Recombinant Enterococcus faecalis GMP synthase [glutamine-hydrolyzing] (guaA), partial

What is the function of GMP synthase (guaA) in Enterococcus faecalis?

GMP synthase (glutamine-hydrolyzing), encoded by the guaA gene in E. faecalis, catalyzes the final step in guanine nucleotide biosynthesis, converting XMP (xanthosine monophosphate) to GMP (guanosine monophosphate) using glutamine as an amide donor. This enzyme plays a critical role in purine nucleotide metabolism, which is essential for DNA and RNA synthesis. In E. faecalis, guaA appears to be chromosomally encoded and serves as an important genomic landmark, with various mobile genetic elements, including integrase genes, frequently found downstream of this locus . Methodologically, researchers investigating guaA function typically employ gene knockout studies followed by complementation assays to confirm phenotypic changes related to nucleotide metabolism.

How is the guaA gene organized in the E. faecalis genome?

The guaA gene in E. faecalis is chromosomally located and serves as an insertion site for mobile genetic elements. Genomic analyses reveal that integrase genes (such as int410 and int583) are frequently positioned downstream of guaA . This genomic organization appears to be conserved across different strains, as evidenced by similar arrangements in E. faecalis strains N00-410 and V583 . When designing experiments to study guaA, researchers should consider this genomic context, as the downstream elements may influence gene expression or function. PCR amplification of this region typically requires primers designed to capture both guaA and its flanking sequences to understand potential regulatory elements.

What expression systems are most effective for recombinant production of E. faecalis guaA?

While the search results don't specifically address expression systems for E. faecalis guaA, recombinant protein expression in E. faecalis typically employs E. coli-based expression systems, similar to those used for other E. faecalis proteins like O-Glycosidase . For optimal expression, consider using BL21(DE3) or similar E. coli strains with a His-tag fusion for purification. The expression construct should include the coding sequence (CDS) of guaA with appropriate regulatory elements. Based on protocols for similar enterococcal proteins, expression can be induced with IPTG (0.5-1.0 mM) at mid-log phase, followed by growth at 25-30°C for 4-6 hours to maximize soluble protein yield. Purification via nickel-affinity chromatography typically yields functional protein when followed by buffer exchange into a stabilizing formulation containing Tris-HCl (pH 7.5-8.0) and NaCl (150-300 mM).

What is the relationship between guaA and mobile genetic elements in E. faecalis?

The guaA locus in E. faecalis serves as a chromosomal landmark that appears to be a preferred insertion site for mobile genetic elements, particularly those carrying integrase genes . Research has identified that in E. faecalis strain N00-410, an integrase gene (int410) is located downstream of guaA, showing significant homology (69% identity) to the integrase of Tn5801 from Staphylococcus aureus Mu50 and the Int459 integrase from Clostridium perfringens . Similarly, in E. faecalis V583, an integrase gene (int583) with 53% identity to Int410 is also found downstream of guaA . This pattern suggests that guaA might be associated with a chromosomal hotspot for integration of mobile genetic elements. For experimental investigation of this relationship, researchers should employ whole genome sequencing followed by comparative genomics across multiple strains, and potentially utilize RecT-based recombineering methods for targeted genetic manipulation of these regions .

How does guaA expression correlate with biofilm formation in E. faecalis?

While direct correlations between guaA expression and biofilm formation are not explicitly stated in the search results, research on E. faecalis biofilm formation highlights the importance of nucleoside and nucleotide biosynthesis pathways in this process . Proteomics studies comparing strong and weak biofilm-forming E. faecalis clinical isolates have shown that proteins associated with nucleoside and nucleotide biosynthesis are differentially regulated between these isolates . Given that guaA is central to guanine nucleotide biosynthesis, it likely influences biofilm formation through its impact on cellular metabolism and energy production.

To experimentally investigate this correlation, researchers should consider:

• Quantitative proteomics comparing guaA expression levels between strong and weak biofilm formers using iTRAQ or similar approaches

• Creation of guaA conditional mutants to observe the effect of controlled expression on biofilm formation

• Metabolic profiling to measure GMP levels and correlate them with biofilm formation capacity

The methodology should include biofilm formation assays where E. faecalis isolates are grown in brain-heart infusion broth, followed by seeding into polystyrene microtiter plates and incubation at 37°C for 48 hours to allow biofilm formation .

