Recombinant Geobacter uraniireducens Translation initiation factor IF-2 (infB), partial

What is the basic function of Translation Initiation Factor IF-2 in Geobacter uraniireducens?

Translation Initiation Factor IF-2 in G. uraniireducens, like in other bacteria, plays an essential role in the translation initiation process by delivering the initiator formylmethionyl-tRNA (fMet-tRNA) to the ribosomal pre-initiation complex. It promotes ribosomal subunit association, recruitment, and binding of fMet-tRNA to the ribosomal P-site, and facilitates initiation dipeptide formation . The infB gene encodes this protein, which is critical for proper protein synthesis initiation in G. uraniireducens.

What growth conditions are optimal for expressing recombinant G. uraniireducens proteins?

For optimal growth and expression of recombinant proteins from G. uraniireducens, several factors need consideration. G. uraniireducens grows best at 30°C, with its growth rate varying depending on the electron acceptor used. The fastest growth occurs with fumarate as the electron acceptor (specific growth rate of 0.106 h⁻¹), while growth with sediment as the electron acceptor is considerably slower (0.013 h⁻¹ at 30°C) . For recombinant protein expression, it's important to note that G. uraniireducens cannot grow with Fe(III) citrate . The table below summarizes growth rates under various conditions:

These conditions should be considered when designing expression systems for recombinant G. uraniireducens IF-2 .

What are the recommended protocols for purifying recombinant G. uraniireducens IF-2 while maintaining its functional integrity?

When purifying recombinant G. uraniireducens IF-2, maintaining its functional integrity requires careful consideration of the protein's structural domains and activity requirements. Based on methodologies used for bacterial IF-2, a multi-step purification approach is recommended:

• Initial clarification of cell lysate through centrifugation and filtration

• Affinity chromatography using either:

  • Ni-NTA chromatography for His-tagged constructs

  • GTP-agarose affinity chromatography, exploiting IF-2's natural affinity for nucleotides

• Ion-exchange chromatography to separate different isoforms

• Size-exclusion chromatography as a final polishing step

To maintain functional integrity, all buffers should contain:

• 1-5 mM DTT or 2-mercaptoethanol to maintain thiol groups

• 5-10% glycerol to stabilize protein structure

• Low concentrations of Mg²⁺ (1-2 mM) which is essential for nucleotide binding

• GDP or GTP at low concentrations (0.1-0.5 mM) depending on which conformational state is desired

Throughout purification, it's essential to monitor the GTP-binding capacity and fMet-tRNA binding activity to ensure functional integrity is maintained.

How can one differentiate between the potential isoforms of G. uraniireducens IF-2 in experimental settings?

Bacterial IF-2 commonly exists in multiple isoforms. Based on studies of other bacterial species, the infB gene codes for at least two forms of translational initiation factor IF-2: IF-2 alpha and IF-2 beta, which differ at their N-terminus . To differentiate between these potential isoforms in G. uraniireducens:

• SDS-PAGE and Western Blotting: Use antibodies specific to the N-terminal region of the full-length IF-2 alpha, which would not recognize IF-2 beta. Alternatively, use antibodies against conserved C-terminal regions to detect both isoforms with different molecular weights.

• N-terminal Sequencing: Apply Edman degradation to determine the N-terminal amino acid sequences of purified isoforms, as was done to differentiate IF-2 isoforms in other bacteria .

• Mass Spectrometry: Utilize LC-MS/MS analysis of tryptic digests to identify peptides unique to each isoform, particularly focusing on the N-terminal differences.

• Genetic Approaches: Create fusion constructs between the proximal half of the infB gene and a reporter gene (such as lacZ), which would express fusion proteins corresponding to each isoform, allowing their detection and differentiation .

• In vitro Translation: Conduct in vitro dipeptide synthesis assays using templates containing the entire infB gene to confirm initiation at different start sites .

These methodologies, when applied systematically, can effectively differentiate between potential IF-2 isoforms in G. uraniireducens.

