Recombinant Geobacter sulfurreducens 8-amino-7-oxononanoate synthase (bioF)

What is Geobacter sulfurreducens and why is it significant for bioelectrochemical research?

Geobacter sulfurreducens is a metal-reducing microorganism predominantly found in anaerobic subsurface environments. It plays a critical role in the bioremediation of both organic and metal contaminants through its unique electron transfer mechanisms. G. sulfurreducens can transfer respiratory electrons to extracellular acceptors via direct contact with minerals such as iron and manganese oxides, making it particularly valuable for bioelectrochemical studies .

The significance of G. sulfurreducens extends to its nitrogen fixation capabilities, which may be essential for its competitive success in petroleum-contaminated subsurface environments that are carbon-rich but nitrogen-poor . This metabolic versatility, combined with its ability to reduce various metal oxides and interface with electrode surfaces, makes G. sulfurreducens a model organism for research in environmental microbiology and bioelectrochemical systems.

How has a genetic system been developed for G. sulfurreducens?

A comprehensive genetic system for G. sulfurreducens has been established through several methodological advancements:

• Characterization of antibiotic sensitivity profiles to identify suitable selection markers

• Establishment of optimal plating conditions for high efficiency colony formation

• Development of electroporation protocols for introducing foreign DNA

• Identification of compatible broad-host-range vectors, particularly IncQ and pBBR1 classes

• Validation of expression vectors like pCD342 for functional gene expression

These developments have enabled targeted gene disruptions, as demonstrated with the nifD gene, which eliminated G. sulfurreducens' ability to grow in media lacking fixed nitrogen . The genetic system also allows for complementation experiments, where restoration of function occurs through introduction of functional gene copies in trans, providing a powerful tool for gene characterization studies.

What is the function of 8-amino-7-oxononanoate synthase in biological systems?

8-Amino-7-oxononanoate synthase (AONS) catalyzes the first committed step in biotin biosynthesis. This pyridoxal 5′-phosphate-dependent enzyme performs a decarboxylative condensation reaction between L-alanine and pimeloyl-CoA, producing 8(S)-amino-7-oxononanoate, coenzyme A, and carbon dioxide in a stereospecific manner .

Additionally, AONS has been shown to catalyze the carboxylation of acetyl-CoA to produce malonyl-CoA, which represents the initial step in fatty acid biosynthesis . This dual functionality highlights the enzyme's versatility in metabolic pathways. The enzyme's role in biotin synthesis is particularly significant as biotin serves as an essential cofactor for carboxylases, decarboxylases, and transcarboxylases involved in various metabolic processes.

What vectors are suitable for expressing recombinant proteins in G. sulfurreducens?

For successful recombinant protein expression in G. sulfurreducens, researchers should consider the following vector systems:

• IncQ plasmids: These broad-host-range vectors have demonstrated reliable replication in G. sulfurreducens. In particular, pCD342 has proven effective as an expression vector for this organism .

• pBBR1-derived vectors: This class of broad-host-range vectors also replicates in G. sulfurreducens, providing an alternative platform for gene expression .

• pk18mobsacB vectors: These have been successfully employed for markerless gene deletions in G. sulfurreducens, as demonstrated in the creation of the pgcA deletion mutant .

• pRK2-Geo2 vectors: This system has been used for complementation testing, featuring constitutive promoters such as that from the G. sulfurreducens acpP gene (GSU1604) .

• Arabinose-inducible expression systems: Vectors like pBAD202/D-TOPO® have been adapted for inducible expression of G. sulfurreducens proteins with histidine tags for purification purposes .

The choice of vector should be based on specific experimental requirements, including the need for constitutive versus inducible expression, copy number considerations, and whether complementation or protein purification is the primary objective.

What is the optimal protocol for introducing foreign DNA into G. sulfurreducens?

