Recombinant Desulfovibrio vulgaris Probable GTP-binding protein EngB (engB)

What genetic systems are available for expressing recombinant proteins in Desulfovibrio vulgaris?

Desulfovibrio vulgaris has seen enormous progress in genetic manipulation techniques in recent years. For recombinant protein expression, researchers can utilize several systems, with the markerless genetic exchange system being particularly valuable. This system employs the uracil phosphoribosyltransferase gene (upp) as a counterselectable marker combined with 5-fluorouracil (5-FU) resistance for selection . The advantage of this approach is that it allows for multiple sequential genetic modifications without accumulating antibiotic resistance markers, which is crucial when engineering strains for recombinant protein expression .

For optimal expression of recombinant proteins like EngB, researchers should consider using the Δupp strain JW710, which has been developed specifically to facilitate genetic manipulation in D. vulgaris . This strain allows for a two-step integration and excision strategy that enables precise genetic modifications without polar effects that could interfere with protein expression.

What is the typical transformation efficiency when introducing recombinant EngB constructs into D. vulgaris?

The JW7035 strain, which contains deletions in both the upp gene and the hsdR gene (encoding a type I restriction endonuclease), demonstrates 100-1,000 times greater transformation efficiency compared to wild-type when introducing stable plasmids via electroporation . This makes it an excellent choice for recombinant EngB expression studies.

Transformation efficiency comparison for different D. vulgaris strains:

This data indicates that JW7035 is the optimal strain for introducing recombinant EngB constructs .

How can I verify the cellular localization of recombinant EngB in D. vulgaris?

To determine the cellular localization of recombinant EngB in D. vulgaris, you can employ a GFP fusion approach similar to that used for studying CbiKᴾ localization . By fusing GFP to the C-terminus of EngB, you can visualize its localization using fluorescence microscopy.

The methodology involves:

• Creating a genetic construct with the engB gene fused to gfp

• Introducing this construct into an appropriate D. vulgaris strain (preferably JW7035 for higher transformation efficiency)

• Confirming expression using Western blotting

• Visualizing localization using fluorescence microscopy

• Conducting subcellular fractionation to confirm microscopy results

This approach successfully confirmed the periplasmic localization of CbiKᴾ in previous studies and would be applicable to determining EngB localization .

What is the optimal protocol for constructing markerless deletion mutants to study EngB function in D. vulgaris?

The optimal protocol for constructing markerless deletion mutants to study EngB function in D. vulgaris involves a two-step integration and excision strategy using the upp/5-FU counterselection system. This methodology allows for the creation of clean, in-frame deletions without antibiotic markers that could interfere with subsequent genetic manipulations .

The detailed protocol includes:

• Construct a suicide plasmid vector containing:

  • Approximately 1kb of DNA sequence upstream of engB

  • Approximately 1kb of DNA sequence downstream of engB

  • The wild-type upp gene expressed constitutively from a suitable promoter (e.g., the aph(3')-II promoter)

• Transform the JW710 (Δupp) strain with this suicide plasmid vector

• Select for integration of the plasmid (first recombination event) by screening for 5-FU sensitivity

• Verify integration by PCR analysis

• Select for excision of the plasmid (second recombination event) by plating on medium containing 5-FU

• Screen 5-FU resistant colonies by PCR to identify those with the desired deletion

• Confirm the deletion by sequencing

This approach has been successfully used to generate deletion strains in D. vulgaris with mutation frequencies of approximately 50% .

What are the most effective methods for purifying recombinant EngB from D. vulgaris for structural studies?

For purifying recombinant EngB from D. vulgaris for structural studies, a combination of approaches based on established protocols for similar proteins can be used:

• Expression optimization:

  • Use the JW7035 strain for higher transformation efficiency

  • Add a histidine tag to facilitate purification while minimizing interference with protein function

  • Consider periplasmic targeting if native localization is periplasmic

• Cell lysis and initial purification:

  • Use anaerobic techniques throughout purification due to D. vulgaris being an obligate anaerobe

  • Perform cell disruption by sonication or French press under anaerobic conditions

  • Clarify lysate by centrifugation at 15,000 × g for 30 minutes

• Chromatography sequence:

  • Immobilized metal affinity chromatography (IMAC) as the initial capture step

  • Ion exchange chromatography for intermediate purification

  • Size exclusion chromatography as a final polishing step

• Quality assessment:

  • SDS-PAGE to assess purity

  • Dynamic light scattering to assess homogeneity

  • Activity assays to confirm GTPase activity

• Structure determination considerations:

  • Test protein stability in various buffers before crystallization attempts

  • Consider protein-GTP complexes for crystallization

  • Assess oligomerization state as it may affect crystal formation

This approach is based on successful purification methods used for other D. vulgaris proteins like CbiKᴾ, which was purified for structure-function relationship studies .

