Recombinant Proteus mirabilis GMP synthase [glutamine-hydrolyzing] (guaA), partial

What is GMP synthase (guaA) and what is its function in Proteus mirabilis?

GMP synthase (EC 6.3.4.1), encoded by the guaA gene, is a glutamine amidotransferase that catalyzes the synthesis of GMP from XMP in the purine biosynthesis pathway . In Proteus mirabilis, as in other bacteria, this enzyme plays a critical role in nucleotide metabolism, which is essential for DNA and RNA synthesis. The enzymatic reaction involves converting xanthosine monophosphate (XMP) to guanosine monophosphate (GMP) in an ATP-dependent reaction that utilizes glutamine as the amino group donor .

The reaction can be represented as:
XMP + ATP + Glutamine → GMP + AMP + PPi + Glutamate

This reaction represents the final step in the de novo biosynthesis of guanine nucleotides, making guaA essential for cellular function and bacterial survival, particularly in environments where purine salvage is limited .

What is the genetic structure of the guaA gene in P. mirabilis?

The guaA gene in Proteus mirabilis shares structural similarities with its counterparts in other gram-negative bacteria. Based on studies in related bacteria, the guaA gene typically encodes a protein of approximately 525 amino acid residues with a calculated molecular weight of around 58,604 Da .

Within bacterial operons, guaA is often part of the polycistronic guaBA operon, where a 68-base pair intercistronic region separates guaA from the upstream guaB gene . The 3' end of the guaA mRNA is typically located 36-37 nucleotides downstream of the translation stop codon within a region of dyad symmetry that resembles a rho-independent transcription termination site .

The genomic context of guaA is particularly important for understanding its regulation and expression patterns during infection and stress conditions. In P. mirabilis specifically, the gene is expressed in conjunction with other genes involved in purine metabolism, creating a coordinated response to nucleotide demands.

How does guaA function within the purine biosynthesis pathway?

GuaA functions as an integral component in the purine biosynthesis pathway, catalyzing the final step in de novo GMP synthesis. The pathway involves a multi-step process:

• The pathway begins with the formation of phosphoribosyl pyrophosphate (PRPP)

• Through a series of enzymatic reactions, inosine monophosphate (IMP) is formed

• IMP dehydrogenase (GuaB) converts IMP to XMP

• GMP synthase (GuaA) then converts XMP to GMP

The guaA enzyme operates through a two-domain mechanism:

• The N-terminal domain catalyzes the ATP-dependent activation of XMP to form an adenylated intermediate

• The C-terminal domain, containing the glutaminase activity, hydrolyzes glutamine and transfers the resulting ammonia to the activated XMP intermediate

This two-step process is tightly regulated, ensuring appropriate levels of guanine nucleotides for cellular processes. The enzyme's activity is subject to feedback inhibition by guanine nucleotides, allowing bacteria to modulate purine biosynthesis according to metabolic needs.

What is the relationship between guaA and guaB in bacterial operons?

In many bacteria, including those related to P. mirabilis, the guaA and guaB genes are organized in a polycistronic operon (the guaBA operon), reflecting their sequential functions in the purine biosynthesis pathway . This genetic organization facilitates coordinated expression and regulation of these functionally related enzymes.

Within this operon structure:

• GuaB (IMP dehydrogenase) catalyzes the conversion of IMP to XMP

• GuaA (GMP synthase) then converts XMP to GMP

• The genes are typically separated by a 68-base pair intercistronic region

• Transcription typically occurs as a single mRNA unit, allowing proportional synthesis of both enzymes

This arrangement ensures proper stoichiometry of the enzymes in the purine biosynthesis pathway and allows for efficient regulatory control. Bacteria can modulate the expression of the entire operon in response to purine availability or metabolic demands, ensuring energy-efficient production of these essential enzymes.

What experimental approaches can be used to generate and characterize guaA mutants in P. mirabilis?

Several approaches can be employed to generate and characterize guaA mutants in P. mirabilis:

• Homologous recombination-based mutagenesis:

  • Amplify the guaA gene or fragment from P. mirabilis genomic DNA

  • Introduce a deletion or disruption and insert an antibiotic resistance marker

  • Transform the construct into P. mirabilis and select for recombinants

  • Verify disruption via PCR and/or Southern blotting

• Transposon mutagenesis:

  • Use mini-Tn5 or similar transposons to generate a library of P. mirabilis mutants

  • Screen for insertions in guaA using PCR or Southern blot analysis

  • Verify insertion sites by sequencing

  • Assess transposon mutants for phenotypic changes

• CRISPR-Cas9 genome editing:

  • Design guide RNAs targeting specific regions of the guaA gene

  • Introduce CRISPR-Cas9 components with appropriately designed repair templates

  • Select and verify edited clones

Characterization of the resulting mutants should include:

Complementation studies, reintroducing a functional copy of guaA on a plasmid with its native promoter, are essential to confirm that observed phenotypes are specifically due to guaA inactivation rather than polar effects or secondary mutations .