Based on established protocols, the optimal method for introducing foreign DNA into G. sulfurreducens is electroporation . While specific electroporation parameters were not detailed in the search results, the following general methodology has proven effective:

• Development of an electrocompetent cell preparation protocol specific to G. sulfurreducens

• Optimization of DNA concentration and purity for transformation

• Fine-tuning of electroporation settings (voltage, resistance, and capacitance)

• Implementation of appropriate recovery conditions following electroporation

• Selection on suitable antibiotic-containing media based on the vector's resistance marker

For more complex genetic manipulations, such as markerless deletions, a two-step selection process has been established:

• Initial selection for recombinants using antibiotic resistance (e.g., kanamycin at 200 μg/mL)

• Secondary selection on sucrose (10%) plates to identify subsequent recombination events resulting in either gene deletion or reversion to wild type

• PCR verification of the desired genetic modification

This approach has been successfully employed to create deletion mutants such as ΔpgcA, demonstrating its effectiveness for targeted genetic manipulation in G. sulfurreducens.

How can the BioF tag be utilized for protein immobilization in biotechnology applications?

The BioF tag represents a versatile tool for protein immobilization on polyhydroxyalkanoate (PHA) supports, offering several methodological advantages:

• Direct protein immobilization: The BioF tag enables straightforward in vitro attachment of recombinant proteins to PHA particles, including poly-3-hydroxybutyrate (PHB), without requiring chemical crosslinking .

• Fusion protein strategy: BioF can be genetically fused to proteins of interest, creating chimeric proteins that retain both BioF binding properties and the functional activity of the target protein .

• Stability across conditions: BioF-tagged proteins demonstrate remarkable stability when bound to PHB across a wide range of experimental conditions:

  • Temperature stability from 25-37°C

  • pH stability from pH 2-9

  • Temporal stability for 48-96 hours

  • Resistance to various detergents

• Protection from degradation: The BioF-PHA interaction provides substantial protection against protein degradation, as demonstrated with BioF-C-LytA fusions .

• Modifiable binding strength: The strength of protein adsorption can be adjusted by varying the coating of the support material, allowing for controlled protein loading and release properties .

This immobilization system provides researchers with a robust platform for creating bioactive materials with tailored properties for various biotechnological applications.

What factors affect the binding of BioF-tagged proteins to PHA surfaces?

Several key factors influence the binding efficiency and stability of BioF-tagged proteins to PHA surfaces:

• Temperature: The BioF-PHB interaction remains stable between 25-37°C, making it compatible with most biological research applications .

• pH range: BioF binding is maintained across pH values from 2 to 9, providing exceptional versatility for different experimental conditions and potential applications in varying biochemical environments .

• Support coating: The binding strength of BioF-tagged proteins can be modulated by coating the PHA support with amphiphilic compounds, allowing researchers to fine-tune protein loading density .

• Incubation time: Stable binding is observed over extended periods (48-96 hours), enabling long-term experimental applications .

• Detergent presence: The BioF-PHA interaction demonstrates remarkable resistance to detergent action, maintaining binding integrity even under conditions that would typically disrupt protein-surface interactions .

• Surface chemistry of PHA: While BioF naturally recognizes medium-chain-length PHAs, it also binds effectively to short-chain-length PHAs like PHB, suggesting a broad substrate range that may extend to various PHA blends, copolymers, or chemically modified derivatives .

Understanding these parameters enables researchers to optimize immobilization protocols for specific experimental objectives, whether developing biocatalysts, biosensors, or other bioactive materials.

What experimental approaches demonstrate the enzymatic stability of BioF-immobilized proteins?

The enzymatic stability of BioF-immobilized proteins has been experimentally demonstrated through several methodological approaches:

• Continuous activity-plus-washing cycles: BioF-β-galactosidase immobilized in a minibioreactor maintained very stable enzymatic activity after multiple cycles of activity measurement followed by washing steps. This demonstrates both the stability of the enzyme in its immobilized state and the durability of the BioF-PHA interaction .

• Protection from self-degradation: Comparative studies have shown that proteins bound via the BioF tag are strongly protected from degradation compared to their free counterparts. This was specifically demonstrated with BioF-C-LytA, where the immobilized protein exhibited significantly enhanced stability over time .

