Recombinant Escherichia fergusonii Triosephosphate isomerase (tpiA)

What is the role of triosephosphate isomerase in Escherichia fergusonii metabolism?

Triosephosphate isomerase (TpiA) in E. fergusonii, similar to its E. coli counterpart, plays a central role in carbon metabolism by catalyzing the reversible interconversion between dihydroxyacetone phosphate (DHAP) and glyceraldehyde 3-phosphate (GA3P). This enzyme is essential for glycolysis, gluconeogenesis, and the efficient utilization of three-carbon substrates. In E. coli, TpiA is encoded as a stand-alone gene that is independently transcribed from adjacent genes, and knockout studies have demonstrated that the tpiA gene is essential for cellular growth on glycerol but not on glucose . This is because standard glycolysis of 6C-sugars can proceed without DHAP to GA3P conversion, whereas the enzyme is required for gluconeogenic synthesis of hexoses from 3C compounds .

How does the structure of E. fergusonii TpiA compare to other bacterial TpiAs?

E. fergusonii TpiA likely shares the highly conserved (βα)8-barrel superfold structure characteristic of triosephosphate isomerases across species. This archetypal fold consists of eight βα units linked together by loops that form a cylinder of parallel β-strands (β-barrel) surrounded by a layer of parallel α-helices . Based on structural studies of E. coli TpiA, this enzyme exhibits remarkable structural resilience, with permissiveness for insertions not only in loop regions but unexpectedly also in highly structured domains . This structural conservation combined with functional plasticity suggests evolutionary optimization while maintaining adaptability.

What are the most effective methods for cloning and expressing recombinant E. fergusonii TpiA?

For efficient expression of recombinant E. fergusonii TpiA, researchers should consider the following methodological approach:

• Gene isolation: PCR-amplify the tpiA gene from E. fergusonii genomic DNA using primers designed based on conserved regions compared with E. coli tpiA.

• Vector construction: Clone the amplified gene into an IPTG-inducible expression vector with a C-terminal epitope tag (such as E-tag) to facilitate protein detection and purification. This approach was successfully used for E. coli TpiA expression, where the E-tagged construct retained full enzymatic activity .

• Expression system: Transform the construct into an E. coli strain lacking endogenous TpiA activity (ΔtpiA strain). This allows for functional complementation assays and ensures that measured activity comes exclusively from the recombinant protein .

• Induction conditions: Optimize IPTG concentration and induction temperature (typically 0.5-1.0 mM IPTG at 30°C for 4-6 hours) to maximize soluble protein yield.

• Verification: Confirm successful expression through SDS-PAGE analysis and immunodetection using antibodies against the epitope tag .

How can the enzymatic activity of recombinant E. fergusonii TpiA be measured accurately?

Triosephosphate isomerase activity can be measured using the following protocol adapted from methods used for E. coli TpiA :

• Prepare cell lysates: Harvest cells expressing recombinant TpiA, lyse by sonication or commercial lysis reagents, and obtain soluble fractions through centrifugation.

• Protein quantification: Determine total protein concentration in soluble fractions using Bradford assays to ensure equivalent loading between samples.

• Activity assay options:
  a) Direct measurement: Monitor the isomerization of GA3P to DHAP or DHAP to GA3P spectrophotometrically.
  b) Coupled enzyme assay: Link TpiA activity to glycerol-3-phosphate dehydrogenase (GPDH) and NADH oxidation, measuring decrease in absorbance at 340 nm.

• Kinetic parameters determination: Calculate kcat and Km values using varying substrate concentrations (typically 0.01-10 mM range) and Michaelis-Menten kinetics.

• Controls: Include wild-type E. coli TpiA and no-enzyme controls for baseline comparison. When using complemented ΔtpiA strains, a growth-based assay on minimal medium with glycerol as sole carbon source provides functional validation .

Which amino acid residues are critical for the catalytic activity of E. fergusonii TpiA?

Based on mutagenesis studies of triosephosphate isomerase in other organisms, several amino acid residues are likely critical for E. fergusonii TpiA catalytic activity:

• Glutamic acid residue (equivalent to Glu-165 in chicken TpiA): Site-directed mutagenesis studies replacing this residue with aspartic acid resulted in drastically reduced catalytic efficiency, with kcat values 1/1500th of the wild-type enzyme for GA3P and 1/240th for DHAP . This suggests this glutamic acid residue is crucial for catalysis, affecting transition state stability rather than substrate binding.

• Active site residues: Based on structural conservation, the active site likely contains a catalytic base (glutamate) that abstracts a proton from the substrate and a histidine that facilitates proton transfer during the isomerization reaction.

• Loop regions: Particularly loop 6, which undergoes conformational changes during catalysis, contains essential residues for substrate binding and catalytic efficiency.

Researchers studying E. fergusonii TpiA should focus on these regions when designing mutagenesis experiments to investigate catalytic mechanisms .

How structurally permissive is E. fergusonii TpiA to amino acid insertions or modifications?

E. fergusonii TpiA likely exhibits structural permissiveness similar to that observed in E. coli TpiA. Recent studies on E. coli TpiA have revealed unexpected tolerance to structural perturbations:

This structural resilience has significant implications for protein engineering approaches using E. fergusonii TpiA as a scaffold for novel functions or as a model for studying protein folding and evolution.