What methodologies can be employed to express and purify recombinant P. mirabilis guaA?

The expression and purification of recombinant P. mirabilis guaA requires careful optimization of several parameters:

• Expression system selection:

  • E. coli BL21(DE3) or similar strains are commonly used for bacterial protein expression

  • Alternative systems include yeast (P. pastoris) for potentially improved solubility

  • Cell-free protein synthesis systems may be considered for toxic proteins

• Vector design and construction:

  • PCR amplification of the guaA coding sequence from P. mirabilis genomic DNA

  • Addition of appropriate tags (His6, GST, MBP) to facilitate purification

  • Optimization of codon usage if necessary

  • Inclusion of precision protease sites for tag removal

• Purification strategy:

  • Initial capture: Affinity chromatography (Ni-NTA for His-tagged protein)

  • Intermediate purification: Ion exchange chromatography

  • Polishing: Size exclusion chromatography

  • Buffer optimization to maintain enzyme stability and activity

• Quality control assessments:

  • SDS-PAGE for purity evaluation

  • Mass spectrometry for identity confirmation

  • Dynamic light scattering for aggregation analysis

  • Activity assays to confirm functional enzyme production

Common challenges in guaA expression include protein insolubility and proper folding of the multi-domain enzyme. These can be addressed through fusion partners (MBP, SUMO), co-expression with chaperones, or refolding protocols if the protein forms inclusion bodies.

What techniques can be used to analyze the structure-function relationship of P. mirabilis guaA?

Investigating the structure-function relationship of P. mirabilis guaA requires a multifaceted approach combining structural biology, molecular genetics, and biochemical analyses:

• Structural determination methods:

  • X-ray crystallography of purified recombinant guaA

  • Cryo-electron microscopy for visualization of larger complexes

  • Homology modeling based on related GMP synthases with known structures

  • Molecular dynamics simulations to study conformational changes

• Domain and motif analysis:

  • Bioinformatic identification of conserved domains (glutaminase domain, ATP-binding domain)

  • Sequence alignment with characterized GMP synthases from other species

  • Secondary structure prediction to identify critical structural elements

• Site-directed mutagenesis studies:

  • Mutation of predicted catalytic residues

  • Alteration of substrate binding sites

  • Modification of interdomain regions

  • Creation of truncated variants to assess domain contributions

• Functional characterization of variants:

  • Enzyme kinetics with purified mutant proteins

  • Complementation studies in guaA-deficient bacterial strains

  • Thermal stability analysis to assess structural integrity

  • Ligand binding studies using isothermal titration calorimetry or surface plasmon resonance

A systematic experimental approach might include:

Correlating structural features with enzymatic parameters provides insights essential for understanding guaA function and potentially developing specific inhibitors.

How can researchers investigate the regulation of guaA expression in P. mirabilis?

Understanding the regulation of guaA expression in P. mirabilis requires investigation at multiple levels:

• Transcriptional regulation analysis:

  • Promoter mapping using primer extension and 5' RACE

  • Construction of reporter fusions (guaA promoter-lacZ/GFP)

  • Identification of transcription factors through DNA-protein interaction studies

  • Chromatin immunoprecipitation (ChIP) to identify binding sites

• Environmental and nutritional regulation:

  • RNA-Seq to identify co-regulated genes under different conditions

• Post-transcriptional regulation:

  • mRNA stability assays using rifampicin treatment and time-course analysis

  • Identification of potential regulatory RNAs affecting guaA expression

  • Analysis of translation efficiency using ribosome profiling

• Genetic approaches:

  • Deletion analysis of the guaA promoter region to identify regulatory elements

  • Mutagenesis of potential regulatory sequences

  • Suppressor screens to identify genes affecting guaA expression

• In vivo expression studies:

  • Analysis of guaA expression during infection using animal models

  • Ex vivo studies using urine samples or bladder epithelial cell co-culture

A particular focus should be placed on understanding the relationship between guaA expression and virulence factor production in P. mirabilis. Correlation analyses between guaA expression levels and virulence phenotypes such as swarming motility, biofilm formation, and urease activity would provide valuable insights into the role of guaA in pathogenicity .

