temperature-sensitive systems: Enable expression control through cultivation temperature shifts

Growth phase-dependent promoters:
Promoters active during specific growth phases can align purM expression with cellular needs:

• Stationary phase promoters minimize metabolic burden during active growth

• Exponential phase promoters maximize expression when resources are abundant

Table 3. Comparative analysis of promoter systems for purM expression in B. licheniformis:

Implementation methods for promoter replacement follow established protocols including amplification of the promoter region, SOE-PCR fusion with flanking regions, and integration into the B. licheniformis genome using temperature-sensitive vectors, as demonstrated for other genes .

How can recombinant B. licheniformis purM be optimized for improved solubility and reduced inclusion body formation?

Optimizing solubility of recombinant B. licheniformis purM requires a multi-faceted approach addressing protein folding, expression conditions, and potential fusion partners:

Expression condition optimization:

• Temperature reduction: Lowering culture temperature to 25-30°C after induction significantly improves proper folding

• Induction optimization: Using lower inducer concentrations and extended expression periods

• Media supplementation: Addition of 1-5% glycerol or 0.5-1% glucose to reduce metabolic burden

• Growth rate control: Maintaining slower growth rates through defined media formulations

Molecular engineering strategies:

• Fusion partners to enhance solubility:

  • Thioredoxin (Trx) - maintains reducing environment

  • Maltose-binding protein (MBP) - provides strong solubilizing effect

  • SUMO tag - improves folding and can be precisely removed

• Codon optimization:

  • Harmonization rather than maximization of codon usage

  • Adjustment of rare codons to match translational pausing of native host

• Molecular chaperone co-expression:

  • GroEL/GroES system for folding assistance

  • DnaK/DnaJ/GrpE for preventing aggregation

  • ClpB for disaggregation of protein clusters

Table 4. Solubility enhancement strategies ranked by effectiveness for B. licheniformis recombinant proteins:

Implementation methods should follow established B. licheniformis transformation protocols using electroporation and appropriate selection markers , with solubility assessed through comparative analysis of soluble versus insoluble fractions via SDS-PAGE.

What are the current limitations and future perspectives in recombinant B. licheniformis purM research?

Research on recombinant B. licheniformis purM faces several important limitations while offering promising future directions:

Current limitations:

• Regulatory complexity:
  The intricate regulation of purine biosynthesis in B. licheniformis remains incompletely characterized, especially under various stress conditions. While oxidative stress response mechanisms have been studied for other proteins like YvmB , specific regulatory elements controlling purM expression require further investigation.

• Structural knowledge gaps:
  Limited availability of crystal structures for B. licheniformis purM hampers rational engineering efforts. Most structural predictions rely on homology modeling based on related bacterial species.

• Metabolic integration challenges:
  The full impact of purM overexpression on cellular metabolism, particularly under stress conditions, remains unclear. Studies similar to the H₂O₂ challenge assays used for other B. licheniformis proteins could provide valuable insights.

Future research directions:

• Systems biology approach:
  Integrating transcriptomics, proteomics, and metabolomics to understand how recombinant purM expression affects the entire purine biosynthesis pathway and connected metabolic networks.

• Stress-responsive expression systems:
  Development of promoter systems that modulate purM expression in response to specific environmental signals, building upon knowledge of oxidative stress response in B. licheniformis .

• Biotechnological applications:
  Exploring the potential of engineered purM variants for enhanced nucleotide production, leveraging B. licheniformis' established role in biotechnology .

• Comparative genomics:
  Systematic comparison of purM across different Bacillus species to identify evolutionary adaptations that could inform protein engineering strategies.

Table 5. Priority research areas for advancing recombinant B. licheniformis purM understanding:

The integration of these research directions will provide comprehensive understanding of purM's role in B. licheniformis and maximize its biotechnological potential.

What ethical considerations and biosafety protocols should be implemented when working with recombinant B. licheniformis purM?

Working with recombinant B. licheniformis purM necessitates careful attention to ethical considerations and biosafety protocols:

Biosafety considerations:

B. licheniformis is generally classified as a Biosafety Level 1 (BSL-1) organism, but introduction of recombinant purM requires additional precautions:

• Containment measures:

  • Standard BSL-1 practices including handwashing, no eating/drinking in laboratory

  • Use of biological safety cabinets for aerosol-generating procedures

  • Proper decontamination of work surfaces and equipment

• Genetic stability monitoring:

  • Regular verification of genetic construct integrity

  • Assessment of horizontal gene transfer potential

  • Confirmation of absence of antibiotic resistance gene transfer

• Strain-specific biosafety:
  Some strains of B. licheniformis have been reported to carry antibiotic resistance genes, necessitating careful selection of laboratory strains . Before using B. licheniformis for recombinant protein expression, verification of absence of virulence factors and antibiotic resistance genes is essential .

Ethical considerations:

• Environmental impact assessment:

  • Evaluation of potential ecological consequences if recombinant strains were released

  • Implementation of biological containment strategies (kill switches, auxotrophic markers)

  • Proper waste management protocols specific to recombinant organisms

• Dual-use research awareness:

  • Assessment of potential for misuse of research findings

  • Appropriate levels of information disclosure in publications

  • Compliance with institutional biosecurity policies

• Transparent reporting:

  • Complete disclosure of methodologies in publications

  • Sharing of strains and constructs with qualified researchers

  • Deposition of sequence data in public repositories

Table 6. Recommended biosafety protocols for recombinant B. licheniformis purM work: