Recombinant Vibrio vulnificus tRNA-modifying protein ygfZ (VV2842)

What is the primary function of the YgfZ protein in Vibrio vulnificus?

YgfZ is a folate-dependent protein that participates in the synthesis and repair of iron-sulfur (Fe-S) clusters in bacterial systems. In Vibrio vulnificus, as in other bacteria like E. coli, YgfZ appears to play a critical role in maintaining the functionality of Fe-S enzymes such as MiaB. The protein belongs to a family of enzymes that use folate to capture formaldehyde units, suggesting its involvement in one-carbon metabolism. Research indicates that YgfZ counteracts damage to Fe-S cluster proteins that may occur during normal cellular processes or under stress conditions .

How does YgfZ deletion affect V. vulnificus virulence and survival?

Deletion of the ygfZ gene results in multiple phenotypic changes that impact bacterial fitness. Experimental data show that ygfZ ablation reduces bacterial growth rates, increases sensitivity to oxidative stress, and significantly lowers the activities of MiaB and other Fe-S enzymes . These effects may indirectly influence virulence by compromising bacterial metabolism and stress resistance. The relationship between YgfZ function and V. vulnificus pathogenicity is particularly relevant given that this pathogen is associated with high mortality rates worldwide and its geographical range is expanding due to climate warming .

What is the relationship between YgfZ and tRNA modification in bacterial systems?

YgfZ appears to have an interconnected relationship with tRNA modification pathways, particularly those involving the MnmEG complex. Research demonstrates that deleting mnmE (a component of the MnmEG complex) can suppress the growth phenotype observed in ygfZ deletant cells. Furthermore, deletion of mnmE substantially restores MiaB activity in ygfZ-deficient cells . MiaB is responsible for the methylthiolation of i⁶A to ms²i⁶A in tRNA, and the ms²i⁶A/i⁶A ratio serves as a measure of MiaB activity. This relationship suggests that YgfZ functions in opposition to certain activities of the MnmEG complex, potentially correcting errors in tRNA modification or protecting tRNA-modifying enzymes from damage.

What are the optimal methods for expressing and purifying recombinant VV2842 (YgfZ) from V. vulnificus?

Recombinant expression of V. vulnificus YgfZ (VV2842) can be achieved using established bacterial expression systems. A methodological approach involves:

• Gene cloning: Amplify the ygfZ gene from V. vulnificus genomic DNA using specific primers designed to include appropriate restriction sites, followed by insertion into a suitable expression vector (e.g., pET series for E. coli expression).

• Expression optimization: Transform the construct into an expression host such as E. coli BL21(DE3) and optimize expression conditions:

  • Test various induction temperatures (16-37°C)

  • Vary IPTG concentrations (0.1-1.0 mM)

  • Adjust induction time (3-18 hours)

  • Consider supplementing with folate or iron, as YgfZ is a folate-dependent protein involved in Fe-S cluster formation

• Purification strategy: Implement a multi-step purification process:

  • Initial capture using affinity chromatography (His-tag purification)

  • Intermediate purification via ion exchange chromatography

  • Polishing step using size exclusion chromatography

  • Maintain reducing conditions throughout purification to protect Fe-S clusters

• Quality assessment: Evaluate protein purity by SDS-PAGE and confirm identity through mass spectrometry and Western blotting.

Ensuring anaerobic conditions during purification may be beneficial for maintaining protein stability and activity, particularly due to YgfZ's role in Fe-S cluster biochemistry.

How can researchers accurately measure YgfZ activity in vitro and in vivo?

Measuring YgfZ activity requires multiple complementary approaches due to its indirect effects on Fe-S enzymes:

In vitro activity assays:

• Fe-S cluster transfer assay: Monitor the transfer or repair of Fe-S clusters to recipient apo-proteins using UV-visible spectroscopy to track characteristic absorption peaks of Fe-S clusters.

• MiaB activity assay: Measure the conversion of i⁶A to ms²i⁶A in tRNA substrates using HPLC or LC-MS methods. The ms²i⁶A/i⁶A ratio provides a quantitative measure of MiaB activity, which is indirectly influenced by YgfZ function .

• Folate-dependent formaldehyde capture: Assess the ability of purified YgfZ to bind folate and capture formaldehyde units using isotope-labeled formaldehyde ([¹³C]formaldehyde) and NMR spectroscopy .

In vivo assessment methods:

• Genetic complementation: Measure the restoration of growth rate and stress resistance in ygfZ deletion strains after complementation with recombinant YgfZ.

• Fe-S enzyme activity profiles: Compare activities of multiple Fe-S enzymes in wild-type, ΔygfZ, and complemented strains.

• Oxidative stress resistance: Evaluate bacterial survival under oxidative stress conditions using growth curve analysis or viability assays.

A comprehensive YgfZ activity assessment would combine these methods to develop a complete profile of its biochemical function.

What approaches can be used to study the interaction between YgfZ and the MnmEG complex in V. vulnificus?

Investigating the functional relationship between YgfZ and MnmEG requires multiple biochemical and genetic approaches:

Protein-protein interaction studies:

• Co-immunoprecipitation: Use antibodies against YgfZ to precipitate potential protein complexes from bacterial lysates, followed by Western blotting to detect MnmE and MnmG.

• Bacterial two-hybrid assays: Construct fusion proteins to assess direct interactions between YgfZ and MnmE or MnmG components.

• Proximity labeling: Utilize BioID or APEX2 fusion proteins to identify proteins in close proximity to YgfZ in vivo.

Genetic interaction analysis:

• Epistasis studies: Generate single and double knockouts (ΔygfZ, ΔmnmE, ΔmnmG, ΔygfZ ΔmnmE, ΔygfZ ΔmnmG) and measure phenotypic effects on growth and Fe-S enzyme activities .

• Overexpression studies: Examine the effect of overexpressing MnmEG in a ΔygfZ background and vice versa to understand antagonistic or synergistic relationships.

• Folate dependency: Assess the requirement for folate in the YgfZ-MnmEG relationship by creating folE deletion strains (ΔfolE) that eliminate all folates, as has been demonstrated in prior research .

Biochemical analysis:

• tRNA modification analysis: Compare tRNA modification profiles (particularly ms²i⁶A formation) in various genetic backgrounds using LC-MS/MS.

• Formaldehyde transfer tracking: Use isotope-labeled formaldehyde to track transfer events between folate, MnmEG, and potential protein targets.

How does the molecular mechanism of YgfZ differ between V. vulnificus and other bacterial species?

The YgfZ protein belongs to a conserved family found across many bacterial species, but its function may have species-specific adaptations. Comparative analysis reveals:

Sequence and structural conservation:
While the core domain structure of YgfZ is preserved across species, V. vulnificus YgfZ shows specific amino acid variations compared to E. coli and other model organisms. These variations may affect:

• Folate binding affinity

• Interaction with partner proteins

• Substrate specificity

• Resistance to environmental stressors

Functional conservation across species:
Research indicates that YgfZ homologs generally participate in Fe-S cluster metabolism, but their detailed mechanisms may vary. Notably, YgfZ occurs in many genomes that lack MnmEG (e.g., Actinobacteria), suggesting that in V. vulnificus and other species, YgfZ likely has additional functions beyond counteracting MnmEG-related damage .

Species-specific adaptations:
V. vulnificus, as a marine pathogen facing unique environmental challenges including salinity fluctuations and host defense mechanisms, may have evolved specialized features in its YgfZ protein. Research suggests that in the context of V. vulnificus virulence, YgfZ may play roles in:

• Adaptation to oxidative stress during host infection

• Maintenance of pathogen metabolism under iron-limited conditions

• Regulation of virulence factor expression through effects on Fe-S proteins

A comprehensive comparative genomics and structural biology approach would be required to fully characterize these species-specific differences.

What is the relationship between YgfZ function and antibiotic resistance in V. vulnificus?

The connection between YgfZ and antibiotic resistance represents a complex relationship that may involve both direct and indirect mechanisms:

Potential direct connections:
While YgfZ itself is not an antibiotic resistance determinant, its role in maintaining Fe-S cluster proteins may influence specific antibiotic resistance mechanisms. Fe-S cluster proteins are involved in various cellular processes including DNA repair and metabolism, which can impact antibiotic susceptibility.

Indirect influence through stress responses:
YgfZ deletion increases sensitivity to oxidative stress , which is a common secondary effect of many antibiotics. This suggests that YgfZ might contribute to intrinsic antibiotic tolerance through general stress response pathways.

Relationship to known resistance patterns in V. vulnificus:
V. vulnificus clinical isolates show varying patterns of antibiotic resistance genes (ARGs). Common ARGs detected in V. vulnificus include PBP3, parE, adeF, varG, and CRP, which confer resistance to beta-lactams, fluoroquinolones, and carbapenems . Research could explore whether YgfZ function affects the expression or activity of these resistance determinants.

The table below summarizes the potential intersection between YgfZ function and antibiotic resistance mechanisms in V. vulnificus:

How might environmental factors affect YgfZ expression and function in V. vulnificus?

Environmental factors significantly influence V. vulnificus biology, and YgfZ expression and function are likely responsive to these conditions:

Temperature effects:
Climate warming is expanding the geographical range of V. vulnificus . Research suggests that temperature shifts may influence YgfZ expression and activity through:

• Heat-shock response pathways affecting ygfZ transcription

• Temperature-dependent changes in protein folding and stability

• Altered demand for Fe-S cluster repair at elevated temperatures

Iron availability:
As YgfZ functions in Fe-S cluster metabolism, iron availability may regulate its expression and activity:

• Iron limitation might increase YgfZ expression to maximize efficiency of Fe-S cluster formation

• Iron overload could generate oxidative stress, increasing the need for YgfZ function

• Research approaches should include growth in iron-limited media and iron chelation experiments

Oxidative stress conditions:
Marine environments and host immune responses expose V. vulnificus to varying levels of oxidative stress:

• Hydrogen peroxide, superoxide, and other ROS may induce ygfZ expression

• YgfZ activity may be modulated by the redox state of the cell

• Experimental designs should incorporate oxidative stress challenges with measurement of ygfZ expression and YgfZ activity

Methodological approaches to study environmental effects:

• Transcriptional analysis: qRT-PCR and RNA-seq to measure ygfZ expression under different environmental conditions

• Protein level assessment: Western blotting with anti-YgfZ antibodies to quantify protein abundance

• Activity measurements: Fe-S enzyme activity assays (as proxies for YgfZ function) under varying environmental conditions

• Reporter constructs: ygfZ promoter fusions to reporter genes for real-time monitoring of expression

How does YgfZ contribute to the pathogenesis of V. vulnificus infections?

YgfZ appears to play an important role in V. vulnificus pathogenesis through multiple mechanisms:

Maintenance of essential metabolic functions:
By ensuring proper Fe-S cluster formation and repair, YgfZ supports core metabolic processes required for bacterial growth and virulence. Fe-S proteins participate in:

• Energy generation

• Amino acid biosynthesis

• Nucleotide metabolism

• Cofactor synthesis

Oxidative stress resistance during infection:
YgfZ deletion increases sensitivity to oxidative stress , which bacteria encounter during host infection through:

• Phagocyte-generated reactive oxygen species

• Host-induced nutritional immunity (iron restriction)

• Environmental stressors in the infection site

Potential effects on virulence factor expression:
V. vulnificus virulence depends on numerous factors including:

• Capsular polysaccharide (CPS) genes such as cpsA/B, kpsF, cysC, and others

• Hemolysin/cytolysin genes including cylA, hlyD, hlyB, and hlyA (vvh)

• MARTX gene clusters rtxABCD

• Metalloprotease strcE

YgfZ function may indirectly regulate these virulence factors through effects on Fe-S proteins involved in their expression or activity.

Connection to iron acquisition systems:
V. vulnificus pathogenesis relies on efficient iron acquisition, often through specialized systems containing Fe-S proteins. YgfZ may contribute to the functioning of:

• Heme uptake systems (chuX/W/V/T, shuU/A)

• Siderophore-based iron acquisition (enterobactin fepC, entA/F/E/D, ligiobactin lbtC)

What strategies can be employed to target YgfZ function as a potential therapeutic approach?

Considering YgfZ's role in V. vulnificus metabolism and virulence, several therapeutic strategies could be explored:

Direct inhibition approaches:

• Small molecule inhibitors: Structure-based design of compounds that interfere with:

  • Folate binding to YgfZ

  • YgfZ interaction with damaged Fe-S proteins

  • YgfZ conformational changes required for activity

• Peptide-based inhibitors: Design of peptides that mimic YgfZ interaction partners to competitively inhibit its function.

• Allosteric modulation: Identification of allosteric sites on YgfZ that could be targeted to alter protein function without directly competing with substrates.

Methodological evaluation of inhibitors:

Combination therapy potential:
Targeting YgfZ could sensitize V. vulnificus to:

• Conventional antibiotics, particularly those that generate oxidative stress

• Host immune defenses

• Environmental stressors

Considerations for therapeutic development:

• Selectivity for bacterial YgfZ over human homologs or related proteins

• Bioavailability in infection sites (particularly wounds and bloodstream)

• Stability under conditions found in infection environments

• Potential for resistance development through compensatory mutations

How does the interaction between YgfZ and MnmEG affect V. vulnificus virulence during host infection?

The relationship between YgfZ and MnmEG represents a novel aspect of bacterial physiology that may impact virulence:

Antagonistic relationship impact on virulence:
Research demonstrates that YgfZ counteracts the potentially harmful activities of MnmEG . During infection, this relationship may influence virulence through:

• Maintenance of optimal tRNA modification patterns required for efficient translation of virulence factors

• Protection of Fe-S cluster proteins from damage during stress conditions encountered in the host

• Balancing of folate utilization between one-carbon metabolism and tRNA modification

Molecular mechanism during infection:
The current understanding suggests that MnmEG, which normally transfers formaldehyde units from folate to tRNA, may occasionally mistakenly transfer these units to proteins, potentially damaging them. YgfZ appears to counter this damage . In the infection context:

• Host-induced stress might increase the frequency of MnmEG errors

• The balance between YgfZ and MnmEG activities could determine bacterial fitness

• Folate availability in infection sites may modulate this relationship

Experimental approaches to study this interaction during infection:

• In vivo transcriptomics: Compare expression of ygfZ and mnmEG genes during different stages of infection

• Genetic manipulation: Test virulence of ΔygfZ, ΔmnmE, and double mutant strains in animal models

• Proteomics: Identify proteins damaged in ΔygfZ strains during infection that might represent MnmEG targets

• Metabolomics: Analyze folate metabolite profiles during infection in different genetic backgrounds

Research suggests that elucidating this relationship could provide insights into bacterial adaptation during infection and potentially identify new therapeutic targets.

What are the key technical challenges in studying YgfZ function and how can they be overcome?

Research on YgfZ presents several technical challenges that require specialized approaches:

Challenges in protein purification and stability:

• Fe-S cluster preservation: YgfZ's role in Fe-S cluster metabolism means that purified protein may require special handling to maintain activity.

  • Solution: Perform all purification steps under anaerobic conditions using glove boxes or specialized equipment.

  • Alternative: Reconstitute Fe-S clusters in vitro after purification under standard conditions.

• Folate binding: As a folate-dependent protein, YgfZ may lose activity if folate cofactors dissociate during purification.

  • Solution: Supplement buffers with folate or folate derivatives during purification.

  • Validation: Verify folate binding by spectroscopic methods or isothermal titration calorimetry.

Challenges in functional assays:

• Indirect nature of activity: YgfZ effects are often measured indirectly through Fe-S enzyme activities.

  • Solution: Develop direct assays for YgfZ function, such as measuring formaldehyde transfer from proteins to folate.

  • Complementary approach: Use multiple readouts (growth, stress resistance, multiple Fe-S enzyme activities) to build a comprehensive activity profile.

• Complex interactions with MnmEG: The antagonistic relationship with MnmEG complicates interpretation of results.

  • Solution: Design experiments with appropriate genetic controls (ΔmnmE, ΔmnmG, double mutants) to isolate YgfZ-specific effects.

