Recombinant Desulfovibrio vulgaris GMP synthase [glutamine-hydrolyzing] (guaA), partial

What is the functional role of GMP synthase (guaA) in D. vulgaris metabolic pathways?

GMP synthase (guaA) catalyzes the ATP-dependent conversion of xanthosine 5'-phosphate (XMP) to guanosine 5'-monophosphate (GMP) using glutamine as an amide donor. In D. vulgaris, as in other bacteria, guaA is a critical enzyme in the guanine nucleotide biosynthetic pathway. According to comparative genomic analyses, guaA works alongside guaB (IMP dehydrogenase) and gmk (guanylate kinase) to facilitate GTP synthesis, which is essential for various cellular processes including protein synthesis, signal transduction, and energy metabolism . The guaA gene appears to be one of the essential components in nucleotide metabolism, particularly relevant for an anaerobic organism like D. vulgaris that must carefully regulate energy utilization.

How is the guaA gene organized in the D. vulgaris genome?

While specific information about guaA's genomic organization in D. vulgaris isn't explicitly detailed in the current literature, genomic analyses of D. vulgaris Hildenborough have mapped many metabolic genes. The guaA gene is likely part of a purine biosynthesis gene cluster, potentially organized in an operon structure with other nucleotide metabolism genes. Based on comparative genomics with other bacterial species, it may be co-localized with genes involved in purine metabolism, similar to the relationship seen in H. pylori where guaA (HP0409) functions alongside guaB (HP0829) and gmk (HP0321) . A comprehensive genetic characterization study of D. vulgaris has generated genome-wide resources that could help determine the precise genomic context of guaA .

What are the key structural and functional domains of the D. vulgaris guaA protein?

The D. vulgaris GMP synthase likely follows the conserved two-domain architecture found in other bacterial homologs:

• N-terminal glutamine amidotransferase (GAT) domain: Responsible for glutamine binding and hydrolysis to release ammonia

• C-terminal synthetase domain: Catalyzes the ATP-dependent incorporation of the ammonia into XMP to form GMP

These domains work in a coordinated manner, with the ammonia generated in the GAT domain traveling through an intramolecular tunnel to the synthetase domain where it reacts with XMP. The protein requires ATP and Mg²⁺ as cofactors for activity. While D. vulgaris-specific structural adaptations haven't been fully characterized, as an anaerobe, its GMP synthase may contain unique features that optimize function in low-oxygen environments.

What expression systems are most effective for producing recombinant D. vulgaris guaA?

For expressing recombinant D. vulgaris guaA, researchers should consider several expression systems with their respective advantages:

Optimization parameters for E. coli expression include using lower induction temperatures (16-25°C), adding solubility-enhancing fusion tags (His, MBP, GST), and employing anaerobic expression conditions to improve folding. The 70-mer oligonucleotide design approach used for D. vulgaris microarrays provides insight into sequence optimization considerations .

What are the most reliable methods for purifying active recombinant D. vulgaris guaA?

A robust purification protocol for obtaining active recombinant D. vulgaris guaA should include:

• Affinity chromatography: Using His-tag or other fusion partners (GST, MBP) as the primary capture step

• Ion-exchange chromatography: For removing impurities based on charge differences

• Size-exclusion chromatography: As a polishing step to achieve high purity

Key considerations for maintaining enzyme activity during purification:

• Perform all steps at 4°C to minimize proteolysis

• Include protease inhibitors in lysis buffers

• Maintain reducing conditions (5-10 mM DTT or 2-3 mM β-mercaptoethanol) to prevent oxidation of cysteine residues

• Consider including stabilizing agents specific for nucleotide-binding proteins (glycerol, low concentrations of substrates)

• For D. vulgaris proteins, consider anaerobic purification to maintain native conformation

The purification approach should be validated by activity assays at each step to ensure the enzyme remains functional.

How can I assess the activity and kinetics of purified recombinant D. vulgaris guaA?

Multiple complementary methods can be employed to assess GMP synthase activity:

A standard kinetic characterization would determine:

• Km values for substrates (XMP, glutamine, ATP)

• kcat and catalytic efficiency (kcat/Km)

• Effects of pH, temperature, and ionic strength

• Potential allosteric regulators (GTP, other nucleotides)

For anaerobic D. vulgaris enzymes, comparing activity under aerobic vs. anaerobic conditions can provide insights into oxygen sensitivity and structural adaptations.

