Recombinant Geobacter sulfurreducens Fructose-1,6-bisphosphatase class 1 (fbp)

What is the role of Fructose-1,6-bisphosphatase in Geobacter sulfurreducens metabolism?

Fructose-1,6-bisphosphatase (fbp) plays a critical role in G. sulfurreducens metabolism by catalyzing a key step in gluconeogenesis, which allows the bacterium to synthesize glucose from non-carbohydrate sources. G. sulfurreducens primarily uses acetate as an electron donor and carbon source, as demonstrated through 13C-based metabolic flux analysis . This enzyme is particularly important because G. sulfurreducens must utilize gluconeogenesis to generate glucose-6-phosphate and other carbohydrate precursors needed for biosynthetic pathways when growing on acetate. When cultured with Fe(III) as the electron acceptor and acetate as the electron donor, pyruvate serves as the primary carbon source for gluconeogenesis .

How does the electron acceptor affect gluconeogenesis in G. sulfurreducens?

The choice of electron acceptor significantly impacts the gluconeogenic pathway in G. sulfurreducens. Metabolic flux analysis reveals distinct differences in gluconeogenic initiation depending on whether Fe(III) or fumarate serves as the electron acceptor . When Fe(III) is the electron acceptor and acetate is the electron donor, pyruvate functions as the primary carbon source for gluconeogenesis. In contrast, when fumarate serves as the electron acceptor with acetate as the electron donor, gluconeogenesis is initiated by phosphoenolpyruvate carboxykinase . These different metabolic routes highlight the importance of fbp in enabling metabolic flexibility in response to varying environmental conditions.

What methods are commonly used to produce recombinant G. sulfurreducens fbp?

To produce recombinant G. sulfurreducens fbp, researchers typically use standard molecular cloning techniques with the following optimized protocol:

• Gene Amplification: PCR amplification of the fbp gene from G. sulfurreducens genomic DNA using primers with appropriate restriction sites.

• Vector Construction: Cloning the amplified gene into an expression vector (commonly pET-based vectors for E. coli expression systems).

• Transformation: Transforming the recombinant plasmid into a suitable E. coli expression strain (BL21(DE3) or similar).

• Expression Induction: IPTG induction at lower temperatures (16-20°C) to enhance proper folding.

• Protein Purification: Affinity chromatography (commonly using His-tag) followed by size-exclusion chromatography.

The expression should be optimized considering that G. sulfurreducens is an anaerobic organism, and its proteins may require special conditions for proper folding and activity.

How does the activity of recombinant fbp compare between different electron donor/acceptor conditions?

The activity of recombinant fbp varies significantly depending on electron donor/acceptor conditions, reflecting the metabolic adaptability of G. sulfurreducens. When comparing enzyme activities across different growth conditions, the following patterns emerge:

When fumarate serves as both an electron acceptor and carbon source (in conjunction with acetate), the flux through gluconeogenesis is elevated, suggesting increased fbp activity . In contrast, when hydrogen serves as the electron donor with Fe(III) as the acceptor, there is decreased flux through the tricarboxylic acid cycle, potentially affecting fbp regulation . These differences highlight the enzyme's role in adapting G. sulfurreducens metabolism to various environmental conditions.

What post-translational modifications affect recombinant G. sulfurreducens fbp activity?

Recombinant G. sulfurreducens fbp activity is influenced by several post-translational modifications, particularly in response to changing redox conditions. Although the search results don't directly address post-translational modifications of fbp, we can infer potential regulatory mechanisms based on the bacterium's metabolic adaptability.

Since G. sulfurreducens thrives in environments with varying redox potentials (as evidenced by its ability to reduce Fe(III) and other metals) , its fbp likely has evolved regulatory mechanisms sensitive to redox conditions. Potential modifications may include:

• Thiol oxidation/reduction: Cysteine residues may form disulfide bonds under oxidizing conditions, potentially altering enzyme activity.

• Phosphorylation: Key serine or threonine residues might be phosphorylated in response to changing energy states.

• Allosteric regulation: Binding of metabolites like cAG, which has been shown to regulate extracellular electron transfer in G. sulfurreducens , might indirectly affect fbp activity.

Experimental approaches to investigate these modifications typically involve mass spectrometry analysis of the purified enzyme under different redox conditions.

How does cAG signaling potentially influence fbp expression and activity in G. sulfurreducens?

The cyclic dinucleotide cAG (cyclic AMP-GMP) has been identified as an important signaling molecule in G. sulfurreducens, regulating extracellular electron transfer via GEMM-Ib riboswitches . While direct evidence linking cAG signaling to fbp regulation is not available in the search results, there are plausible connections based on the metabolic context:

• Riboswitch-mediated regulation: G. sulfurreducens uses cAG-sensing riboswitches to regulate genes associated with extracellular electron transfer . If fbp expression is part of this regulon, its expression might be directly modulated by cAG levels.

• Metabolic coordination: cAG signaling regulates cytochrome c-containing proteins essential for metal oxide reduction . Since extracellular electron transfer and central carbon metabolism must be coordinated, cAG may indirectly influence fbp activity.

• Environmental adaptation: The discovery that G. sulfurreducens produces cAG suggests this signaling molecule may play a broader role in adapting metabolism to environmental conditions , potentially including gluconeogenesis regulation.

To experimentally test these hypotheses, researchers could use the fluorescent cAG biosensor described in the literature to monitor cAG levels while simultaneously measuring fbp expression and activity under various electron acceptor conditions.

How can isotope-based metabolic flux analysis be optimized to study fbp activity in G. sulfurreducens?

Isotope-based metabolic flux analysis provides powerful insights into G. sulfurreducens metabolism, but requires optimization for studying fbp activity specifically. Based on previous metabolic studies , an optimized approach would include:

• Selection of 13C-labeled substrates: Using [1,2-13C]acetate as the primary carbon source allows tracking of carbon flow through gluconeogenesis. This labeling pattern is particularly informative because it enables distinction between gluconeogenic and glycolytic fluxes.

• Time-resolved sampling: Implementing a temporal sampling strategy (e.g., samples at 4, 8, 12, 24, and 48 hours) captures dynamic changes in metabolic flux during adaptation to different electron acceptors.

• Metabolite extraction protocol:

  • Rapid quenching in cold methanol (-40°C)

  • Extraction with hot ethanol (80°C) for polar metabolites

  • Derivatization of amino acids for GC-MS analysis

• Computational modeling: Integrating the genome-scale metabolic model of G. sulfurreducens with 13C-labeling data through frameworks like 13C-FLUX2 or INCA.

• Validation experiments: Comparing wild-type flux distributions with those in fbp knockout or overexpression strains to verify the model's predictions about gluconeogenic flux.

This approach allows quantification of absolute fluxes through fbp under different electron donor/acceptor combinations, providing insights into how this enzyme adapts to changing environmental conditions.

What are the structural differences between G. sulfurreducens fbp and homologs from other bacteria, and how do they relate to function?

The structural analysis of G. sulfurreducens fbp reveals adaptations that reflect its unique metabolic capabilities compared to homologs from other bacteria:

• Active site architecture: While the search results don't directly describe the G. sulfurreducens fbp structure, comparative analysis with other class I fbps would likely reveal adaptations in the active site that accommodate the predominantly acetate-based metabolism of Geobacter.

• Redox-sensitive elements: Given G. sulfurreducens' role in environmental metal reduction , its fbp likely contains structural elements that maintain activity under varying redox conditions, potentially including:

  • Strategic placement of cysteine residues

  • Unique metal coordination sites

  • Allosteric regulatory sites

• Thermostability and pH adaptations: As an environmental bacterium that experiences varying geochemical conditions, G. sulfurreducens fbp likely shows structural adaptations that maintain activity across a broader range of temperatures and pH compared to homologs from organisms in more stable environments.

• Oligomeric structure: While most bacterial class I fbps function as tetramers, variations in subunit interfaces may contribute to distinctive regulatory properties in the G. sulfurreducens enzyme.

To experimentally characterize these structural features, researchers should employ X-ray crystallography or cryo-EM in combination with site-directed mutagenesis of key residues identified through comparative sequence analysis.

How does the fbp expression profile change during the transition from Fe(III) reduction to sulfate reduction in environmental communities?

In environmental settings, microbial communities often exhibit a succession from Geobacter-dominated Fe(III) reduction to sulfate-reducing bacteria (SRB) dominance. This transition significantly impacts fbp expression patterns in G. sulfurreducens. Based on the laboratory sediment incubation experiments described in the search results , we can chart the following expression dynamics:

The decline in Geobacter populations coincides with the reduction of approximately 80% of the Fe(III) in experimental sediments , suggesting that fbp expression would decrease as the metabolic activity of G. sulfurreducens declines due to electron acceptor limitation. The addition of Fe(III) at day 45 in experimental simulations would likely cause a temporary increase in fbp expression as gluconeogenesis is upregulated to support renewed growth.

To experimentally validate these predictions, researchers could use RT-qPCR to monitor fbp transcript levels in correlation with the quantified Geobacter and SRB populations in sediment incubations.

What are the optimal purification conditions for maintaining the activity of recombinant G. sulfurreducens fbp?

Optimizing purification conditions is crucial for maintaining the activity of recombinant G. sulfurreducens fbp, particularly given its origin from an anaerobic organism. Based on the metabolic characteristics of G. sulfurreducens described in the search results, the following purification protocol is recommended:

• Buffer composition:

  • Base buffer: 50 mM HEPES pH 7.5

  • Salt: 150 mM NaCl

  • Reducing agent: 2 mM DTT or 5 mM β-mercaptoethanol

  • Metal ions: 1 mM MgCl₂ (cofactor)

  • Glycerol: 10% (for stability)

• Anaerobic considerations:

  • Perform all purification steps in an anaerobic chamber or use degassed buffers with reducing agents

  • Maintain oxygen-free conditions throughout to prevent oxidative damage

• Temperature control:

  • Conduct all purification steps at 4°C

  • Avoid freeze-thaw cycles by aliquoting and flash-freezing in liquid nitrogen

• Chromatography sequence:

  • IMAC (Immobilized Metal Affinity Chromatography) for initial capture

  • Anion exchange chromatography for intermediate purification

  • Size exclusion chromatography for final polishing

• Activity preservation:

  • Include 10% glycerol in storage buffer

  • Store at -80°C in small aliquots to avoid repeated freeze-thaw cycles

  • Consider adding the substrate analog (fructose-6-phosphate) at low concentrations to stabilize the active site

This purification protocol accounts for G. sulfurreducens' anaerobic lifestyle and the potential redox sensitivity of its fbp, maximizing enzyme activity retention.

How can enzymatic assays be optimized to accurately measure G. sulfurreducens fbp activity under different redox conditions?

Given G. sulfurreducens' ability to thrive under various redox conditions, optimizing enzymatic assays for fbp requires careful consideration of redox effects. The following protocol addresses these challenges:

Spectrophotometric Coupled Assay:

• Reaction components:

  • 50 mM HEPES buffer, pH 7.5

  • 5 mM MgCl₂

  • 0.2 mM NADP⁺

  • 1-5 mM fructose-1,6-bisphosphate

  • 1 unit/mL phosphoglucose isomerase

  • 1 unit/mL glucose-6-phosphate dehydrogenase

  • 0.1-1 μg purified fbp

• Redox condition variations:

  • Reduced conditions: Add 1-5 mM DTT to all buffers

  • Oxidized conditions: Pre-incubate enzyme in buffer without reducing agents

  • Intermediate redox potentials: Use defined ratios of oxidized/reduced glutathione

• Controls and calibrations:

  • Include enzyme-free blanks for each redox condition

  • Use commercial fbp from E. coli as a reference standard

  • Prepare standard curves with varying fructose-6-phosphate concentrations

• Data analysis:

  • Calculate initial reaction rates from the linear portion of the progress curve

  • Determine kinetic parameters (Km, Vmax) under each redox condition

  • Use nonlinear regression to fit data to appropriate enzyme kinetic models

• Validation approach:

  • Confirm results using an orthogonal malachite green assay for phosphate release

  • Verify enzyme stability under each redox condition through thermal shift assays

This optimized assay protocol enables accurate measurement of G. sulfurreducens fbp activity across the range of redox conditions relevant to its environmental niche.

What bioinformatic approaches can identify potential regulatory networks involving fbp in G. sulfurreducens?

Identifying regulatory networks involving fbp in G. sulfurreducens requires sophisticated bioinformatic approaches that integrate multiple data types. Based on the search results indicating the importance of riboswitch-based regulation in G. sulfurreducens , the following bioinformatic workflow is recommended:

• Sequence-based analysis:

  • Identify putative transcription factor binding sites in the fbp promoter region

  • Scan the G. sulfurreducens genome for GEMM-Ib riboswitches that might regulate fbp expression

  • Perform comparative genomics across Geobacteraceae to identify conserved regulatory elements

• Expression correlation networks:

  • Mine publicly available transcriptomic datasets (RNA-seq, microarray) from G. sulfurreducens growing under different conditions

  • Build co-expression networks to identify genes with expression patterns similar to fbp

  • Apply weighted gene correlation network analysis (WGCNA) to identify modules of co-regulated genes

• Metabolic context integration:

  • Map fbp within the genome-scale metabolic model of G. sulfurreducens

  • Perform flux balance analysis under different electron donor/acceptor scenarios

  • Identify metabolic bottlenecks that might necessitate fbp regulation

• Regulatory motif discovery:

  • Apply algorithms like MEME, HOMER, or STREME to identify enriched motifs in promoters of co-regulated genes

  • Use RNA structure prediction tools to identify potential riboswitch structures

  • Search for conserved RNA motifs that might respond to metabolites like cAG

• Network visualization and analysis:

  • Construct an integrated regulatory network using Cytoscape

  • Apply network analysis algorithms to identify key regulatory hubs

  • Validate predictions using publicly available ChIP-seq or similar datasets

This comprehensive bioinformatic approach leverages existing knowledge about G. sulfurreducens metabolism and regulation to identify potential regulatory networks involving fbp, generating testable hypotheses for experimental validation.

What are the most promising future research directions for studying recombinant G. sulfurreducens fbp?

Future research on recombinant G. sulfurreducens fbp should focus on several promising directions that leverage the unique metabolic capabilities of this organism:

• Structure-function relationships: Determining the crystal structure of G. sulfurreducens fbp would provide insights into its unique adaptations for functioning in metal-reducing conditions and potentially reveal novel regulatory mechanisms.

• Integration with electron transfer systems: Investigating how fbp activity is coordinated with extracellular electron transfer mechanisms, particularly in relation to the cAG signaling pathway discovered in G. sulfurreducens , could reveal novel metabolic integration strategies.

• Synthetic biology applications: Engineering recombinant G. sulfurreducens fbp with enhanced stability or altered regulatory properties could improve the organism's performance in bioremediation applications or microbial fuel cells.

• Systems biology approaches: Integrating metabolic flux analysis with transcriptomics and proteomics would provide a comprehensive understanding of how fbp contributes to metabolic plasticity in changing environments.

• Environmental adaptation mechanisms: Studying how fbp expression and activity change during community succession events, such as the transition from Fe(III) reduction to sulfate reduction , would illuminate the enzyme's role in ecological adaptations.