Recombinant Geobacter uraniireducens Serine hydroxymethyltransferase (glyA)

What is Serine hydroxymethyltransferase (SHMT) and what cellular functions does it perform?

Serine hydroxymethyltransferase (SHMT), encoded by the glyA gene, is a ubiquitous pyridoxal 5'-phosphate (PLP)-dependent enzyme that catalyzes the reversible interconversion of serine and tetrahydrofolate (THF) to glycine and 5,10-methylene tetrahydrofolate (MTHF) . This reaction is pivotal in cellular metabolism for several reasons:

• It generates MTHF, which serves as a major source of cellular one-carbon units

• It provides essential precursors for purine and thymidylate biosynthesis

• It plays a central role in amino acid metabolism through glycine-serine interconversion

Beyond its primary function, SHMT has also been demonstrated to catalyze THF-independent reactions including aldolytic cleavage, decarboxylation, and transamination reactions under specific conditions . This enzymatic versatility makes SHMT a key metabolic intersection point worthy of detailed investigation across bacterial species.

How does Geobacter uraniireducens differ from other Geobacter species, particularly G. sulfurreducens?

Geobacter uraniireducens exhibits several distinctive characteristics that set it apart from the more extensively studied G. sulfurreducens:

• PilA structure: G. uraniireducens possesses a substantially longer PilA sequence (193 amino acids) compared to the truncated PilA of G. sulfurreducens (61 amino acids)

• Pili conductivity: The conductivity of G. uraniireducens pili is approximately 0.3 ± 0.09 mS/cm, which is more than two orders of magnitude lower than G. sulfurreducens pili (approximately 50 mS/cm at pH 7)

• Electron transfer mechanism: Unlike G. sulfurreducens, G. uraniireducens can reduce Fe(III) oxides occluded within microporous beads, suggesting it produces a soluble electron shuttle rather than relying on conductive pili

• Gene regulation: G. uraniireducens does not upregulate pilA expression when grown on Fe(III) oxide, unlike G. sulfurreducens and G. metallireducens

Why is SHMT considered a potential target for antimicrobial development?

SHMT has attracted significant attention as a potential target for antimicrobial development due to several key factors:

• It plays a pivotal role in DNA synthesis through thymidylate production

• High levels of SHMT activity are observed in rapidly proliferating cells

• Its central position in one-carbon metabolism makes it essential for cellular function

• Structural differences between bacterial and human SHMT enzymes may allow for selective targeting

In the context of Geobacter species, understanding SHMT function may provide insights into metabolic adaptation during host-pathogen interactions, supporting the concept of "nutritional virulence" where metabolic capabilities influence bacterial survival and pathogenicity .

What are the recommended approaches for cloning and expressing recombinant G. uraniireducens SHMT?

Based on successful approaches with related bacterial SHMT enzymes, the following methodology is recommended:

• Gene amplification:

  • Design primers targeting the G. uraniireducens glyA gene sequence with appropriate restriction sites

  • Use high-fidelity polymerase to amplify the gene from genomic DNA

• Vector construction:

  • Select an expression vector with an inducible promoter (e.g., pPLT173 or pQE60 as used for similar constructs)

  • Include appropriate affinity tags (His-tag or similar) for purification

  • Verify the construct by sequencing before transformation

• Expression system:

  • E. coli is typically suitable as demonstrated in complementation studies with H. pylori SHMT

  • Optimize expression conditions including temperature (typically 30°C based on Geobacter cultivation conditions), induction timing, and concentration of inducer

• Purification strategy:

  • Implement affinity chromatography followed by size exclusion chromatography

  • Include PLP in purification buffers to maintain cofactor association

  • Verify enzymatic activity following each purification step

This strategy leverages approaches that have proven successful with other bacterial SHMT enzymes while accounting for specific properties of Geobacter species.

How can functional complementation be used to verify SHMT activity?