How can site-directed mutagenesis be optimized for studying structure-function relationships in EngB?

Site-directed mutagenesis for studying structure-function relationships in EngB can be optimized using a systematic approach similar to that employed for CbiKᴾ characterization . The methodology should focus on identifying key functional residues and determining their specific roles.

Optimized methodology includes:

• In silico analysis to identify target residues:

  • Sequence alignment with homologous GTP-binding proteins to identify conserved residues

  • Structural prediction to identify residues in the GTP-binding pocket

  • Identification of potential metal coordination sites

• Primer design considerations:

  • Use primers with a minimum of 20 bp complementary sequence flanking the mutation site

  • Ensure GC content of 40-60% for stable annealing

  • Verify primer specificity using in silico PCR tools

• Mutagenesis protocol optimization:

  • Use high-fidelity polymerase to minimize unintended mutations

  • Optimize extension time based on template size

  • Consider using methylation-dependent selection to eliminate template DNA

• Functional characterization of mutants:

  • GTPase activity assays to measure kinetic parameters (Km, Vmax)

  • Binding studies to assess GTP interaction (ITC, fluorescence-based assays)

  • Oligomerization analysis using size exclusion chromatography

  • Structural studies (if possible) to confirm the effects of mutations

This systematic approach has been successfully applied to D. vulgaris proteins, identifying key residues such as His154 and His216 in CbiKᴾ that are essential for its metal-chelation activity .

How does the deletion of the hsdR restriction endonuclease affect expression of recombinant EngB in D. vulgaris?

The deletion of the hsdR gene, encoding the type I restriction endonuclease in D. vulgaris, significantly impacts recombinant protein expression by enhancing transformation efficiency. Research has demonstrated that the JW7035 strain (Δupp ΔhsdR) exhibits 100-1,000 times greater transformation efficiency compared to wild-type D. vulgaris .

For recombinant EngB expression, this has several important implications:

• Higher plasmid uptake efficiency leads to increased transformation success rates, reducing experimental variability and enhancing reproducibility .

• The increased number of transformants allows for more efficient screening of expression constructs, facilitating optimization of expression conditions.

• The absence of the restriction-modification system reduces the degradation of introduced DNA, allowing for more stable maintenance of expression vectors.

• Transformation data indicates that with the JW7035 strain, researchers can expect approximately 2.4 × 10³ transformants/μg of plasmid DNA when using pSC27-based vectors, making it significantly more efficient than working with wild-type strains .

This enhancement in transformation efficiency is particularly valuable when working with challenging proteins like EngB, where expression optimization may require multiple rounds of construct modification and testing.

What approaches can resolve conflicting data regarding EngB oligomerization states in structural studies?

Resolving conflicting data regarding EngB oligomerization states requires a multi-technique approach that can provide complementary information. Based on methodologies used for studying other D. vulgaris proteins, the following strategies are recommended:

• Size exclusion chromatography with multi-angle light scattering (SEC-MALS):

  • Provides direct measurement of absolute molecular weight independent of shape

  • Can detect multiple oligomeric species and their proportion in solution

  • Should be performed under various buffer conditions and protein concentrations

• Analytical ultracentrifugation (AUC):

  • Sedimentation velocity experiments can resolve different oligomeric states

  • Provides information about shape and homogeneity

  • Less affected by protein-column interactions than SEC

• Native mass spectrometry:

  • Can precisely determine oligomeric states and their distribution

  • Allows detection of ligand binding (e.g., GTP) that might influence oligomerization

  • Provides information about complex stability

• Cross-linking mass spectrometry:

  • Identifies interaction interfaces between subunits

  • Can "freeze" transient interactions for analysis

  • Provides structural information about the oligomers

• Hydrogen-deuterium exchange mass spectrometry (HDX-MS):

  • Maps regions involved in protein-protein interactions

  • Can reveal conformational changes associated with oligomerization

  • Particularly useful when combined with mutagenesis studies

This comprehensive approach has been valuable in characterizing the tetrameric form of CbiKᴾ in D. vulgaris, identifying specific residues like Arg54 and Glu76 that stabilize the oligomeric structure through hydrogen bonding between subunits .

How can potential cofactors or binding partners of EngB be identified in D. vulgaris?

Identifying potential cofactors or binding partners of EngB in D. vulgaris requires a systematic approach combining in vivo and in vitro techniques. Based on successful methods used for studying D. vulgaris proteins, the following strategy is recommended:

• Affinity purification coupled with mass spectrometry (AP-MS):

  • Express tagged EngB in D. vulgaris JW7035 strain

  • Perform gentle lysis under anaerobic conditions to preserve protein-protein interactions

  • Purify using an appropriate affinity tag (His-tag or FLAG-tag)

  • Identify co-purifying proteins by mass spectrometry

  • Validate interactions using reciprocal pull-downs

• Bacterial two-hybrid screening:

  • Construct a D. vulgaris genomic library in a bacterial two-hybrid vector

  • Screen against EngB as bait

  • Validate positive interactions through secondary screening

• Protein co-evolution analysis:

  • Perform bioinformatic analysis to identify proteins with similar phylogenetic profiles

  • Focus on proteins whose genes are consistently co-located with engB across bacterial genomes

  • Validate predicted interactions experimentally

• Metabolite screening:

  • Use differential scanning fluorimetry to identify small molecules that stabilize EngB

  • Focus on GTP analogs and potential metal cofactors

  • Validate binding using isothermal titration calorimetry (ITC)

• Crosslinking studies:

  • Perform in vivo crosslinking using formaldehyde or other crosslinkers

  • Isolate EngB complexes and identify crosslinked partners by mass spectrometry

This approach is inspired by successful studies of D. vulgaris proteins like CbiKᴾ, which identified unexpected cofactors such as haem binding through His96 and His103 residues that were absent in homologous proteins .

What strategies can overcome the low transformation efficiency in D. vulgaris when expressing recombinant EngB?

Low transformation efficiency is a significant challenge when working with D. vulgaris, but several strategies can help overcome this limitation:

• Use optimized host strains:

  • The JW7035 strain (Δupp ΔhsdR) has demonstrated 100-1,000 times greater transformation efficiency compared to wild-type D. vulgaris .

  • The JW801 strain (cured of native plasmid pDV1) also shows improved transformation efficiency and may be suitable for certain applications .

• Optimize electroporation conditions:

  • Use freshly prepared competent cells harvested in early to mid-log phase

  • Ensure complete removal of salts during washing steps

  • Optimize field strength, pulse duration, and temperature

  • Use pre-warmed recovery media immediately after electroporation

• Plasmid considerations:

  • Use plasmids derived from the native D. vulgaris cryptic plasmid pBG1 for stable maintenance

  • Minimize plasmid size to improve transformation efficiency

  • Ensure plasmids are free of DNA methylation patterns recognized by D. vulgaris restriction systems

• Protocol modifications:

  • Include a heat-shock step (42°C for 10 minutes) before electroporation

  • Add glycine betaine or DMSO as osmoprotectants during electroporation

  • Extend recovery time under anaerobic conditions

Comparative transformation efficiency data shows that with these optimizations, researchers can improve transformation rates from 2-5 transformants/μg plasmid DNA (wild-type) to over 2,000 transformants/μg (JW7035) .

How can anaerobic conditions be maintained throughout purification to ensure proper folding and activity of recombinant EngB?

Maintaining anaerobic conditions throughout the purification of recombinant EngB from D. vulgaris is crucial for preserving protein structure and activity. A comprehensive strategy includes:

• Specialized equipment setup:

  • Use an anaerobic chamber or glove box maintained at <1 ppm O₂ for all purification steps

  • Equip the chamber with necessary chromatography systems and fraction collectors

  • Install a refrigeration unit within the chamber for temperature-sensitive steps

  • Use oxygen-scavenging catalysts and palladium catalysts to remove trace oxygen

• Buffer preparation:

  • Degas all buffers by vacuum followed by sparging with nitrogen or argon

  • Include oxygen scavengers such as dithiothreitol (DTT) or β-mercaptoethanol

  • Consider adding sodium dithionite (0.5-2 mM) as a strong reducing agent

  • Pre-equilibrate buffers in the anaerobic chamber for at least 24 hours before use

• Cell disruption considerations:

  • Harvest cells anaerobically and transfer to the chamber without exposure to oxygen

  • Use pressure-based lysis methods rather than sonication when possible

  • Maintain low temperature during lysis to prevent protein denaturation

• Chromatography adaptations:

  • Use degassed mobile phases for all chromatography steps

  • Maintain a positive pressure of inert gas above buffer reservoirs

  • Keep flow rates moderate to minimize introduction of trace oxygen

  • Collect fractions directly into tubes containing additional oxygen scavengers

• Activity preservation:

  • Perform activity assays within the anaerobic environment

  • Include GTP and appropriate metal ions in storage buffers to stabilize EngB

  • Consider flash-freezing aliquots in liquid nitrogen within the chamber for long-term storage

This comprehensive approach is essential for maintaining the native structure and function of oxygen-sensitive proteins from anaerobic organisms like D. vulgaris .

What analytical techniques can differentiate between properly folded and misfolded recombinant EngB protein?

Differentiating between properly folded and misfolded recombinant EngB requires a multi-technique analytical approach that examines different aspects of protein structure and function:

• Functional assays:

  • GTPase activity measurements using colorimetric or fluorescence-based assays

  • GTP binding assays using fluorescent GTP analogs

  • Comparison of kinetic parameters (Km, kcat) with those of native or homologous proteins

• Spectroscopic techniques:

  • Circular dichroism (CD) spectroscopy to assess secondary structure content

  • Fluorescence spectroscopy to monitor tertiary structure through intrinsic tryptophan fluorescence

  • Fourier-transform infrared spectroscopy (FTIR) to detect aggregation and non-native β-sheet formation

• Hydrodynamic methods:

  • Size-exclusion chromatography to detect aggregation or aberrant oligomerization

  • Dynamic light scattering to assess size distribution and polydispersity

  • Analytical ultracentrifugation to determine shape parameters and oligomeric state

• Thermal stability analysis:

  • Differential scanning calorimetry (DSC) to measure conformational stability

  • Differential scanning fluorimetry (DSF) using environment-sensitive dyes

  • Temperature-dependent activity assays to correlate structure with function

• Protease susceptibility:

  • Limited proteolysis followed by mass spectrometry to identify exposed, potentially misfolded regions

  • Comparison of digestion patterns between purified protein and native controls

This approach has been valuable in characterizing properly folded D. vulgaris proteins, as demonstrated in structure-function studies of CbiKᴾ, where both functional assays and structural analyses were used to confirm proper protein folding after mutagenesis .

How can cryo-EM be optimized for structural studies of EngB and its interaction partners in D. vulgaris?

Cryo-electron microscopy (cryo-EM) offers significant advantages for studying EngB structure and interactions, particularly for capturing dynamic states. Optimizing cryo-EM for this specific application requires addressing several key considerations:

• Sample preparation optimization:

  • Protein concentration: Test concentration ranges (typically 0.5-5 mg/ml) to identify optimal particle distribution

  • Buffer composition: Screen different buffers to minimize background and maximize contrast

  • Grid treatment: Test different grid types (Quantifoil, C-flat) and surface treatments (glow discharge parameters, graphene coating)

  • Vitrification conditions: Optimize blotting time and temperature to achieve ideal ice thickness

• Data collection strategy:

  • Use energy filters to improve signal-to-noise ratio for a relatively small protein like EngB

  • Implement beam-tilt data collection to increase throughput and improve reconstruction quality

  • Consider collecting data in super-resolution mode with subsequent binning

  • Use beam-image shift strategy for faster data collection while maintaining quality

• EngB-specific considerations:

  • Capture different nucleotide-bound states (GDP vs. GTP vs. apo)

  • Study EngB in complex with interaction partners identified through pull-down experiments

  • Use GTPase-deficient mutants to trap transition states

  • Consider using nanodiscs to study membrane-associated forms if relevant

• Processing workflow optimization:

  • Implement 2D classification strategies suitable for smaller proteins

  • Use 3D classification to separate different conformational states

  • Apply focused refinement on regions of interest

  • Consider Bayesian polishing and CTF refinement to maximize resolution

This approach builds on techniques used for structural studies of other D. vulgaris proteins, adapting them to the specific challenges of EngB as a probable GTP-binding protein with potential dynamic conformational states .

What insights can comparative genomics provide about the evolution and function of EngB across different strains of Desulfovibrio?

Comparative genomics can provide valuable insights into the evolution and function of EngB across Desulfovibrio species, revealing conservation patterns, potential functional adaptations, and evolutionary relationships:

• Conservation analysis:

  • Multiple sequence alignment of EngB proteins across Desulfovibrio species and other bacterial genera

  • Identification of universally conserved residues that likely play critical roles in GTP binding and hydrolysis

  • Mapping conservation patterns onto structural models to identify functional surfaces

  • Analysis of selection pressure (dN/dS ratios) to identify residues under positive or purifying selection

• Genomic context analysis:

  • Examination of gene neighborhoods around engB across different Desulfovibrio species

  • Identification of consistently co-localized genes that may be functionally related

  • Comparison with other sulfate-reducing bacteria to identify shared genomic architectures

  • Investigation of potential operon structures and co-regulation patterns

• Phylogenetic profiling:

  • Construction of phylogenetic trees based on EngB sequences

  • Correlation with species phylogeny to identify potential horizontal gene transfer events

  • Identification of lineage-specific adaptations in EngB structure and function

  • Correlation of EngB variants with ecological niches of different Desulfovibrio species

• Structural variation analysis:

  • Prediction of structural differences in EngB proteins across Desulfovibrio species

  • Identification of insertions/deletions that may confer specialized functions

  • Analysis of surface properties and potential interaction interfaces

  • Correlation of structural variations with biochemical characteristics of different species

This comprehensive comparative genomics approach draws inspiration from studies of other D. vulgaris proteins, such as CbiKᴾ, which revealed unique evolutionary adaptations like the acquisition of haem-binding capabilities through specific histidine residues (His96 and His103) that are absent in homologous proteins from other bacteria .

How can system-wide approaches integrate EngB function into the broader metabolic network of D. vulgaris?

Integrating EngB function into the broader metabolic network of D. vulgaris requires sophisticated system-wide approaches that combine multiple omics technologies with computational modeling:

• Multi-omics data integration:

  • Transcriptomics: Analyze co-expression patterns between engB and other genes under various conditions

  • Proteomics: Identify changes in the proteome when engB is deleted or overexpressed

  • Metabolomics: Measure metabolic shifts associated with altered EngB function

  • Interactomics: Map protein-protein interactions involving EngB using techniques like AP-MS

• Network analysis approaches:

  • Construct protein-protein interaction networks centered on EngB

  • Develop gene regulatory networks to identify factors controlling engB expression

  • Create metabolic flux models incorporating EngB-dependent processes

  • Identify network motifs and modules associated with EngB function

• Perturbation studies:

  • Generate conditional engB mutants using the markerless deletion system

  • Perform time-resolved multi-omics analyses after EngB depletion

  • Conduct synthetic lethality screens to identify genes functionally related to engB

  • Implement CRISPR interference for targeted down-regulation of engB and potential partners

• Computational model development:

  • Integrate experimental data into genome-scale metabolic models of D. vulgaris

  • Perform flux balance analysis with and without functional EngB

  • Develop kinetic models of GTP-dependent processes involving EngB

  • Use machine learning approaches to predict condition-specific roles of EngB

This systems biology approach builds on the genetic manipulation techniques available for D. vulgaris, particularly the markerless deletion system that allows for the creation of clean genetic backgrounds suitable for system-wide studies . The approach is particularly valuable for understanding proteins like EngB that may have multiple cellular roles or participate in complex regulatory networks.

What are the most effective strategies for optimizing expression of soluble, active recombinant EngB in D. vulgaris?

Optimizing expression of soluble, active recombinant EngB in D. vulgaris requires a systematic approach addressing multiple aspects of protein production:

• Strain selection and optimization:

  • Use the JW7035 strain (Δupp ΔhsdR) for its significantly higher transformation efficiency (100-1,000 fold improvement over wild-type)

  • Consider deleting problematic proteases if degradation occurs

  • Evaluate different growth media formulations to optimize expression conditions

• Expression construct design:

  • Test different promoter systems (constitutive vs. inducible)

  • Optimize the ribosome binding site sequence for efficient translation

  • Consider codon optimization based on D. vulgaris codon usage

  • Evaluate N-terminal vs. C-terminal tags based on protein topology

  • Include a TEV protease cleavage site for tag removal

• Expression condition optimization:

  • Test different growth temperatures (typically 30-37°C)

  • Evaluate various induction times and durations

  • Optimize cell density at induction (mid-log vs. late-log phase)

  • Supplement growth media with appropriate cofactors (GTP, metal ions)

• Solubility enhancement strategies:

  • Co-express with molecular chaperones if folding issues are observed

  • Test fusion partners known to enhance solubility (SUMO, MBP, TrxA)

  • Evaluate the effect of osmolytes in the growth medium

  • Consider periplasmic targeting if appropriate

• Purification strategy optimization:

  • Maintain anaerobic conditions throughout purification

  • Include stabilizing agents (GTP, metal ions) in all buffers

  • Optimize purification buffer composition (pH, salt concentration, additives)

  • Implement a multi-step purification strategy tailored to EngB properties

This systematic approach draws on the successful genetic manipulation techniques developed for D. vulgaris, particularly the advanced transformation systems that allow for efficient introduction of expression constructs .

How can in vivo imaging techniques be adapted to study the localization and dynamics of EngB in D. vulgaris cells?

Adapting in vivo imaging techniques to study EngB localization and dynamics in D. vulgaris requires specialized approaches that account for both the anaerobic nature of the organism and its specific biology:

• Fluorescent protein optimization:

  • Test oxygen-independent fluorescent proteins (e.g., flavin-based fluorescent proteins)

  • Evaluate split-GFP systems where one fragment is fused to EngB and the other expressed separately

  • Consider photoconvertible fluorescent proteins for pulse-chase experiments

  • Validate that the GFP fusion approach successfully used for CbiKᴾ localization is also suitable for EngB

• Microscopy setup adaptation:

  • Implement anaerobic chambers that interface with microscope stages

  • Use sealed, gas-impermeable imaging chambers with oxygen scavengers

  • Optimize illumination parameters to minimize phototoxicity

  • Consider light sheet microscopy for reduced photobleaching and photodamage

• Dynamic imaging approaches:

  • Employ fluorescence recovery after photobleaching (FRAP) to measure protein mobility

  • Use single-particle tracking for studying EngB movement within cells

  • Implement fluorescence correlation spectroscopy (FCS) to measure diffusion rates

  • Apply stimulated emission depletion (STED) microscopy for super-resolution imaging

• Multi-color imaging strategies:

  • Co-express EngB fusion with markers for different cellular compartments

  • Study co-localization with potential interaction partners

  • Use fluorescence resonance energy transfer (FRET) to detect protein-protein interactions

  • Implement fluorescent timers to track protein age and turnover

• Data analysis considerations:

  • Develop automated image analysis pipelines specific for D. vulgaris cell morphology

  • Implement deconvolution algorithms optimized for anaerobic imaging conditions

  • Use machine learning approaches for feature detection and classification

  • Apply quantitative analysis methods to extract kinetic parameters

This approach builds on the successful C-terminal GFP fusion strategy that confirmed the periplasmic localization of CbiKᴾ in D. vulgaris, adapting it for the specific requirements of studying EngB dynamics .

What are the most appropriate assays for measuring the GTPase activity of recombinant EngB under anaerobic conditions?

Measuring GTPase activity of recombinant EngB under strictly anaerobic conditions requires specialized assays that can function in the absence of oxygen. The following approaches are recommended:

• Colorimetric phosphate detection methods:

  • Malachite green assay:

    • Can be performed anaerobically in a glove box

    • Measures free phosphate released during GTP hydrolysis

    • Sample protocol:
      a) Incubate purified EngB (0.1-5 μM) with GTP (0.1-1 mM) in reaction buffer
      b) At timed intervals, remove aliquots and add to malachite green solution
      c) Measure absorbance at 630 nm after color development

    • Include standard curves prepared under identical conditions

  • PiColorLock Gold assay:

    • Higher sensitivity than malachite green

    • Lower interference from reducing agents commonly used in anaerobic buffers

    • Can be adapted for microplate format for higher throughput

• HPLC-based nucleotide analysis:

  • Separate and quantify GTP and GDP using ion-pair reversed-phase HPLC

  • Sample preparation can be performed anaerobically with analysis under aerobic conditions

  • Allows direct measurement of substrate consumption and product formation

  • Can detect other potential nucleotide intermediates

• Coupled enzyme assays:

  • Pyruvate kinase/lactate dehydrogenase coupled system:

    • Links GTP hydrolysis to NADH oxidation (measurable at 340 nm)

    • All enzymes must be oxygen-stable or used within an anaerobic chamber

    • Requires confirmation that coupling enzymes aren't affected by anaerobic conditions

• Fluorescence-based methods:

  • BODIPY-GTP assays:

    • Measures changes in fluorescence upon GTP hydrolysis

    • Can be performed in sealed, oxygen-impermeable cuvettes

    • Allows continuous, real-time monitoring of activity

• Radioactive assays:

  • [γ-³²P]GTP hydrolysis:

    • Extremely sensitive detection of GTPase activity

    • Can be performed under strictly anaerobic conditions

    • Separation of radioactive Pi from GTP by thin-layer chromatography

    • Requires appropriate radioactive material handling facilities

These methods should be calibrated using known GTPase proteins and appropriate controls to ensure reliability under anaerobic conditions. The approach is informed by successful enzymatic assays developed for other D. vulgaris proteins, adapted for the specific requirements of measuring GTPase activity .

How might CRISPR-Cas9 genome editing systems be adapted for more precise genetic manipulation of engB in D. vulgaris?

Adapting CRISPR-Cas9 genome editing for precise manipulation of engB in D. vulgaris would represent a significant advancement over current genetic manipulation techniques. The following approach outlines how this could be accomplished:

• Development of a D. vulgaris-optimized CRISPR-Cas9 system:

  • Engineer Cas9 expression with codon optimization for D. vulgaris

  • Test various promoters for optimal Cas9 expression levels

  • Evaluate different guide RNA (gRNA) delivery methods

  • Use the JW7035 strain as the base strain due to its improved transformation efficiency

• Design considerations for targeting engB:

  • Analyze the engB locus for optimal protospacer adjacent motif (PAM) sites

  • Design gRNAs with minimal off-target effects across the D. vulgaris genome

  • Create repair templates that include desired modifications (point mutations, tags, etc.)

  • Consider using nickase variants for higher specificity

• Integration with existing genetic systems:

  • Combine CRISPR-Cas9 with the upp/5-FU counterselection system for marker-free editing

  • Develop a two-step process:
    a) First recombination event introduces Cas9, gRNA, and repair template
    b) Second step removes the CRISPR components after successful editing

• Validation and optimization:

  • Develop efficient screening methods to identify successful edits

  • Optimize homology-directed repair efficiency through manipulation of DNA repair pathways

  • Establish protocols for multiplexed editing of engB and related genes

  • Create a library of validated gRNAs for different regions of engB

• Advanced applications:

  • Develop CRISPRi systems for tunable repression of engB expression

  • Create CRISPRa systems for enhanced expression when needed

  • Establish base editing capabilities for precise single nucleotide modifications

  • Develop protocols for large-scale functional genomics studies

This approach would significantly advance beyond the current markerless deletion system by offering more precise and versatile genetic manipulation capabilities for studying engB function in D. vulgaris .

What potential biotechnological applications could emerge from detailed structural and functional characterization of EngB?

Detailed structural and functional characterization of EngB from D. vulgaris could lead to several innovative biotechnological applications:

• Novel antimicrobial development:

  • If EngB proves essential for D. vulgaris survival, structural data could guide the design of selective inhibitors

  • Comparative analysis with homologs from pathogenic bacteria could identify conserved targetable features

  • Structure-based virtual screening could identify compounds that interfere with GTP binding

  • Rational design of peptide inhibitors targeting unique structural features of bacterial EngB proteins

• Protein engineering applications:

  • Development of GTP-responsive molecular switches based on EngB conformational changes

  • Creation of biosensors for detecting GTP or related nucleotides in complex biological samples

  • Engineering EngB variants with altered nucleotide specificity for specialized applications

  • Design of EngB-based scaffold proteins for organizing multi-enzyme complexes

• Biocatalysis innovations:

  • Exploration of potential promiscuous enzymatic activities that could be enhanced through directed evolution

  • Development of EngB variants capable of catalyzing novel chemical transformations

  • Creation of chimeric enzymes combining EngB domains with other catalytic modules

  • Engineering oxygen-tolerant variants for broader biotechnological applications

• Bioremediation applications:

  • If EngB plays a role in stress responses, engineered variants could enhance D. vulgaris survival in contaminated environments

  • Development of biosensors for monitoring environmental conditions based on EngB activity

  • Creation of immobilized EngB systems for specific biotransformations relevant to environmental cleanup

  • Engineering D. vulgaris strains with enhanced EngB function for improved metal precipitation or reduction

• Structural biology tools:

  • Development of EngB as a crystallization chaperone for difficult-to-crystallize proteins

  • Creation of EngB-based affinity tags for protein purification under anaerobic conditions

  • Design of EngB fusion proteins for enhancing solubility of recalcitrant proteins

  • Development of engineered EngB domains as molecular probes for studying GTP-dependent processes

These applications would build upon the detailed structural and functional insights gained from studies similar to those conducted on other D. vulgaris proteins like CbiKᴾ, where unexpected functions (haem binding) were discovered through rigorous structural and biochemical analysis .

How might synthetic biology approaches be used to engineer novel functions into EngB for biotechnological applications?

Synthetic biology approaches offer exciting possibilities for engineering novel functions into EngB from D. vulgaris, potentially creating proteins with unique and valuable properties:

• Domain fusion engineering:

  • Create chimeric proteins by fusing EngB with domains from other proteins:

    • EngB-fluorescent protein fusions for in vivo biosensing applications

    • EngB-enzyme fusions for GTP-dependent catalysis regulation

    • EngB-binding domain fusions for controlled protein recruitment

  • Optimize linker regions between domains for optimal function

  • Test various domain orientations to minimize steric hindrance

• Directed evolution strategies:

  • Develop high-throughput screening systems compatible with anaerobic conditions

  • Apply error-prone PCR to generate EngB variant libraries

  • Use PACE (phage-assisted continuous evolution) adapted for D. vulgaris

  • Implement targeted mutagenesis of GTP-binding regions to alter nucleotide specificity

  • Select for variants with novel properties such as:

    • Altered nucleotide specificity (ATP, UTP instead of GTP)

    • Enhanced stability under various conditions

    • New catalytic activities

• Computational design approaches:

  • Use Rosetta-based computational tools to redesign the active site

  • Apply machine learning algorithms to predict beneficial mutations

  • Design de novo binding sites for novel ligands

  • Engineer allosteric regulation mechanisms into EngB structure

• Genetic circuit integration:

  • Incorporate engineered EngB variants into synthetic genetic circuits

  • Design GTP-responsive gene expression systems using EngB as a sensor

  • Create oscillatory circuits based on EngB activity

  • Develop complex logical operations using multiple engineered EngB variants

• Non-canonical amino acid incorporation:

  • Introduce reactive handles for bioorthogonal chemistry

  • Incorporate photocrosslinkable amino acids for controlled protein interactions

  • Add fluorescent amino acids for direct activity monitoring

  • Introduce metal-binding residues for novel catalytic activities

This synthetic biology approach would build upon the genetic manipulation tools available for D. vulgaris, particularly the markerless deletion system that allows for clean genetic backgrounds suitable for synthetic biology applications . The approach is also informed by successful protein engineering studies like those conducted on CbiKᴾ, where specific residues (His96, His103) were identified as critical for novel functions .