Recombinant Geobacter sulfurreducens 2-C-methyl-D-erythritol 2,4-cyclodiphosphate synthase (ispF)

What genetic tools are available for expressing recombinant proteins in G. sulfurreducens?

G. sulfurreducens offers several well-established genetic tools for protein expression studies. A comprehensive genetic system has been developed that includes protocols for antibiotic sensitivity characterization and optimal plating conditions. Foreign DNA can be introduced into G. sulfurreducens through electroporation, with two classes of broad-host-range vectors - IncQ and pBBR1 - demonstrating successful replication in this organism . The IncQ plasmid pCD342 has proven particularly effective as an expression vector for G. sulfurreducens, making it suitable for ispF expression studies . To introduce recombinant constructs, researchers should optimize electroporation parameters according to established protocols, typically using freshly harvested cells in exponential growth phase with careful post-electroporation recovery in specialized media.

How does G. sulfurreducens serve as a model organism for studying bacterial enzymes like ispF?

G. sulfurreducens functions as an excellent model organism for several reasons relevant to enzyme studies. It serves as the primary model for investigating extracellular electron transport mechanisms within the Geobacter species . The organism has significant biogeochemical and technological applications, including reduction of Fe(III) oxides in soils, bioelectrochemical applications for generating electric current from organic waste, and driving useful processes with renewable electricity . These diverse metabolic capabilities make it valuable for studying enzymes involved in central metabolism and biosynthetic pathways like the MEP pathway where ispF functions. Its well-characterized genome and established genetic manipulation techniques allow for targeted gene deletions and complementation studies to elucidate enzyme function in vivo .

What are the key considerations when designing primers for cloning the ispF gene from G. sulfurreducens?

When designing primers for cloning the ispF gene from G. sulfurreducens, several critical factors must be considered to ensure successful amplification and subsequent protein expression:

• Codon optimization: Review the G. sulfurreducens genome for codon usage patterns that might affect heterologous expression.

• Restriction site selection: Include appropriate restriction sites that are absent in the target gene but present in your chosen expression vector. The IncQ plasmid pCD342 has proven suitable as an expression vector for G. sulfurreducens and should be considered when selecting compatible restriction sites .

• Tag incorporation: Design primers to include sequences for affinity tags (His, FLAG, etc.) if protein purification is planned. Notably, immunogold labeling has been successfully used with His-tagged proteins in G. sulfurreducens, suggesting this approach is viable for ispF studies .

• Termination signals: Ensure proper transcriptional termination sequences are incorporated to prevent read-through transcription.

• Annealing temperature optimization: Design primers with similar melting temperatures (Tm), ideally between 55-65°C, to facilitate efficient PCR amplification under anaerobic conditions often required for G. sulfurreducens work.

What expression conditions optimize the production of functionally active recombinant ispF from G. sulfurreducens?

Optimizing expression conditions for functionally active recombinant ispF requires careful consideration of multiple parameters:

To confirm functional activity, enzyme assays should be performed using purified protein to measure the conversion of 4-diphosphocytidyl-2-C-methyl-D-erythritol 2-phosphate to 2-C-methyl-D-erythritol 2,4-cyclodiphosphate. Activity can be monitored through coupled assays or direct product detection using HPLC or LC-MS methods.

How can genetic knockout/complementation strategies be implemented to study ispF function in G. sulfurreducens?

Implementing genetic knockout/complementation strategies for ispF in G. sulfurreducens requires a systematic approach:

For gene knockout:

• Design homologous recombination constructs with 500-1000 bp flanking regions surrounding the ispF gene.

• Clone these regions into a suicide vector containing a selectable marker (e.g., kanamycin resistance).

• Introduce the construct into G. sulfurreducens via electroporation using established protocols .

• Select transformants on appropriate antibiotics and confirm gene deletion through PCR and sequencing.

• Validate the phenotype by assessing growth rates, metabolic profiles, and specific pathway intermediates.

For complementation:

• Clone the wild-type ispF gene into the IncQ plasmid pCD342, which has been shown effective for G. sulfurreducens .

• Introduce the complementation vector into the knockout strain via electroporation.

• Select transformants and verify expression through RT-PCR or Western blotting.

• Assess restoration of phenotype through growth analysis and metabolic profiling.

This approach mirrors successful strategies used for other G. sulfurreducens genes, such as the nifD gene knockout and complementation, which demonstrated the nitrogen fixation capabilities of this organism . Similar approaches with the fumarate reductase gene (frdCAB) have revealed dual functionality in central metabolism .

What are the challenges in purifying recombinant ispF from G. sulfurreducens and how can they be addressed?

Purifying recombinant ispF from G. sulfurreducens presents several challenges that require specialized approaches:

• Anaerobic conditions: G. sulfurreducens is an anaerobe, necessitating oxygen-free purification methods. Solution: Perform all purification steps in an anaerobic chamber with oxygen scavengers in buffers.

• Membrane association: If ispF associates with membranes, solubilization becomes difficult. Solution: Screen detergents (CHAPS, DDM, Triton X-100) at varying concentrations for optimal extraction without denaturation.

• Low expression levels: Bacterial enzyme expression can be limited. Solution: Optimize codon usage for G. sulfurreducens and utilize the IncQ plasmid pCD342, which has been established as an effective expression vector for this organism .

• Protein stability: Enzymes from G. sulfurreducens may have specific stability requirements. Solution: Include stabilizing agents (glycerol 10-20%, reducing agents like DTT) in all buffers.

• Co-purifying contaminants: G. sulfurreducens produces conductive pili and extracellular appendages that may contaminate preparations. Solution: Implement multiple purification steps, combining affinity chromatography (using His-tag labeling, which has been successfully demonstrated in G. sulfurreducens ) with size exclusion and ion exchange methods.

• Activity preservation: Maintaining enzyme function throughout purification is critical. Solution: Supplement buffers with substrate analogs or stabilizing cofactors and minimize time between purification steps.

How can enzyme kinetics of recombinant ispF from G. sulfurreducens be accurately measured?

Accurately measuring enzyme kinetics of recombinant ispF requires specialized approaches that address the unique challenges of this enzyme:

• Substrate preparation: Synthesize or acquire pure 4-diphosphocytidyl-2-C-methyl-D-erythritol 2-phosphate (CDP-ME2P) as the substrate for ispF.

• Reaction monitoring: The reaction can be monitored through:

  • Direct measurement of 2-C-methyl-D-erythritol 2,4-cyclodiphosphate formation using HPLC

  • Tracking pyrophosphate release using coupled enzyme assays

  • Employing LC-MS for direct product quantification

• Anaerobic considerations: Prepare all buffers and reagents under anaerobic conditions, as G. sulfurreducens proteins may be oxygen-sensitive. This parallels approaches used for characterizing other G. sulfurreducens enzymes .

• Optimal assay conditions:

• Data analysis: Apply Michaelis-Menten kinetics to determine KM and Vmax, with careful consideration of potential substrate inhibition effects. Plot both Lineweaver-Burk and Eadie-Hofstee transformations to identify any deviations from standard kinetic models.

• Controls: Include heat-denatured enzyme controls and reactions without substrate or enzyme to account for background and non-enzymatic reactions.

What structural analysis techniques are most appropriate for characterizing recombinant G. sulfurreducens ispF?

For comprehensive structural characterization of recombinant G. sulfurreducens ispF, multiple complementary techniques should be employed:

• X-ray crystallography: The gold standard for atomic-level resolution, requiring:

  • High-purity protein (>95%) at concentrations of 5-15 mg/mL

  • Screening of 500-1000 crystallization conditions

  • Optimization of crystal growth parameters

  • Data collection at synchrotron facilities for maximum resolution

• Small-angle X-ray scattering (SAXS): For solution-state structural information:

  • Provides insights into protein shape, size, and oligomerization

  • Requires monodisperse protein samples (verified by dynamic light scattering)

  • Can reveal conformational changes upon substrate binding

• Circular dichroism (CD) spectroscopy: For secondary structure content:

  • Far-UV CD (190-250 nm) for α-helix and β-sheet content

  • Near-UV CD (250-350 nm) for tertiary structure fingerprinting

  • Thermal denaturation studies to assess stability

• Mass spectrometry-based approaches:

  • Hydrogen-deuterium exchange mass spectrometry (HDX-MS) for conformational dynamics

  • Native MS for oligomerization state determination

  • Cross-linking MS for interface mapping

• Molecular modeling:

  • Homology modeling based on known ispF structures from other bacteria

  • Molecular dynamics simulations to explore conformational flexibility

  • Docking studies with substrates and potential inhibitors

This multi-technique approach follows the pattern of thorough characterization seen in other G. sulfurreducens protein studies, such as the detailed analyses performed on extracellular electron transfer components .

How should conflicting experimental results regarding ispF function in G. sulfurreducens be reconciled?

Reconciling conflicting experimental results regarding ispF function requires a systematic approach similar to that used in resolving contradictory findings in G. sulfurreducens research:

• Verify genetic constructs: Confirm the integrity of all genetic modifications through sequencing and expression analysis. This is particularly important as gene deletion studies in G. sulfurreducens have sometimes produced unexpected results, as demonstrated in research on pilB deletion mutants where phenotypes differed from predictions .

• Cross-validate with multiple techniques: When conflicting results emerge, employ orthogonal methods to confirm findings. For example, if enzyme activity assays show contradictory results, combine biochemical assays with in vivo metabolite profiling and genetic complementation studies.

• Examine experimental conditions: Minor variations in growth conditions, media composition, or electron acceptors can significantly impact G. sulfurreducens metabolism. The organism's ability to switch between electron acceptors like fumarate and Fe(III) can influence central metabolism , potentially affecting ispF function indirectly.

• Consider genetic compensation: G. sulfurreducens may possess redundant pathways or compensatory mechanisms. For example, the discovery that G. sulfurreducens uses the same enzyme for both fumarate reduction and succinate oxidation demonstrates metabolic versatility not seen in other organisms .

• Evaluate strain background effects: Different laboratory strains of G. sulfurreducens may contain genetic differences that influence experimental outcomes. Document the specific strain lineage and any known modifications.

• Publish comprehensive methods: Ensure all experimental parameters are fully documented to allow proper reproduction by other laboratories, following the example of studies that resolved contradictory findings regarding the role of pilB in G. sulfurreducens extracellular electron transfer .

What bioinformatic approaches can predict functional partners of ispF in G. sulfurreducens metabolic networks?

Predicting functional partners of ispF in G. sulfurreducens metabolic networks requires sophisticated bioinformatic approaches:

• Co-expression analysis:

  • Analyze transcriptomic data across different growth conditions

  • Identify genes with expression patterns closely correlated with ispF

  • Focus particularly on genes upregulated under conditions where isoprenoid production would be essential

• Genomic context analysis:

  • Examine the chromosomal neighborhood of ispF in G. sulfurreducens

  • Look for conserved gene clusters across related species

  • Identify operonic structures that may indicate functional relationships

• Protein-protein interaction prediction:

  • Employ algorithms like STRING, PSICQUIC, and InterPreTS

  • Focus on domain-domain interaction predictions

  • Use homology-based inference from known interactions in related species

• Metabolic pathway reconstruction:

  • Map ispF within the MEP pathway in G. sulfurreducens

  • Identify upstream suppliers and downstream consumers of metabolites

  • Look for potential regulatory connections to electron transport systems

• Phylogenetic profiling:

  • Identify genes with similar phylogenetic distributions to ispF

  • Focus on genes that co-occur specifically in organisms with similar metabolic capabilities

  • Pay special attention to genes that co-evolve in delta-proteobacteria

• Structural modeling for interaction prediction:

  • Generate homology models of ispF and potential partners

  • Perform protein-protein docking simulations

  • Validate predictions with co-immunoprecipitation or bacterial two-hybrid assays

This comprehensive approach mirrors successful strategies used to elucidate metabolic networks in G. sulfurreducens, such as those that revealed the dual functionality of the fumarate reductase/succinate dehydrogenase system .

How can isotope labeling be used to trace ispF-dependent metabolic pathways in G. sulfurreducens?

Isotope labeling provides powerful insights into metabolic pathways involving ispF in G. sulfurreducens:

• 13C-glucose labeling pattern design:

  • Feed G. sulfurreducens with 13C-labeled glucose at specific carbon positions

  • Track isotope distribution in MEP pathway intermediates using LC-MS/MS

  • Analyze labeling patterns to determine carbon flux through the ispF-catalyzed reaction

• Metabolic flux analysis (MFA):

  • Develop a metabolic model incorporating the MEP pathway

  • Measure isotopic enrichment in pathway intermediates and products

  • Calculate flux distributions using computational algorithms like 13CFLUX2

  • Compare flux distributions between wild-type and ispF-modified strains

• Time-course experiments:

  • Capture dynamic metabolic responses by sampling at multiple time points after isotope introduction

  • Determine the order of label incorporation in pathway intermediates

  • Calculate turnover rates and pool sizes of MEP pathway metabolites

• Orthogonal labeling strategies:

  • Combine 13C labeling with 2H or 15N labeling

  • Use multiple isotope tracers to simultaneously track different aspects of metabolism

  • Apply advanced MS techniques to resolve complex labeling patterns

• In vivo and in vitro comparison:

  • Perform parallel isotope tracing in whole cells and with purified enzymes

  • Identify potential differences in pathway regulation

  • Detect any unforeseen metabolic branching

This metabolic tracing approach follows methodological principles similar to those employed in studies of other G. sulfurreducens metabolic pathways, such as the investigations that revealed the bifunctionality of fumarate reductase/succinate dehydrogenase .

What are the implications of ispF function for electron transfer mechanisms in G. sulfurreducens?

Understanding the relationship between ispF and electron transfer mechanisms in G. sulfurreducens requires investigation of several potential connections:

• Isoprenoid-derived electron carriers:

  • Isoprenoids form the side chains of menaquinones and ubiquinones

  • These quinones function as electron carriers in respiratory chains

  • Modulation of ispF activity could directly impact electron carrier availability

• Membrane composition effects:

  • Isoprenoids contribute to membrane lipid biosynthesis

  • Membrane composition affects the organization and function of electron transport complexes

  • Changes in ispF activity may alter membrane properties critical for extracellular electron transfer

• Energy conservation coupling:

  • The MEP pathway consumes ATP and reducing equivalents

  • This energy demand may compete with electron transfer processes

  • Co-regulation may exist to balance these metabolic requirements

• Biofilm formation impacts:

  • Isoprenoid-derived molecules may influence cell-cell communication

  • Biofilm structure significantly affects extracellular electron transfer efficiency

  • ispF activity could indirectly modulate community electron transfer capabilities

• Experimental approaches:

  • Compare electron transfer rates in wild-type versus ispF-modified strains

  • Measure current production in bioelectrochemical systems

  • Analyze cellular redox state during ispF modulation

This research direction builds upon established understanding of G. sulfurreducens as a model for extracellular electron transport mechanisms and the organism's importance in bioelectrochemical applications .