What methods are recommended for targeted mutagenesis of guaA in E. faecalis?

For efficient targeted mutagenesis of guaA in E. faecalis, RecT recombinase-mediated recombineering combined with CRISPR-Cas9 offers the most effective approach based on recent advancements in enterococcal genetic engineering . This methodology enables rapid and efficient integration of mutagenic DNA templates to generate substitutions, deletions, and insertions.

The experimental protocol should include:

• Expression of RecT recombinase in E. faecalis to facilitate recombination

• Design of single-stranded DNA (ssDNA) oligonucleotides with 35-45 bp homology arms flanking the desired mutation site in guaA

• Co-expression of CRISPR-Cas9 with a guide RNA targeting the wild-type guaA sequence

• Transformation of the ssDNA template and screening for successful recombinants

For complete gene deletion, researchers can design double-stranded DNA templates carrying antibiotic selection markers flanked by homology regions to the guaA locus. This approach has been demonstrated to achieve high efficiency in various Enterococcus species . Following transformation, curing of CRISPR and recombineering plasmids can be achieved by passaging cells and screening for antibiotic sensitivity over approximately three days .

This methodology significantly improves upon traditional approaches that require passive homologous recombination from plasmid DNA, which can take multiple weeks to perform.

How can the enzymatic activity of recombinant E. faecalis guaA be measured in vitro?

The enzymatic activity of recombinant E. faecalis GMP synthase can be measured through several complementary approaches:

• Spectrophotometric assay: Monitor the conversion of XMP to GMP by measuring the decrease in absorbance at 290 nm, which corresponds to the consumption of XMP.

• Coupled enzyme assay: Link GMP synthase activity to NADH oxidation through pyruvate kinase and lactate dehydrogenase, monitoring the decrease in absorbance at 340 nm.

• HPLC analysis: Quantify the production of GMP using reverse-phase HPLC with UV detection at 254 nm.

For optimal assay conditions, use a buffer containing:

• 50 mM Tris-HCl (pH 8.0)

• 100 mM KCl

• 5 mM MgCl₂

• 1 mM DTT

• 1 mM ATP

• 0.5 mM XMP

• 5 mM glutamine

A typical reaction mixture would contain 0.1-1 μg of purified recombinant guaA protein. Incubate at 37°C and take measurements at regular intervals (0, 5, 10, 15, 30 minutes) to determine initial velocity rates.

What structural domains characterize E. faecalis GMP synthase and how do they compare to other bacterial GMP synthases?

While specific structural information for E. faecalis GMP synthase is not provided in the search results, bacterial GMP synthases typically contain two functional domains:

• N-terminal glutaminase domain: Catalyzes the hydrolysis of glutamine to glutamate and ammonia

• C-terminal synthetase domain: Mediates the amination of XMP to GMP using the ammonia generated by the glutaminase domain

Based on sequence homology with other bacterial GMP synthases, E. faecalis guaA likely shares these conserved domains. Comparative sequence analysis would reveal the degree of conservation with other enterococcal species as well as more distant bacterial pathogens.

To experimentally determine domain structure and function, researchers should consider:

• Limited proteolysis experiments to identify domain boundaries

• Expression of individual domains to test for independent functionality

• Site-directed mutagenesis of conserved residues in each domain to assess their contribution to catalytic activity

Structural studies including X-ray crystallography or cryo-EM would provide definitive information about domain organization and potential interfaces with inhibitory compounds.

How can E. faecalis guaA be targeted for antimicrobial development?

GMP synthase represents a potential antimicrobial target due to its essential role in nucleotide biosynthesis. To develop inhibitors targeting E. faecalis guaA:

• Structure-based drug design: Utilize homology modeling based on known bacterial GMP synthase structures to identify potential binding sites for inhibitors. Focus on regions that differ from human GMP synthase to ensure selectivity.

