Recombinant Vibrio vulnificus GTPase obg (obg)

What is Vibrio vulnificus ObgE and what are its primary functions?

ObgE (GTP-binding protein) in Vibrio vulnificus is a member of the highly conserved Obg family of P-loop GTPases found across bacteria to humans. This protein plays multiple roles in bacterial physiology:

• Functions as a ribosome-dependent GTPase that regulates translation by preventing ribosomal subunit association

• Acts as a molecular switch in stress response mechanisms

• Participates in the regulation of bacterial dormancy and persistence

• Mediates interactions with host defense mechanisms

The protein consists of multiple domains, with the evolutionarily conserved N-terminal domain (NTD) serving as a tRNA structural mimic that interacts specifically with the peptidyl-transferase center, similar to Class I release factors .

How does ObgE contribute to Vibrio vulnificus pathogenesis?

ObgE plays significant roles in V. vulnificus pathogenesis through multiple mechanisms:

• Regulates bacterial persistence and transition to viable but non-culturable (VBNC) state during stress conditions

• Mediates interactions with gut microbiome through molecular mechanisms involving cyclo-Phe-Pro (cFP)

• Contributes to bacterial survival within host environments by modulating translation and stress responses

• Potentially influences virulence factor expression through its role in translation regulation

Studies demonstrate that ObgE's interactions with molecular signals like (p)ppGpp enhance V. vulnificus adaptability during infection, contributing to its pathogenic potential .

What are the optimal conditions for expressing and purifying recombinant V. vulnificus ObgE?

For optimal expression and purification of recombinant V. vulnificus ObgE:

• Expression system selection:

  • Yeast expression systems have been successfully used for commercial production

  • E. coli expression systems are suitable for laboratory-scale production using pBAD expression vectors (arabinose-inducible) as demonstrated in published protocols

• Expression conditions:

  • Induce expression when cultures reach A600 of 0.3

  • Add arabinose to a final concentration of 0.1 mM for pBAD vector systems

  • Maintain expression at 30°C for optimal protein folding

• Purification strategy:

  • Use Ni-NTA affinity chromatography for His-tagged constructs

  • Include GTP or non-hydrolyzable GTP analogs (GMPPNP) in purification buffers to stabilize protein conformation

  • Consider size exclusion chromatography as a polishing step to remove aggregates

The purity and activity of purified ObgE should be verified through SDS-PAGE and GTPase activity assays .

What experimental approaches are most effective for studying ObgE interactions with bacterial ribosomes?

To study ObgE-ribosome interactions effectively:

• Biochemical approaches:

  • Pre-steady state fast kinetics to measure binding kinetics and affinities

  • Equilibrium ultracentrifugation to determine complex formation

  • Filter binding assays to quantify ribosome binding

• Structural approaches:

  • Cryo-electron microscopy (cryo-EM) of 50S·ObgE·GMPPNP complexes has proven effective for determining interaction sites

  • X-ray crystallography for high-resolution structural details

  • Hydrogen-deuterium exchange mass spectrometry to map interaction surfaces

• Functional assays:

  • Ribosomal subunit association assays

  • GTPase activity measurements in the presence of ribosomal components

  • Translation inhibition assays

When designing these experiments, it's critical to use non-hydrolyzable GTP analogs (GMPPNP) to capture stable complexes, as ObgE is a ribosome-dependent GTPase .

How does ObgE induce bacterial persistence and transition to the VBNC state?

ObgE mediates the transition to persistence and VBNC states through several mechanisms:

• Protein aggregation induction:

  • Overexpression of obgE strongly accelerates protein aggregation

  • Creates larger and more intense protein aggregates compared to normal conditions

  • This aggregation is specifically linked to the transition to dormant states and is not merely an artifact of protein overexpression

• Temporal dynamics:

  • ObgE accelerates persister development rather than increasing maximal persister levels

  • The transition shows a clear progression: normal cells → persisters → VBNC state

  • Persister development correlates with increasing IbpA-msfGFP fluorescence (a marker for protein aggregation)

• Molecular mechanisms:

  • ObgE specifically increases aggregation of translation-related proteins

  • This effect may be mediated through ObgE's known interaction with the 50S ribosomal subunit

  • Flow cytometry data demonstrates a direct correlation between cellular ObgE levels and protein aggregation (IbpA-msfGFP) at wild-type expression levels

These findings suggest ObgE functions as a molecular switch controlling the transition between active growth and dormancy in response to stress conditions .

What methods can be used to differentiate between persistence and VBNC states when studying ObgE function?

To effectively differentiate between persistence and VBNC states:

• Combined viability assays:

  • Measure colony-forming units (CFUs) to determine culturable cells

  • Use live/dead staining (e.g., SYTO9/propidium iodide) to enumerate total viable cells

  • Calculate VBNC cells as the difference between total viable cells and culturable cells

• Time-lapse microscopy approaches:

  • Monitor cells grown for different periods (24h, 40h, 72h) using time-lapse microscopy

  • Classify cells as culturable or VBNC based on division resumption within 16 hours

  • Correlate with fluorescent markers such as IbpA-msfGFP for protein aggregation

• Antibiotic challenge method:

  • Treat samples with ofloxacin to enumerate persister cells

  • Monitor absolute persister numbers over time

  • Combine with CFU counts to track transitions between states

• Protein aggregation markers:

  • Use IbpA-msfGFP fusion as a fluorescent reporter for protein aggregation

  • Quantify phase-bright structures using phase contrast microscopy

  • Apply flow cytometry to correlate ObgE levels with aggregation states

These methods should be applied dynamically over time to capture the transition between persistence and VBNC states, as ObgE accelerates this transition rather than simply increasing one state .

How does ObgE interact with (p)ppGpp during stringent response, and what are the implications for bacterial survival?

ObgE interacts with ppGpp (guanosine tetraphosphate) through several mechanisms with significant implications for bacterial survival:

• Binding characteristics:

  • ObgE directly binds ppGpp, the global regulator of stringent response

  • Upon binding ppGpp, ObgE exhibits enhanced interaction with the 50S ribosomal subunit

  • This leads to increased equilibrium dissociation of 70S ribosomes into subunits

• Functional consequences:

  • Acts as a ppGpp effector protein that regulates translation in response to stress

  • Functions as an anti-association factor preventing ribosomal subunit association

  • Controls the 50S subunit production and participation in translation under stressed conditions

• Regulatory implications:

  • Serves as a checkpoint in the final stages of 50S subunit assembly

  • Creates a mechanism for translation control in response to environmental cues

  • Potentially mediates cross-talk between stringent response and translation machinery

Interestingly, while ObgE-induced protein aggregation occurs normally in a ΔrelA ΔspoT strain (unable to produce ppGpp), the persistence phenotype is significantly affected. This suggests a complex relationship between ppGpp, ObgE, and the persistence/VBNC transition pathways .

What is the relationship between V. vulnificus ObgE, cyclo-Phe-Pro (cFP), and interactions with gut microbiota?

The relationship between V. vulnificus ObgE, cFP, and gut microbiota involves several key interactions:

• cFP-ObgE binding dynamics:

  • Recombinant ObgE exhibits high affinity binding to cFP at a 1:1 ratio

  • cFP is a major compound secreted by V. vulnificus that mediates antagonistic effects against gut commensals

  • The binding potentially influences ObgE's GTPase activity and cellular functions

• Effects on gut microbiota:

  • V. vulnificus-derived cFP significantly modulates the abundance of predominant gut commensal species

  • Specifically reduces Bacteroides vulgatus population in the intestinal microbiome

  • The antagonistic effect follows a dose-dependent pattern in co-culture experiments

• Membrane disruption mechanism:

  • cFP-treated B. vulgatus shows collapsed cellular morphology with undulated cell surface

  • Exhibits enlarged periplasmic space and lysed membranes indicating membrane disruption

  • The degree of disruption is directly dependent on cellular levels of ObgE in B. vulgatus

• Pathogenicity implications:

  • Oral administration of cFP to mice reduces B. vulgatus levels in feces

  • This reduction correlates with increased susceptibility to V. vulnificus infection

  • Suggests ObgE-cFP interaction represents a novel virulence mechanism

These findings reveal that V. vulnificus exploits ObgE-cFP interactions to modulate gut microbiota composition, enhancing its pathogenicity in the host environment .

How can researchers effectively measure ObgE's GTPase activity and its modulation by different factors?

To effectively measure ObgE's GTPase activity and its modulation:

• Biochemical GTPase assays:

  • Malachite green assay to measure inorganic phosphate release

  • HPLC-based methods to monitor GTP hydrolysis directly

  • Coupled enzyme assays that link GTP hydrolysis to NADH oxidation for continuous monitoring

• Ribosome-dependent activity assessment:

  • Compare GTPase activity with and without purified ribosomal subunits

  • Use ribosomal fractions (50S, 30S, 70S) to determine specificity

  • Control for ribosome quality and concentration in assays

• Modulating factors testing:

  • Examine effects of ppGpp at physiologically relevant concentrations (0.1-2 mM)

  • Test cFP effects on GTPase activity at various concentrations

  • Assess influence of ionic conditions, pH, and temperature

• Data analysis approaches:

  • Determine kinetic parameters (kcat, Km) using Michaelis-Menten kinetics

  • Analyze inhibition/activation patterns using appropriate models (competitive, non-competitive)

  • Apply thermal shift assays to assess ligand binding and stability effects

For meaningful results, maintain consistent experimental conditions and include appropriate controls like heat-inactivated ObgE and non-hydrolyzable GTP analogs (GMPPNP) as negative controls .

What strategies should be employed to study the relationship between ObgE, protein aggregation, and bacterial dormancy?

To effectively study the relationship between ObgE, protein aggregation, and dormancy:

• Fluorescent reporter systems:

  • Create dual reporter strains expressing both ObgE-mCherry and IbpA-msfGFP

  • Use flow cytometry to quantify correlation between ObgE levels and protein aggregation

  • Apply time-lapse microscopy to visualize aggregate formation and cell fate

• Proteomics approaches:

  • Perform differential proteomics on aggregate fractions from control vs. ObgE-overexpressing cells

  • Use COG enrichment analysis to identify overrepresented functional categories

  • Apply SILAC or TMT labeling for quantitative comparison

• Temporal dynamics analysis:

  • Monitor persistence, VBNC transition, and aggregation simultaneously over extended time periods

  • Sample at multiple time points (8h, 16h, 24h, 40h, 72h) to capture the complete transition process

  • Correlate ObgE expression levels, aggregate formation, and dormancy phenotypes

• Genetic manipulation strategy:

  • Create ObgE variants with mutations in key functional domains

  • Test complementation with wild-type vs. mutant ObgE in deletion strains

  • Generate conditional expression systems with tunable ObgE levels

• Stress response integration:

  • Examine effects of various stressors (oxidative stress, nutrient limitation) on ObgE-mediated aggregation

  • Test the role of (p)ppGpp using ΔrelA ΔspoT strains

  • Investigate potential cross-talk with other stress response pathways

These approaches will help elucidate the causal relationships between ObgE expression, protein aggregation patterns, and the transition to dormancy states .

How do the structural and functional properties of V. vulnificus ObgE compare to ObgE homologs in other bacterial species?

Comparative analysis of V. vulnificus ObgE with homologs in other species reveals:

• Structural conservation and divergence:

  • The N-terminal domain (NTD) is highly conserved across bacterial species and functions as a tRNA structural mimic

  • Central GTPase domain maintains the characteristic P-loop GTPase fold with conserved motifs

  • C-terminal regions show greater sequence divergence, potentially reflecting species-specific functions

  • V. vulnificus ObgE shares significant structural homology with V. cholerae GbpA, particularly in domain organization

• Functional comparison:

  • E. coli ObgE functions as an anti-association factor preventing ribosomal subunit association

  • V. cholerae GbpA serves as a lectin-like mucus adhesin with a four-domain structure

  • V. vulnificus ObgE appears to combine roles in both ribosome regulation and host interaction

  • The ribosomes-binding capabilities appear conserved across species, while host-interaction mechanisms show species-specific adaptations

• Regulatory mechanisms:

  • V. vulnificus ObgE expression is regulated by IscR, CRP, and SmcR in a growth phase-dependent manner

  • E. coli ObgE is regulated by stringent response via ppGpp

  • V. cholerae GbpA expression is induced by mucin and negatively regulated by cyclic di-GMP and quorum sensing

  • These differences suggest adaptations to distinct ecological niches and host environments

• Pathogenesis roles:

  • V. vulnificus ObgE contributes to virulence through both persistence mechanisms and gut microbiota modulation

  • V. cholerae GbpA functions as a colonization factor binding to GlcNAc residues of mucin

  • E. coli ObgE primarily contributes to antibiotic tolerance and stress survival

These comparative insights highlight both the conserved ancestral functions and the species-specific adaptations of ObgE proteins across bacterial pathogens .

What are the unresolved contradictions in our understanding of ObgE function in bacterial persistence and virulence?

Several significant contradictions and knowledge gaps exist in our understanding of ObgE's role in bacterial persistence and virulence:

• ppGpp dependency contradiction:

  • ObgE-induced protein aggregation occurs normally in ΔrelA ΔspoT strains lacking ppGpp

  • Yet ObgE-induced persistence is significantly reduced in these same strains

  • This suggests a bifurcation in the pathways connecting ObgE, aggregation, and persistence

  • Unresolved question: How does ppGpp influence ObgE-mediated persistence independent of aggregation?

• Temporal dynamics paradox:

  • ObgE overexpression accelerates both entry into and exit from the VBNC state

  • This contradicts the simple model of ObgE as a persistence/VBNC inducer

  • Suggests complex temporal regulation or potential negative feedback mechanisms

  • Unresolved question: What determines the switch back to culturability in ObgE-overexpressing cells?

• Translation-related protein aggregation:

  • ObgE specifically increases aggregation of translation-related proteins (COG category J)

  • Yet ObgE binds to ribosomes and potentially stabilizes them against dissociation

  • These seemingly contradictory functions suggest context-dependent activities

  • Unresolved question: How does ObgE distinguish between ribosomal proteins to be aggregated versus protected?

• Species-specific functions:

  • V. vulnificus ObgE appears to have both intracellular (ribosome regulation) and extracellular (microbiota modulation) functions

  • How these diverse functions are coordinated or compartmentalized remains unclear

  • The evolutionary path to these dual functions is not well understood

  • Unresolved question: Are these truly distinct functions or mechanistically linked processes?

• Contradictory regulation patterns:

  • V. vulnificus gbpA is both induced by oxidative stress (via IscR) and repressed by quorum sensing (via SmcR)

  • This suggests complex integration of multiple environmental signals

  • The hierarchy of these regulatory inputs remains unclear

  • Unresolved question: How are these opposing regulatory signals integrated under different infection conditions?

Resolving these contradictions will require integrative approaches combining structural biology, systems-level analysis, and in vivo infection models .

What emerging technologies could advance our understanding of ObgE's role in V. vulnificus pathogenesis?

Emerging technologies with potential to transform ObgE research include:

• Cryo-electron tomography:

  • Visualize ObgE-ribosome complexes in their native cellular context

  • Map spatial distribution of protein aggregates in persister/VBNC cells

  • Correlate aggregate formation with cellular ultrastructure changes

• Single-cell transcriptomics and proteomics:

  • Profile gene expression in ObgE-high versus ObgE-low bacterial subpopulations

  • Identify cell-to-cell variability in persistence development

  • Correlate ObgE levels with global expression patterns at single-cell resolution

• Microfluidics and live-cell imaging:

  • Track individual bacterial cells through persistence/VBNC transitions in real-time

  • Correlate ObgE dynamics with phenotypic changes using fluorescent reporters

  • Apply controlled stress gradients to determine response thresholds

• CRISPR-based technologies:

  • Apply CRISPRi for tunable knockdown of ObgE expression

  • Use CRISPR-based screens to identify genetic interactors of ObgE

  • Deploy CRISPR-based imaging to visualize ObgE localization dynamics

• In situ structural techniques:

  • Apply in-cell NMR to probe ObgE conformational changes

  • Use protein-protein interaction reporters (FRET, BiFC) to map interaction networks

  • Deploy cross-linking mass spectrometry to capture transient interactions

• Intestinal organoid models:

  • Study V. vulnificus-host interactions in complex 3D tissue models

  • Examine ObgE-dependent effects on epithelial barrier function

  • Incorporate gut microbiome components to assess microbial community dynamics

These technologies will enable researchers to dissect the complex multifunctionality of ObgE in V. vulnificus pathogenesis with unprecedented resolution and precision .

How might targeting ObgE function be exploited for novel antimicrobial strategies against V. vulnificus?

Targeting ObgE functions offers several promising antimicrobial approaches:

• GTPase inhibition strategies:

  • Develop small molecule inhibitors targeting the GTPase domain of ObgE

  • Screen for compounds that lock ObgE in either GTP- or GDP-bound states

  • Design nucleotide analogs that compete with GTP binding

• Ribosome interaction disruption:

  • Target the interface between ObgE's N-terminal domain and the 50S ribosomal subunit

  • Develop peptide mimetics based on the tRNA-like structure of ObgE NTD

  • Screen for compounds that prevent ObgE-mediated ribosome dissociation

• Anti-persistence approaches:

  • Develop compounds that prevent ObgE-induced protein aggregation

  • Target the transition to persistence/VBNC states to maintain bacterial susceptibility to conventional antibiotics

  • Design combination therapies that simultaneously target active and persistent cells

• cFP-ObgE interaction targeting:

  • Develop competitive inhibitors of the cFP-ObgE interaction

  • Design cFP analogs that bind ObgE but don't trigger downstream effects

  • Create antibody-based approaches that sequester cFP

• Regulatory circuit intervention:

  • Target the IscR-CRP-SmcR regulatory network controlling ObgE expression

  • Modulate the oxidative stress response to prevent ObgE induction

  • Develop artificial gene regulatory systems to control ObgE expression

• Microbiome-protective strategies:

  • Develop probiotics expressing modified ObgE proteins that bind cFP but resist effects

  • Design microbiome-targeted approaches to maintain Bacteroides populations

  • Create engineered strains with enhanced resistance to V. vulnificus antagonism