• pH and temperature stability analysis: Functional testing across various pH values (2-9) and temperatures (25-37°C) has confirmed that BioF-immobilized enzymes retain catalytic activity under diverse conditions .

• Detergent resistance testing: Despite exposure to detergents that would typically denature proteins or disrupt protein-surface interactions, BioF-immobilized proteins maintained their binding and functional properties .

The following table summarizes key stability parameters for BioF-immobilized enzymes:

These experimental approaches collectively validate the BioF tag system as a robust platform for enzyme immobilization with exceptional stability characteristics.

How does the deletion of electron transfer components affect G. sulfurreducens function in different contexts?

The deletion of electron transfer components in G. sulfurreducens reveals fascinating insights into the specificity of extracellular electron transfer mechanisms. Research on the deletion of pgcA, which encodes a triheme c-type cytochrome, demonstrates context-dependent functional impacts:

• Fe(III) and Mn(IV) oxide reduction: Deletion of pgcA results in mutants unable to transfer electrons to insoluble Fe(III) and Mn(IV) oxides, indicating its essential role in these specific electron transfer pathways .

• Electrode and soluble Fe(III) reduction: Interestingly, the same ΔpgcA mutants retain full ability to respire to electrode surfaces and reduce soluble Fe(III) citrate. When cultivated using +0.24 V vs. SHE poised graphite electrodes, wild-type and ΔpgcA cells demonstrated nearly identical doubling times (5.6 h vs. 5.5 h) and reached similar current densities of approximately 550 μA/cm² .

• Complementation studies: The Fe(III) oxide reduction deficiency in ΔpgcA mutants can be restored either by:

  • Expressing pgcA from a constitutive promoter in trans

  • Adding purified PgcA protein to cell suspensions

• Functional specificity: Cyclic voltammetry scans over a wide potential range (-0.4 V to +0.3 V) showed no differences between wild-type and ΔpgcA mutants, further confirming that PgcA plays no role in electron transfer to electrodes at any redox potential .

These findings highlight an important distinction between the molecular mechanisms responsible for electron transfer to metal oxides versus those involved in electron transfer to poised electrodes. This suggests that G. sulfurreducens possesses multiple, specialized extracellular electron transfer pathways that can be selectively targeted for research or biotechnological applications.

What approaches can be used to study extracellular electron transfer mechanisms in G. sulfurreducens?

Investigation of extracellular electron transfer mechanisms in G. sulfurreducens requires a multi-faceted experimental approach:

• Markerless gene deletion methodology:

  • Using vectors like pk18mobsacB for creating clean deletions without antibiotic markers

  • Two-stage selection process with initial kanamycin selection followed by sucrose counter-selection

  • PCR verification of deletion mutants

• Complementation analysis:

  • Expressing deleted genes from plasmids with constitutive or inducible promoters

  • Testing restoration of phenotypes to confirm gene function

  • Using vectors like pRK2-Geo2 with constitutive promoters (e.g., from acpP gene)

• Heterologous expression and purification:

  • Expression of G. sulfurreducens proteins in alternate hosts (e.g., Shewanella oneidensis)

  • Fusion with affinity tags (His-tag) for purification

  • Structural and functional characterization of purified proteins

• Electron acceptor-specific activity assays:

  • Comparing growth and activity with different electron acceptors:

    • Fe(III) citrate (soluble)

    • Fe(III) oxide (insoluble)

    • Mn(IV) oxide

    • Poised electrodes at defined potentials

• Electrochemical characterization:

  • Chronoamperometry to measure current production over time

  • Cyclic voltammetry to identify redox-active components

  • Comparison of wild-type and mutant electrochemical signatures

• Protein-mineral interaction studies:

  • Testing binding of purified proteins to various mineral forms

  • Assessing acceleration of reduction rates upon protein addition

  • Determining specificity of protein-mineral interactions

These approaches collectively provide a comprehensive toolkit for dissecting the complex electron transfer mechanisms in G. sulfurreducens, enabling researchers to understand the specific roles of individual components in different environmental contexts.

How can researchers optimize expression and purification of recombinant proteins from G. sulfurreducens?

Optimizing expression and purification of recombinant proteins from G. sulfurreducens requires careful consideration of several factors:

• Vector selection:

  • For expression within G. sulfurreducens, IncQ plasmids like pCD342 have proven effective

  • For higher yield purposes, heterologous expression in organisms like Shewanella oneidensis may be advantageous, as demonstrated with PgcA

• Promoter optimization:

  • Constitutive promoters like that from the G. sulfurreducens acpP gene (GSU1604) provide reliable expression

  • Inducible systems (e.g., arabinose-inducible promoters) offer controlled expression levels

• Expression conditions:

  • Growth media composition affects protein expression levels

  • For anaerobic proteins, maintaining strict anaerobic conditions throughout cultivation is crucial

  • Temperature, pH, and growth phase must be optimized for each target protein

• Protein tagging strategies:

  • C-terminal histidine tags (6X-His) facilitate purification via affinity chromatography

  • When using BioF as an affinity tag, consider its strong binding to PHA supports for potential single-step purification

• Processing considerations:

  • Some proteins may undergo processing during expression, as observed with PgcA which was processed into a shorter 41 kDa form lacking the lipodomain when expressed in S. oneidensis

  • Verify protein integrity through mass spectrometry or N-terminal sequencing

• Purification approaches:

  • Immobilized metal affinity chromatography (IMAC) for His-tagged proteins

  • Ion exchange chromatography based on theoretical isoelectric point

  • Size exclusion chromatography for final polishing and buffer exchange

  • For extracellular proteins, initial concentration from culture supernatants may be required

• Activity preservation:

  • Include appropriate cofactors in purification buffers (e.g., pyridoxal 5′-phosphate for AONS)

  • Optimize buffer composition, pH, and ionic strength to maintain native protein conformation

  • Consider anaerobic purification methods for oxygen-sensitive proteins

Systematic optimization of these parameters will enhance the likelihood of obtaining functionally active recombinant proteins from G. sulfurreducens for subsequent characterization and application studies.

What experimental design is recommended for validating the functionality of BioF-tagged proteins?

A comprehensive experimental design for validating BioF-tagged protein functionality should include the following components:

• Expression verification:

  • SDS-PAGE analysis to confirm protein expression at the expected molecular weight

  • Western blotting with antibodies against the target protein or tag

  • Mass spectrometry to verify protein identity and integrity

• Binding efficiency assessment:

  • Quantify protein binding to PHA supports under standard conditions

  • Determine binding capacity (maximum protein loading per unit of support)

  • Measure binding kinetics to establish optimal incubation times

  • Compare binding to different PHA types (PHB, mcl-PHA, copolymers)

• Stability testing across conditions:

  • Evaluate stability at different temperatures (25-37°C)

  • Test pH stability across a wide range (pH 2-9)

  • Assess long-term stability (48-96 hours)

  • Determine resistance to detergents

• Enzymatic activity measurements:

  • Compare specific activity of immobilized enzyme vs. free enzyme

  • Measure activity after extended storage periods

  • Perform multiple activity-plus-washing cycles to assess operational stability

  • Determine kinetic parameters (Km, Vmax) in immobilized vs. free state

• Structural integrity analysis:

  • Circular dichroism to evaluate secondary structure maintenance

  • Fluorescence spectroscopy to assess tertiary structure

  • Thermal shift assays to determine stability changes upon immobilization

• Control experiments:

  • Include non-tagged versions of the target protein

  • Test BioF tag alone without the target protein

  • Use alternative immobilization methods for comparison

  • Include inactivated enzyme controls to distinguish between specific and non-specific effects

A specific example from the literature demonstrates this approach with BioF-β-galactosidase, which was validated through stability testing and enzymatic activity measurements after multiple continuous activity-plus-washing cycles when immobilized in a minibioreactor . This comprehensive validation strategy ensures that both the BioF tag and the target protein maintain their respective functions in the fusion construct.

What are promising research avenues combining G. sulfurreducens capabilities with protein immobilization technologies?

The intersection of G. sulfurreducens research and protein immobilization technologies offers several promising research directions:

• Bioelectrochemical systems enhancement:

  • Immobilization of G. sulfurreducens electron transfer proteins (like PgcA) on electrode surfaces to enhance electron transfer rates

  • Development of enzyme cascades combining multiple redox proteins for improved bioelectrocatalysis

  • Creation of structured biofilms with defined composition for optimized current production

• Bioremediation applications:

  • Immobilization of G. sulfurreducens metal-reducing proteins on stable carriers for enhanced heavy metal remediation

  • Development of field-deployable bioreactors with immobilized proteins for contaminated groundwater treatment

  • Engineering of protein variants with improved metal reduction capabilities through directed evolution approaches

• Biosensing platforms:

  • Integration of BioF-tagged sensing proteins with electroactive G. sulfurreducens components for electrical detection of analytes

  • Development of whole-cell biosensors with enhanced stability through surface immobilization technologies

  • Creation of multiplex sensing platforms combining different immobilized proteins

• Nitrogen fixation engineering:

  • Leveraging G. sulfurreducens' natural nitrogen fixation capabilities through immobilized nitrogenase components

  • Development of artificial nitrogen-fixing systems for agricultural applications

  • Engineering of oxygen-protected nitrogenase systems using protein immobilization strategies

• Bioactive material development:

  • Creation of self-regenerating catalytic surfaces combining immobilized enzymes with living G. sulfurreducens biofilms

  • Development of "smart" materials responsive to electrical stimuli through integration of G. sulfurreducens components

  • Engineering of biocompatible interfaces for medical applications using non-immunogenic protein coatings

• Fundamental research:

  • Investigation of structure-function relationships in electron transfer proteins through systematic immobilization studies

  • Exploration of protein-protein interactions in artificial assemblies mimicking natural electron transfer chains

  • Development of in vitro systems for studying extracellular electron transfer mechanisms under controlled conditions

These research directions could significantly advance our understanding of microbial electrochemistry while developing novel biotechnological applications leveraging the unique capabilities of G. sulfurreducens combined with advanced protein immobilization technologies.

How might genetic engineering approaches enhance the functionality of BioF-based immobilization systems?

Genetic engineering approaches offer substantial opportunities to enhance BioF-based immobilization systems through several strategies:

• Affinity enhancement:

  • Directed evolution of the BioF tag to generate variants with increased binding affinity for specific PHA types

  • Site-directed mutagenesis targeting key residues involved in PHA recognition

  • Creation of BioF libraries with varying binding properties for different applications

• Orientation control:

  • Engineering of BioF fusion proteins with defined linker regions to control the orientation of immobilized proteins

  • Dual-tagging strategies combining BioF with other affinity tags for directional immobilization

  • Development of split-BioF systems for assembly of multi-protein complexes with defined geometry

• Substrate range expansion:

  • Modification of BioF to recognize additional polymer types beyond PHAs

  • Engineering of chimeric binding domains combining BioF with other polymer-binding modules

  • Adaptation of BioF for binding to non-biological surfaces such as metals or ceramics

• Responsive binding systems:

  • Development of engineered BioF variants with binding properties responsive to environmental triggers (pH, temperature, light)

  • Creation of allosteric BioF systems where binding is regulated by small molecule effectors

  • Engineering of redox-sensitive BioF variants for electrochemically controlled immobilization

• Multifunctional fusion designs:

  • Creation of multi-domain fusion proteins combining BioF with catalytic and sensing modules

  • Development of self-assembling protein architectures using BioF as building blocks

  • Engineering of protein scaffolds with multiple BioF domains for co-localization of different enzymes

• Release-on-demand systems:

  • Engineering of protease-sensitive linkers between BioF and target proteins for controlled release

  • Development of photo-cleavable or chemically-cleavable linkages for triggered protein detachment

  • Creation of competing-ligand responsive systems for regulated protein release

These genetic engineering approaches could significantly expand the utility of BioF-based immobilization technologies beyond their current capabilities, enabling more sophisticated and controllable bioactive materials for various biotechnological applications.