  • Advanced approach: Reconstitute the YgfZ-MnmEG system in vitro with purified components to directly study their interactions.

Challenges in in vivo studies:

• Pleiotropic effects of ygfZ deletion: Multiple phenotypes make it difficult to isolate specific functions.

  • Solution: Use point mutations in YgfZ that affect specific activities rather than complete gene deletion.

  • Alternative: Create conditional expression systems to study acute effects of YgfZ depletion.

• Variability in V. vulnificus strains: Different clinical isolates show varying genetic backgrounds.

  • Solution: Study YgfZ function across multiple clinical isolates to distinguish strain-specific from conserved functions.

  • Standardization: Establish reference strains for YgfZ research to facilitate comparison between studies.

How can researchers resolve contradictory findings regarding YgfZ function in different experimental systems?

Scientific literature may contain apparently contradictory findings about YgfZ function due to differences in experimental approaches:

Systematic approach to resolving contradictions:

• Standardize experimental conditions:

  • Define standard growth media, temperature, and aeration conditions

  • Establish reference strains with verified genotypes

  • Develop standardized assay protocols for key YgfZ-related phenotypes

• Comprehensive genetic analysis:

  • Create a standard set of deletion strains (ΔygfZ, ΔmnmE, ΔmnmG, ΔfolE, etc.)

  • Verify deletions by both PCR and functional assays

  • Ensure complementation studies use physiological expression levels

• Address methodological differences:

  Experimental Variable	Potential Impact on Results	Standardization Approach
  Growth phase	YgfZ function may vary with growth phase	Compare results at multiple defined growth points
  Oxygen availability	Affects Fe-S cluster stability	Control oxygen levels precisely in all experiments
  Media composition	Folate availability affects YgfZ function	Use defined media with known folate content
  Strain background	Genetic modifiers may exist	Use isogenic strains for all comparisons

• Collaborative cross-validation:

  • Establish multi-laboratory studies with shared materials

  • Perform blind validation of key findings across different research groups

  • Create a repository of standardized reagents (plasmids, strains, antibodies)

Resolution of specific contradictions:
When contradictory findings are reported, a systematic approach involves:

• Identifying the exact experimental differences between studies

• Replicating both conditions in parallel

• Testing intermediate conditions to identify transition points

• Determining whether discrepancies represent different aspects of YgfZ function rather than true contradictions

What emerging technologies could advance our understanding of YgfZ biology?

Several cutting-edge technologies show promise for elucidating YgfZ function:

Advanced structural biology approaches:

• Cryo-electron microscopy (Cryo-EM): Determine high-resolution structures of YgfZ in complex with interaction partners, particularly during formaldehyde transfer events.

• Hydrogen-deuterium exchange mass spectrometry (HDX-MS): Map conformational changes in YgfZ upon folate binding or interaction with partner proteins.

• Integrative structural biology: Combine X-ray crystallography, NMR, and computational methods to generate complete models of YgfZ functional states.

Single-molecule techniques:

• Single-molecule FRET: Monitor real-time conformational changes in YgfZ during its functional cycle.

• Optical tweezers or atomic force microscopy: Measure binding forces between YgfZ and its interaction partners.

• Single-cell microscopy: Track YgfZ localization and dynamics in living cells using fluorescent protein fusions.

Systems biology approaches:

• Multi-omics integration: Combine transcriptomics, proteomics, and metabolomics data from ΔygfZ strains to build comprehensive models of YgfZ function.

• Genome-wide interaction screens: Use CRISPRi or transposon libraries to identify genetic interactions with ygfZ.

• Metabolic flux analysis: Trace the flow of metabolites through pathways affected by YgfZ using stable isotope labeling.

Chemical biology methods:

• Activity-based protein profiling: Develop probes that specifically label active YgfZ to quantify functional protein.

• Photo-crosslinking: Create photo-activatable folate analogs to capture transient YgfZ interactions.

• PROTAC/degrader approach: Generate molecules that selectively degrade YgfZ to study acute effects of protein loss.

Implementation of these technologies could resolve long-standing questions about YgfZ function and potentially reveal new aspects of its biology that are currently inaccessible with conventional methods.