• High-throughput screening: Develop an in vitro enzymatic assay suitable for screening chemical libraries to identify compounds that inhibit E. faecalis guaA activity.

• Fragment-based approach: Screen small molecular fragments that bind to recombinant guaA and then elaborate these fragments into more potent inhibitors.

• Natural product exploration: Test extracts from plants, fungi, or bacteria for inhibitory activity against recombinant guaA.

Methodologically, researchers should express and purify the recombinant E. faecalis guaA using carrier-free preparations to avoid interference from BSA or other carrier proteins in inhibitor screening assays . Selected inhibitors should then be tested against both planktonic cultures and biofilms of E. faecalis to assess their efficacy in different growth states, particularly given the importance of biofilm formation in E. faecalis pathogenicity .

What is the role of guaA in E. faecalis antibiotic resistance mechanisms?

While the search results don't directly address guaA's role in antibiotic resistance, the genomic context of guaA provides insights into potential connections. The proximity of guaA to integrase genes and mobile genetic elements in E. faecalis suggests it may be associated with genomic regions involved in the acquisition of resistance determinants . In E. faecalis N00-410, the vanE operon (conferring vancomycin resistance) is found in proximity to the guaA-associated integrase .

To investigate the potential role of guaA in antibiotic resistance:

• Compare guaA expression levels between antibiotic-resistant and susceptible strains using RT-qPCR or proteomics

• Generate guaA knockdown mutants and assess changes in minimum inhibitory concentrations (MICs) for various antibiotics

• Analyze genomic contexts across multiple antibiotic-resistant E. faecalis isolates to identify patterns of resistance gene acquisition in relation to the guaA locus

This research direction is particularly relevant given the emergence of vancomycin-resistant enterococci (VRE) as significant nosocomial pathogens. E. faecalis strain N00-410, for example, exhibits intermediate vancomycin resistance (MIC = 24 μg/ml) associated with the vanE operon , highlighting the clinical significance of understanding resistance mechanisms in this organism.

What are the optimal storage conditions for recombinant E. faecalis guaA preparations?

Based on recommendations for similar recombinant proteins from E. faecalis, the following storage conditions would likely be optimal for recombinant guaA:

• Short-term storage (1-2 weeks): Store at 4°C in a buffer containing 50 mM Tris-HCl (pH 7.5-8.0), 150-300 mM NaCl, and optionally 10% glycerol.

• Long-term storage: Use a manual defrost freezer at -20°C or preferably -80°C and avoid repeated freeze-thaw cycles . Aliquot the protein solution before freezing to minimize freeze-thaw cycles.

• Formulation considerations:

  • For carrier-free preparations, supply as a 0.2 μm filtered solution in Tris buffer with NaCl

  • For enhanced stability, consider adding stabilizing agents such as 1-5 mM DTT or 2-mercaptoethanol to maintain reduced cysteine residues

• Shipping recommendations: Ship with polar packs and upon receipt, store immediately at the recommended temperature .

What quality control methods should be employed to verify recombinant E. faecalis guaA identity and purity?

To ensure the identity, purity, and functionality of recombinant E. faecalis guaA preparations, implement the following quality control methods:

• Purity assessment:

  • SDS-PAGE with Coomassie staining (target: >95% purity)

  • Size exclusion chromatography to verify monodispersity and absence of aggregates

• Identity confirmation:

  • Western blotting using anti-His tag antibodies (if His-tagged)

  • Mass spectrometry:

    • MALDI-TOF for molecular weight verification

    • LC-MS/MS for peptide fingerprinting and sequence coverage analysis

• Functional validation:

  • Enzymatic activity assay measuring the conversion of XMP to GMP

  • Binding assays for key substrates (XMP, ATP, glutamine)

• Endotoxin testing:

  • LAL (Limulus Amebocyte Lysate) assay for preparations intended for cell culture experiments

• Stability testing:

  • Thermal shift assays to determine melting temperature and buffer optimization

  • Activity retention after storage at different temperatures and time points

• Protein concentration determination:

  • BCA or Bradford assay, with considerations for potential interference from buffer components

  • Absorbance at 280 nm using the calculated extinction coefficient

A standard quality control data table should include: