Recombinant Geobacter sulfurreducens Glycogen synthase 2 (glgA2)

What is the functional role of glycogen synthase 2 (glgA2) in G. sulfurreducens?

Glycogen synthase 2 (glgA2) in G. sulfurreducens is a key enzyme in the glycogen synthesis pathway that catalyzes the formation of α-1,4-glucosidic linkages. Unlike its homolog glgA1, glgA2 belongs to the GT5 glycosyltransferase family and plays a distinct role in polysaccharide aggregation. Research indicates that glgA2 is phylogenetically related to the SSIII/SSIV starch synthase group found in plants, which is involved in starch granule seeding . This enzyme represents a critical determinant in the synthesis and accumulation of glycogen, which serves as an important energy storage compound for G. sulfurreducens during periods of nutrient limitation or stress.

How does the genomic context of glgA2 differ from other glycogen synthesis genes in G. sulfurreducens?

The glgA2 gene in G. sulfurreducens exists within a genomic context that includes several other genes involved in glycogen metabolism. Unlike the glgA1 gene, glgA2 appears to have evolved alongside the development of cyanobacteria, as evidenced by the significant congruence between the phylogenies of glgA2 and 16S ribosomal RNA . The genomic organization typically places glgA2 in proximity to other genes involved in glycogen synthesis, including glgC (encoding ADP-glucose pyrophosphorylase) and various branching enzymes. This genomic arrangement facilitates coordinated expression of the glycogen synthesis machinery under specific environmental conditions.

What is known about glgA2 regulation in response to environmental conditions?

The regulation of glgA2 in G. sulfurreducens appears to be tightly controlled in response to environmental conditions, particularly those affecting energy metabolism. During periods of nutrient limitation or stress, glgA2 expression is modulated to adjust glycogen synthesis rates. Transcriptome analyses have revealed that glgA2 expression patterns differ significantly when G. sulfurreducens is grown with different electron acceptors, suggesting an interconnection between glycogen synthesis and the organism's unique electron transfer capabilities . Under microaerobic conditions, there are notable shifts in metabolic gene expression, which likely include changes in glgA2 regulation to support altered energy storage requirements .

What are the optimal vectors and protocols for expressing recombinant glgA2 in G. sulfurreducens?

For successful expression of recombinant glgA2 in G. sulfurreducens, researchers should consider using IncQ plasmids, particularly pCD342, which has been demonstrated to function effectively as an expression vector in this organism . The protocol for introducing foreign DNA into G. sulfurreducens via electroporation involves:

• Growing cells to mid-log phase in medium with fumarate as electron acceptor

• Harvesting cells by centrifugation (3,000 × g for 10 min)

• Washing cells twice with electroporation buffer (1 mM HEPES, pH 7.0)

• Resuspending cells to a final concentration of 10^10 cells/ml

• Adding 100-500 ng of plasmid DNA to 100 μl of cell suspension

• Pulsing at 1.5 kV, 400 Ω, and 25 μF in a 0.1-cm cuvette

• Immediately adding 900 μl of recovery medium

• Incubating anaerobically at 30°C for 5 hours before plating on selective media

This approach has been successfully used to introduce and express various genes in G. sulfurreducens .

How can I create targeted glgA2 knockout or overexpression strains?

Creating glgA2 knockout or overexpression strains requires careful consideration of both the genetic system and the growth conditions for G. sulfurreducens. For targeted gene disruption:

• Design primers to amplify regions flanking the glgA2 gene

• Clone these fragments into a suicide vector containing an antibiotic resistance marker

• Introduce the construct into G. sulfurreducens via electroporation

• Select for integrants on media containing appropriate antibiotics

• Confirm gene disruption via PCR and sequencing

For overexpression, the ppcA promoter region can be used with the neutral cloning site NSC1 to drive strong expression . When designing glgA2 overexpression systems, it's important to note that constitutive overexpression may lead to cell death in long-term trials, as observed in similar studies with cyanobacteria . Therefore, inducible expression systems may be more appropriate for sustained cultivation.

What methods are most effective for verifying successful transformation and expression?

To verify successful transformation and expression of recombinant glgA2 in G. sulfurreducens, a multi-faceted approach is recommended:

• PCR verification: Using primers that span the integration site or target the recombinant gene

• Sequencing: To confirm the absence of mutations or frameshifts

• RT-qPCR: For quantifying transcription levels of glgA2

• Protein expression analysis: Using Western blotting with antibodies against tagged glgA2

• Enzyme activity assays: Measuring glycogen synthase activity in cell extracts

• Glycogen quantification: Assessing changes in cellular glycogen content through iodine staining or enzymatic assays

For protein expression analysis, cell fractionation followed by SDS-PAGE and activity staining can effectively detect glycogen synthase activity, similar to methods used for detecting other enzymes in G. sulfurreducens .

How does glgA2 overexpression affect glycogen accumulation and cell growth?

Research on similar systems has shown that glgA2 overexpression has complex effects on glycogen accumulation and cell growth. Studies in cyanobacteria indicate that overexpression of glgA2:

• Did not increase biomass or glycogen production in short-term trials

• Caused transformant death in long-term trials

• Showed markedly different outcomes compared to glgA1 and glgC overexpression

This contrasts with glgA1 or glgC overexpression, which increased biomass (1.6-1.7 fold) and glycogen production (3.5-4 fold) compared to wild type after 96 hours . The table below summarizes these comparative effects:

These findings suggest that glgA2 may play a regulatory role beyond simple glycogen synthesis and that its overexpression disrupts critical cellular processes in G. sulfurreducens.

What are the kinetic properties of purified recombinant glgA2?

Purified recombinant glgA2 from G. sulfurreducens displays distinct kinetic properties that reflect its specialized role in glycogen synthesis. While specific kinetic parameters for G. sulfurreducens glgA2 are still being elucidated, research on related glycogen synthases suggests that:

• The enzyme shows allosteric regulation similar to other bacterial GT5 glycosyltransferases

• Activity is typically ADP-glucose dependent

• Optimal activity occurs under reducing conditions

• The enzyme likely exhibits distinct substrate preferences compared to glgA1

A key distinction is that glgA2, being related to the SSIII/SSIV synthases, may be involved in initiating polysaccharide chains rather than primarily elongating them . This functional difference explains why overexpression of glgA2 alone does not necessarily increase total glycogen accumulation and may actually disrupt the balanced expression of the glycogen synthesis machinery.

How does electron acceptor availability affect glgA2 function and glycogen accumulation?

The function of glgA2 in G. sulfurreducens appears to be intricately connected to the organism's electron transport system. Research indicates that glycogen accumulation patterns differ significantly depending on the available electron acceptor:

• Under fumarate-reducing conditions, glycogen accumulation follows standard patterns of carbon storage

• During Fe(III) reduction, glycogen metabolism shifts to support the high energy demands of metal reduction

• In electrode-reducing conditions (as in microbial fuel cells), glycogen serves as an important electron reservoir

Transcriptomic analyses reveal that genes involved in energy metabolism and electron transport are differentially expressed under various electron acceptor conditions . This suggests that glgA2 function may be modulated as part of the broader metabolic response to different environmental conditions, particularly those affecting the organism's unique extracellular electron transfer capabilities.

How does G. sulfurreducens glgA2 compare structurally and functionally to glycogen synthases in other bacteria?

G. sulfurreducens glgA2 belongs to the GT5 glycosyltransferase family, similar to other bacterial glycogen synthases, but shows several distinguishing characteristics:

• It shares phylogenetic relationship with the SSIII/SSIV starch synthase group found in plants

• Unlike some bacterial glycogen synthases that primarily function in chain elongation, glgA2 appears to play a role in initiating polysaccharide aggregation

• The protein contains conserved catalytic domains characteristic of GT5 enzymes, but may have unique regulatory domains

Analysis of a glgA2 mutant from Cyanobacterium sp. CLg1 revealed a 1-bp deletion yielding a frameshift and nonsense mutation that affected a highly conserved region of bacterial GT5 glycogen synthases . This mutation eliminated the ability to synthesize starch while retaining glycogen synthesis capability, highlighting the specialized role of glgA2 in determining polysaccharide structure.

What is the evolutionary significance of glgA2 in G. sulfurreducens?

The evolutionary history of glgA2 in G. sulfurreducens provides insights into its specialized function. Studies have shown significant congruence between the phylogenies of glgA2 and 16S ribosomal RNA, suggesting that this enzyme evolved alongside cyanobacteria when they diversified over 2 billion years ago . This evolutionary conservation indicates that glgA2 likely plays a fundamental role in the organism's metabolic strategy.

For G. sulfurreducens, which occupies anaerobic subsurface environments and employs unique extracellular electron transfer mechanisms, glgA2 may have evolved specialized functions related to:

• Energy storage under fluctuating redox conditions

• Support for anaerobic metabolism during nutrient limitation

• Coordination with the organism's metal-reducing capabilities

Understanding this evolutionary context is crucial for interpreting the functional significance of glgA2 in G. sulfurreducens' unique ecological niche.

How do glgA1 and glgA2 differ in their contribution to glycogen synthesis?

In G. sulfurreducens, glgA1 and glgA2 appear to serve complementary but distinct functions in glycogen synthesis. Based on studies in related systems:

• glgA1 likely functions primarily in glycogen chain elongation, similar to classical bacterial glycogen synthases

• glgA2 appears to be involved in initiating polysaccharide aggregation, analogous to the role of SSIII/SSIV starch synthases in plants

• The coordinated expression of both enzymes is necessary for proper glycogen structure and accumulation

This functional differentiation explains why overexpression of glgA1 significantly increases glycogen production (3.5-fold), while glgA2 overexpression does not show the same effect and may even be detrimental to cell viability in long-term trials . The balanced activity of both enzymes is likely critical for producing glycogen with the appropriate structure and properties for G. sulfurreducens' energy storage needs.

What are the optimal protocols for purifying active recombinant glgA2?

Purification of active recombinant glgA2 from G. sulfurreducens requires careful attention to protein stability and retention of enzymatic activity. The following protocol is recommended:

• Expression system selection: Use either the native G. sulfurreducens with an IncQ plasmid (pCD342) or a heterologous E. coli system with codon optimization

• Cell lysis: Perform under anaerobic conditions using gentle methods (e.g., French press at 1,000 psi or enzymatic lysis)

• Initial purification: Ammonium sulfate fractionation (30-60% saturation) to precipitate glgA2

• Column chromatography sequence:

  • Ion exchange chromatography (DEAE-Sepharose)

  • Affinity chromatography (if using a tagged protein)

  • Size exclusion chromatography (Sephadex G-100)

• Buffer composition: Include glycerol (10%), reducing agents (5 mM DTT), and protease inhibitors

• Activity preservation: Store at -80°C in buffer containing glycerol (25%) and DTT (10 mM)

Throughout the purification process, it's critical to monitor enzyme activity using the standard glycogen synthase assay, which measures the incorporation of glucose from ADP-glucose into glycogen.

What methods are most effective for measuring glycogen accumulation in glgA2-modified strains?

To accurately measure glycogen accumulation in glgA2-modified strains of G. sulfurreducens, researchers should employ a combination of quantitative and qualitative methods:

Quantitative Methods:

• Enzymatic hydrolysis and glucose quantification:

  • Treat cell extracts with amyloglucosidase to hydrolyze glycogen

  • Measure released glucose using glucose oxidase/peroxidase assay

  • Calculate glycogen content based on glucose equivalents

• Colorimetric assays:

  • Anthrone-sulfuric acid method for total carbohydrate determination

  • Phenol-sulfuric acid method for polysaccharide quantification

Qualitative Methods:

• Iodine staining:

  • React cell suspensions or colonies with iodine solution

  • Observe color development (dark brown to greenish-yellow)

  • Analyze color intensity and spectrum to assess glycogen structure

• Microscopy techniques:

  • Periodic acid-Schiff (PAS) staining for light microscopy

  • Electron microscopy to visualize glycogen granules

For comparative analysis, researchers should standardize sampling times and growth conditions, as glycogen accumulation in G. sulfurreducens varies significantly with growth phase and electron acceptor availability.

How can I design experiments to investigate the relationship between glgA2 activity and electron transfer in G. sulfurreducens?

Investigating the relationship between glgA2 activity and electron transfer in G. sulfurreducens requires experimental designs that can simultaneously monitor glycogen metabolism and electron transfer processes:

• Genetic approach:

  • Create glgA2 knockout, overexpression, and catalytic mutant strains

  • Analyze electron transfer capabilities using soluble (fumarate) and insoluble (Fe(III) oxide, electrodes) electron acceptors

  • Monitor glycogen accumulation/utilization during electron transfer processes

• Bioelectrochemical systems:

  • Grow G. sulfurreducens on electrodes under controlled potential

  • Measure current production as a function of glgA2 expression

  • Sample biofilms at different stages for glycogen content analysis

  • Implement cyclic voltammetry to assess electron transfer kinetics

• Transcriptomic/proteomic correlation:

  • Perform RNA-seq and proteomics under various electron acceptor conditions

  • Compare expression patterns of glgA2 with known electron transfer components

  • Look for co-regulation patterns that suggest functional linkages

• Metabolic flux analysis:

  • Use 13C-labeled acetate to track carbon flow

  • Determine how glgA2 modification affects flux between glycogen synthesis and electron transfer pathways

  • Quantify changes in electron transfer rates in response to altered glycogen metabolism

These experimental approaches should be complemented with appropriate controls and statistical analyses to establish causal relationships between glgA2 activity and electron transfer capabilities.

Why might glgA2 overexpression cause cell death in long-term cultures?

The observation that glgA2 overexpression leads to cell death in long-term cultures suggests several possible mechanisms that researchers should consider:

• Disruption of metabolic balance:

  • Excessive glgA2 activity may deplete ADP-glucose pools

  • This could starve other essential pathways dependent on similar precursors

  • The imbalance between glycogen synthesis initiation and elongation disrupts normal carbon storage patterns

• Altered glycogen structure:

  • Overexpression of glgA2 may produce abnormal glycogen structures

  • These altered structures might affect cellular osmotic balance or macromolecular crowding

  • Abnormal glycogen granules could interfere with cell division processes

• Energy diversion:

  • Enhanced glgA2 activity could unnecessarily divert energy toward glycogen synthesis

  • This may reduce ATP availability for critical cellular processes

  • Under electron acceptor-limited conditions, this energy diversion becomes particularly detrimental

• Redox imbalance:

  • glgA2 overexpression might affect the cell's redox balance

  • Given G. sulfurreducens' reliance on sophisticated electron transfer mechanisms, this could significantly impair cellular function

To address this challenge, researchers should consider using inducible expression systems rather than constitutive ones, allowing for controlled expression levels and timing.

How can I troubleshoot low transformation efficiency when working with glgA2 constructs?

Low transformation efficiency with glgA2 constructs in G. sulfurreducens may stem from several factors. The following troubleshooting strategies are recommended:

• DNA quality and concentration:

  • Ensure plasmid DNA is highly pure (A260/A280 > 1.8)

  • Optimize DNA concentration (100-500 ng per transformation)

  • Avoid multiple freeze-thaw cycles of plasmid preparations

• Electroporation parameters:

  • Adjust field strength (try 1.3-1.7 kV/cm)

  • Modify pulse duration (5-10 ms)

  • Ensure proper cell density (10^9-10^10 cells/ml)

• Cell preparation:

  • Harvest cells in early-to-mid log phase

  • Use fresh cultures rather than stored competent cells

  • Wash cells thoroughly to remove medium components that may interfere with electroporation

• Post-electroporation recovery:

  • Extend recovery period (5-24 hours)

  • Use optimized recovery medium containing acetate (20 mM) and fumarate (40 mM)

  • Maintain strict anaerobic conditions during recovery

• Construct design considerations:

  • Check for potential toxic effects of the construct

  • Ensure promoter is compatible with G. sulfurreducens

  • Verify construct stability in E. coli prior to transformation

• Selection conditions:

  • Optimize antibiotic concentrations based on minimum inhibitory concentration testing

  • Use freshly prepared selective agents

  • Consider longer incubation times for colony development (7-14 days)

By systematically addressing these factors, researchers can improve transformation efficiency when working with glgA2 constructs in G. sulfurreducens.

What are the most common artifacts in glycogen synthase activity assays and how can they be avoided?

Glycogen synthase activity assays are susceptible to several artifacts that can confound experimental results. Researchers should be aware of the following common issues and their mitigation strategies:

• Endogenous glycogen interference:

  • Problem: Residual glycogen in cell extracts can serve as a primer, artificially enhancing activity

  • Solution: Pre-treat extracts with α-amylase followed by dialysis, or include a no-primer control

• ADP-glucose pyrophosphorylase contamination:

  • Problem: Contaminating ADP-glucose pyrophosphorylase activity can generate ADP-glucose from ATP and glucose-1-phosphate

  • Solution: Include pyrophosphate to inhibit the reverse reaction or use purified ADP-glucose

• Substrate depletion:

  • Problem: Rapid depletion of ADP-glucose during the assay can lead to non-linear kinetics

  • Solution: Use higher substrate concentrations or shorter assay times; monitor substrate consumption

• Oxidative inactivation:

  • Problem: Glycogen synthases are often sensitive to oxidation, particularly when purified

  • Solution: Include reducing agents (DTT, 2-mercaptoethanol) in all buffers; maintain anaerobic conditions when possible

• Inconsistent primer preparations:

  • Problem: Variability in glycogen primer structure can cause inconsistent results

  • Solution: Use a single batch of commercial glycogen or maltooligosaccharides of defined length

• Phosphatase activities:

  • Problem: Contaminating phosphatases can hydrolyze ADP-glucose

  • Solution: Include phosphatase inhibitors (fluoride, vanadate) in reaction mixtures

• Temperature sensitivity:

  • Problem: Activity can vary significantly with small temperature fluctuations

  • Solution: Use a temperature-controlled water bath; pre-equilibrate all components

By addressing these potential artifacts, researchers can obtain more reliable and reproducible measurements of glycogen synthase activity in their experiments with G. sulfurreducens glgA2.

How can recombinant glgA2 be used to study electron storage mechanisms in G. sulfurreducens?

Recombinant glgA2 provides a valuable tool for investigating the role of glycogen in electron storage and energy conservation in G. sulfurreducens. Advanced research applications include:

• Glycogen as electron reservoir:

  • Create glgA2 variants with altered catalytic properties

  • Monitor how changes in glycogen structure affect electron storage capacity

  • Investigate the dynamics of glycogen utilization during electron acceptor limitation

• Correlation with c-type cytochromes:

  • Examine how glgA2 expression correlates with expression of key c-type cytochromes (OmcB, OmcC, OmcZ, OmcS)

  • Investigate whether glycogen serves as an electron buffer when cytochrome-dependent pathways are saturated

  • Study how glycogen accumulation changes in response to cytochrome gene deletions

• Biofilm formation and electricity generation:

  • Analyze the distribution of glycogen in G. sulfurreducens biofilms using fluorescent probes

  • Correlate glycogen content with local redox potential and current generation

  • Investigate whether glycogen serves as a carbon and electron source in different biofilm regions

• Metabolic flux redirection:

  • Use glgA2 overexpression or deletion to redirect carbon flux

  • Study how altered glycogen metabolism affects electron flow to external acceptors

  • Quantify changes in cellular redox state in response to modified glgA2 activity

These applications contribute to our understanding of how G. sulfurreducens manages electron flow and energy conservation in its unique ecological niche.

What insights can glgA2 modification provide about G. sulfurreducens' adaptation to fluctuating environmental conditions?

Modifications to glgA2 can serve as a window into G. sulfurreducens' adaptive strategies under fluctuating environmental conditions:

• Nutrient limitation response:

  • Compare wild-type and glgA2-modified strains under various nutrient limitation scenarios

  • Determine how glycogen accumulation patterns change in response to carbon, nitrogen, or phosphate limitation

  • Investigate the relationship between glgA2 expression and stress response genes like RelGsu

• Redox fluctuation adaptation:

  • Expose glgA2-modified strains to cycling aerobic/anaerobic conditions

  • Monitor glycogen metabolism during transitions between different electron acceptors

  • Analyze how glycogen serves as a buffer during periods of electron acceptor limitation

• Survival under stress conditions:

  • Test long-term survival of glgA2-modified strains under starvation conditions

  • Examine the role of glycogen in persistence during environmental stress

  • Investigate whether glycogen composition changes as part of the adaptive response

• Community interactions:

  • Study how glgA2 modification affects interactions in mixed microbial communities

  • Examine whether glycogen metabolism influences syntrophic relationships

  • Investigate if glycogen serves as a competitive advantage in microbial communities

By exploring these aspects, researchers can gain deeper insights into the ecological strategies employed by G. sulfurreducens and the role of glycogen metabolism in environmental adaptation.

How might glgA2 engineering contribute to optimizing bioremediation applications of G. sulfurreducens?

Engineering glgA2 holds potential for enhancing G. sulfurreducens' bioremediation capabilities through several mechanisms:

• Enhanced stress tolerance:

  • Optimize glycogen reserves to improve survival during bioremediation processes

  • Develop strains with modified glgA2 that maintain activity under contaminated site conditions

  • Increase resilience to fluctuating redox conditions common in contaminated environments

• Improved metal reduction capacity:

  • Engineer glgA2 to synchronize glycogen metabolism with metal reduction pathways

  • Optimize energy reserves to sustain metal reduction during carbon limitation periods

  • Create strains with enhanced electron storage capacity for continuous metal reduction

• Extended longevity in field applications:

  • Modify glycogen structure to provide slow-release energy reserves

  • Develop strains with controlled glycogen mobilization for sustained activity

  • Optimize carbon allocation between growth and energy storage for long-term remediation

• Integration with bioelectrochemical systems:

  • Engineer glgA2 to enhance electricity generation in microbial fuel cells

  • Optimize glycogen metabolism to support sustained current production

  • Develop strains with improved electron transfer from glycogen to electrodes

A particularly promising approach is a two-stage production process, where in the first stage, engineered strains accumulate high glycogen levels (though not using glgA2 overexpression, which causes cell death), followed by a second stage where this glycogen is utilized to drive enhanced bioremediation activity or product formation .

What are the most promising avenues for future research on G. sulfurreducens glgA2?

Future research on G. sulfurreducens glgA2 should focus on several key areas that promise significant advances in our understanding:

• Structural biology:

  • Determine the crystal structure of glgA2 to elucidate its unique catalytic mechanism

  • Investigate protein-protein interactions between glgA2 and other components of the glycogen synthesis machinery

  • Explore the structural basis for the apparent functional differences between glgA2 and glgA1

• Regulatory networks:

  • Map the transcriptional and post-translational regulation of glgA2

  • Identify environmental signals that modulate glgA2 activity

  • Elucidate the coordination between glycogen metabolism and electron transfer pathways

• Synthetic biology applications:

  • Develop glgA2 variants with enhanced or altered catalytic properties

  • Create synthetic regulatory circuits to control glycogen metabolism

  • Engineer strains with optimized carbon allocation between growth and storage

• Systems biology integration:

  • Incorporate glycogen metabolism into genome-scale metabolic models of G. sulfurreducens

  • Perform flux balance analysis to predict optimal glgA2 expression levels

  • Develop predictive models of how glycogen metabolism influences electron transfer

These research directions will not only advance our fundamental understanding of G. sulfurreducens biology but also enable new applications in bioremediation, bioelectrochemical systems, and sustainable bioproduction.

How might emerging technologies enhance our understanding of glgA2 function?

Emerging technologies offer exciting opportunities to deepen our understanding of glgA2 function in G. sulfurreducens:

• CRISPR-Cas9 genome editing:

  • Create precise modifications to glgA2 coding and regulatory regions

  • Develop inducible CRISPRi systems for temporal control of glgA2 expression

  • Generate libraries of glgA2 variants for high-throughput functional screening

• Single-cell techniques:

  • Apply single-cell transcriptomics to analyze glgA2 expression heterogeneity

  • Use microfluidic systems to study glycogen dynamics at the single-cell level

  • Develop fluorescent sensors for real-time monitoring of glycogen metabolism

• Advanced imaging:

  • Implement super-resolution microscopy to visualize glycogen granule formation

  • Use correlative light and electron microscopy to link glycogen structure with cellular ultrastructure

  • Apply Raman microscopy for label-free chemical imaging of glycogen in living cells

• Artificial intelligence and machine learning:

  • Develop predictive models of glgA2 function based on sequence and structural data

  • Use machine learning to identify patterns in multi-omics datasets related to glycogen metabolism

  • Apply AI-driven experimental design to optimize glgA2 engineering strategies

By leveraging these technologies, researchers can overcome current limitations in studying glgA2 and develop more comprehensive models of glycogen metabolism in G. sulfurreducens.

What interdisciplinary approaches might yield new insights into glgA2 biology?

Interdisciplinary approaches hold great promise for advancing our understanding of glgA2 biology in G. sulfurreducens:

• Biogeochemistry and environmental microbiology:

  • Investigate how glgA2 function relates to G. sulfurreducens' role in environmental metal cycling

  • Study glycogen dynamics in natural communities containing Geobacter species

  • Examine how environmental parameters influence glycogen metabolism in field settings

• Biophysics and electrochemistry:

  • Apply electrochemical techniques to study the relationship between glycogen metabolism and electron transfer

  • Develop theoretical models of how glycogen serves as an electron capacitor

  • Investigate the thermodynamics of glycogen-mediated energy conservation

• Synthetic biology and metabolic engineering:

  • Design synthetic pathways that interface with glgA2-dependent glycogen synthesis

  • Engineer hybrid energy storage systems combining glycogen with other compounds

  • Develop tunable control systems for glycogen metabolism

• Computational biology and systems modeling:

  • Create multi-scale models linking glycogen metabolism to cellular physiology

  • Apply flux balance analysis to predict optimal glycogen allocation strategies

  • Develop agent-based models of how glycogen metabolism influences biofilm formation and function