Recombinant Agrobacterium vitis GMP synthase [glutamine-hydrolyzing] (guaA), partial

What is the function of GMP synthase (GuaA) in Agrobacterium vitis?

GMP synthase (GuaA) in Agrobacterium vitis, like in other bacteria, catalyzes the conversion of xanthine monophosphate (XMP) to guanosine monophosphate (GMP) using glutamine as an amide donor in the final step of de novo GMP biosynthesis. This reaction is ATP-dependent and represents a critical junction in purine nucleotide metabolism. The enzyme belongs to the glutamine amidotransferase family and contains two functional domains: an N-terminal glutaminase domain that hydrolyzes glutamine to glutamate and ammonia, and a C-terminal synthetase domain that transfers the ammonia to XMP to form GMP .

How is guaA gene expression regulated in Agrobacterium species?

In many bacteria, guaA expression is regulated by guanine riboswitches, which are RNA elements that bind guanine with high affinity (nanomolar range). When guanine concentrations are high, the riboswitch undergoes conformational changes that typically result in premature transcription termination. Based on studies in other bacteria like C. difficile, these riboswitches exhibit high specificity, with guanine binding causing structural changes that affect transcription elongation. While specific data for A. vitis is limited, it's likely that similar regulatory mechanisms exist, potentially with species-specific variations in riboswitch sensitivity and response to different purine metabolites .

What is the role of GuaA in bacterial fitness and metabolism?

GuaA plays an essential role in bacterial fitness, particularly under nutrient-limiting conditions. Research in various bacterial species has shown that GuaA activity becomes critical when bacteria cannot salvage guanine or guanosine from their environment. In C. difficile, for example, guaA mutants could survive in rich media where guanine sources are available but showed significant growth defects in minimal media that require de novo GMP synthesis. This suggests that GuaA in A. vitis would similarly be important for bacterial survival in plant tissues or soil environments where purine sources may be limited .

How do structural variations in GuaA across bacterial species affect enzyme function?

Structural analysis of GuaA across bacterial species reveals conserved catalytic domains but with species-specific variations that may affect substrate binding efficiency and catalytic rates. Comparative studies indicate that while the active site architecture remains largely conserved, differences in peripheral loops and regulatory domains can influence enzyme dynamics. For recombinant A. vitis GuaA, crystallographic studies comparing it with enzymes from other bacterial species could illuminate how structural differences correlate with substrate specificity, catalytic efficiency, and regulatory control. Molecular dynamics simulations suggest that even small variations in binding pocket residues can significantly alter enzyme kinetics and response to inhibitors .

What experimental approaches best characterize the riboswitch control of guaA in Agrobacterium vitis?

For characterizing riboswitch control of guaA in A. vitis, a multi-faceted approach is recommended:

• In-line probing assays: These assays exploit the chemical instability of RNA under physiological conditions to determine structural changes upon ligand binding. For guanine riboswitches, this technique can reveal the binding affinity (Kd) for guanine and related metabolites.

• Reporter gene assays: Constructing transcriptional fusions using reporter genes like gusA (β-glucuronidase) can quantify riboswitch activity in vivo. The riboswitch sequence along with its native promoter should be fused to the reporter gene, and expression measured under varying concentrations of guanine and analogs.

• Structure determination: Techniques such as X-ray crystallography, cryo-EM, or NMR can provide atomic-level details of riboswitch-ligand interactions.

Data from C. difficile studies showed guanine riboswitches had Kd values in the nanomolar range (1.2-21 nM), with the guaA riboswitch specifically exhibiting a Kd of 21 ± 2 nM for guanine .

How does mutation of guaA affect bacterial virulence and host colonization?

Mutation studies in pathogenic bacteria provide insights into GuaA's role in virulence. In C. difficile, guaA mutants showed significantly reduced capacity to colonize the mouse gut, with up to 3-log reduction in colonization efficiency compared to wild-type strains. This suggests that de novo GMP biosynthesis is critical during infection. For A. vitis, which causes crown gall disease in grapevines, similar in planta experiments could determine if GuaA is essential for plant colonization and virulence.