What are the optimal conditions for analyzing the GTPase activity of recombinant G. uraniireducens IF-2?

Analyzing the GTPase activity of recombinant G. uraniireducens IF-2 requires carefully optimized conditions to accurately measure intrinsic and ribosome-stimulated GTP hydrolysis. Based on studies of bacterial IF-2 GTPase activity:

• Buffer Composition:

  • HEPES or Tris buffer (50 mM, pH 7.5-7.8)

  • Monovalent ions: 70-100 mM NH₄Cl or KCl

  • Divalent ions: 7-10 mM MgCl₂ (critical for GTPase activity)

  • 1-2 mM DTT to maintain reduced state

• Temperature Considerations:

  • Primary assays should be conducted at 30°C (optimal growth temperature for G. uraniireducens)

  • Comparative assays at 15°C, 18°C, and 37°C to assess temperature dependence (as G. uraniireducens shows different growth characteristics at these temperatures)

• Assay Methods:

  • Colorimetric malachite green assay for phosphate release

  • HPLC-based nucleotide analysis

  • Radiometric assays using [γ-³²P]-GTP

• Kinetic Parameter Determination:

  • Measure activity across GTP concentrations (1-200 μM)

  • Determine Km and kcat values

  • Assess the effects of GDP competition

• Ribosome-Stimulated Activity:

  • Compare intrinsic GTPase activity with activity in presence of:

    • 70S ribosomes

    • 30S subunits

    • 50S subunits

    • Various combinations with initiation factors IF1 and IF3

• Data Analysis:

  • Initial velocity measurements

  • Michaelis-Menten kinetics

  • Lineweaver-Burk plots for inhibition studies

These conditions will allow for comprehensive characterization of G. uraniireducens IF-2 GTPase activity, particularly important given the evidence that GTP hydrolysis and structural dynamics may not be directly coupled to fMet-tRNA positioning .

How does the N-terminal IDR of G. uraniireducens IF-2 contribute to its function at low temperatures?

The N-terminal intrinsically disordered region (IDR) of bacterial IF-2 plays a crucial role in cold adaptation, and this is likely true for G. uraniireducens IF-2 as well. Based on studies of bacterial IF-2:

The IDR of IF-2 is required for growth at cold temperatures (15°C), though it appears dispensable at normal growth temperatures (37°C) . In G. uraniireducens, this adaptation may be particularly important as the organism shows different growth characteristics at various temperatures, with specific growth rates of 0.018 h⁻¹ at 10°C and 0.026 h⁻¹ at 15°C when using fumarate as an electron acceptor .

The N-terminal IDR drives phase separation of IF-2, particularly in cold conditions, suggesting that IF-2 condensation is an adaptive strategy to promote fitness during cold stress . This phase separation behavior appears to be a conserved property across bacterial species, observed in both E. coli and C. crescentus .

Mechanistically, the IDR-driven condensation likely enhances translation initiation efficiency at low temperatures by:

• Increasing the local concentration of IF-2 near ribosomes

• Facilitating more efficient delivery of initiator tRNA

• Potentially protecting the translation machinery from cold-induced inhibition

• Creating microenvironments that optimize the thermodynamics of translation initiation reactions

This cold-adaptive function represents a specialized aspect of IF-2 biology that goes beyond its canonical role in translation initiation and highlights the multifunctional nature of this protein in bacterial environmental adaptation .

What experimental approaches can be used to investigate IF-2 phase separation in G. uraniireducens?

Investigating phase separation properties of G. uraniireducens IF-2 requires multidisciplinary approaches spanning from in vitro biochemical studies to in vivo cellular analyses:

• In vitro Phase Separation Assays:

  • Purify recombinant full-length IF-2 and truncated variants (with and without the N-terminal IDR)

  • Monitor turbidity using spectrophotometry across temperature ranges (5-37°C)

  • Utilize differential interference contrast (DIC) microscopy to directly visualize condensate formation

  • Apply fluorescence microscopy with fluorescently labeled IF-2 to track protein dynamics within condensates

• Physical Property Characterization:

  • Determine critical concentration thresholds for phase separation

  • Measure temperature-dependency of phase transition

  • Analyze salt and pH sensitivity of condensate formation

  • Implement fluorescence recovery after photobleaching (FRAP) to assess condensate material properties

• Cellular Imaging:

  • Create fluorescently tagged IF-2 constructs for expression in G. uraniireducens

  • Apply confocal microscopy at various temperatures to visualize condensate formation in vivo

  • Use electron microscopy to examine the ultrastructure of condensates at high resolution

• Functional Analyses:

  • Develop translation activity assays in the presence and absence of IF-2 condensates

  • Compare translation efficiencies at various temperatures with wild-type and IDR-deleted IF-2

  • Assess correlation between condensate formation and growth rates at low temperatures

• Sequence-Function Relationships:

  • Generate chimeric constructs with IDRs from different organisms to identify sequence determinants of phase separation

  • Perform systematic mutagenesis of the IDR to identify critical residues

  • Utilize bioinformatic analyses to predict phase separation propensity based on amino acid composition

These methodologies, when applied systematically, can provide comprehensive insights into the phase separation properties of G. uraniireducens IF-2 and its biological significance in cold adaptation .

How might the phase separation properties of IF-2 impact translation initiation efficiency in G. uraniireducens during environmental stress?

The phase separation properties of IF-2 likely represent a sophisticated adaptation mechanism that modulates translation initiation efficiency in G. uraniireducens during environmental stress:

• Cold Stress Response Mechanism:
  The N-terminal IDR of bacterial IF-2 drives phase separation particularly in cold conditions . For G. uraniireducens, which can grow at temperatures as low as 10°C , this property may be critical for maintaining translation initiation during cold exposure. The formation of IF-2 condensates likely creates microenvironments with elevated local concentrations of translation initiation components, compensating for the reduced molecular kinetics at low temperatures.

• Translational Efficiency Modulation:
  IF-2 condensates may serve as dynamic translation initiation hubs that:

  • Concentrate translation components (ribosomes, mRNAs, tRNAs)

  • Shield the translation machinery from inhibitory conditions

  • Accelerate the rate-limiting steps of initiation complex formation

  • Prioritize translation of specific mRNA subsets needed for stress adaptation

• Coordination with Metabolic Adaptation:
  G. uraniireducens demonstrates different growth rates depending on the electron acceptor and temperature . The phase separation properties of IF-2 may synchronize translation rates with these metabolic adaptations, ensuring energy conservation during stress by:

  • Limiting bulk protein synthesis during severe stress

  • Preferentially translating stress-response proteins

  • Adjusting translation rates to match available energy resources

• Ecological Implications:
  As G. uraniireducens is involved in uranium bioremediation , its ability to maintain cellular functions through IF-2 phase separation during environmental fluctuations may impact its bioremediation efficacy. Optimized translation during stress could enhance:

  • Survival during environmental transitions

  • Expression of metal reduction machinery under suboptimal conditions

  • Adaptation to seasonal temperature variations in subsurface environments

• Evolutionary Conservation Context:
  The observation that IF-2 condensation appears to be a conserved property across bacterial species suggests it represents an ancient and fundamental adaptation strategy. The specific properties of G. uraniireducens IF-2 condensates may be fine-tuned to its environmental niche, reflecting evolutionary adaptation to its specific ecological context.

This multifaceted impact of IF-2 phase separation represents an emerging paradigm in understanding bacterial stress adaptation at the molecular level .

What is known about the regulation of infB expression in G. uraniireducens and related Geobacter species?

The regulation of infB expression in G. uraniireducens and related Geobacter species involves complex regulatory networks, though specific information about infB regulation is limited in the provided context. Based on what is known about regulatory mechanisms in Geobacter:

• Transcriptional Regulation Mechanisms:
  While direct information on infB regulation is not provided, Geobacter species utilize enhancer-binding proteins (EBPs) that recognize specific promoter regions to regulate gene expression. For instance, EBP2 and EBP13 in Geobacter species bind to promoter regions and regulate flagellar genes . Similar mechanisms might control infB expression, particularly in response to environmental conditions.

• Two-Component Signal Transduction:
  Geobacter species employ a multicomponent His-Asp phosphorelay system for signal transduction. This system involves histidine kinases like GHK3 and GHK4, which participate in phosphorylation cascades . These signaling pathways likely influence translation-related genes, including infB, especially during environmental adaptations.

• Environmental Response Regulation:
  G. uraniireducens demonstrates different growth rates under varying conditions, suggesting sophisticated regulatory mechanisms that respond to electron acceptor availability and temperature . The regulation of infB likely integrates into these broader environmental response networks to synchronize translation capacity with metabolic state.

• Growth Phase-Dependent Regulation:
  In bacterial systems, translation initiation factors often show growth phase-dependent expression patterns. The transcript abundance of ribosomal proteins in G. uraniireducens correlates with growth rates , suggesting similar regulatory mechanisms might control infB expression to match translation initiation capacity with growth demands.

• Comparative Genomic Insights:
  Analysis of related Geobacter species indicates conservation of certain regulatory elements. For example, the RpoN-dependent promoter elements control certain genes in G. sulfurreducens . Similar promoter architectures might contribute to infB regulation across Geobacter species.

Further research specifically targeting infB regulation in G. uraniireducens would be valuable to elucidate these regulatory mechanisms in greater detail.

How can one investigate the role of IF-2 in coordinating translation with cellular responses to changing electron acceptor availability?

Investigating the role of IF-2 in coordinating translation with cellular responses to changing electron acceptor availability in G. uraniireducens requires sophisticated experimental approaches:

• Transcriptomic Profiling Across Electron Acceptor Conditions:

  • Perform RNA-Seq analysis of G. uraniireducens grown with different electron acceptors (sediment, Fe(III)-oxide, Mn(IV)-oxide, fumarate)

  • Analyze differential expression of infB alongside other translation-related genes

  • Correlate infB expression patterns with specific growth rates observed under different electron acceptors

  • Identify potential co-regulated gene clusters that might form functional modules

• Translational Efficiency Measurement:

  • Implement ribosome profiling to assess translation efficiency genome-wide

  • Compare translational landscapes across electron acceptor conditions

  • Quantify ribosome occupancy on infB mRNA to determine its translation regulation

  • Analyze translation of specific mRNAs that may depend on IF-2 activity under different growth conditions

• Genetic Manipulation Approaches:

  • Create conditional IF-2 depletion strains to monitor translation under different electron acceptor conditions

  • Engineer strains expressing tagged IF-2 to facilitate immunoprecipitation studies

  • Develop reporter systems fused to promoters of interest to monitor real-time gene expression changes

  • Construct strains with modified IF-2 lacking specific domains to assess their importance

• Biochemical Interaction Studies:

  • Perform pull-down assays to identify proteins that interact with IF-2 under different electron acceptor conditions

  • Analyze changes in the IF-2 interactome when switching between electron acceptors

  • Investigate potential post-translational modifications of IF-2 that might regulate its activity

  • Assess changes in IF-2 phase separation properties across conditions

• Integrative Systems Biology:

  • Develop mathematical models that integrate transcriptomic, proteomic, and metabolic data

  • Map IF-2 activity within the broader cellular response network

  • Identify potential regulatory motifs that coordinate translation with electron acceptor utilization

  • Predict cellular outcomes based on IF-2 activity levels and validate experimentally

These methodologies will provide a comprehensive understanding of how IF-2 functions within the regulatory networks that coordinate translation with electron acceptor availability in G. uraniireducens .

What is the relationship between the His-Asp phosphorelay signaling systems and translation regulation in Geobacter species?

The relationship between His-Asp phosphorelay signaling systems and translation regulation in Geobacter species represents an intriguing but underexplored area of bacterial physiology. While direct evidence for this relationship is limited in the provided context, several potential connections can be inferred:

• Integration of Environmental Sensing and Translation:
  The multicomponent His-Asp phosphorelay system in Geobacter species functions as an environmental sensing mechanism, with histidine kinases like GHK3 and GHK4 participating in phosphorylation cascades . These signaling pathways likely influence translation regulation mechanisms to synchronize protein synthesis with environmental conditions. This integration would enable Geobacter to adapt its translational machinery, including IF-2 activity, in response to changing environmental parameters.

• Response Regulator Effects on Gene Expression:
  Response regulators in the His-Asp phosphorelay system, such as FgrM in G. sulfurreducens, control the expression of specific gene sets . While these have been primarily studied in the context of flagellar gene expression, similar regulatory mechanisms might extend to translation-related genes, potentially including infB, which encodes IF-2.

• Coordinative Regulation During Stress Adaptation:
  When Geobacter species encounter environmental stressors or changing electron acceptor availability, both signaling systems and translation must be coordinately regulated. The phosphorelay system likely contributes to this coordination by modulating gene expression patterns that prepare the translational machinery for changed conditions.

• Growth Rate Control Mechanisms:
  G. uraniireducens exhibits different growth rates depending on electron acceptors and temperature . The His-Asp phosphorelay system may contribute to growth rate regulation by influencing translation initiation factor availability or activity, potentially through direct or indirect effects on IF-2.

• Potential Direct Interaction:
  Although not directly evidenced in the provided context, bacterial signaling systems sometimes directly modulate translation through protein-protein interactions or post-translational modifications. The His-Asp phosphorelay components might interact with translation factors, including IF-2, to fine-tune translation initiation under specific conditions.

• Experimental Approaches to Investigate This Relationship:

  • Phosphoproteomics to identify potential phosphorylation of translation factors

  • Co-immunoprecipitation studies to detect physical interactions

  • Genetic studies with phosphorelay system mutants to observe effects on translation

  • Ribosome profiling in wild-type versus signaling system mutants

This relationship represents an important area for future research to fully understand how Geobacter species integrate environmental sensing with translational control .

How does G. uraniireducens IF-2 compare evolutionarily to IF-2 from other Geobacter species and more distant bacterial taxa?

The evolutionary comparison of G. uraniireducens IF-2 with those from other bacterial taxa reveals important insights about conservation and specialization of this essential factor:

• Conservation Within Geobacter Genus:
  While specific comparative analyses of IF-2 across Geobacter species are not directly addressed in the provided context, related Geobacter species like G. sulfurreducens, G. metallireducens, and G. bemidjiensis share significant genomic similarities . These similarities likely extend to the infB gene and its product, suggesting conservation of core IF-2 functions within the genus, particularly in domains responsible for GTP binding and fMet-tRNA interaction.

• Structural Domain Conservation:
  Bacterial IF-2 proteins generally contain conserved structural domains, including a G-domain responsible for GTP binding and hydrolysis, and domains involved in fMet-tRNA binding . The consistent arrangement of these domains across bacterial taxa suggests fundamental functional constraints on IF-2 evolution. G. uraniireducens IF-2 likely maintains these highly conserved domains while potentially exhibiting species-specific variations in less constrained regions.

• N-terminal IDR Variation:
  The N-terminal intrinsically disordered region (IDR) of bacterial IF-2 is present across species yet shows considerable sequence variation . This region drives phase separation properties that appear conserved functionally despite sequence divergence. The specific sequence characteristics of G. uraniireducens IF-2's IDR may reflect adaptation to its environmental niche, particularly regarding temperature adaptation.

• Multiple Isoform Evolution:
  The presence of multiple IF-2 isoforms (alpha and beta) observed in bacterial systems represents an evolutionary strategy for functional diversification. The specific arrangements of translation initiation sites in the G. uraniireducens infB gene may reveal evolutionary pressures specific to its lifestyle and environmental adaptations.

• Adaptation to Environmental Conditions:
  G. uraniireducens demonstrates growth across a range of temperatures and with various electron acceptors . The evolutionary trajectory of its IF-2 likely reflects adaptations to these specific environmental parameters, particularly in regions that influence temperature sensitivity and activity regulation.

• Phylogenetic Context:
  Comparative phylogenetic analysis would likely place G. uraniireducens IF-2 within the broader context of proteobacterial translation factors, with closest relationships to other delta-proteobacterial IF-2 proteins, while maintaining the core features essential for bacterial translation initiation.

This evolutionary perspective provides insights into how G. uraniireducens IF-2 maintains essential functions while potentially exhibiting specialized adaptations to its ecological niche .

What methods are most effective for analyzing the sequence-structure-function relationships of IF-2 across bacterial species?

Analyzing sequence-structure-function relationships of IF-2 across bacterial species, including G. uraniireducens, requires an integrated methodological approach:

• Comparative Sequence Analysis:

  • Multiple sequence alignment of IF-2 proteins from diverse bacterial taxa

  • Phylogenetic tree construction to visualize evolutionary relationships

  • Identification of conserved motifs using tools like MEME and GLAM2

  • Analysis of selection pressures using dN/dS ratios to identify sites under positive or purifying selection

  • Domain architecture comparison using tools like SMART and Pfam

• Structural Bioinformatics:

  • Homology modeling based on available IF-2 structures (e.g., B. stearothermophilus IF2-G2)

  • Molecular dynamics simulations to analyze structural dynamics across temperature ranges

  • Prediction of intrinsically disordered regions using specialized tools (PONDR, IUPred)

  • Protein-protein interaction interface prediction

  • Analysis of conformational changes upon GTP/GDP binding

• Functional Domain Mapping:

  • Generation of chimeric proteins with domains from different bacterial IF-2 sources

  • Systematic mutagenesis of conserved residues across different bacterial IF-2 proteins

  • In vitro translation assays to measure functional impacts of domain swapping

  • GTPase activity assays comparing catalytic properties across species variants

  • tRNA binding assays to evaluate functional conservation of C-terminal domains

• Integrated Structural Biology:

  • X-ray crystallography of IF-2 domains from multiple bacterial sources

  • Cryo-EM studies of IF-2 in ribosomal complexes from different bacteria

  • Nuclear magnetic resonance (NMR) spectroscopy to analyze dynamics of isolated domains

  • Small-angle X-ray scattering (SAXS) to characterize solution conformations

  • Hydrogen-deuterium exchange mass spectrometry to identify flexible regions

• Systems Biology Approaches:

  • Analysis of genetic context and operon structure of infB across species

  • Transcriptomic comparison of IF-2 expression patterns across growth conditions

  • Correlation of IF-2 sequence features with organismal traits (optimal growth temperature, metabolic capabilities)

  • Network analysis of IF-2 genetic interactions across bacterial species

  • Phenotypic analysis of cross-species complementation experiments

• Phase Separation Analysis:

  • Sequence determinants of phase separation across bacterial IF-2 proteins

  • Comparative analysis of phase separation properties in relation to ecological niches

  • Structure-based prediction of interaction motifs driving condensate formation

  • Correlation between sequence features and phase separation temperature sensitivity

These methodologies, when applied systematically across bacterial species including G. uraniireducens, will provide comprehensive insights into the sequence-structure-function relationships of IF-2 and their evolutionary significance .

How might the functional adaptations of G. uraniireducens IF-2 reflect its ecological niche in uranium bioremediation environments?

The functional adaptations of G. uraniireducens IF-2 likely reflect sophisticated evolutionary responses to its specialized ecological niche in uranium bioremediation environments:

• Metal Stress Adaptation:
  G. uraniireducens thrives in environments containing uranium and other heavy metals . Its IF-2 may have evolved specific features that confer resilience to metal-induced translational stress, potentially including:

  • Structural modifications that reduce metal binding to functionally critical regions

  • Regulatory mechanisms that maintain translation initiation efficiency despite metal stress

  • Domain adaptations that protect the factor from metal-induced misfolding or aggregation

• Temperature-Responsive Translation Control:
  G. uraniireducens demonstrates growth across a range of temperatures, with distinct growth rates at 10°C, 15°C, 20°C, and 30°C . The phase separation properties of its IF-2, driven by the N-terminal IDR , likely represent an adaptation to maintain translational efficiency across these temperature ranges, particularly important in subsurface environments where temperatures can fluctuate seasonally.

• Metabolic Versatility Support:
  The ability of G. uraniireducens to utilize various electron acceptors (sediment, Fe(III)-oxide, Mn(IV)-oxide, fumarate) with different growth efficiencies suggests its translational machinery, including IF-2, has adapted to support rapid metabolic switching. This versatility would be advantageous in heterogeneous subsurface environments where electron acceptor availability can change spatially and temporally.

• Energy Conservation Mechanisms:
  In electron acceptor-limited environments, efficient energy utilization becomes critical. G. uraniireducens IF-2 may have evolved features that optimize the energy expenditure of translation initiation, particularly important given the significant energy cost of protein synthesis. This adaptation would enhance survival during periods of energy limitation in subsurface environments.

• Stress Response Integration:
  The uranium bioremediation environment represents a stressful ecological niche. IF-2's phase separation properties may integrate with stress response pathways specific to G. uraniireducens, potentially prioritizing the translation of proteins involved in uranium reduction and detoxification during environmental stress.

• Redox Sensitivity Adaptations:
  Given the importance of redox reactions in G. uraniireducens metabolism and uranium reduction , its IF-2 may have evolved specific sensitivities to cellular redox state, allowing translation initiation to respond to changing redox conditions encountered during uranium bioremediation processes.

These adaptive features would collectively enhance G. uraniireducens' fitness in its specialized ecological niche, contributing to its effectiveness in uranium bioremediation applications .

How can recombinant G. uraniireducens IF-2 be utilized to enhance in vitro translation systems for specialized applications?

Recombinant G. uraniireducens IF-2 offers unique properties that can enhance in vitro translation systems for specialized applications:

• Temperature-Adaptive Translation Systems:
  The phase separation properties of bacterial IF-2 at low temperatures make G. uraniireducens IF-2 particularly valuable for developing cold-adaptive in vitro translation systems. Such systems would benefit:

  • Low-temperature protein expression applications

  • Translation of proteins that require cold conditions for proper folding

  • Coupled transcription-translation systems that need to operate across temperature ranges

• Metal-Tolerant Cell-Free Protein Synthesis:
  Given G. uraniireducens' natural habitat in environments containing uranium and other metals , its IF-2 may possess inherent resistance to metal-induced inhibition. This property could be exploited to develop:

  • Cell-free protein synthesis systems with enhanced tolerance to metal contaminants

  • In vitro translation platforms for expressing metalloproteins

  • Biosensors that maintain functionality in metal-containing environments

• Energy-Efficient Translation Platforms:
  G. uraniireducens has evolved to grow efficiently with various electron acceptors and under energy-limited conditions . Its IF-2 may contribute to:

  • Development of translation systems with reduced energy requirements

  • Optimization of ATP/GTP consumption in continuous cell-free protein synthesis

  • Enhancement of translation yield under resource-limited conditions

• Methodology for Implementation:
  To utilize G. uraniireducens IF-2 in in vitro translation systems:

  • Clone and express the full-length infB gene with proper consideration of potential isoforms

  • Purify the protein maintaining its functional integrity through careful buffer composition

  • Replace standard E. coli IF-2 in commercial cell-free systems with titrated amounts of G. uraniireducens IF-2

  • Optimize reaction conditions (temperature, ionic strength, nucleotide concentrations) specific to G. uraniireducens IF-2 function

  • Characterize translation efficiency using reporter proteins across various conditions

• Hybrid Translation Systems:
  Creating hybrid systems with components from G. uraniireducens and other organisms:

  • Combine G. uraniireducens IF-2 with ribosomes from thermophilic organisms for systems with broad temperature ranges

  • Integrate with specialized tRNAs for enhanced incorporation of non-canonical amino acids

  • Develop chimeric IF-2 proteins with domains from different species for novel functionalities

• Diagnostic Applications:
  The phase separation properties of IF-2 could be exploited for developing diagnostics:

  • Temperature-responsive biosensors that utilize controlled phase separation

  • Visible detection systems based on IF-2 condensation properties

  • Enrichment mechanisms for dilute analytes based on condensate formation

These applications leverage the unique evolutionary adaptations of G. uraniireducens IF-2 to enhance the capabilities of in vitro translation technologies for specialized research and biotechnological purposes .

What experimental strategies are most effective for investigating the role of G. uraniireducens IF-2 in environmental adaptation and stress response?

Investigating G. uraniireducens IF-2's role in environmental adaptation and stress response requires multifaceted experimental strategies:

• Genetic Manipulation Approaches:

  • Generate conditional IF-2 depletion strains using inducible promoter systems

  • Create domain deletion variants to assess the contribution of specific regions

  • Develop complementation systems with IF-2 variants from different species

  • Utilize CRISPR-Cas9 for precise genomic editing of the infB gene

  • Construct reporter fusions to monitor IF-2 expression under different stress conditions

  Methodology: Transform G. uraniireducens with appropriate constructs, verify transformants using PCR and sequencing, and analyze phenotypes under various environmental conditions .

• Transcriptomic and Proteomic Profiling:

  • Perform RNA-Seq analysis comparing wild-type and IF-2 variant strains across stress conditions

  • Implement ribosome profiling to assess translational changes during stress

  • Use quantitative proteomics to identify proteins differentially expressed in IF-2 mutants

  • Apply selective ribosome profiling to identify mRNAs preferentially translated by different IF-2 isoforms

  • Conduct RNA immunoprecipitation to identify mRNAs directly associated with IF-2

  Methodology: Extract RNA/protein under standardized conditions, process for appropriate high-throughput analysis, and apply bioinformatic pipelines for data interpretation .

• Biochemical and Biophysical Analysis:

  • Characterize phase separation properties of IF-2 across temperature ranges

  • Measure GTPase activity under various stress conditions

  • Assess fMet-tRNA binding efficiency in the presence of environmental stressors

  • Determine structural changes using circular dichroism or fluorescence spectroscopy

  • Analyze post-translational modifications that might regulate IF-2 during stress

  Methodology: Purify recombinant IF-2, expose to controlled stress conditions, and measure functional parameters using established biochemical assays .

• Environmental Simulation Studies:

  • Develop laboratory bioreactors mimicking uranium-contaminated subsurface environments

  • Monitor IF-2 expression and activity during shifts in electron acceptor availability

  • Assess translation rates during temperature fluctuations using pulse-chase methods

  • Measure growth rates and translation efficiency during exposure to multiple stressors

  • Compare wild-type and IF-2 variant strains in simulated environmental transitions

  Methodology: Establish controlled environmental parameters reflecting natural conditions, introduce G. uraniireducens cultures, and monitor relevant parameters over time .

• Single-Cell Analysis Techniques:

  • Implement fluorescent translational reporters to visualize translation activity

  • Use fluorescently tagged IF-2 to track localization and condensate formation

  • Apply microfluidics to observe real-time cellular responses to changing conditions

  • Perform single-cell RNA-Seq to capture cell-to-cell variability in stress responses

  • Use FISH-based methods to visualize mRNA localization patterns

  Methodology: Develop appropriate reporter constructs, establish imaging protocols optimized for G. uraniireducens, and apply quantitative image analysis.

These complementary strategies will provide comprehensive insights into how G. uraniireducens IF-2 contributes to environmental adaptation and stress response mechanisms .