How can researchers investigate the potential of guaA as an antimicrobial target?

Evaluation of guaA as a potential antimicrobial target against P. mirabilis requires a systematic drug discovery approach:

• Target validation studies:

  • Essentiality assessment through gene knockout/knockdown studies

  • Growth inhibition in minimal media vs. rescue with guanine supplementation

  • In vivo importance using animal infection models

  • Assessment of human homolog differences to predict selectivity

• High-throughput screening approaches:

  • Development of recombinant enzyme assays suitable for screening

  • Fluorescence-based or colorimetric activity assays

  • Fragment-based screening using thermal shift assays

  • Virtual screening using guaA homology models

• Structure-based drug design:

  • Crystallization of P. mirabilis guaA with substrate analogs

  • Computational docking studies to identify binding pockets

  • Design of compounds targeting catalytic or allosteric sites

  • Rational modification of known nucleotide analogs

• Lead compound characterization:

  • Determination of inhibition constants (Ki) and mechanism

  • Selectivity profiling against human enzymes

  • Cytotoxicity assessment using mammalian cell lines

  • ADME properties evaluation

• In vitro and in vivo efficacy studies:

  • Minimum inhibitory concentration (MIC) determination

  • Time-kill kinetics against P. mirabilis

  • Efficacy against biofilms and persister cells

  • Animal infection model testing

A data table for hit compound evaluation might include:

This comprehensive approach would identify whether guaA inhibition represents a viable strategy for developing new antimicrobials against P. mirabilis, potentially addressing the growing concern of antibiotic resistance in this uropathogen .

How can researchers overcome expression and solubility issues with recombinant P. mirabilis guaA?

Expression and solubility challenges are common when working with recombinant P. mirabilis guaA. Several strategies can help overcome these obstacles:

• Optimization of expression conditions:

  • Reduce induction temperature (16-20°C) to slow protein folding

  • Use lower inducer concentrations to prevent overwhelming cellular machinery

  • Extend expression time at lower temperatures

  • Test different growth media formulations

• Protein engineering approaches:

  • Domain-based expression approach (expressing individual domains separately)

  • Surface entropy reduction through site-directed mutagenesis

• Host strain selection:

  • Use strains with extra tRNAs for rare codons (e.g., Rosetta)

  • Strains with enhanced chaperone expression (e.g., Arctic Express)

  • Strains lacking specific proteases (e.g., BL21)

  • Consideration of eukaryotic expression systems for complex proteins

• Buffer and additive optimization:

  • Screen various buffer systems (HEPES, Tris, phosphate)

  • Test stabilizing additives (glycerol, arginine, sucrose)

  • Include appropriate cofactors (Mg2+, ATP)

  • Use mild detergents below critical micelle concentration

• Refolding strategies (if inclusion bodies form):

  • Solubilization in denaturants (urea, guanidinium)

  • Gradual dilution or dialysis-based refolding

  • On-column refolding during purification

  • Chaperone-assisted refolding

A systematic approach to optimization using a structured experimental design is recommended:

The selected optimization strategy should be tailored to the specific characteristics of P. mirabilis guaA and the intended experimental applications.

How can researchers ensure reproducibility in P. mirabilis virulence studies involving guaA?

Ensuring reproducibility in P. mirabilis virulence studies involving guaA requires careful experimental design and rigorous controls:

• Strain management and validation:

  • Maintain frozen stock cultures of original strains

  • Limit passage number to prevent accumulation of mutations

  • Regular verification of genetic modifications (PCR, sequencing)

  • Assessment of strain stability under experimental conditions

• Standardized growth conditions:

  • Consistent media preparation protocols

  • Defined inoculum preparation method

  • Standardized growth phase for experiments

  • Environmental condition control (temperature, pH, oxygen)

• Virulence factor assessment protocols:

  • Quantitative urease activity assays with appropriate standards

  • Standardized swarming assays on consistent media formulations

  • Biofilm quantification using multiple complementary methods

  • Hemolysin production measured using validated protocols

• Infection model consistency:

  • Defined animal strains, age, and gender

  • Consistent infection protocols and inoculum preparation

  • Blinded assessment of infection outcomes

  • Appropriate sample sizes based on power analysis

• Comprehensive control groups:

  • Wild-type parent strain controls

  • Complemented mutant strains to confirm phenotype specificity

  • Multiple independent guaA mutant clones

  • Positive and negative controls for each assay

A data reporting framework should include:

Meticulous documentation of all methodological details, including strain construction, media composition, growth conditions, and analytical procedures, is essential for enabling other researchers to reproduce the results .