Functional complementation represents a powerful approach to confirm SHMT enzymatic activity in vivo, as demonstrated with H. pylori SHMT:

• Host strain preparation:

  • Generate an E. coli ΔglyA deletion strain by interrupting the chromosomal glyA gene with an antibiotic resistance cassette

  • Verify glycine auxotrophy of the deletion strain (inability to grow on minimal medium without glycine)

• Complementation setup:

  • Transform the E. coli ΔglyA strain with an expression plasmid carrying the G. uraniireducens glyA gene under an inducible promoter

  • Include appropriate controls: wild-type E. coli and the deletion strain with empty vector

• Growth assessment:

  • Test growth on minimal medium with and without glycine supplementation

  • Include inducer (e.g., IPTG) to ensure expression of the recombinant enzyme

  • Monitor growth rates under different conditions

• Data interpretation:

  • Successful complementation is indicated by restored growth on minimal medium without glycine

  • Quantitative assessment of growth rates provides information about the efficiency of the recombinant enzyme

This methodology provides functional evidence of enzymatic activity within a cellular context before proceeding to biochemical characterization.

What techniques are most effective for characterizing the kinetic properties of recombinant G. uraniireducens SHMT?

A comprehensive kinetic characterization of recombinant G. uraniireducens SHMT should include:

• Spectroscopic analysis:

  • Monitor PLP binding through absorption spectra (characteristic peaks at 425-435 nm)

  • Assess formation of enzyme-PLP-glycine-folate complexes, which produce distinctive spectral shifts

• Steady-state kinetics:

  • Determine Km and Vmax values for key substrates (serine, glycine, THF)

  • Evaluate the effect of pH and temperature on enzymatic activity

  • Assess cofactor (PLP) binding affinity through titration experiments

• Reaction directionality:

  • Compare forward (serine to glycine) and reverse (glycine to serine) reaction rates

  • Determine the equilibrium constant under physiological conditions

• Alternative reaction pathways:

  • Test THF-independent reactions including aldolytic cleavage and transamination

  • Quantify relative efficiencies of different reaction pathways

• Inhibition studies:

  • Evaluate the effect of known SHMT inhibitors

  • Identify specific inhibitory mechanisms (competitive, noncompetitive, etc.)

These approaches will provide comprehensive understanding of the enzymatic properties of G. uraniireducens SHMT and allow comparison with enzymes from other species.

How can the three-dimensional structure of G. uraniireducens SHMT be determined and what insights might it provide?

Determination of the G. uraniireducens SHMT structure can follow this methodological approach:

• Protein preparation:

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

  • Ensure sample monodispersity through dynamic light scattering

  • Optimize buffer conditions for stability during crystallization

• Crystallization:

  • Perform initial screening using commercial crystallization kits

  • Optimize promising conditions for diffraction-quality crystals

  • Consider both apo-enzyme and holo-enzyme (with PLP) crystallization

• X-ray diffraction:

  • Collect high-resolution diffraction data at synchrotron facilities

  • Process data and determine phase information (molecular replacement using other bacterial SHMT structures is likely feasible)

  • Build and refine the structural model

The resulting structure would provide valuable insights including:

• PLP binding pocket architecture and key residues involved in cofactor interaction

• Substrate binding sites and catalytic residues

• Structural basis for any observed differences in cofactor affinity compared to other SHMTs, similar to observations in H. pylori SHMT which showed weak PLP binding and structural features that may explain this property

• Oligomeric state and subunit interfaces

• Potential unique features related to G. uraniireducens metabolism

The structural analysis could be particularly informative given the unique metabolic characteristics of G. uraniireducens and its distinct electron transport mechanisms.

What is the relationship between SHMT function and the electron transfer mechanisms in G. uraniireducens?

The relationship between SHMT and electron transfer in G. uraniireducens presents an intriguing research avenue that may be explored through:

• Metabolic pathway analysis:

  • SHMT generates glycine and one-carbon units essential for nucleotide synthesis

  • These biosynthetic processes require reducing equivalents that may interact with electron transfer pathways

• Comparative expression studies:

  • Analyze glyA expression under different electron acceptor conditions

  • Compare expression patterns with G. sulfurreducens, which shows different electron transfer mechanisms

• Metabolic flux analysis:

  • Trace carbon flow through SHMT-dependent pathways under different electron acceptor conditions

  • Determine if altered electron transfer mechanisms affect SHMT-dependent metabolism

How does PLP binding affinity in G. uraniireducens SHMT compare to other bacterial enzymes and what are the implications?

While specific data for G. uraniireducens SHMT is not directly available, insights can be gained from observations of other bacterial SHMTs:

• PLP binding characteristics:

  • H. pylori SHMT shows unexpectedly weak binding affinity for PLP

  • The structural basis for this weak binding was revealed through crystallographic studies at 2.8Å resolution

• Structural considerations:

  • Key residues in the PLP binding pocket likely determine cofactor affinity

  • The three-dimensional structure of G. uraniireducens SHMT would reveal whether similar structural features exist

• Functional implications:

  • Variable PLP binding affinity may reflect adaptation to different intracellular environments

  • Weak PLP binding could potentially serve as a regulatory mechanism by making enzyme activity more sensitive to cofactor availability

• Inhibitor development potential:

  • Differences in PLP binding sites between bacterial and human enzymes could be exploited for selective inhibition

  • As noted with H. pylori SHMT, "stabilization of the proposed inactive configuration using small molecules has potential to provide a specific way for inhibiting SHMT"

Comparative analysis of PLP binding across different bacterial SHMTs could provide insights into evolutionary adaptation and potential species-specific regulatory mechanisms.

What approaches are recommended for creating a G. uraniireducens glyA knockout strain?

Creating a G. uraniireducens glyA knockout strain would require:

• Knockout construct design:

  • Create a construct with an antibiotic resistance cassette (e.g., kanamycin resistance gene aphA-3) flanked by regions homologous to sequences upstream and downstream of the glyA gene

  • Ensure the construct design prevents polar effects on downstream genes

• Transformation methodology:

  • Adapt established transformation protocols for Geobacter species

  • Consider natural competence, electroporation, or conjugation approaches

  • Optimize transformation conditions based on G. uraniireducens-specific requirements

• Selection and verification:

  • Select transformants on appropriate antibiotic-containing media

  • Verify gene disruption through PCR, sequencing, and possibly Western blotting

  • Confirm the absence of SHMT enzymatic activity in cell extracts

• Phenotypic characterization:

  • Assess growth rates under various conditions (as demonstrated with H. pylori ΔglyA, which showed significantly impaired growth)

  • Determine metabolic requirements (e.g., glycine auxotrophy)

  • Evaluate electron transfer capabilities and Fe(III) reduction

This methodological approach would need to address potential challenges associated with genetic manipulation of G. uraniireducens, which may differ from more genetically tractable species.

How can researchers assess the phenotypic consequences of glyA inactivation in Geobacter species?

Based on studies with other bacterial systems, a comprehensive assessment of glyA inactivation phenotypes should include:

• Growth characteristics:

  • Measure growth rates in both rich and minimal media

  • Construct growth curves under aerobic and anaerobic conditions

  • Compare doubling times between wild-type and ΔglyA strains

  For example, in H. pylori, the ΔglyA strain showed dramatically reduced growth with a doubling time of 21 hours compared to 4 hours for the wild-type strain .

• Nutritional requirements:

  • Determine whether glycine supplementation rescues growth defects

  • Assess the effect of serine and other potential metabolic precursors

  • Test alternative carbon and nitrogen sources

• Electron transfer capabilities:

  • Measure Fe(III) reduction rates with various forms of iron

  • Assess reduction of Fe(III) oxide sequestered in alginate beads

  • Compare current production in microbial electrochemical systems

• Gene expression analysis:

  • Perform RNA-seq to identify compensatory gene expression changes

  • Focus on pathways potentially related to one-carbon metabolism

  • Compare expression profiles under different growth conditions

• Virulence factor expression:

  • Assess production of key cellular components and potential virulence factors

  • In H. pylori, glyA inactivation led to loss of the virulence factor CagA

This multi-faceted approach would provide comprehensive understanding of the metabolic role of SHMT in Geobacter species.

What are the expected metabolic effects of glyA gene complementation in knockout strains?

Complementation studies with glyA provide valuable insights into SHMT function and can be designed to answer specific questions:

• Restoration of growth:

  • Full complementation should restore wild-type growth rates

  • Partial complementation may indicate requirements for precise expression levels or additional factors

• Heterologous complementation:

  • Testing whether SHMT from other species can complement G. uraniireducens ΔglyA

  • This approach could reveal species-specific requirements or functions

• Structure-function analysis:

  • Complementation with mutated versions of glyA to identify essential residues

  • Testing chimeric SHMT proteins to pinpoint functionally important domains

• Regulated expression:

  • Using inducible promoters to control SHMT levels

  • Determining the minimum SHMT activity required for normal growth

• In vitro vs. in vivo activity correlation:

  • Comparing enzymatic properties of purified mutant SHMTs with their ability to complement in vivo

  • This could reveal additional functions beyond the canonical enzymatic activity

Successful complementation methodology has been demonstrated with H. pylori SHMT in an E. coli ΔglyA strain, confirming functional conservation of the enzyme across bacterial species . Similar approaches could be applied to study G. uraniireducens SHMT.

How might transcriptional regulation of glyA differ between Geobacter species with different electron transfer mechanisms?

Investigating differential regulation of glyA between Geobacter species requires:

• Comparative promoter analysis:

  • Identify regulatory elements in the glyA promoter regions across Geobacter species

  • Look for binding sites of known transcription factors related to metabolism and electron transfer

• Expression profiling:

  • Compare glyA expression patterns under various growth conditions

  • G. sulfurreducens and G. metallireducens highly express pilA when growing with extracellular electron acceptors, while G. uraniireducens does not upregulate pilA under the same conditions

  • Similar differential expression patterns might exist for glyA

• Regulatory network mapping:

  • Construct transcriptional networks including glyA and electron transfer components

  • Identify potential co-regulated genes that may connect these pathways

• Chromatin immunoprecipitation (ChIP) studies:

  • Identify transcription factors binding to the glyA promoter

  • Compare binding patterns across different species and conditions

This approach would help elucidate how metabolic pathways involving SHMT are integrated with the distinct electron transfer mechanisms observed in different Geobacter species.

What role might SHMT play in thymidylate synthesis in Geobacter uraniireducens?

SHMT's role in thymidylate synthesis pathways can be investigated through:

• Pathway comparison:

  • Determine whether G. uraniireducens utilizes ThyA or ThyX for thymidylate synthesis

  • This distinction is critical as it affects folate cycling:

    • ThyA-containing organisms typically have SHMT, thymidylate synthase, and dihydrofolate reductase as part of the thymidylate/folate cycle

    • ThyX-containing organisms produce tetrahydrofolate directly and often lack dihydrofolate reductase

• Metabolic flux analysis:

  • Trace one-carbon units from serine through SHMT to thymidylate synthesis

  • Quantify the contribution of SHMT-derived one-carbon units to total thymidylate production

• Growth studies with labeled precursors:

  • Use isotopically labeled serine to track carbon flow through SHMT to thymidylate

  • Compare incorporation patterns between wild-type and ΔglyA strains

• Synthetic lethality testing:

  • Attempt to create double mutants affecting both SHMT and alternative pathways for one-carbon unit generation

  • Determine whether SHMT becomes essential under specific metabolic conditions

Understanding SHMT's role in thymidylate synthesis is particularly important as "the presence of ThyX may provide growth benefits under conditions where the level of reduced folate derivatives is compromised" , suggesting potential metabolic adaptations that may be relevant to G. uraniireducens' unique ecological niche.

How can isotope labeling experiments be designed to trace one-carbon metabolism through SHMT in G. uraniireducens?

Isotope labeling experiments to investigate SHMT-dependent metabolism can follow this methodology:

• Experimental design:

  • Culture G. uraniireducens with isotopically labeled substrates (13C-serine, 13C-glycine, or 15N-labeled amino acids)

  • Compare wild-type and ΔglyA strains to identify SHMT-dependent pathways

  • Harvest cells at different growth phases to capture temporal dynamics

• Analytical techniques:

  • Apply GC-MS or LC-MS/MS to detect labeled metabolites

  • Use NMR for detailed structural characterization of key metabolites

  • Implement metabolic flux analysis software to quantify pathway activities

• Target metabolites:

  • Monitor incorporation into nucleotides (particularly thymidylate)

  • Track label distribution in amino acids and protein

  • Analyze incorporation into potential electron shuttle compounds

• Data interpretation framework:

  • Construct metabolic models incorporating G. uraniireducens-specific pathways

  • Account for the unique electron transfer mechanisms and their metabolic requirements

  • Compare results with similar studies in G. sulfurreducens to identify species-specific features

This experimental approach would provide detailed insights into the integration of SHMT activity with G. uraniireducens' distinctive metabolism and electron transfer mechanisms.

What are common challenges in expressing and purifying recombinant bacterial SHMT enzymes?

Researchers working with recombinant bacterial SHMT enzymes frequently encounter these challenges and solutions:

• Inclusion body formation:

  • Challenge: Overexpressed SHMT often forms insoluble aggregates

  • Solutions:

    • Reduce expression temperature (e.g., 18-25°C)

    • Use solubility-enhancing fusion tags (SUMO, MBP)

    • Co-express with molecular chaperones

    • Include PLP in growth medium and lysis buffer

• Cofactor loss during purification:

  • Challenge: PLP can dissociate during purification, leading to inactive enzyme

  • Solutions:

    • Add PLP to all purification buffers

    • Monitor absorbance ratio (280 nm vs. 425 nm) to track cofactor retention

    • Implement reconstitution protocols if cofactor is lost

• Protein instability:

  • Challenge: Bacterial SHMTs can show limited stability during storage

  • Solutions:

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

    • Test glycerol, arginine, or other stabilizing agents

    • Determine appropriate storage conditions (-80°C vs. liquid nitrogen)

• Heterogeneous oligomeric states:

  • Challenge: Variable oligomerization affecting activity measurements

  • Solutions:

    • Implement size exclusion chromatography as a final purification step

    • Analyze oligomeric state by native PAGE or analytical ultracentrifugation

    • Stabilize preferred oligomeric form through buffer optimization

These approaches address issues commonly encountered with bacterial SHMTs and would likely be applicable to G. uraniireducens SHMT expression and purification.

How can researchers troubleshoot unexpected results in SHMT activity assays?

When troubleshooting unexpected SHMT activity results, consider this systematic approach:

• Enzyme quality assessment:

  • Verify protein purity by SDS-PAGE (>95% recommended)

  • Check PLP content through absorbance spectrum (characteristic peak at 425-435 nm)

  • Assess oligomeric state by native PAGE or size exclusion chromatography

• Assay validation:

  • Include positive controls (commercial SHMT or well-characterized recombinant enzyme)

  • Test buffer components individually for interference

  • Verify linear range of detection method and enzyme concentration

• Common interference factors:

  • Metal ions: some may inhibit activity or promote aggregation

  • Oxidation: cysteine residues may be sensitive to oxidation

  • Product inhibition: accumulation of products may cause feedback inhibition

  • Temperature sensitivity: activity can be highly temperature-dependent

• Specific issues for G. uraniireducens SHMT:

  • Based on observations with other bacterial SHMTs, consider:

    • Potential weak PLP binding requiring higher cofactor concentrations

    • Species-specific pH optima that may differ from other SHMTs

    • Possible alternative substrate preferences

• Data interpretation challenges:

  • Non-linear kinetics may indicate cooperative binding or multiple catalytic sites

  • Apparent substrate inhibition at high concentrations

  • Time-dependent changes in activity suggesting protein instability

This systematic troubleshooting approach will help identify and address specific issues affecting SHMT activity measurements.

What controls are essential when designing experiments to compare SHMT function across different Geobacter species?

Rigorous experimental design for comparative SHMT studies should include these critical controls:

• Expression system consistency:

  • Use identical expression vectors and host strains

  • Apply consistent induction and growth conditions

  • Verify protein expression levels by Western blotting

• Purification protocol standardization:

  • Implement identical purification steps

  • Maintain consistent buffer compositions

  • Verify final purity by SDS-PAGE and specific activity measurements

• Enzymatic assay controls:

  • Perform enzyme kinetics at multiple enzyme concentrations to ensure linearity

  • Include time-course measurements to verify steady-state conditions

  • Test multiple substrate concentrations spanning below and above predicted Km values

• Structure-based comparisons:

  • Include spectroscopic analysis of PLP binding

  • Measure thermal stability using differential scanning fluorimetry

  • Assess oligomeric state under identical conditions

• Specific controls for G. uraniireducens vs. G. sulfurreducens comparison:

  • Given the different electron transfer mechanisms between these species , include:

    • Assays under varying redox conditions

    • Tests with potential electron shuttle compounds

    • Comparisons in both aerobic and strictly anaerobic environments

These controls will ensure that observed differences in SHMT properties reflect genuine species-specific adaptations rather than experimental variables.