How does E. fergusonii TpiA sequence and function compare with TpiA from other Escherichia species?

E. fergusonii belongs to a diverse genus that includes several cryptic lineages with varying genetic characteristics . When comparing TpiA across Escherichia species:

• Sequence conservation: TpiA is highly conserved across Escherichia species due to its essential metabolic role, though specific amino acid variations may exist between E. fergusonii and other species like E. coli.

• Functional conservation: The catalytic mechanism and substrate specificity are likely preserved across species, with kinetic parameters potentially showing subtle variations reflecting ecological adaptations.

• Genomic context: While E. coli tpiA is expressed as a single cistron transcribed independently of adjacent genes , comparative genomic analysis would be valuable to determine if this arrangement is conserved in E. fergusonii.

• Evolutionary implications: The remarkable structural permissiveness observed in E. coli TpiA suggests that similar properties might exist in E. fergusonii TpiA, potentially providing evolutionary advantages through tolerance to mutations and structural adaptability.

Researchers interested in evolutionary aspects should perform detailed phylogenetic analyses comparing TpiA sequences across multiple Escherichia lineages, including cryptic clades identified through multilocus sequence typing (MLST) .

What is the relationship between E. fergusonii antimicrobial resistance mechanisms and recombinant protein expression?

E. fergusonii has been identified as an important reservoir of antimicrobial resistance (AMR) genes, with studies showing high prevalence of resistance to multiple antibiotics . This characteristic has several implications for recombinant protein expression:

• Selection marker considerations: When designing expression systems for E. fergusonii TpiA, researchers should carefully select antibiotic resistance markers that are not already present in the E. fergusonii strain being studied. Studies have shown that E. fergusonii isolates frequently carry resistance to sulfafurazole (97.74%) and tetracycline (94.74%) .

• Expression host selection: For recombinant expression, using a well-characterized E. coli strain lacking antimicrobial resistance genes minimizes potential complications from horizontally transferred resistance determinants.

• Plasmid stability: If the E. fergusonii strain contains multiple plasmids carrying resistance genes (as detected in some isolates) , this could affect compatibility with expression vectors and plasmid stability during recombinant protein production.

• Horizontal gene transfer risks: When working with E. fergusonii as a source organism, researchers should implement appropriate containment measures due to its potential role in AMR gene dissemination, particularly for mcr-1 and extended-spectrum beta-lactamase (ESBL) genes (found in 51.88% of isolates in one study) .

How can site-directed mutagenesis of E. fergusonii TpiA advance our understanding of enzyme evolution?

Site-directed mutagenesis of E. fergusonii TpiA offers valuable insights into enzyme evolution:

• Comparative mutational analysis: By replicating mutations studied in other species (such as the Glu to Asp substitution at position 165 studied in chicken TpiA) , researchers can investigate conserved catalytic mechanisms and evolutionary constraints.

• Testing structural permissiveness: Based on findings from E. coli TpiA , researchers can introduce insertions at various positions to map structurally permissive regions. This approach revealed that E. coli TpiA can tolerate five-amino acid insertions even in highly structured regions, challenging traditional views on protein structure-function relationships.

• Ancestral state reconstruction: Introducing mutations that revert specific residues to ancestral states can help understand the evolutionary trajectory of TpiA and the selective pressures that shaped it.

• Adaptive laboratory evolution: Combined with directed evolution approaches, site-directed mutagenesis can probe the adaptive landscape of TpiA under different selective pressures.

• Fitness landscape mapping: Systematic mutagenesis across the protein can generate data on how sequence variations affect enzyme kinetics, stability, and in vivo fitness, revealing the constraints and opportunities in TpiA evolution.

Such studies could reveal whether the remarkable structural resilience observed in E. coli TpiA is a conserved feature across the Escherichia genus or specific to certain lineages.

What methodological approaches are most effective for studying the in vivo activity of E. fergusonii TpiA variants?

For studying the in vivo activity of E. fergusonii TpiA variants, researchers should consider the following methodological approaches:

• Complementation assays: Express TpiA variants in an E. coli ΔtpiA strain and assess growth on minimal medium with glycerol as sole carbon source. This approach effectively discriminates between functional and non-functional variants .

• Growth rate analysis: Quantitative growth curve measurements in various carbon sources can reveal subtle differences in enzyme efficiency that may not be apparent in endpoint assays.

• In vivo activity assays: Measure intracellular accumulation of pathway intermediates (DHAP, GA3P) using metabolomic approaches to directly assess TpiA activity in living cells.

• Competition experiments: Co-culture of strains expressing different TpiA variants can reveal fitness differences under various selective conditions.

• Chaperone dependency testing: Express TpiA variants in strains deficient in major chaperone systems (e.g., ΔdnaK) to assess whether variant stability and function depend on chaperone assistance in vivo .

• In vitro transcription/translation systems: Use cell-free expression systems to evaluate protein folding and activity without cellular quality control mechanisms, as demonstrated for E. coli TpiA variants .

This multi-faceted approach allows researchers to distinguish between effects on enzyme catalysis, protein stability, and cellular fitness, providing a comprehensive understanding of how mutations impact TpiA function in living systems.