Recombinant Desulfovibrio vulgaris Translation initiation factor IF-2 (infB), partial

Basic Research Questions

• What is the role of translation initiation factor IF-2 in Desulfovibrio vulgaris?

  Translation initiation factor IF-2 plays a critical role in bacterial protein synthesis by facilitating the binding of initiator tRNA (fMet-tRNAfMet) to the 30S ribosomal subunit. In sulfate-reducing bacteria like D. vulgaris, this process operates under anaerobic conditions. Based on studies of related translation factors in D. vulgaris, the protein likely functions within the complex energy metabolism network that enables this organism to thrive in anaerobic environments. The genome sequencing of D. vulgaris Hildenborough has revealed important insights into the organism's translational machinery , suggesting that IF-2 contributes to the bacterium's adaptation to specific environmental niches.

• How do expression systems for recombinant D. vulgaris proteins compare?

  The expression of recombinant D. vulgaris proteins is typically conducted in Escherichia coli systems, though specific considerations are needed due to D. vulgaris' anaerobic nature. Baculovirus expression systems have been successfully used for recombinant D. vulgaris translation factors, as demonstrated with translation initiation factor IF-1 2 . For optimal results, researchers should consider:

  Expression System	Advantages	Disadvantages	Yield
  E. coli	Cost-effective, rapid growth	Potential inclusion body formation	Variable (strain-dependent)
  Baculovirus	Proper folding, post-translational modifications	Higher cost, longer timeframe	Typically higher purity
  D. desulfuricans G201	Native-like conditions	Technical complexity	Demonstrated for cytochrome subunits

  The choice depends on the specific experimental requirements and the structural complexity of the target protein.

• What are the recommended storage conditions for recombinant D. vulgaris proteins?

  Based on documented protocols for D. vulgaris recombinant proteins, the following storage conditions are recommended:

  • Liquid form: 6 months shelf life at -20°C/-80°C

  • Lyophilized form: 12 months shelf life at -20°C/-80°C

  • Glycerol addition: 5-50% (final concentration) for long-term storage

  • Working aliquots: Store at 4°C for up to one week

  Repeated freezing and thawing should be avoided to maintain protein integrity . For translation factors specifically, storage in buffer containing reducing agents may help preserve activity.

Advanced Research Applications

• How can genetic manipulation systems be applied to study IF-2 function in D. vulgaris?

  D. vulgaris has several established genetic tools that can be applied to study IF-2 function:

  • Markerless genetic exchange system: This allows for sequential gene deletions without accumulating antibiotic resistance genes . This approach would be ideal for creating precise modifications in the infB gene without disrupting nearby genes.

  • Marker exchange deletion method: While limited by available selectable markers, this system has been used successfully for targeted gene deletions in D. vulgaris .

  • Transposon mutagenesis: Large-scale genetic characterization using randomly barcoded transposon mutant libraries has been established for D. vulgaris Hildenborough, enabling systematic phenotypic analysis .

  When studying essential genes like infB, conditional expression systems or partial deletions would be necessary to maintain cell viability while investigating protein function.

• What methodologies are most effective for purifying recombinant D. vulgaris translation factors?

  Based on successful purification of other D. vulgaris recombinant proteins, the following protocol is recommended:

  1. Initial preparation: Centrifuge vial briefly before opening to bring contents to the bottom

  2. Reconstitution: Use deionized sterile water to a concentration of 0.1-1.0 mg/mL

  3. Glycerol addition: Add glycerol to 5-50% final concentration for stability

  4. Chromatography: Employ affinity chromatography based on the specific tag used

  5. Quality control: Verify purity via SDS-PAGE (aim for >85% purity)

  For iron-containing D. vulgaris proteins, special reconstitution protocols may be needed, as demonstrated with rubrerythrin, where iron was incorporated in vitro by dissolving the protein in 3 M guanidinium chloride, adding Fe(II) anaerobically, and diluting the denaturant .

• How does D. vulgaris adapt its translation machinery under stress conditions?

  D. vulgaris shows specific adaptations in gene expression under stress conditions that likely affect translation initiation:

  • Metal toxicity response: Exposure to Cu(II) and Hg(II) causes upregulation of ATP-dependent mechanisms, with 4-6 fold increases in expression for certain proteins under Hg(II) exposure and 1.4-3 fold increases with Cu(II) . Similar mechanisms may regulate IF-2 under stress.

  • Oxidative stress: D. vulgaris employs specialized mechanisms to handle reactive oxygen species, including bacterioferritin that uses H₂O₂ as a co-substrate and protects DNA from hydroxyl radical damage . Translation factors may be regulated in coordination with these stress responses.

  • Metabolic adaptation: The bacterium's complex anaerobic respiration pathways are likely coordinated with translational regulation during resource limitation, potentially involving IF-2 activity modulation.

  Transcriptomic and proteomic analyses under various stress conditions would provide insights into how translation initiation factors respond to environmental challenges.

Experimental Design and Methodology

• What are the key considerations for designing site-directed mutagenesis experiments with D. vulgaris IF-2?

  When designing mutagenesis experiments for D. vulgaris IF-2, researchers should consider:

  1. Target domain selection: Focus on conserved regions identified through sequence alignment with well-characterized bacterial IF-2 proteins

  2. Genetic system selection: Utilize the markerless genetic exchange system developed for D. vulgaris to generate clean mutations without antibiotic markers

  3. Functional assays: Develop assays specific to each domain (GTP binding, tRNA interaction, ribosome binding)

  4. Growth condition variations: Test mutant performance under different electron donors/acceptors, as D. vulgaris can utilize various substrates including hydrogen and formate

  5. Complementation studies: Include restoration of wild-type phenotype through complementation with plasmid-borne wild-type gene to confirm phenotype specificity

  This approach enables detailed structure-function analysis while maintaining physiological relevance.

• How can researchers investigate the interaction between D. vulgaris IF-2 and the bacterial ribosome?

  A multi-method approach is recommended:

  1. Biochemical methods:

     • Gradient ultracentrifugation to isolate ribosome-IF-2 complexes

     • Filter binding assays to measure binding kinetics

     • Chemical cross-linking followed by mass spectrometry

  2. Structural approaches:

     • Cryo-electron microscopy of ribosome-IF-2 complexes

     • Hydrogen-deuterium exchange mass spectrometry

     • NMR spectroscopy for mapping interaction interfaces

  3. Genetic approaches:

     • Suppressor mutation analysis

     • Construction of chimeric IF-2 proteins with domains from other bacteria

  4. Computational methods:

     • Homology modeling based on known bacterial IF-2 structures

     • Molecular docking simulations

     • Molecular dynamics studies of the complex

  Each method provides complementary information about structural and functional aspects of the interaction.

• What techniques are appropriate for analyzing the GTP hydrolysis activity of recombinant D. vulgaris IF-2?

  Several complementary methods can be employed:

  1. Colorimetric assays:

     • Malachite green assay for released inorganic phosphate

     • EnzChek phosphate assay for continuous monitoring

  2. Chromatographic methods:

     • HPLC separation of GTP and GDP

     • Thin-layer chromatography with radiolabeled GTP

  3. Spectroscopic approaches:

     • Fluorescence-based assays using FRET or fluorescent GTP analogs

     • Circular dichroism to monitor conformational changes upon GTP binding/hydrolysis

  4. Stopped-flow kinetics:

     • Rapid kinetic measurements of GTP hydrolysis and conformational changes

  When conducting these assays, consider the anaerobic nature of D. vulgaris and maintain appropriate reducing conditions to ensure physiological relevance.

• How can researchers differentiate between direct and indirect effects when studying IF-2 function in D. vulgaris?

  To establish direct causality:

  1. In vitro reconstitution: Assemble purified components (ribosomes, tRNAs, initiation factors) to demonstrate direct effects

  2. Domain-specific mutations: Create targeted mutations affecting specific functions rather than eliminating the entire protein

  3. Time-resolved experiments: Monitor the sequence of events following perturbation of IF-2 function

  4. Complementation analysis: Use both homologous and heterologous complementation with wild-type and mutant versions

  5. Interaction-specific inhibitors: Employ small molecules or aptamers that specifically disrupt IF-2 interactions

  The genetic tools developed for D. vulgaris, including the markerless deletion system and transposon mutagenesis approaches , provide methods for these sophisticated genetic manipulations.

Data Analysis and Advanced Applications

• What approaches can be used to study the evolutionary conservation of IF-2 across Desulfovibrio species?

  Several comparative genomic and evolutionary analysis methods are relevant:

  1. Multiple sequence alignment: Compare IF-2 sequences across diverse Desulfovibrio species and other sulfate-reducing bacteria

  2. Phylogenetic analysis: Construct trees to understand the evolutionary history of IF-2 within Desulfovibrio

  3. Selection pressure analysis: Calculate dN/dS ratios to identify conserved functional domains under purifying selection

  4. Genome context analysis: Examine the genomic neighborhood of infB genes across species to identify conserved gene clusters

  5. Structural comparison: Use homology modeling to predict structural conservation across species

  These approaches can reveal unique adaptations in the translation machinery of sulfate-reducing bacteria compared to other bacterial groups, similar to the way biotin synthesis pathways have been found to be conserved specifically in anaerobic bacteria including Desulfovibrio .

• How can isotope labeling of recombinant D. vulgaris IF-2 be optimized for structural studies?

  Effective isotope labeling strategies include:

  1. Growth media optimization:

     • Minimal media supplemented with 15N-ammonium sulfate and/or 13C-glucose

     • For anaerobic D. vulgaris proteins, maintain reducing conditions throughout

  2. Expression system selection:

     • E. coli BL21(DE3) with rare codon supplementation

     • Consider adaptations to D. vulgaris codon usage

  3. Labeling schemes:

     • Uniform labeling for complete structure determination

     • Selective amino acid labeling for specific interaction studies

     • Segmental labeling for large proteins

  4. Purification considerations:

     • Maintain anaerobic conditions during purification

     • Include reducing agents in buffers

     • Verify proper folding through activity assays

  These approaches can facilitate structural studies using NMR spectroscopy, enabling detailed analysis of IF-2 conformational changes during its functional cycle.

• What are the implications of D. vulgaris IF-2 research for understanding bacterial adaptation to extreme environments?

  Research on D. vulgaris IF-2 contributes to understanding bacterial adaptation through:

  1. Environmental adaptation: As an anaerobic, sulfate-reducing bacterium, D. vulgaris has evolved unique metabolic and regulatory systems. Translation initiation factors likely contain adaptations for function under these conditions.

  2. Stress response mechanisms: D. vulgaris shows specific adaptations to metal toxicity and oxidative stress , which may involve coordinated regulation of translation.

  3. Evolutionary insights: Comparative analysis of translation machinery across diverse Desulfovibrio species can reveal convergent or divergent evolutionary adaptations.

  4. Biotechnological applications: Understanding these adaptations may inform the development of recombinant protein expression systems optimized for anaerobic conditions.

  5. Pathogenesis relevance: D. vulgaris has been implicated in gut inflammation and colitis , and understanding its translation machinery may provide insights into its pathogenic potential.

  This research connects to broader ecological and evolutionary questions about how fundamental cellular processes adapt to specialized environmental niches.

• How can chemotaxis and motility studies in D. vulgaris inform research on translation initiation factors?

  While seemingly unrelated, chemotaxis/motility and translation initiation are connected in interesting ways:

  1. Coordinated regulation: Both systems respond to environmental cues and must be coordinately regulated during adaptation

  2. Energy allocation: D. vulgaris must balance energy expenditure between motility and protein synthesis, especially under nutrient limitation

  3. Experimental approaches: Methods developed for studying the CheA3 kinase and flagellar systems in D. vulgaris provide valuable approaches for investigating regulatory networks

  4. Stress responses: Understanding how translation factors and motility systems respond to environmental stressors offers insights into integrated cellular responses

  5. Genetic tools: The marker exchange and markerless deletion systems developed for motility studies can be applied to create precise modifications in translation factor genes

  This cross-disciplinary perspective enriches the understanding of how D. vulgaris coordinates multiple cellular systems during environmental adaptation.

• What role might D. vulgaris IF-2 play in the bacterium's response to iron limitation and iron-mediated processes?

  D. vulgaris has sophisticated iron-related processes that may involve translation regulation:

  1. Iron storage and utilization: D. vulgaris contains specialized iron storage proteins like bacterioferritin that uses H₂O₂ as a co-substrate , suggesting complex iron regulation

  2. Iron-mediated corrosion: D. vulgaris mediates iron corrosion primarily through a hydrogen-dependent mechanism , which may require coordinated regulation of translation

  3. Translational response: Iron limitation likely triggers translational reprogramming involving IF-2 to prioritize essential iron-containing proteins

  4. Experimental approaches: Studies could examine IF-2 activity and ribosome association under varying iron concentrations

  5. Comparative analysis: Comparing translation initiation in D. vulgaris with non-iron-dependent bacteria may reveal specific adaptations

  This research direction connects translation initiation to the bacterium's distinctive iron-dependent metabolism.