What strategies can be used to create specific guaA knockout or knockdown strains in D. vulgaris?

Based on established genetic manipulation techniques for D. vulgaris, researchers can employ several approaches:

• Gene deletion via homologous recombination: The most effective method involves:

  • Construction of a knockout cassette by fusion PCR to delete guaA via double recombination

  • Electroporation of the cassette with lambda DNA to improve transformation efficiency

  • Selection with G418 (gentamicin) at 400 μg/ml in LS4D medium, which provides cleaner selection than chloramphenicol

  • Verification by PCR and Southern analysis

  This approach has demonstrated transformation efficiency of 1.25 × 10⁻⁷ in D. vulgaris .

• Conditional knockdown alternatives:

  • Inducible antisense RNA expression

  • CRISPR interference (CRISPRi) systems adapted for anaerobes

  • Riboswitch-controlled expression systems

If guaA proves essential (as might be expected for nucleotide metabolism genes), conditional approaches may be necessary. The large-scale genetic characterization of D. vulgaris has identified essential genes using transposon mutagenesis, which could inform the approach .

How can I monitor guaA expression in D. vulgaris under different growth conditions?

Multiple techniques can be employed to analyze guaA expression:

• Microarray analysis using custom 70-mer oligonucleotide probes:

  • Design criteria include melting temperature within 5°C of target (82-92°C), absence of secondary binding, no hairpin/dimer formation exceeding 19 kcal/mol stability, and avoidance of high-G+C stretches

  • This approach was successfully used to analyze D. vulgaris gene expression under different growth conditions

• RT-qPCR (Reverse Transcription Quantitative PCR):

  • More sensitive for specific gene quantification

  • Requires careful primer design and optimization

  • Needs stable reference genes for normalization

• RNA-Seq:

  • Provides comprehensive transcriptome analysis

  • Allows identification of potential antisense transcripts or small RNAs affecting guaA

  • Enables co-expression network analysis

When analyzing expression data, researchers should account for D. vulgaris growth under anaerobic conditions and potential metal-dependent regulation systems like the Fur regulator described in the literature .

What tools are available for studying protein-protein interactions involving D. vulgaris guaA?

Several complementary approaches can be used to identify and characterize protein-protein interactions:

For anaerobic organisms like D. vulgaris, interactions might be oxygen-sensitive, necessitating anaerobic conditions during experimentation. The genome-wide resources developed for D. vulgaris, including transposon mutant libraries, provide valuable tools for validating functional relevance of identified interactions .

How does guaA function integrate with other metabolic pathways in D. vulgaris?

GMP synthase (guaA) in D. vulgaris operates at a critical intersection of multiple metabolic pathways:

• Purine nucleotide synthesis: Part of the pathway converting IMP → XMP → GMP → GDP → GTP, working with guaB (IMP dehydrogenase) and gmk (guanylate kinase)

• Nitrogen metabolism: Utilizes glutamine as nitrogen donor, linking to amino acid metabolism and nitrogen assimilation pathways

• Energy metabolism: Requires ATP, connecting purine synthesis to cellular energetics

• Pentose phosphate pathway: Provides ribose-5-phosphate precursors for nucleotide synthesis through a series of enzymatic reactions involving rpe, devB, g6pD, tal, and tkt genes

The interconnection with these pathways makes guaA a potential metabolic regulation point. In bacteria, purine nucleotide synthesis is often coordinated with carbon and nitrogen metabolism, responding to nutrient availability and energy status.

What is the importance of guaA in stress response and survival mechanisms of D. vulgaris?

