Recombinant Geobacter sulfurreducens UPF0102 protein GSU0650 (GSU0650)

What is the role of the UPF0102 protein GSU0650 in Geobacter sulfurreducens metabolism?

The UPF0102 protein GSU0650 is part of the extensive network of proteins in Geobacter sulfurreducens that likely contributes to its unique metabolism. While specific information about GSU0650 is limited in the current literature, G. sulfurreducens is known for its distinctive metabolism that is heavily dependent on an extensive network of cytochromes, enabling it to respire metals and electrodes to produce measurable electric current . The bacterium contains a large amount of iron (2 ± 0.2 μg/g dry weight), attributed to abundant cytochrome production . As a UPF (Uncharacterized Protein Family) member, GSU0650 may be involved in electron transfer pathways or other metabolic functions essential for the organism's unique capabilities, though its precise function requires further characterization through targeted studies.

How does GSU0650 compare to other UPF0102 family proteins in related bacteria?

Comparative analysis of UPF0102 family proteins across related bacteria requires sequence alignment and structural comparison methodologies. While the search results don't specifically address GSU0650 comparisons, research approaches would involve:

• Multiple sequence alignment of GSU0650 with homologs from related species

• Phylogenetic analysis to determine evolutionary relationships

• Structural prediction to identify conserved domains

G. sulfurreducens has a unique cell composition compared to similar microorganisms, with significantly higher iron concentration attributed to cytochrome production . This distinctive characteristic suggests that proteins involved in its metabolism, potentially including GSU0650, might have evolved specific features to support the organism's electroactive capabilities.

What expression patterns does GSU0650 exhibit under different growth conditions?

Expression patterns of proteins in G. sulfurreducens vary significantly depending on electron acceptor availability and growth conditions. Transcriptomic analyses have revealed differential gene expression patterns between biofilms grown on conductive versus non-conductive surfaces . While specific GSU0650 expression data isn't provided in the search results, the methodological approach would include:

• RNA-seq analysis comparing expression across various growth conditions (anaerobic vs. microaerobic, different electron acceptors)

• RT-qPCR confirmation of differential expression

• Correlation analysis with other known genes involved in electron transfer

Researchers studying GSU0650 should consider that G. sulfurreducens exhibits significant transcriptional differences when grown with different electron acceptors, as demonstrated in studies comparing electrode-grown versus fumarate-grown biofilms .

What are the optimal conditions for recombinant expression of GSU0650?

Optimizing recombinant expression of GSU0650 requires consideration of G. sulfurreducens' unique metabolic requirements. Based on the organism's growth characteristics, researchers should consider:

Expression Host Selection:

• Traditional hosts (E. coli) may lack post-translational modification capabilities

• Consider expression in related Geobacteraceae for proper folding and modification

Growth Medium Composition:
Based on G. sulfurreducens nutritional requirements, expression media should account for:

Induction Conditions:

• Temperature: 30°C (standard for G. sulfurreducens growth)

• Oxygen: Microaerobic or anaerobic conditions (as G. sulfurreducens can tolerate limited oxygen )

• Induction timing: Mid-log phase to avoid nutrient limitations

Given that G. sulfurreducens growth can be limited by iron, copper, and zinc availability at concentrations of ~0.1 g/L , expression protocols should ensure adequate nutrient supply for optimal protein yield.

What purification methods are most effective for isolating recombinant GSU0650?

Effective purification of recombinant GSU0650 would require protocols adapted to its biochemical properties. While specific information about GSU0650 is not available in the search results, general considerations based on G. sulfurreducens proteins would include:

• Initial Clarification:

  • Cell lysis under anaerobic conditions

  • Centrifugation to remove cell debris

  • Membrane fractionation if GSU0650 is membrane-associated

• Chromatographic Methods:

  • Affinity chromatography (if expressed with a tag)

  • Ion exchange chromatography based on predicted pI

  • Size exclusion for final polishing

• Considerations Specific to G. sulfurreducens Proteins:

  • Maintain reducing conditions throughout purification

  • Account for possible high iron content if GSU0650 binds metals

  • Consider detergent requirements if membrane-associated

The purification strategy should be informed by the high lipid content (32 ± 0.5% dry weight) of G. sulfurreducens cells , which may necessitate specialized approaches for protein extraction from membrane fractions.

How can researchers verify the structural integrity and function of purified GSU0650?

Verification of structural integrity and function requires multiple complementary approaches:

Structural Integrity Assessment:

• Circular dichroism spectroscopy to confirm secondary structure

• Thermal shift assays to assess stability

• Size exclusion chromatography to verify oligomeric state

• Dynamic light scattering for homogeneity analysis

Functional Verification:
Without specific information about GSU0650's function, researchers should consider:

• Electron transfer assays if involved in redox processes (common for G. sulfurreducens proteins)

• Binding assays with potential interaction partners

• Activity assays based on predicted function from structural homology

G. sulfurreducens-Specific Considerations:
Researchers should consider potential roles in the bacterium's unique extracellular electron transfer capabilities, using methodologies similar to those employed for cytochromes like OmcS and OmcZ, which have been analyzed using heme-staining and western blotting techniques .

Does GSU0650 contribute to extracellular electron transfer in G. sulfurreducens?

Extracellular electron transfer (EET) is a defining characteristic of G. sulfurreducens, enabling it to "breathe" metals and electrodes . While specific information about GSU0650's role in EET is not provided in the search results, research approaches to investigate this question would include:

• Knockout Studies:

  • Generate ∆GSU0650 mutants

  • Compare electron transfer rates to wild-type using:

    • Chronoamperometry with electrode as electron acceptor

    • Fe(III) reduction assays

• Localization Studies:

  • Immunogold labeling coupled with electron microscopy

  • Cell fractionation and western blotting

  • GFP fusion protein tracking

• Interaction Analysis:

  • Co-immunoprecipitation with known EET components

  • Two-hybrid screening for protein-protein interactions

Studies of G. sulfurreducens have identified key components involved in EET, including c-type cytochromes like OmcB and OmcZ, which show increased expression in current-producing biofilms . If GSU0650 interacts with these components, it may contribute to the EET capabilities that make G. sulfurreducens valuable for microbial fuel cells and bioremediation applications.

How does GSU0650 expression change during biofilm formation on electrodes?

Biofilm formation on electrodes represents a specialized growth mode for G. sulfurreducens that involves substantial transcriptional changes. While specific GSU0650 expression data is not provided in the search results, research approaches would mirror those used in existing studies:

• Comparative Transcriptomics:
  RNA-seq analysis comparing GSU0650 expression between:

  • Planktonic cells vs. electrode biofilms

  • Biofilms on conductive vs. non-conductive surfaces

  • Different stages of biofilm development

• Temporal Expression Analysis:

  • Real-time quantitative PCR at different biofilm growth stages

  • Reporter gene constructs to visualize expression dynamics

Previous transcriptomic studies have identified significant differences between biofilms grown on conductive (graphite electrode) versus non-conductive (glass) surfaces, with 119 differentially expressed genes in the conductive condition . Additionally, microarray analysis has revealed 13 genes with significantly higher transcript levels in current-harvesting biofilms, including genes for outer membrane cytochromes and pili components . Similar methodologies could determine if GSU0650 shows comparable regulation patterns.

What is the relationship between GSU0650 and other key electron transfer proteins in G. sulfurreducens?

Understanding protein interaction networks is crucial for characterizing GSU0650's role. While specific information about GSU0650's relationships is not provided in the search results, methodological approaches would include:

• Protein-Protein Interaction Studies:

  • Pull-down assays with known electron transfer components

  • Surface plasmon resonance to measure binding kinetics

  • Bacterial two-hybrid screening

• Co-expression Analysis:

  • Cluster analysis of transcriptomic data

  • Correlation of expression patterns with known components

• Structural Biology Approaches:

  • Co-crystallization attempts with interaction partners

  • Cryo-EM of protein complexes

Key electron transfer proteins in G. sulfurreducens include c-type cytochromes OmcB and OmcZ, which show increased expression in current-producing biofilms . The GSU1771 transcriptional regulator has been identified as controlling extracellular electron transfer in G. sulfurreducens . Investigating whether GSU0650 is regulated by GSU1771 or interacts with these cytochromes would provide valuable insights into its potential role in electron transfer mechanisms.

How does GSU0650 function under microaerobic conditions compared to strict anaerobic conditions?

G. sulfurreducens was originally considered a strict anaerobe but has been shown to tolerate oxygen exposure and even use it as a terminal electron acceptor . Investigating GSU0650's function under varying oxygen conditions would involve:

• Comparative Expression Analysis:

  • RNA-seq comparing anaerobic vs. microaerobic growth

  • Proteomics to quantify protein levels

  • Post-translational modification analysis

• Functional Assays:

  • Enzyme activity measurements under different oxygen tensions

  • Oxygen consumption rates in wild-type vs. ∆GSU0650 mutants

  • Redox state analysis of the protein under varying conditions

• Regulatory Analysis:

  • Identification of oxygen-responsive regulatory elements in the GSU0650 promoter

  • ChIP-seq to identify transcription factors binding under different oxygen conditions

The microaerobic capabilities of G. sulfurreducens represent an important area of research , and understanding how individual proteins like GSU0650 contribute to this metabolic flexibility could provide insights into the organism's adaptability and potential biotechnological applications.

What metabolic flux changes occur in GSU0650 knockout mutants during suboptimal growth conditions?

Metabolic flux analysis in G. sulfurreducens has revealed important insights about its current-generating mechanisms under different growth conditions . For GSU0650 knockout studies, researchers should consider:

• Flux Balance Analysis (FBA):

  • Comparison of wild-type vs. ∆GSU0650 metabolic models

  • Simulation of optimal and suboptimal growth conditions

  • Prediction of respiratory rate changes

• Experimental Validation:

  • 13C-labeled substrate tracing

  • Measurement of key metabolite concentrations

  • Respiratory rate quantification

• Integration with Growth Parameters:

  • Correlation between growth rate and respiratory activity

  • Analysis of acetate consumption efficiency

Previous research has demonstrated that when G. sulfurreducens grows suboptimally, more substrate is completely oxidized to generate electrons, resulting in higher respiration rates . If GSU0650 is involved in central metabolism or electron transfer, its knockout could significantly impact these relationships between growth and respiration.

How can computational modeling predict the functional domains and potential binding sites of GSU0650?

Computational approaches offer valuable tools for predicting protein function when experimental data is limited:

• Structural Prediction Methodologies:

  • Homology modeling using related protein structures

  • Ab initio modeling for unique domains

  • Molecular dynamics simulations to assess flexibility

• Functional Domain Analysis:

  • Conserved domain database searches

  • Motif identification and comparison with characterized proteins

  • Secondary structure prediction and classification

• Binding Site Prediction:

  • Electrostatic surface potential mapping

  • Identification of conserved pockets or clefts

  • Docking simulations with potential substrates or partners

• Integration with Experimental Data:

  • Validation of predictions through targeted mutagenesis

  • Correlation with proteomic interaction data

  • Refinement based on biochemical assays

For UPF0102 family proteins like GSU0650, computational predictions can provide initial hypotheses about function that guide experimental design, particularly important for understudied proteins in organisms with unique metabolic capabilities like G. sulfurreducens.

What are the common challenges in maintaining GSU0650 stability during purification and storage?

Protein stability challenges are common in research, particularly for proteins from organisms with unique physiological characteristics like G. sulfurreducens:

• Purification Challenges:

  • Maintaining anaerobic conditions throughout purification

  • Preventing aggregation during concentration steps

  • Selecting appropriate detergents if membrane-associated

• Storage Considerations:

  • Optimal buffer composition to maintain native structure

  • Cryoprotectant selection for freeze-thaw stability

  • Temperature sensitivity assessment

• Stability Monitoring Methods:

  • Thermal shift assays to identify stabilizing conditions

  • Activity retention tests over time

  • Size exclusion chromatography to detect aggregation

Given G. sulfurreducens' unique cell composition with high iron content (2 ± 0.2 μg/g dry weight) and lipid content (32 ± 0.5% dry weight) , proteins from this organism may require specialized handling to maintain their native properties outside the cellular environment.

How can researchers address the challenge of low GSU0650 expression levels in recombinant systems?

Low expression of recombinant proteins requires systematic troubleshooting:

• Expression System Optimization:

  • Codon optimization for expression host

  • Testing different promoter strengths

  • Evaluation of various fusion tags to enhance solubility

• Growth Condition Modifications:

  • Media composition based on G. sulfurreducens nutritional requirements

  • Temperature and induction timing optimization

  • Scale-up strategies for increased biomass

• Addressing Potential Toxicity:

  • Inducible expression systems with tight regulation

  • Use of specialized host strains

  • Co-expression of chaperones or folding assistants

Researchers should consider that G. sulfurreducens growth can be limited by metal availability, particularly iron, copper, and zinc, as demonstrated by nutrient limitation studies showing maximum cell density constraints of approximately 0.10 g/L due to iron limitation . Ensuring adequate supplementation of these elements may be crucial for successful recombinant expression.

What strategies can resolve conflicting experimental data about GSU0650 function?

Conflicting experimental results require comprehensive reconciliation approaches:

• Methodological Standardization:

  • Detailed protocol comparison to identify variations

  • Collaborative cross-laboratory validation

  • Development of standard operating procedures

• Multi-technique Verification:

  • Employ complementary methods to assess the same parameter

  • Quantitative analysis of result variability

  • Statistical approaches to determine significance of differences

• Contextual Considerations:

  • Growth condition standardization (exact media composition, growth phase)

  • Strain background verification

  • Control for experimental variables like oxygen exposure

• Integrative Analysis:

  • Systems biology approaches to place conflicting results in context

  • Computational modeling to predict conditions where different results would be expected

  • Meta-analysis of published data

The complexity of G. sulfurreducens' metabolism, with its extensive cytochrome network and unique electron transfer capabilities , means that small variations in experimental conditions could potentially lead to different functional outcomes for individual proteins.

How might GSU0650 be engineered to enhance electron transfer capabilities in microbial fuel cells?

Protein engineering for enhanced electron transfer represents an exciting frontier:

• Structure-Guided Engineering Approaches:

  • Identification of rate-limiting steps in electron transfer

  • Targeted mutagenesis of key residues

  • Domain swapping with more efficient homologs

• System-Level Engineering:

  • Co-expression with complementary electron transfer components

  • Regulatory modifications for increased expression

  • Integration into synthetic electron transfer pathways

• Performance Evaluation Methods:

  • Chronoamperometry to measure current production

  • Coulombic efficiency calculations

  • Long-term stability assessment

Previous research has demonstrated that G. sulfurreducens variants can be developed with enhanced current generation capabilities by modifying their metabolism to favor suboptimal growth with maximum respiration . Similar principles could be applied to GSU0650 engineering if it plays a role in electron transfer pathways.

What is the potential role of GSU0650 in G. sulfurreducens adaptation to diverse environmental conditions?

Understanding protein roles in environmental adaptation requires integrative approaches:

• Comparative Genomics:

  • Analysis of GSU0650 conservation across Geobacter species from different environments

  • Identification of environment-specific sequence variations

  • Correlation with habitat characteristics

• Transcriptional Response Studies:

  • Expression analysis under various stressors (temperature, pH, salinity)

  • Response to different electron acceptor availability

  • Temporal dynamics during adaptation

• Fitness Contribution Assessment:

  • Competition experiments between wild-type and ∆GSU0650 strains

  • Growth rate comparisons under stress conditions

  • Survival monitoring during environmental transitions

G. sulfurreducens has demonstrated remarkable adaptability, including the ability to switch between anaerobic and microaerobic metabolism and form electroactive biofilms on various surfaces . Understanding how individual proteins like GSU0650 contribute to this adaptability could provide insights into the organism's ecological role and biotechnological potential.

How does GSU0650 interact with the regulatory networks controlling biofilm formation and electron transfer?

Regulatory network analysis requires integrated experimental approaches:

• Transcriptional Regulation Studies:

  • Promoter analysis to identify regulatory elements

  • ChIP-seq to identify transcription factors binding to the GSU0650 promoter

  • Response to known regulatory perturbations

• Global Regulatory Context:

  • Integration with known regulators like GSU1771

  • Network analysis to identify co-regulated genes

  • Epistasis studies with regulatory mutants

• Signal Transduction Pathways:

  • Phosphoproteomics to identify post-translational regulation

  • Second messenger involvement (c-di-GMP, cAMP)

  • Environmental sensing mechanisms

Studies have identified GSU1771 as a global regulator controlling extracellular electron transfer and exopolysaccharide synthesis in G. sulfurreducens . Investigating whether GSU0650 is part of this regulatory network would provide valuable insights into its role in the organism's sophisticated electron transfer mechanisms and biofilm formation capabilities.