The table below summarizes findings from guaA mutation studies that might inform A. vitis research:

What are the optimal conditions for recombinant expression of A. vitis GuaA?

For optimal recombinant expression of A. vitis GuaA, consider the following approach:

• Expression system selection: E. coli BL21(DE3) strains typically yield good expression for bacterial proteins. For GuaA, which may have toxic effects when overexpressed, consider using tightly regulated systems like pET with T7lac promoter.

• Codon optimization: Analyze the codon usage of A. vitis guaA and optimize for the expression host to enhance translation efficiency.

• Expression conditions: Initial expression trials should test multiple conditions:

  • Induction temperature: 16°C, 25°C, and 37°C

  • IPTG concentration: 0.1 mM to 1.0 mM

  • Duration: 4-18 hours

• Solubility enhancement: To improve protein solubility:

  • Use fusion tags such as MBP, SUMO, or TrxA

  • Co-express with chaperones like GroEL/GroES

  • Add low concentrations (1-5%) of solubilizing agents like glycerol or sorbitol to the culture medium

For purification, a multi-step approach using affinity chromatography followed by size exclusion chromatography typically yields highest purity .

How can enzyme activity assays be optimized for recombinant A. vitis GuaA?

Optimizing enzyme activity assays for recombinant A. vitis GuaA requires attention to both direct and coupled assay systems:

• Direct assay approach:

  • Monitor the conversion of XMP to GMP spectrophotometrically at 290 nm (ΔƐ = 1.08 mM⁻¹cm⁻¹)

  • Reaction buffer: 50 mM HEPES (pH 7.5), 100 mM KCl, 5 mM MgCl₂, 2 mM ATP

  • Substrates: XMP (0.01-0.5 mM), glutamine (0.1-5 mM)

  • Temperature range: 25-30°C

• Coupled assay system:

  • Link GMP production to NADH oxidation through coupled enzymes

  • Monitor decrease in absorbance at 340 nm

  • Additional components: pyruvate kinase, lactate dehydrogenase, phosphoenolpyruvate

• High-throughput screening method:

  • Malachite green assay to detect inorganic phosphate released during the reaction

  • Suitable for inhibitor screening and kinetic parameter determination

Based on studies of GuaA from other bacteria, optimal activity typically occurs at pH 7.5-8.0 with requirements for divalent metal ions (Mg²⁺ or Mn²⁺). Kinetic parameters for bacterial GuaA enzymes typically show Km values in the range of 10-50 μM for XMP and 0.2-1 mM for glutamine .

What approaches effectively resolve difficulties in purifying active recombinant GuaA?

Common challenges in purifying active recombinant GuaA include protein aggregation, low solubility, and loss of activity during purification. Effective solutions include:

• Addressing aggregation issues:

  • Use mild detergents (0.05% Triton X-100 or 0.01% DDM) in lysis buffers

  • Include stabilizing agents like 10% glycerol, 1 mM DTT, or 5 mM β-mercaptoethanol

  • Maintain low protein concentrations (<1 mg/ml) during concentration steps

• Enhancing protein stability:

  • Add substrate analogs or product (0.1-0.5 mM GMP) to all buffers

  • Include 1-5 mM MgCl₂ to stabilize the nucleotide-binding domain

  • Optimize ionic strength (typically 100-300 mM NaCl)

• Activity preservation:

  • Minimize freeze-thaw cycles by flash-freezing aliquots in liquid nitrogen

  • Store at -80°C with 15-20% glycerol as cryoprotectant

  • When possible, maintain protein at 4°C and use within 1-2 weeks

• Purification strategy optimization:

  • IMAC (Immobilized Metal Affinity Chromatography) using Ni-NTA columns for His-tagged protein

  • Ion exchange chromatography (typically Q-Sepharose) at pH 8.0

  • Size exclusion chromatography as a final polishing step

The average yield from optimized purification protocols for bacterial GuaA proteins is typically 5-15 mg of purified protein per liter of bacterial culture .

How can site-directed mutagenesis of A. vitis GuaA inform structure-function relationships?

Site-directed mutagenesis of A. vitis GuaA can provide valuable insights into structure-function relationships through systematic modification of key residues. Based on homology with other bacterial GuaA enzymes, several target regions should be prioritized:

• Glutaminase domain residues: The conserved Cys-His-Glu catalytic triad is essential for glutamine hydrolysis. Mutating these residues (typically C86, H183, E185, using E. coli numbering as reference) would help confirm their roles in A. vitis GuaA.

• ATP-binding site: Residues that coordinate ATP binding, such as those in the P-loop motif, can be mutated to assess their contribution to catalysis.

• XMP-binding site: Residues interacting with XMP should be identified through homology modeling and systematically mutated to analyze substrate specificity.

• Interdomain communication: Residues at the interface between glutaminase and synthetase domains play crucial roles in ammonia channeling. Mutations in these regions can reveal how the two catalytic activities are coordinated.

Each mutant should be characterized for:

Correlation of these functional data with structural information can provide comprehensive understanding of the catalytic mechanism .

What role does GuaA play in Agrobacterium-plant interactions during infection?

GuaA likely plays a significant role in Agrobacterium-plant interactions during infection, particularly in adapting to the plant environment. Several experimental approaches can investigate this relationship:

• Construction of guaA conditional mutants: Since guaA may be essential, creating conditional mutants using inducible promoters allows for controlled depletion of GuaA during different infection stages.

• Plant infection assays: Comparing wild-type, guaA mutant, and complemented strains in their ability to:

  • Attach to plant cells

  • Transfer T-DNA

  • Form crown galls

  • Persist in planta

• Metabolomic analysis: Comparing purine metabolite profiles between:

  • Bacteria growing in culture medium

  • Bacteria during early plant infection

  • Bacteria established in plant tissues

• Transcriptomic studies: RNA-seq analysis comparing guaA expression levels and the entire purine biosynthesis pathway during:

  • Saprophytic growth

  • Early infection stages

  • Established plant colonization

Based on studies in other plant pathogens, we would expect GuaA activity to be particularly important during the transition from initial infection to established colonization, when bacteria must adapt to nutrient limitations in plant tissues. Research in C. difficile showed that guaA mutants exhibited significantly reduced colonization ability, suggesting similar importance may exist for A. vitis in plant hosts .

How does environmental sensing affect guaA expression in Agrobacterium vitis?

Environmental sensing likely plays a crucial role in regulating guaA expression in A. vitis through both riboswitch-dependent and independent mechanisms. Research approaches to investigate this relationship include:

• Reporter system construction: Developing fluorescent or colorimetric reporter fusions to monitor guaA expression under various environmental conditions:

  • Nutrient availability (carbon, nitrogen sources)

  • Plant-derived signals (phenolic compounds, sugars)

  • pH and oxygen levels mimicking plant environments

  • Presence of competing microorganisms

• Riboswitch characterization: Determining how environmental factors affect riboswitch function through:

  • In-line probing under varying conditions

  • Structure determination using chemical probing techniques

  • Measuring intracellular purine concentrations under different growth conditions

• Transcription factor identification: Identifying proteins that regulate guaA expression beyond riboswitch control using:

  • DNase footprinting

  • Electrophoretic mobility shift assays (EMSA)

  • Chromatin immunoprecipitation (ChIP-seq)

The table below summarizes predicted environmental responses of guaA expression in A. vitis based on studies in related bacteria:

Understanding these regulatory mechanisms could lead to novel strategies to control A. vitis infections in grapevines and other hosts .

How conserved is GuaA structure and function across Agrobacterium species and related bacteria?

GuaA is highly conserved across Agrobacterium species and related bacteria, reflecting its essential metabolic function. Comparative genomic analyses reveal several important patterns:

• Sequence conservation: Core catalytic domains show 70-90% amino acid sequence identity across Rhizobiaceae family members, with particularly high conservation in the active site residues of both glutaminase and synthetase domains.

• Domain architecture: The two-domain structure (N-terminal glutaminase domain and C-terminal synthetase domain) is maintained across all species, though interdomain linker regions show greater variability.

• Genomic context: While the guaA gene is functionally conserved, its genomic context varies. In some Agrobacterium species, guaA exists as a standalone gene with its own promoter and riboswitch, while in others it may be part of an operon with purine metabolism genes.

• Evolutionary relationships: Phylogenetic analysis of GuaA sequences often recapitulates species relationships, suggesting vertical inheritance with minimal horizontal gene transfer despite its metabolic importance.

What bioinformatic approaches best predict regulatory elements controlling guaA in Agrobacterium genomes?

Several bioinformatic approaches are particularly effective for predicting regulatory elements controlling guaA in Agrobacterium genomes:

• Riboswitch identification:

  • Covariance models based on known guanine riboswitch structures

  • RNA secondary structure prediction tools (Mfold, RNAfold)

  • Conserved motif searches using tools like Infernal and Rfam database

  • Comparative genomics approaches that identify conserved non-coding RNAs

• Promoter analysis:

  • Position weight matrices for sigma factor binding sites

  • Identification of -10 and -35 elements

  • Conservation-based approaches comparing intergenic regions

  • Machine learning approaches trained on known Agrobacterium promoters

• Transcription factor binding site prediction:

  • MEME suite for motif discovery

  • RSAT for regulatory sequence analysis

  • FIMO for motif occurrence mapping

  • Comparative genomics to identify conserved regulatory elements

• Integrated approaches:

  • Combining RNA-seq data with sequence analysis

  • Using ATACseq data to identify accessible chromatin regions

  • Correlating expression patterns with putative regulatory elements

Studies in other bacteria have shown that guanine riboswitches exhibit high-affinity binding (Kd values in the nanomolar range) and cause premature transcription termination upon binding guanine. The C. difficile genome encodes multiple guanine riboswitches, each controlling a single gene involved in purine metabolism and transport, suggesting similar complexity might exist in Agrobacterium species .

What metabolomic approaches can effectively track purine metabolism in recombinant A. vitis systems?

Comprehensive metabolomic approaches to track purine metabolism in recombinant A. vitis systems should incorporate multiple complementary techniques:

• Targeted LC-MS/MS analysis:

  • Utilize multiple reaction monitoring (MRM) for quantification of known purine metabolites

  • Focus on key intermediates: IMP, XMP, GMP, guanosine, guanine, xanthine

  • Include labeled internal standards for accurate quantification

  • Typical sensitivity: low nanomolar range

• Untargeted metabolomics:

  • High-resolution MS (QTOF or Orbitrap) for global metabolite profiling

  • Identification of unexpected metabolites and potential regulatory molecules

  • Pattern recognition using multivariate statistical analysis

  • Pathway enrichment analysis to identify broader metabolic effects

• Flux analysis using stable isotopes:

  • ¹⁵N-labeled precursors to track nitrogen incorporation

  • ¹³C-labeled precursors to follow carbon skeleton metabolism

  • Calculate flux using isotopomer distribution analysis

  • Determine pathway preference under different conditions

• In vivo NMR spectroscopy:

  • Real-time monitoring of metabolic changes

  • Non-destructive measurement of metabolite pools

  • ³¹P-NMR for nucleotide triphosphates and energy status

  • ¹³C-NMR for carbon flux through purine pathways

The table below outlines a recommended LC-MS/MS method for purine metabolite analysis in A. vitis:

These metabolomic approaches can effectively track changes in purine metabolism in wild-type versus recombinant strains, or in response to environmental stresses and inhibitor treatments .