While guaA's specific role in D. vulgaris stress response isn't directly addressed in the literature, its function in nucleotide metabolism suggests several potential roles:

• Oxidative stress response: Maintenance of nucleotide pools is crucial for DNA repair mechanisms following oxidative damage, particularly relevant for an anaerobe like D. vulgaris

• Nutrient limitation: During purine starvation, guaA activity becomes critical for maintaining essential GTP pools

• Energy stress: As a GTP-producing pathway component, guaA may influence energy-sensing pathways during metabolic stress

• Metal stress response: Given D. vulgaris' metal-dependent metabolism and the presence of metal-responsive regulators like Fur , guaA could be part of metal homeostasis networks

The large-scale genetic characterization study of D. vulgaris identified conditional phenotypes for 1,137 non-essential genes under various stress conditions , providing a framework for investigating guaA's stress-specific functions.

How does guaA contribute to D. vulgaris survival in its ecological niche?

As a sulfate-reducing bacterium, D. vulgaris occupies anaerobic environmental niches with specific metabolic requirements:

• Energy efficiency: In anaerobic environments with limited energy availability, efficient nucleotide synthesis through guaA activity is crucial for conserving ATP

• Adaptation to microoxic fluctuations: D. vulgaris must maintain metabolic function during occasional oxygen exposure, potentially requiring regulation of nucleotide-dependent stress response mechanisms

• Biofilm formation: Nucleotide signaling (involving GTP-derived second messengers) regulates biofilm formation in many bacteria, potentially linking guaA to community behaviors

• Interspecies interactions: In microbial communities, D. vulgaris produces acetate and interacts metabolically with partner species, processes that may involve signaling pathways dependent on GTP availability

The genetic and metabolic adaptations of D. vulgaris for its ecological niche, including potential specializations in guaA function or regulation, represent an important area for further research.

How can structural biology approaches enhance our understanding of D. vulgaris guaA function?

Structural biology offers several approaches to gain insights into D. vulgaris guaA:

• X-ray crystallography or cryo-EM studies to determine:

  • Three-dimensional structure of D. vulgaris guaA

  • Substrate binding sites and catalytic residues

  • Conformational changes during catalysis

  • Potential anaerobic adaptations in protein structure

• Hydrogen-deuterium exchange mass spectrometry (HDX-MS) to:

  • Map protein dynamics and flexibility

  • Identify regions involved in substrate binding

  • Detect conformational changes upon ligand binding

• Molecular dynamics simulations to investigate:

  • Substrate channeling between domains

  • Effects of mutations on protein stability and function

  • Influence of anaerobic conditions on protein dynamics

These approaches would be particularly valuable for understanding potential adaptations in D. vulgaris guaA that optimize function in anaerobic environments compared to aerobic bacterial homologs.

What insights can systems biology approaches provide into guaA's role in D. vulgaris metabolism?

Systems biology approaches can integrate guaA function into a comprehensive understanding of D. vulgaris metabolism:

• Genome-scale metabolic modeling:

  • Incorporation of guaA-catalyzed reactions into metabolic flux models

  • Simulation of metabolic outcomes under varying guaA activity

  • Integration with experimental data on growth and metabolite levels

• Multi-omics data integration:

  • Correlation of guaA expression with global transcriptome, proteome, and metabolome data

  • Identification of co-regulated genes and proteins

  • Detection of metabolic bottlenecks and regulatory nodes

• Flux analysis:

  • Measurement of metabolic fluxes through purine synthesis pathways using isotope tracers

  • Determination of how guaA activity influences downstream metabolic fluxes

The large-scale fitness dataset and RB-TnSeq mutant library developed for D. vulgaris provide valuable resources for systems biology approaches, as they can help identify condition-specific roles for guaA and its genetic interactions .

How can comparative genomics inform our understanding of guaA evolution and specialization in D. vulgaris?

Comparative genomic analyses can reveal evolutionary insights about D. vulgaris guaA:

• Sequence conservation analysis:

  • Identification of highly conserved catalytic residues versus variable regions

  • Detection of lineage-specific adaptations in sulfate-reducing bacteria

  • Correlation of sequence features with biochemical properties

• Phylogenetic studies:

  • Reconstruction of evolutionary history of guaA across bacterial lineages

  • Identification of potential horizontal gene transfer events

  • Detection of co-evolutionary patterns with interacting proteins

• Genomic context analysis:

  • Comparison of guaA genomic neighborhoods across species

  • Identification of conserved gene clusters or operons

  • Detection of regulatory elements that differ between aerobic and anaerobic bacteria