Recombinant Desulfovibrio vulgaris Bifunctional enzyme IspD/IspF (ispDF)

Why is the bifunctional nature of IspD/IspF in D. vulgaris significant for research?

The bifunctional nature of IspD/IspF in D. vulgaris represents an interesting case of protein evolution where two enzymatic functions have been combined into a single polypeptide chain, in contrast to separate IspD and IspF enzymes found in many other bacterial species. This fusion offers several research advantages:

• Pathway efficiency: The fusion potentially increases the efficiency of the MEP pathway by channeling intermediates between active sites without releasing them into the cytoplasm

• Evolutionary perspective: Provides insights into the evolution of metabolic pathways and enzyme architecture

• Drug target potential: Offers a unique target for antimicrobial development, as inhibitors could potentially disrupt two enzymatic steps simultaneously

• Structure-function relationships: Allows study of how two catalytic domains maintain their individual functions while being part of the same protein

Researchers investigating this bifunctional enzyme should consider comparing its catalytic efficiency with the individual IspD and IspF enzymes from other organisms to understand potential advantages conferred by this fusion .

What are the optimal conditions for recombinant expression of D. vulgaris IspD/IspF?

For optimal recombinant expression of D. vulgaris IspD/IspF:

Expression System:

• Host: E. coli BL21(DE3) or similar expression strains

• Vector: pET-based vectors with T7 promoter system

• Tags: N-terminal His6-tag or alternative affinity tags (consider TEV protease cleavage site)

Culture Conditions:

• Media: LB or 2×YT supplemented with appropriate antibiotics

• Temperature: Induction at lower temperatures (16-18°C) overnight after reaching OD600 of 0.6-0.8

• IPTG concentration: 0.1-0.5 mM (lower concentrations favor soluble protein)

• Supplementation: Consider adding 0.5-1 mM ZnCl2 to the culture medium as the IspF domain requires zinc for activity

Lysis Buffer Components:

• 25-50 mM Tris-HCl, pH 8.0

• 100-300 mM NaCl

• 5-10% glycerol

• 1 mM DTT or 2-5 mM β-mercaptoethanol

• Protease inhibitor cocktail

• 0.1-0.5 mM ZnCl2

• Consider 0.1% Triton X-100 to aid solubilization

Careful optimization of these parameters is essential as the bifunctional nature of the enzyme may present folding challenges .

What purification strategy yields the highest activity for recombinant D. vulgaris IspD/IspF?

A multi-step purification strategy is recommended to obtain high-activity recombinant D. vulgaris IspD/IspF:

Step 1: Affinity Chromatography

• If His-tagged: Ni-NTA or IMAC chromatography

• Buffer: 25 mM Tris-HCl pH 8.0, 300 mM NaCl, 10% glycerol

• Imidazole gradient: 20 mM (wash), 50-300 mM (elution)

• Include 0.1 mM ZnCl2 in all buffers to maintain IspF domain integrity

Step 2: Tag Removal (Optional)

• TEV protease cleavage (1:50 ratio, overnight at 4°C)

• Second IMAC to remove cleaved tag and TEV protease

Step 3: Ion Exchange Chromatography

• Q-Sepharose column (anion exchange)

• Buffer: 25 mM Tris-HCl pH 8.0, 5% glycerol

• NaCl gradient: 0-500 mM

Step 4: Size Exclusion Chromatography

• Superdex 200 column

• Buffer: 25 mM Tris-HCl pH 8.0, 100 mM NaCl, 5% glycerol, 1 mM DTT, 0.1 mM ZnCl2

Final Storage Conditions:

• Concentrate to 1-5 mg/mL

• Add glycerol to 25-50% final concentration

• Flash-freeze in liquid nitrogen

• Store at -80°C

Purity should be assessed by SDS-PAGE (>85% purity is generally acceptable), and activity of both domains should be verified using appropriate enzymatic assays to ensure the bifunctional enzyme has maintained its dual catalytic capabilities .

How can the dual catalytic activities of IspD/IspF be measured independently?

To separately measure the IspD and IspF activities in the bifunctional enzyme:

IspD Activity Assay:

• Reaction mixture:

  • 50 mM Tris-HCl (pH 8.0)

  • 5 mM MgCl2

  • 1-2 mM DTT

  • 100-200 μM CTP

  • 100-200 μM MEP

  • 1-5 μg purified enzyme

• Detection methods:

  • Coupled assay: Monitor release of pyrophosphate using a commercially available pyrophosphate detection kit

  • HPLC method: Separate and quantify CDP-ME formation using a C18 column with UV detection at 254 nm

  • Radioactive assay: Use [α-32P]CTP and measure CDP-ME formation by TLC or scintillation counting

IspF Activity Assay:

• Reaction mixture:

  • 50 mM MOPS (pH 8.0)

  • 5 mM MgCl2

  • 100 μM CDP-ME2P (substrate)

  • 1 mM phosphatase inhibitor

  • 1-5 μg purified enzyme

• Detection methods:

  • Spectrophotometric method: Monitor CMP release using an auxiliary enzyme system that converts CMP to uridine with subsequent UV detection

  • Mass spectrometry: Analyze ME-CPP formation directly using an Agilent 6210 mass spectrometer after reaction termination with 10 mM EDTA

  • HPLC method: Detect ME-CPP formation using appropriate column separation

When analyzing the bifunctional enzyme, it's crucial to run appropriate controls and consider the potential for substrate channeling between domains. For comprehensive characterization, determine kinetic parameters (Km, kcat, kcat/Km) for both activities and compare them to the individual enzymes from other species .

What are the expected kinetic parameters for both IspD and IspF activities?

Based on related research with IspD and IspF enzymes from various bacterial species, the expected kinetic parameters for D. vulgaris bifunctional IspD/IspF activities typically fall within these ranges:

Table 1: Expected Kinetic Parameters for IspD Activity

Table 2: Expected Kinetic Parameters for IspF Activity

Important considerations:

• The bifunctional nature may affect kinetic parameters compared to the individual enzymes

• Substrate channeling between domains might result in apparent kinetic parameters that differ from those of separate enzymes

• Optimal conditions for simultaneously measuring both activities may require compromise conditions

• The kcat/Km value of M. tuberculosis IspF was reported as 5.4×10-4 μM-1min-1, which might serve as a comparison point

When characterizing newly purified recombinant enzyme, establish full kinetic profiles under standardized conditions and compare them with published values for related enzymes .

How does the structure of bifunctional IspD/IspF compare to the individual enzymes?

While the precise crystal structure of D. vulgaris bifunctional IspD/IspF has not been fully determined in the provided search results, comparative structural analysis can be inferred from related enzymes:

Structural Organization:

• Individual IspD enzymes typically form dimers with each monomer comprising a single Rossmann fold domain

• Individual IspF enzymes form homotrimers with a zinc-binding site at each active site

• The bifunctional enzyme likely maintains these structural motifs while connecting them via a linker region

Domain Architecture:

• N-terminal region: Contains the IspD domain with the characteristic nucleotide-binding Rossmann fold

• Central linker region: Provides flexibility between domains

• C-terminal region: Contains the IspF domain that belongs to the IspF family

Key Structural Features:

• Active Sites:

  • IspD active site: Contains conserved residues for MEP and CTP binding

  • IspF active site: Features a zinc-binding site with conserved histidine and aspartate residues

• Quaternary Structure:

  • The bifunctional enzyme likely forms oligomeric structures to maintain the functional states of both domains

  • IspF domain may still form trimeric arrangements while being part of the fusion protein

• Substrate Channeling:

  • The fusion nature suggests possible substrate channeling between domains

  • The linker region length and flexibility would be crucial determinants

Structural Differences from Individual Enzymes:

• Potential constraints on domain movement due to the covalent linkage

• Possible alterations in oligomerization patterns

• Interface regions that may affect substrate access or product release

For definitive structural characterization, X-ray crystallography or cryo-EM studies would be necessary, focusing particularly on the domain interface and potential substrate-channeling pathways .

What role does zinc play in the function of the IspF domain?

Zinc plays a critical role in the function of the IspF domain within the bifunctional IspD/IspF enzyme:

Structural Role:

Catalytic Functions:

• Substrate Binding:

  • Zinc coordinates with the phosphate groups of CDP-ME2P

  • This coordination positions the substrate optimally for catalysis

• Activation of Water:

  • Zinc acts as a Lewis acid, activating a water molecule for nucleophilic attack

  • This facilitates the cyclization reaction converting CDP-ME2P to ME-CPP

• Transition State Stabilization:

  • The zinc ion helps stabilize negative charges that develop during the reaction

Experimental Evidence:

• Treatment with EDTA (a metal chelator) significantly reduces or abolishes IspF activity

• Addition of Zn2+ ions restores activity to the metal-depleted enzyme

• When expressing and purifying recombinant IspD/IspF, supplementation with ZnCl2 (0.1-1 mM) is recommended in culture media and buffers

Practical Considerations for Researchers:

• Include 0.1-0.5 mM ZnCl2 in all purification and storage buffers

• Avoid high concentrations of reducing agents that might displace zinc

• When measuring IspF activity, ensure zinc is present in assay buffers

• Control experiments with EDTA can confirm zinc dependency

The presence of zinc should be confirmed in purified enzyme preparations, potentially using atomic absorption spectroscopy or inductively coupled plasma mass spectrometry (ICP-MS) .

How can targeted mutations be introduced into the D. vulgaris ispDF gene?

Introducing targeted mutations into the D. vulgaris ispDF gene requires specialized techniques due to the anaerobic nature of this organism and its genetic characteristics. Here are methodological approaches:

1. Markerless Deletion System Approach:
The most effective method utilizes the markerless deletion system developed for D. vulgaris Hildenborough:

• Step 1: Generate a Δupp host strain (like JW710) that is resistant to 5-fluorouracil (5-FU)

• Step 2: Create a suicide plasmid containing:

  • Upstream and downstream regions of the target mutation site

  • The wild-type upp gene under control of a constitutive promoter (e.g., aph(3′)-II)

  • Spectinomycin resistance marker

  • Your desired mutation within the ispDF gene

• Step 3: Introduce the plasmid via electroporation and select for spectinomycin resistance

• Step 4: Verify single-crossover integration via PCR

• Step 5: Grow without spectinomycin to allow second recombination

• Step 6: Select for 5-FU resistance (loss of upp)

• Step 7: Screen colonies for the desired mutation by PCR and sequencing

2. Site-Directed Mutagenesis Protocol:
For structure-function studies, use this approach to create specific amino acid substitutions:

• Design primers containing your desired mutation with 15-20 bp flanking sequences

• Use a template containing the ispDF gene from D. vulgaris

• Perform PCR with high-fidelity polymerase

• Treat with DpnI to digest methylated template DNA

• Transform into E. coli

• Verify mutations by sequencing

• Transfer the mutated gene back to D. vulgaris using the markerless system above

Critical Considerations:

• D. vulgaris has a restriction-modification system that can decrease transformation efficiency

• Consider using a restriction-deficient strain (e.g., JW7035) that has ~100-1000× higher transformation efficiency

• The transformation efficiency for D. vulgaris is typically low (2-5 transformants/μg DNA for wild-type)

• All manipulations must account for the organism's anaerobic requirements

This approach allows for precise genetic manipulation without antibiotic marker retention, enabling sequential mutations if needed .

What domains or residues of IspD/IspF are critical targets for structure-function studies?

Based on structural and functional studies of IspD and IspF enzymes from other organisms, several critical domains and residues in the D. vulgaris bifunctional enzyme merit targeted investigation:

IspD Domain Critical Regions:

• CTP-Binding Site:

  • Conserved glycine-rich motif (likely within first 100 amino acids)

  • Lysine residues involved in phosphate coordination

  • Target mutations: Conserved basic residues that interact with CTP phosphates

• MEP-Binding Pocket:

  • Residues interacting with the erythritol moiety

  • Target mutations: Conserved polar residues in the catalytic pocket

• Catalytic Residues:

  • Conserved aspartate or glutamate residues that may coordinate magnesium

  • Target mutations: Acidic residues in the active site

IspF Domain Critical Regions:

• Zinc-Binding Site:

  • Conserved histidines and aspartate that coordinate the zinc ion

  • Based on homology, likely includes residues in the C-terminal portion

  • Target mutations: His→Ala substitutions to disrupt zinc binding

• CDP-ME2P Binding Pocket:

  • Residues interacting with cytidine moiety

  • Residues stabilizing the cyclization transition state

  • Target mutations: Conserved aromatic residues that stack with cytidine

• Dimer/Trimer Interface:

  • Residues involved in oligomerization

  • Target mutations: Hydrophobic residues at subunit interfaces

Domain Interface Region:

• Linker Region:

  • Flexibility may impact substrate channeling

  • Target mutations: Proline insertions to reduce flexibility or linker length modifications

• Interdomain Contacts:

  • Potential residues participating in communication between domains

  • Target mutations: Interface residues to disrupt potential allosteric regulation

Experimental Approach Table:

These structure-function studies would provide valuable insights into how the bifunctional enzyme coordinates its dual catalytic activities and whether substrate channeling occurs between domains .

What are the known inhibitors of IspD/IspF and their mechanisms of action?

While specific inhibitors targeting the D. vulgaris bifunctional IspD/IspF have not been extensively documented in the provided search results, we can extrapolate from studies on related IspD and IspF enzymes:

IspD Inhibitors:

• Cytidine Nucleotide Analogs:

  • Mechanism: Competitive inhibition by mimicking CTP substrate

  • Examples: Cytosine derivatives with modified ribose or phosphate groups

  • Typical IC50 range: 1-100 μM

• MEP-Mimetics:

  • Mechanism: Competitive inhibition at the MEP-binding site

  • Examples: Erythritol derivatives with phosphonate groups

  • Binding features: Interact with conserved residues in the MEP-binding pocket

• Fosmidomycin Derivatives:

  • While fosmidomycin primarily targets DXR (an earlier enzyme in the MEP pathway), some derivatives have shown activity against IspD

  • Mechanism: Likely mixed inhibition

IspF Inhibitors:

• Fragment-Based Hits:

  • FOL7185: Identified as a fragment hit binding to IspD and IspE

  • FOL955: Co-crystallized with Burkholderia pseudomallei IspF (PDB: 3QHD)

  • FOL535: Co-crystallized with BpIspF (PDB: 3K14)

• Imidazole Compounds:

  • Designed based on fragment hit FOL955

  • Likely mechanism: Interaction with zinc-binding site

• Imidazothiazole Compounds:

  • Designed based on FOL535

  • Modifications include replacing ethyl ester with various substituents

  • Some designed to engage both zinc and cytidine binding sites

• L-Tryptophan Hydroxamate:

  • Used as a standard in BpIspF assays

  • Mechanism: Likely involves zinc coordination

Bifunctional Inhibition Potential:

The bifunctional nature of D. vulgaris IspD/IspF presents unique opportunities for inhibitor design:

• Dual-Target Inhibitors:

  • Compounds designed to simultaneously inhibit both catalytic functions

  • Potential for increased potency due to proximity of active sites

• Substrate-Channeling Disruptors:

  • Compounds that interfere with the transfer of CDP-ME between domains

  • May bind at the domain interface

• Allosteric Inhibitors:

  • Target regions that affect communication between domains

  • Could potentially modulate one activity through binding to the other domain

For developing selective inhibitors against D. vulgaris IspD/IspF, researchers should consider screening the above-mentioned compound classes while focusing on unique structural features of this bifunctional enzyme .

What screening methodologies are most effective for identifying novel inhibitors of the bifunctional enzyme?

For effective identification of novel inhibitors targeting the D. vulgaris bifunctional IspD/IspF enzyme, a multi-tiered screening approach is recommended:

Primary Screening Assays:

• High-Throughput Spectrophotometric Assays:

  • For IspD activity: Pyrophosphate-coupled assay using commercial pyrophosphate detection systems

  • For IspF activity: CMP release detection using auxiliary enzymes and spectrophotometric readout

  • Advantages: Quick, continuous measurement, suitable for 96/384-well formats

  • Throughput: 10,000-100,000 compounds per day

• Fluorescence-Based Assays:

  • Dansyl-containing fluorescent compounds that interact with the binding sites

  • FRET-based assays to detect conformational changes upon inhibitor binding

  • Advantages: Higher sensitivity, fewer false positives from colored compounds

  • Throughput: Similar to spectrophotometric assays

Secondary Confirmation Assays:

• Mass Spectrometry-Based Assays:

  • Direct detection of reaction products (CDP-ME or ME-CPP)

  • Lower throughput but higher specificity

  • Example protocol: Reaction mixtures containing 50 mM MOPS (pH 8.0), 5 mM MgCl2, 100 μM substrate, and test compound, incubated with enzyme, terminated with EDTA, then analyzed by MS

• Thermal Shift Assays:

  • Measure changes in protein thermal stability upon inhibitor binding

  • Quick verification of direct binding

  • Can distinguish between inhibitors targeting different domains

Tertiary Mechanistic Characterization:

• Enzyme Kinetics:

  • Determine inhibition mechanisms (competitive, non-competitive, uncompetitive)

  • Generate Ki values and investigate time-dependent inhibition

• Biophysical Methods:

  • Surface Plasmon Resonance (SPR): Direct measurement of binding kinetics

  • Isothermal Titration Calorimetry (ITC): Thermodynamic parameters of binding

  • X-ray Crystallography: Structural confirmation of binding mode

Fragment-Based Approaches:

Given the success with fragment hits like FOL7185, FOL955, and FOL535 for related enzymes:

• Fragment Library Screening:

  • Start with low molecular weight compounds (150-300 Da)

  • Screen at higher concentrations (100 μM - 1 mM)

  • Use NMR or X-ray crystallography to confirm binding

• Fragment Growing/Linking:

  • Develop fragments that bind to adjacent sites

  • Link or merge fragments to create more potent inhibitors

Computational Approaches:

• Virtual Screening:

  • Homology model-based if crystal structure unavailable

  • Dock compound libraries against both active sites

• Structure-Based Design:

  • Leverage knowledge from related IspD and IspF structures

  • Focus on unique features of the bifunctional enzyme

For optimal results, combine these approaches in an integrated workflow, starting with high-throughput methods for initial screening, followed by confirmatory assays and mechanistic studies for promising hits .

How does the bifunctional IspD/IspF enzyme contribute to D. vulgaris pathogenicity?

The bifunctional IspD/IspF enzyme plays a significant role in D. vulgaris pathogenicity through several mechanisms:

Metabolic Contribution to Virulence:

• Isoprenoid Biosynthesis:

  • IspD/IspF is essential for the MEP pathway producing isoprenoid precursors

  • Isoprenoids are critical for:

    • Cell membrane integrity and function

    • Electron transport chain components (menaquinones)

    • Cell wall biosynthesis

• Survival in Host Environments:

  • Enables adaptation to varying nutrient conditions

  • Supports growth in anaerobic gut environments

Direct Pathogenic Mechanisms:

• Hydrogen Sulfide (H2S) Production:

  • D. vulgaris produces H2S as a metabolic byproduct

  • H2S disrupts gut epithelial morphology and function

  • Higher H2S levels have been observed in D. vulgaris-treated mice compared to controls

  • Metabolic functions supported by the MEP pathway indirectly contribute to H2S production capacity

• Inflammatory Response Induction:

  • D. vulgaris transplantation in mice causes:

    • Gut inflammation

    • Disruption of gut barrier function

    • Reduced levels of short-chain fatty acids (SCFAs)

    • Increased expression of inflammatory cytokines (IL-1β, iNOS, TNF-α)

    • Decreased expression of anti-inflammatory markers (IL-10, arginase 1)

• Microbiome Disruption:

  • D. vulgaris significantly alters gut microbiota composition

  • Decreases relative abundance of SCFA-producing bacteria

  • Stimulates growth of Akkermansia muciniphila, possibly via H2S production

  • These changes may be supported by metabolic adaptability enabled by the MEP pathway

Experimental Evidence:

• In mouse models, D. vulgaris exacerbates DSS-induced colitis

• It causes persistent epithelial damage and reduced mucus levels

• It aggravates DSS-induced damage to gut epithelial barriers by decreasing expression of E-cadherin, Occludin, and ZO-1

• These pathogenic effects depend on D. vulgaris metabolic activities, which are supported by essential pathways including the MEP pathway

While the direct contribution of IspD/IspF to these mechanisms has not been specifically isolated in the provided research, its essential role in isoprenoid biosynthesis suggests it is necessary for D. vulgaris survival and pathogenicity. Targeting this enzyme could potentially attenuate D. vulgaris virulence by compromising its metabolic capabilities .

How can IspD/IspF genetic manipulation be used to study D. vulgaris in complex microbial communities?

The bifunctional IspD/IspF enzyme offers a valuable target for genetic manipulation to study D. vulgaris in complex microbial communities, providing insights into its ecological roles and interactions:

Genetic Manipulation Strategies:

• Conditional Expression Systems:

  • Replace native ispDF promoter with inducible promoters

  • Allow controlled expression under specific conditions

  • Methodology: Use the markerless deletion system with upp counterselection

• Reporter Gene Fusions:

  • Create transcriptional/translational fusions with reporter genes (GFP, luciferase)

  • Monitor ispDF expression in different environments or community contexts

  • Methodology: Insert reporter gene downstream of ispDF while maintaining function

• Gene Knockdown Approaches:

  • Create partial loss-of-function variants through targeted mutations

  • Develop antisense RNA constructs targeting ispDF mRNA

  • Methodology: Introduce mutations in catalytic residues using site-directed mutagenesis

Applications in Microbial Ecology Research:

• Tracking D. vulgaris in Complex Communities:

  • Experimental design: Create fluorescent reporter strains with ispDF promoter-driven fluorescence

  • Application: Track spatial distribution and metabolic activity in multi-species biofilms

  • Analysis: Correlate ispDF expression with community structure using microscopy and flow cytometry

• Understanding Metabolic Interactions:

  • Experimental design: Create conditional ispDF mutants with varying expression levels

  • Application: Study how D. vulgaris isoprenoid metabolism affects community composition

  • Analysis: Use 16S rRNA amplicon sequencing to track microbiome shifts when ispDF is modulated

• Host-Microbe Interaction Studies:

  • Experimental design: Create D. vulgaris strains with mutated ispDF affecting catalytic efficiency

  • Application: Colonize gnotobiotic animals with mutant strains

  • Analysis: Measure inflammatory markers, gut barrier integrity, and microbiome composition

• Competitive Fitness Assessment:

  • Experimental design: Co-culture wild-type and ispDF-modified strains in different environments

  • Application: Determine fitness costs of ispDF mutations

  • Analysis: Use quantitative PCR with strain-specific primers to track population dynamics

Technical Implementation Table:

Practical Considerations:

• Use the JW7035 strain with higher transformation efficiency (100-1000× greater than wild-type)

• Account for D. vulgaris' anaerobic requirements in all experimental procedures

• Consider potential polar effects of genetic manipulations on downstream genes

• Validate all genetic constructs through sequencing and functional assays before community studies

These approaches capitalize on the markerless genetic exchange system developed for D. vulgaris, allowing for sophisticated genetic manipulations without antibiotic marker retention .

How does the D. vulgaris bifunctional IspD/IspF compare to homologous enzymes in other bacteria?

The D. vulgaris bifunctional IspD/IspF enzyme presents an interesting case for comparative analysis with homologous enzymes from other bacteria:

Structural Organization Comparison:

Functional Differences:

• Catalytic Efficiency:

  • Separate enzymes may have different kinetic properties compared to the bifunctional enzyme

  • Substrate channeling in the bifunctional enzyme potentially increases efficiency

  • M. tuberculosis IspF shows relatively low catalytic efficiency

• Protein-Protein Interactions:

  • Studies with B. subtilis enzymes show no detectable complex formation among IspD, IspE, and IspF

  • D. vulgaris bifunctional enzyme inherently links IspD and IspF functions

  • E. coli may form transient complexes of pathway enzymes

• Regulatory Mechanisms:

  • Differential regulation possible between bifunctional and separate enzymes

  • D. vulgaris likely has coordinated expression and regulation of both functions

  • Separate enzymes allow independent regulation in other bacteria

Evolutionary Implications:

• Gene Fusion Events:

  • The bifunctional enzyme likely arose through gene fusion events

  • May represent adaptation to specific ecological niches

  • Potential for improved metabolic efficiency through proximity of catalytic domains

• Taxonomic Distribution:

  • Bifunctional arrangement appears less common than separate enzymes

  • May correlate with specific metabolic adaptations in sulfate-reducing bacteria

Sequence Conservation:

The 395-amino acid D. vulgaris IspD/IspF sequence shows recognizable domains corresponding to both IspD and IspF functions, with the C-terminal section belonging to the IspF family. Comparative sequence analysis would likely show:

• Higher conservation in catalytic residues

• Variable regions in domain interfaces

• Unique linker regions in the bifunctional enzyme

The bifunctional nature of D. vulgaris IspD/IspF represents an interesting case of potential evolutionary optimization, possibly providing advantages through substrate channeling or coordinated regulation of consecutive pathway steps .

What insights can be gained from studying the genomic context of the ispDF gene in D. vulgaris?

Analyzing the genomic context of the ispDF gene in D. vulgaris provides valuable insights into its regulation, evolution, and functional relationships within metabolic networks:

Genomic Organization:

• Operon Structure:

  • Determine if ispDF is part of a larger operon containing other MEP pathway genes

  • Identify co-transcribed genes that may reveal functional associations

  • Compare with E. coli where separate ispD and ispF genes are often found in different genomic locations

• Regulatory Elements:

  • Analyze upstream regions for promoter elements and transcription factor binding sites

  • Look for conserved DNA motifs that may be involved in regulation

  • One study derived conserved DNA motifs from potential promoter regions of putative D. vulgaris regulons, although most were apparently unique compared to E. coli

Comparative Genomic Analysis:

• Taxonomic Distribution:

  • Compare ispDF arrangement across related Desulfovibrio species

  • Determine if the bifunctional arrangement is conserved within Deltaproteobacteria

  • Map the evolutionary history of gene fusion events

• Synteny Analysis:

  • Examine conservation of gene order in regions flanking ispDF

  • Identify genomic rearrangements that may have led to the current organization

  • Compare with other sulfate-reducing bacteria

Functional Genomic Insights:

• Co-Expression Patterns:

  • Analyze transcriptomic data to identify genes co-regulated with ispDF

  • Look for condition-specific expression patterns that reveal functional importance

  • Traditional laboratory studies of sulfate-reducing bacteria have focused on biochemistry, but genomic sequences now allow insights into metabolic and regulatory networks

• Metabolic Context:

  • Map connections between the MEP pathway and other metabolic pathways in D. vulgaris

  • Identify potential metabolic dependencies or regulatory cross-talk

  • Understand how isoprenoid biosynthesis integrates with core metabolism

Regulatory Network Analysis:

• Transcription Factor Binding:

  • The GlpR binding site (similar to E. coli) was identified in D. vulgaris

  • Most regulatory motifs in D. vulgaris appear unique compared to E. coli

  • Some expected orthologs for regulatory proteins have not been recognized in D. vulgaris

• Response to Environmental Conditions:

  • Analyze expression changes under different growth conditions

  • Identify factors that upregulate or downregulate ispDF expression

  • Connect to the organism's ecological niche and pathogenic potential

Practical Applications:

• Genetic Tool Development:

  • Use promoter elements for development of expression systems

  • Identify suitable regions for genetic manipulation

  • D. vulgaris has seen significant improvements in genetic manipulation capabilities in recent years

• Target Validation:

  • Assess essentiality based on genomic context and lack of redundant pathways

  • Evaluate the ispDF gene as a potential antimicrobial target

Understanding the genomic context of ispDF provides crucial insights for both fundamental research and applied studies targeting this enzyme, helping to place it within the broader metabolic and regulatory framework of D. vulgaris .

What CRISPR-based approaches can be applied to study ispDF function in D. vulgaris?

CRISPR-based technologies offer powerful new approaches for studying ispDF function in D. vulgaris, though they require adaptation for this anaerobic organism:

CRISPR-Cas9 Gene Editing Approaches:

• Genome Editing Strategy:

  • Design guide RNAs (gRNAs) targeting specific regions of ispDF

  • Deliver CRISPR components via electroporation using optimized protocols for D. vulgaris

  • Consider using the restriction-deficient strain JW7035 for higher transformation efficiency

  • Include appropriate homology-directed repair (HDR) templates for precise modifications

• Specific Applications:

  • Domain-specific knockouts: Target either IspD or IspF domain while keeping the other intact

  • Point mutations: Create catalytic residue mutations to study domain-specific functions

  • Tagged versions: Insert epitope or fluorescent tags for localization and interaction studies

  • Promoter modifications: Alter expression levels by modifying regulatory regions

CRISPR Interference (CRISPRi) for Conditional Regulation:

• System Design:

  • Implement dCas9 (catalytically dead Cas9) under control of inducible promoter

  • Design gRNAs targeting the ispDF promoter or coding region

  • Optimize for D. vulgaris codon usage and regulatory elements

• Experimental Applications:

  • Tunable repression: Create partial knockdown phenotypes

  • Temporal control: Study effects of ispDF depletion at different growth phases

  • Spatial regulation: Control expression in different microenvironments

CRISPR Activation (CRISPRa) for Upregulation:

• System Components:

  • dCas9 fused to transcriptional activators adapted for D. vulgaris

  • gRNAs targeting ispDF promoter region

• Applications:

  • Overexpression studies: Evaluate effects of increased IspD/IspF levels

  • Metabolic engineering: Potentially enhance isoprenoid production

  • Stress response analysis: Test if upregulation confers survival advantages

Base and Prime Editing Applications:

• Precise Nucleotide Modifications:

  • Implement cytosine or adenine base editors for targeted mutations without DSBs

  • Create specific codon changes to alter enzyme properties

• Applications:

  • Structure-function analysis: Create libraries of variants with specific amino acid changes

  • Domain interface modifications: Alter interdomain communication

  • Regulatory element tuning: Modify promoter strength through precise nucleotide changes

Technical Implementation Considerations:

The patent information (search result ) indicates developing CRISPR systems for modifying bacterial genomic sequences, suggesting these approaches are becoming more feasible for organisms like D. vulgaris. Researchers should optimize protocols specifically for this anaerobic bacterium, potentially using the improved markerless genetic exchange system as a foundation .

What emerging technologies could advance our understanding of IspD/IspF function and inhibition?

Several cutting-edge technologies hold promise for deepening our understanding of the D. vulgaris bifunctional IspD/IspF enzyme:

Advanced Structural Biology Approaches:

• Cryo-Electron Microscopy (Cryo-EM):

  • Application: Determine high-resolution structures of the complete bifunctional enzyme

  • Advantage: Captures multiple conformational states without crystallization constraints

  • Key insight potential: Visualize dynamic domain movements during catalysis

  • Implementation: Single-particle analysis with preferably >300kV microscopes

• AlphaFold2 and Machine Learning Structure Prediction:

  • Application: Generate accurate structural models of the bifunctional enzyme

  • Advantage: Complements experimental structures, predicts flexible regions

  • Key insight potential: Model domain interfaces and substrate channeling pathways

  • Implementation: Use multiple sequence alignments from diverse Desulfovibrio species

• Time-Resolved X-ray Crystallography:

  • Application: Capture intermediates during catalytic cycles

  • Advantage: Provides dynamic "snapshots" of enzyme function

  • Key insight potential: Elucidate transition states for inhibitor design

  • Implementation: X-ray free-electron laser (XFEL) facilities for femtosecond imaging

Advanced Functional and Interaction Analysis:

• Single-Molecule Förster Resonance Energy Transfer (smFRET):

  • Application: Track conformational dynamics between domains

  • Advantage: Observes heterogeneous behaviors masked in ensemble measurements

  • Key insight potential: Directly observe substrate channeling between domains

  • Implementation: Strategic fluorophore placement at domain interfaces

• Hydrogen-Deuterium Exchange Mass Spectrometry (HDX-MS):

  • Application: Map protein dynamics and ligand-induced conformational changes

  • Advantage: Provides regional dynamics information without structure determination

  • Key insight potential: Identify allosteric networks between domains

  • Implementation: Compare exchange patterns in various ligand-bound states

• Native Mass Spectrometry:

  • Application: Determine oligomeric states and complex formation

  • Advantage: Preserves non-covalent interactions

  • Key insight potential: Understand quaternary structure arrangements

  • Implementation: Analyze intact enzyme complexes with substrates/inhibitors

Emerging Chemical Biology Approaches:

• Chemoproteomics:

  • Application: Identify binding sites and off-targets of inhibitors

  • Advantage: Whole-proteome scope of interactions

  • Key insight potential: Discover unexpected cross-reactivity of inhibitors

  • Implementation: Activity-based protein profiling with clickable probes

• DNA-Encoded Libraries (DELs):

  • Application: Screen billions of compounds for binding

  • Advantage: Massive compound diversity with minimal material

  • Key insight potential: Discover novel chemical scaffolds targeting specific domains

  • Implementation: Target enzyme immobilization and selection protocols

• Fragment-Based Drug Discovery with NMR:

  • Application: Identify fragment hits against both domains

  • Advantage: Maps precise binding locations of weakly binding fragments

  • Key insight potential: Develop domain-specific inhibitors

  • Implementation: 19F NMR and protein-observed experiments

Systems Biology and Metabolic Integration: