Recombinant Desulfovibrio vulgaris 2,3-bisphosphoglycerate-independent phosphoglycerate mutase (gpmI), partial

What is the role of gpmI in D. vulgaris metabolism?

The 2,3-bisphosphoglycerate-independent phosphoglycerate mutase (gpmI) in D. vulgaris catalyzes the reversible conversion of 3-phosphoglycerate to 2-phosphoglycerate in the glycolytic pathway without requiring 2,3-bisphosphoglycerate as a cofactor. This enzyme represents an alternative to the more common dPGM (dependent) form found in many organisms and is particularly important for energy metabolism in anaerobic bacteria like D. vulgaris.

To investigate its metabolic significance, researchers should:

• Perform enzyme activity assays using purified recombinant protein

• Develop a gene knockout using markerless deletion systems similar to those established for other D. vulgaris genes

• Analyze metabolic flux through glycolysis by measuring intracellular metabolite concentrations in wild-type versus mutant strains

• Examine growth characteristics under various carbon sources and environmental conditions

What are optimal conditions for cultivating D. vulgaris for gpmI studies?

D. vulgaris Hildenborough is a strictly anaerobic, sulfate-reducing bacterium that requires specialized cultivation techniques:

• Use anaerobic chambers or techniques that completely exclude oxygen

• Standard media such as Wall LS4 medium can be employed, with specific modifications based on experimental requirements

• For genetic studies, consider laboratory strains like D. vulgaris Hildenborough or D. desulfuricans G200, which has been successfully used as a host for expression of proteins from the Hildenborough strain

• Monitor growth by measuring optical density without exposing samples to oxygen

• Supplement media with appropriate electron donors (lactate, pyruvate) and electron acceptors (sulfate)

How can the gpmI gene be cloned and expressed in heterologous systems?

For recombinant expression of D. vulgaris gpmI, researchers should consider:

• PCR amplification of the gpmI gene from D. vulgaris genomic DNA using high-fidelity polymerase

• Cloning into appropriate expression vectors such as the T7 RNA polymerase/promoter system for E. coli expression, similar to techniques used for other D. vulgaris proteins

• For expression in Desulfovibrio species, broad-host-range vectors like pJRD215 can be utilized

• Consider placing the gene under control of strong promoters, as replacing the native promoter with the stronger cyc gene promoter has been shown to increase expression up to 20-fold for other D. vulgaris proteins

• Optimize expression conditions by testing different induction parameters, temperatures, and media compositions

What purification strategies are most effective for recombinant gpmI?

Purification of recombinant gpmI requires careful consideration of the enzyme's properties:

• Perform all steps under anaerobic conditions if possible, or include reducing agents to maintain enzyme activity

• Initial purification by affinity chromatography if the protein is expressed with a tag (His6, GST, etc.)

• Further purification by ion-exchange and size-exclusion chromatography

• For proteins expressed in E. coli, preparative SDS-PAGE can be used for isolation, as demonstrated for other D. vulgaris proteins

• When expressed in native Desulfovibrio hosts, immunoprecipitation with specific antibodies can be employed for detection and isolation of the protein

How can I generate specific antibodies against D. vulgaris gpmI?

Generation of specific antibodies follows a systematic approach:

• Express and purify recombinant gpmI or a unique peptide fragment

• Use techniques similar to those employed for DcrA from D. vulgaris, where the C-terminal domain was overexpressed using the T7 RNA polymerase/promoter system and isolated by preparative SDS-PAGE

• Immunize mice or rabbits with approximately 25 μg of purified protein, followed by booster injections

• Test antibody specificity by Western blotting and immunoprecipitation

• For improved specificity, perform affinity purification of antibodies using immobilized antigen

How can I create a markerless deletion of gpmI in D. vulgaris?

Creating a markerless deletion requires a sophisticated genetic approach:

• Utilize the markerless genetic exchange system developed for D. vulgaris that employs the counterselectable marker uracil phosphoribosyltransferase (upp)

• Design a suicide plasmid containing DNA regions flanking the gpmI gene

• Follow the two-step integration and excision strategy:

  • First introduce the plasmid into D. vulgaris by conjugation or electroporation

  • Select for single recombinants using appropriate antibiotic resistance

  • Allow second recombination to occur by growing without selection

  • Select for cells that have lost the plasmid using 5-fluorouracil (5-FU) resistance

• Screen for successful deletion mutants using PCR and confirm by Southern blotting, as demonstrated for other gene deletions in D. vulgaris

What structural features distinguish D. vulgaris gpmI from other phosphoglycerate mutases?

Investigating structural characteristics requires advanced biophysical approaches:

• Express and purify gpmI to high homogeneity (>95%)

• Perform crystallization trials under anaerobic conditions to obtain diffraction-quality crystals

• Determine the three-dimensional structure using X-ray crystallography

• Compare with known structures of:

  • iPGM enzymes from other organisms

  • dPGM enzymes to identify key structural differences

Key structural investigations should focus on:

• Active site architecture and catalytic residues

• Substrate binding pocket characteristics

• Metal coordination sites (many iPGMs require metal cofactors)

• Conformational changes during catalysis

How does environmental oxygen affect gpmI expression and activity?

D. vulgaris is a strictly anaerobic bacterium, making oxygen response particularly relevant:

• Analyze gpmI expression under different oxygen exposures using techniques like those employed for DcrA analysis, including immunoprecipitation of radiolabeled protein

• Investigate regulatory elements in the gpmI promoter region that might respond to oxygen

• Measure enzyme activity after controlled oxygen exposure to determine stability

• Examine potential connections between gpmI and oxygen-sensing proteins like DcrA, which has been shown to sense oxygen in D. vulgaris Hildenborough

Why is my recombinant gpmI expression level low in E. coli?

Low expression levels can result from multiple factors:

• Codon bias - D. vulgaris has different codon usage than E. coli; consider codon optimization

• Protein toxicity - gpmI might be toxic to E. coli when overexpressed; use tightly regulated promoters

• Formation of inclusion bodies - Modify expression conditions:

  • Lower temperature (16-25°C)

  • Reduce inducer concentration

  • Co-express with chaperones

• Promoter strength - When expressing in Desulfovibrio species, replacing the native promoter with stronger promoters like the cyc gene promoter can increase expression up to 20-fold, as demonstrated for other proteins

How can I measure gpmI enzymatic activity accurately?

Accurate enzyme activity measurement requires careful experimental design:

• Develop a coupled enzyme assay system where 2-phosphoglycerate production is linked to measurable signals

• Perform all assays anaerobically since exposure to oxygen may inactivate the enzyme

• Include appropriate controls:

  • No enzyme control

  • Heat-inactivated enzyme

  • Known concentrations of substrates and products for standard curves

What factors affect the transformation efficiency of D. vulgaris?

Transformation of D. vulgaris presents significant challenges:

• Restriction-modification systems - The type I restriction-modification system in D. vulgaris can reduce transformation efficiency by 100-1000 fold; consider using restriction-deficient strains like JW7035 that have the hsdR gene deleted

• DNA methylation - Methylate plasmid DNA appropriately before transformation

• Electroporation parameters - Optimize voltage, resistance, and capacitance

• Recovery conditions - Ensure strictly anaerobic recovery after transformation

• Plasmid design - Use appropriate origins of replication compatible with D. vulgaris, such as the pBG1 replicon from D. desulfuricans G100A

How can metabolic engineering of gpmI enhance bioremediation applications?

D. vulgaris has potential applications in bioremediation, and gpmI engineering could enhance these capabilities:

• Analyze the metabolic relationship between glycolysis (involving gpmI) and pathways relevant to bioremediation

• Create gpmI variants with improved catalytic efficiency or stability

• Overexpress optimized gpmI to potentially enhance energy metabolism during bioremediation

• Integrate gpmI modifications with other genetic changes to develop specialized bioremediation strains

Consider testing engineered strains in relevant environmental conditions, particularly focusing on heavy metal remediation where D. vulgaris has shown promise.

What is the relationship between gpmI activity and D. vulgaris pathogenicity?

Recent research has implicated D. vulgaris in gut inflammation and colitis , raising questions about the role of metabolic enzymes in pathogenicity:

• Compare gpmI expression levels between commensal and pathogenic situations

• Develop gpmI deletion mutants and test their ability to induce inflammation in models like DSS-induced colitis

• Investigate whether metabolites produced through pathways involving gpmI contribute to epithelial damage

• Examine connections between central carbon metabolism and virulence factor production

How can structural data be used to develop specific inhibitors of D. vulgaris gpmI?

Development of specific inhibitors requires a structure-based drug design approach:

• Utilize crystal structures or homology models to identify unique features of the D. vulgaris gpmI active site

• Perform virtual screening of compound libraries against the active site

• Test promising candidates in enzyme inhibition assays

• Optimize lead compounds through medicinal chemistry approaches

• Evaluate specificity by testing against human PGM and other bacterial PGMs

Potential applications include developing new antimicrobials targeting sulfate-reducing bacteria or research tools for studying D. vulgaris metabolism.