Recombinant Pectobacterium carotovorum subsp. carotovorum GMP synthase [glutamine-hydrolyzing] (guaA), partial

What is the molecular structure and enzymatic mechanism of GMP synthase in P. carotovorum?

GMP synthase (encoded by guaA) is an amidotransferase that catalyzes the amination of xanthosine 5'-monophosphate (XMP) to form GMP in the presence of glutamine and ATP. The enzyme contains two distinct functional domains that operate in a coordinated manner :

• Glutaminase domain: Responsible for glutamine hydrolysis to provide the necessary amino group

• Synthetase domain: Responsible for ATP hydrolysis and GMP formation

The reaction occurs in two steps:

• Glutamine is hydrolyzed to produce ammonia

• ATP hydrolysis drives the transfer of the amino group to XMP, forming GMP

Notably, ammonia can also serve as a direct amino group donor when glutamine is unavailable . The enzyme likely functions as a dimer in solution, similar to human GMP synthase which has been crystallized in complex with XMP .

How is the guaA gene organized within the P. carotovorum genome?

The guaA gene in P. carotovorum is part of a polycistronic guaBA operon, with a 68-base pair intercistronic region separating guaA from the upstream guaB gene . According to sequence analyses, the structural gene encodes a protein with a calculated molecular weight similar to other bacterial GMP synthases. The 3' end of the guaA mRNA is located 36-37 nucleotides downstream of the translation stop codon within a region of dyad symmetry that resembles a rho-independent transcription termination site .

In the STRING protein interaction database, guaA is shown to interact with several proteins involved in nucleotide metabolism, including nucleoside-diphosphate kinase (ndk) and other enzymes involved in purine synthesis pathways .

What are the optimal expression systems for producing functional recombinant P. carotovorum guaA?

Based on successful expression methodologies for similar enzymes from bacterial sources, researchers should consider the following expression systems:

E. coli-based expression system:

• Use BL21(DE3) or similar strains with T7 expression vectors

• Induce with IPTG at OD600 = 0.6-0.8

• Grow at temperatures between 25-30°C to minimize inclusion body formation

Bacillus subtilis expression system:

• This system offers advantages for enzymes intended for biotechnological applications

• Using B. subtilis WB800N strain with 2X-YT medium has proven effective for similar enzymes

• Employ IPTG induction (1mM) with a potential second induction after 8 hours

• Culture at 37°C with shaking at 225 rpm

For optimal results, construct the expression vector with appropriate promoters and ribosome binding sites, and consider adding affinity tags (His6, GST) to facilitate purification while ensuring they don't interfere with enzyme activity.

What biochemical assays can be used to verify the dual catalytic activities of purified recombinant guaA?

To properly characterize both functional domains of recombinant P. carotovorum guaA, researchers should employ separate assays for each activity:

Glutaminase activity assays:

• Measure ammonia production using Nessler's reagent or enzymatic methods

• Perform assays in the absence of XMP to isolate glutaminase function

• Use acivicin as a specific inhibitor to confirm this activity

Synthetase activity assays:

• Monitor XMP to GMP conversion spectrophotometrically at 290nm

• Use ammonia as an alternative nitrogen donor to bypass glutaminase activity

• Analyze reaction products via HPLC or TLC (similar to methods in )

Combined activity assessment:

How does guaA function integrate with other metabolic pathways in P. carotovorum?

GMP synthase functions as a critical enzyme in purine biosynthesis, but its activity is interconnected with several other metabolic pathways:

Nucleotide metabolism connections:

• The guaA product (GMP) serves as a precursor for all guanine nucleotides (GDP, GTP)

• GTP is essential for protein synthesis, signal transduction, and as substrate for cyclic di-GMP synthesis

• The STRING database shows high confidence interactions (score >0.8) between guaA and nucleoside-diphosphate kinase (ndk), indicating coordinated nucleotide synthesis

Energy metabolism integration:

• ATP consumption by guaA links purine metabolism to cellular energy status

• Potential coordination with Entner-Doudoroff (ED) pathway enzymes like Eda

Regulatory networks:

• In Pectobacterium, metabolic enzymes can have moonlighting functions in virulence regulation

• Possible connections to c-di-GMP signaling networks that regulate virulence traits

Protein interaction analyses suggest that guaA operates within a network of enzymes involved in nucleotide biosynthesis, with potential coordination with other metabolic enzymes through protein-protein interactions or metabolic flux regulation .

How does guaA expression correlate with virulence factor production in P. carotovorum?

Understanding the relationship between guaA and virulence requires examining expression patterns during infection:

Evidence from proteomics studies:

• Differential protein expression analysis between in vitro and in vivo conditions has identified proteins involved in P. carotovorum pathogenicity

• Similar approaches could reveal correlations between guaA expression and known virulence factors

Potential regulatory connections:

• Quorum sensing mutants (ΔexpI) show altered expression of multiple virulence factors in P. carotovorum

• Nucleotide metabolism may be linked to quorum sensing networks through second messenger signaling

• The cyclic di-GMP signaling pathway, which requires GTP (a downstream product of guaA activity), regulates virulence gene expression in P. carotovorum

Experimental strategies:

• Real-time PCR analysis comparing guaA expression with virulence genes (plant cell wall-degrading enzymes, motility factors) during infection

• Reporter gene fusions (guaA promoter with reporter genes) to track expression patterns in planta

• Comparison of expression patterns in wild-type and virulence-attenuated mutants

Research in P. atrosepticum has demonstrated that metabolic enzymes like Eda (KDPG aldolase) can significantly impact virulence gene expression , suggesting that guaA might similarly influence virulence factor production through direct or indirect mechanisms.

What experimental approaches can determine if guaA is essential for P. carotovorum virulence in plant hosts?

To establish the role of guaA in pathogenicity, researchers should employ multiple complementary approaches:

Genetic manipulation strategies:

• Generate targeted guaA deletion mutants using allelic exchange techniques

• Create conditional mutants if guaA proves essential for viability

• Construct complemented strains carrying wild-type guaA on a plasmid

Virulence assays:

• Plant tissue maceration tests:

  • Potato tuber soft rot assays measuring tissue maceration

  • Quantification of bacterial populations in infected tissues

• Plant colonization assays:

  • GFP-tagged strains to visualize infection progression

  • Comparative colonization between wild-type and mutant strains

• Virulence factor production:

  • Quantitative assays for plant cell wall-degrading enzymes

  • Measurement of motility (swimming, swarming) and biofilm formation

Separation of fitness vs. virulence effects:

• Compare in vitro growth in minimal and rich media with in planta growth

• Provide guanine supplements in planta to distinguish between metabolic deficiencies and specific virulence roles

Comparative analysis with known virulence factors:

• Studies of the Entner-Doudoroff pathway enzyme Eda showed that it affects both metabolism and virulence factor production in P. atrosepticum

• Similar dual roles might exist for guaA in P. carotovorum

How can structural information about P. carotovorum guaA inform antimicrobial development?

GMP synthase represents a potential target for controlling P. carotovorum infections in crops:

Target validation strategies:

• Determine if guaA is essential for pathogen viability or virulence through gene deletion studies

• Identify key catalytic residues through site-directed mutagenesis

• Compare with the human enzyme to identify bacterial-specific features

Structure-based inhibitor design:

• Human GMP synthase crystal structure in complex with XMP provides a template for homology modeling

• The conserved cysteine residue in the glutaminase domain (equivalent to Cys104 in human enzyme) represents a critical target for inhibition

• Acivicin, a glutamine analog, irreversibly inhibits GMP synthase by covalently modifying this cysteine residue

Inhibitor screening methodologies:

• Enzymatic assays with purified recombinant guaA to identify inhibitors

• Bacterial growth inhibition assays to confirm whole-cell efficacy

• Plant protection assays to evaluate disease control potential

Selectivity considerations:

• Design inhibitors that exploit structural differences between bacterial and plant GMP synthases

• Focus on compounds that preferentially inhibit bacterial enzyme while sparing plant homologs

What techniques can explore potential moonlighting functions of guaA in P. carotovorum?

Recent research indicates that metabolic enzymes in bacteria often perform secondary "moonlighting" functions beyond their primary catalytic roles. To investigate such possibilities for guaA:

Protein-protein interaction studies:

• Affinity purification coupled with mass spectrometry to identify interaction partners

• Bacterial two-hybrid screening against genomic libraries

• In vivo crosslinking followed by co-immunoprecipitation

Transcriptional regulation analysis:

• Chromatin immunoprecipitation to detect potential DNA binding

• Reporter gene assays to test transcriptional effects

• RNA-seq comparison between wild-type and guaA mutants

Phenotypic characterization beyond metabolism:

• Evaluate effects on biofilm formation and motility

• Test stress response phenotypes (oxidative stress, pH tolerance)

• Examine changes in cell morphology and envelope properties

Comparative analysis with other moonlighting enzymes:

• The KDPG aldolase Eda in P. atrosepticum was found to independently inhibit expression of virulence determinants

• Similar functional studies could reveal regulatory roles for guaA

What are the key methodological challenges in studying in vivo activity of guaA during plant infection?

Investigating guaA function during actual plant infection presents several technical challenges:

Extraction and activity preservation:

• Plant tissues contain compounds that can interfere with enzyme assays

• Rapid extraction protocols must be developed to preserve enzyme activity

• Methods similar to those used for differential proteomics studies in P. carotovorum can be adapted

Distinguishing bacterial from plant enzymes:

• Plants have their own GMP synthase enzymes

• Use of epitope-tagged recombinant versions can allow specific immunoprecipitation

• RT-PCR with species-specific primers can differentiate bacterial from plant transcripts

Temporal and spatial dynamics:

• Infection progression varies across plant tissues

• Time-course sampling strategies are essential

• Laser capture microdissection may allow isolation of bacteria from specific infection zones

Experimental design considerations:

• Include appropriate controls (uninfected plant tissue, heat-killed bacteria)

• Compare in vitro vs. in planta expressed enzyme characteristics

• Consider environmental factors (temperature, pH, nutrient availability) that differ between laboratory and plant conditions

Data table for sample processing protocol:

How can researchers effectively design experiments to distinguish between direct and indirect effects of guaA on virulence?

Separating direct regulatory roles from indirect metabolic effects requires carefully designed experiments:

Genetic approach strategies:

• Complementation analysis:

  • Test if guaA catalytic mutants (preserving structure but lacking activity) can restore virulence

  • Create domain-specific mutants to separate glutaminase from synthetase functions

• Bypass experiments:

  • Provide guanine nucleotides exogenously to determine if metabolic deficiencies can be complemented

  • Use alternative metabolic pathways to restore GMP levels without guaA function

• Protein engineering:

  • Create fusion proteins to artificially tether guaA to suspected interaction partners

  • Use protein localization tags to redirect guaA within the cell

Biochemical and molecular approaches:

• Direct binding assays:

  • Test guaA binding to promoter regions of virulence genes

  • Evaluate protein-protein interactions with regulatory factors

• Metabolic flux analysis:

  • Use isotope labeling to track nucleotide metabolism in wild-type versus mutant strains

  • Correlate metabolic changes with virulence factor production

• Temporal sequence determination:

  • Establish precise timeline of metabolic changes versus virulence gene expression

  • Use inducible systems to create rapid changes in guaA levels

Comprehensive experimental design table:

How might guaA interact with cyclic di-GMP signaling networks that regulate P. carotovorum virulence?

Recent research has identified cyclic di-GMP as a key second messenger in regulating P. carotovorum virulence . As guaA produces GMP, a precursor for GTP (the substrate for c-di-GMP synthesis), potential connections between these pathways merit investigation:

Metabolic connectivity:

• GMP synthase activity directly influences GTP availability

• GTP serves as the substrate for diguanylate cyclases (DGCs) that produce c-di-GMP

• Changes in guaA activity could alter the cellular GTP pool available for c-di-GMP synthesis

Experimental approaches to explore connections:

• Nucleotide pool analysis:

  • Quantify GTP and c-di-GMP levels in wild-type versus guaA mutant strains

  • Measure changes in these pools during infection progression

• Double mutant analysis:

  • Generate combinations of guaA mutations with alterations in DGC genes

  • Compare phenotypes to identify genetic interactions

• Signaling pathway monitoring:

  • Use c-di-GMP biosensors to track signaling changes when guaA activity is manipulated

  • Monitor expression of c-di-GMP regulated genes in guaA mutant backgrounds

Research implications:

• An oxygen-sensing diguanylate cyclase broadly affects transcript levels in P. carotovorum

• C-di-GMP influences metal transporter expression, motility, and biofilm formation

• guaA activity may provide a metabolic input that influences this regulatory network

This research direction could establish novel connections between nucleotide metabolism and virulence regulation in plant pathogens.

What systems biology approaches can best reveal the position of guaA in the P. carotovorum virulence network?

Understanding guaA's place in the complex virulence regulatory network requires integrated systems biology approaches:

Multi-omics integration strategies:

• Combined transcriptomics and proteomics:

  • RNA-seq and quantitative proteomics in wild-type versus guaA mutants

  • Identify directly and indirectly affected pathways

  • Compare with datasets from other regulatory mutants (e.g., expI quorum sensing mutants )

• Metabolomics analysis:

  • Profile changes in nucleotide pools and central metabolism

  • Connect metabolic alterations to virulence factor production

  • Identify metabolic bottlenecks in guaA mutants

• Protein-protein interaction network mapping:

  • Affinity purification-mass spectrometry to identify guaA interactors

  • Construct interaction networks specific to infection conditions

  • Compare with established P. carotovorum protein networks from STRING database

Advanced computational approaches:

• Network inference algorithms:

  • Bayesian network reconstruction from multi-omics data

  • Identification of regulatory motifs and feedback loops

• Genome-scale metabolic modeling:

  • Incorporate guaA function into flux balance analysis models

  • Predict metabolic consequences of guaA perturbation

  • Identify synthetic lethal interactions

• Comparative systems analysis:

  • Cross-species comparison with other plant pathogens

  • Evolutionary analysis of guaA and associated regulatory networks

These integrative approaches can position guaA within both metabolic and virulence networks, revealing how nucleotide metabolism interfaces with pathogenicity in P. carotovorum.

What are the most promising translational applications of P. carotovorum guaA research?

Research on P. carotovorum guaA has several potential translational applications:

Agricultural disease management:

• Development of small molecule inhibitors targeting bacterial guaA for crop protection

• Design of detection methods for early diagnosis of P. carotovorum infections

• Creation of transgenic plants with enhanced resistance to bacterial soft rot

Biotechnological applications:

• Engineering of guaA for improved nucleotide production in industrial applications

• Development of biosensors using guaA-based detection systems

• Creation of attenuated strains for biological control purposes

Fundamental scientific advances:

• Deeper understanding of nucleotide metabolism in bacterial pathogenesis

• Insights into potential moonlighting functions of metabolic enzymes

• New paradigms for metabolic regulation of virulence traits

By targeting a critical metabolic enzyme with potential regulatory roles, guaA research represents a promising avenue for developing novel strategies to combat bacterial soft rot diseases that cause significant economic losses in agriculture worldwide.

What are the key technical innovations needed to advance P. carotovorum guaA research?

Future progress in understanding P. carotovorum guaA will require several technical innovations:

Structural biology advances:

• High-resolution crystal structures of P. carotovorum guaA to guide inhibitor design

• Cryo-EM studies of guaA complexes with potential interaction partners

• NMR investigations of protein dynamics during catalysis

In planta monitoring technologies:

• Development of non-destructive methods to track guaA activity in living plant tissues

• Biosensors for real-time monitoring of nucleotide pools during infection

• Improved imaging techniques for visualizing bacterial metabolism in plant hosts

Genetic tool development:

• CRISPR-Cas9 genome editing systems optimized for P. carotovorum

• Inducible promoter systems for temporal control of guaA expression

• Site-specific recombination systems for in planta genetic manipulation

High-throughput screening platforms:

• Automated systems for testing guaA inhibitors against multiple Pectobacterium strains

• Plant infection models amenable to high-throughput screening

• Computational pipelines for analyzing multi-omics data from infection studies