Recombinant Streptococcus gordonii 6-phosphofructokinase (pfkA)

What is the role of 6-phosphofructokinase (pfkA) in Streptococcus gordonii metabolism?

6-phosphofructokinase (pfkA) in Streptococcus gordonii is a key glycolytic enzyme that catalyzes the conversion of fructose-6-phosphate (F6P) and ATP to fructose-1,6-bisphosphate (F-1,6-P) and ADP. This reaction represents a critical regulatory point in glycolysis, directly influencing the metabolic rate and energy production in S. gordonii. As a primary colonizer of the oral cavity, S. gordonii relies on efficient energy metabolism to establish itself within the oral microbiome and form biofilms. The pfkA enzyme plays a crucial role in the bacterium's adaptation to changing nutrient conditions in the oral environment. Similar to other PFK1 enzymes, S. gordonii pfkA activity is likely subject to allosteric regulation by various metabolites, including ATP, which can act as both a substrate and an inhibitor at higher concentrations .

What genetic elements regulate pfkA expression in S. gordonii?

The expression of pfkA in Streptococcus gordonii is regulated by multiple genetic elements that respond to environmental and metabolic cues. While specific regulatory mechanisms for S. gordonii pfkA have not been fully characterized, research on streptococcal species suggests that expression is likely controlled by carbon catabolite repression (CCR) systems responding to carbohydrate availability. The promoter region of pfkA typically contains binding sites for global transcriptional regulators that sense energy status and sugar availability. In streptococci, the CcpA (catabolite control protein A) often plays a significant role in regulating glycolytic genes including pfkA, binding to catabolite-responsive elements (cre) in the promoter region. Additionally, the expression may be influenced by oxygen tension and pH, factors that are particularly relevant in the oral environment where S. gordonii naturally resides as part of the commensal microflora . Understanding these regulatory mechanisms is essential for optimizing recombinant expression systems.

What expression systems are most effective for producing recombinant S. gordonii pfkA?

The selection of an appropriate expression system for recombinant S. gordonii pfkA depends on research objectives and downstream applications. Several systems have been successfully employed:

Streptococcal homologous expression: Using modified S. gordonii as both the source and expression host can provide proper folding and post-translational modifications. S. gordonii strain GP251 has been successfully used as a carrier strain for recombinant protein expression, demonstrating the feasibility of utilizing S. gordonii vectors for production of biologically active proteins .

Lactococcal expression systems: Lactococcus lactis provides another Gram-positive alternative with GRAS (Generally Recognized As Safe) status, potentially offering advantages for applications requiring non-pathogenic hosts.

The most effective system often depends on the specific research goals. For structural studies requiring large protein quantities, E. coli systems may be preferable, while for functional studies examining physiological relevance, homologous expression in S. gordonii might yield more biologically representative results.

What are optimal purification strategies for maintaining activity of recombinant S. gordonii pfkA?

Purification of recombinant S. gordonii pfkA requires strategies that preserve enzymatic activity while achieving high purity. The following methodological approach is recommended:

• Cell lysis optimization: Gentle lysis using lysozyme treatment followed by sonication in buffer containing glycerol (10-20%) and reducing agents (1-5 mM DTT or β-mercaptoethanol) helps maintain protein stability.

• Affinity chromatography: Histidine-tagged constructs can be purified using Ni-NTA or TALON resins. Elution should utilize an imidazole gradient rather than step elution to separate pfkA from contaminants with different binding affinities.

• Buffer composition considerations:

  • pH range: 7.0-7.5 (optimal for stability)

  • Salt concentration: 50-150 mM NaCl

  • Stabilizing agents: 10% glycerol, 1 mM DTT

  • Substrate protection: 0.1-0.5 mM ATP

• Size exclusion chromatography: As a polishing step, gel filtration separates oligomeric forms and removes aggregates, which is critical as PFK enzymes typically function as tetramers or octamers.

• Activity preservation: All purification steps should be performed at 4°C, and the enzyme preparation should be supplemented with glycerol (20-25%) for storage at -80°C to preserve activity.

These strategies help ensure that the purified recombinant pfkA maintains its native conformation and catalytic functionality for subsequent enzymatic assays and structural studies.

How can protein engineering enhance stability and activity of recombinant S. gordonii pfkA?

Protein engineering approaches can significantly enhance both stability and catalytic efficiency of recombinant S. gordonii pfkA. Several methodological strategies have proven effective:

• Rational design based on homology modeling: Using structural information from homologous PFK enzymes, targeted mutations can strengthen hydrogen bonding networks or introduce disulfide bridges to enhance thermostability without compromising catalytic function.

• Directed evolution: Libraries of pfkA variants can be generated through error-prone PCR or DNA shuffling, followed by screening for enhanced stability under conditions relevant to intended applications.

• Active site optimization: Modifying residues in the substrate binding pocket can alter the Km and kcat values, potentially reducing substrate inhibition effects observed with ATP at higher concentrations .

• Surface charge engineering: Altering surface charge distribution through targeted mutations can improve solubility and reduce aggregation tendencies.

• Domain stabilization: Introducing mutations at domain interfaces can enhance oligomeric stability, which is particularly important as PFK enzymes typically function as multimers.

• Fusion protein approaches: N-terminal or C-terminal fusion tags beyond those used for purification (e.g., thioredoxin, SUMO) can enhance solubility and expression levels.

These engineering strategies must be balanced against maintaining the native catalytic mechanism, as alterations that enhance stability may sometimes compromise activity or regulatory properties. Iterative rounds of engineering followed by biochemical characterization are typically required to achieve optimal enhancement.

What methods are most accurate for measuring S. gordonii pfkA enzymatic activity?

Several complementary methods can be employed for accurate measurement of S. gordonii pfkA activity, each with distinct advantages:

Coupled spectrophotometric assays: The most widely used approach couples PFK activity to NADH oxidation through auxiliary enzymes (aldolase, triosephosphate isomerase, and glycerol-3-phosphate dehydrogenase). This method allows continuous monitoring of activity by tracking NADH absorbance decrease at 340 nm. For optimal results:

• Maintain excess of coupling enzymes to ensure PFK is rate-limiting

• Include controls to verify coupling enzymes are not inhibited under experimental conditions

• Account for potential lag phases in assay progression

Direct product quantification: Measuring F-1,6-P production directly using chromatographic methods (HPLC or LC-MS) provides an alternative that eliminates concerns about coupling enzyme limitations. This approach is particularly valuable for:

• Determining initial reaction velocities with high precision

• Investigating complex kinetic mechanisms including substrate inhibition

• Comparing data from both initial-velocity and modeling methods

ADP formation assays: Luminescence-based assays that quantify ADP production offer high sensitivity and are amenable to high-throughput screening formats.

Enzyme activity modeling: Beyond simple initial velocity measurements, differential equation-based modeling that explicitly accounts for ATP's dual roles as substrate and inhibitor can provide deeper insights into pfkA behavior under varying conditions .

A comparison of these methods reveals that while the coupled assay is more convenient for routine measurements, direct product quantification provides more reliable data for detailed kinetic analyses, particularly when investigating substrate inhibition phenomena.

How do pH and temperature affect the kinetic parameters of S. gordonii pfkA?

The enzymatic activity of S. gordonii pfkA demonstrates significant dependence on both pH and temperature, with these parameters affecting multiple kinetic constants:

pH effects on kinetic parameters:

• S. gordonii pfkA typically exhibits a bell-shaped pH-activity profile with optimum around pH 7.0-7.5

• At lower pH values (6.0-6.5), both substrate affinity (increased Km) and maximum velocity (decreased Vmax) are negatively affected

• ATP inhibition becomes more pronounced at lower pH values, crucial for understanding enzyme behavior in acidic microenvironments

• The pH sensitivity reflects protonation states of key catalytic residues and impacts both substrate binding and catalytic efficiency

Temperature effects:

• Activity typically increases with temperature up to an optimum (usually 37-42°C for S. gordonii enzymes)

• Higher temperatures initially increase reaction velocity but also accelerate thermal denaturation

• The activation energy (Ea) calculated from Arrhenius plots typically ranges from 30-50 kJ/mol

• Temperature effects are particularly important when considering S. gordonii's adaptation to the oral environment

The table below summarizes the typical changes in kinetic parameters under varying conditions:

These relationships between environmental conditions and enzyme kinetics are essential for understanding S. gordonii metabolism in both natural and experimental contexts.

What mechanisms explain ATP's dual role as both substrate and inhibitor of S. gordonii pfkA?

ATP plays a complex dual role in S. gordonii pfkA function, serving as both an essential substrate and an allosteric inhibitor at higher concentrations. This phenomenon represents a sophisticated regulatory mechanism that can be explained through several molecular mechanisms:

Conformational changes upon inhibitory binding: When ATP binds to the allosteric inhibitory site, it likely induces conformational changes that propagate to the catalytic site, affecting either substrate binding or the catalytic mechanism itself. Structural studies of homologous PFKs suggest that this involves shifting the enzyme from an "R" (relaxed, more active) to "T" (tense, less active) state.

Physiological significance: This substrate inhibition mechanism serves as a negative feedback loop that helps regulate glycolytic flux based on cellular energy status. When ATP levels are high (indicating abundant energy), glycolysis is slowed to prevent unnecessary carbohydrate metabolism. This regulatory feature is particularly important for S. gordonii as a commensal organism that must carefully regulate its metabolism within the competitive oral microenvironment .

Understanding this mechanism is crucial for accurate interpretation of experimental data and for designing inhibitors or activators that might target specific conformational states of the enzyme.

How can recombinant S. gordonii expressing modified pfkA be used as a vaccine delivery system?

Recombinant S. gordonii strains expressing modified pfkA or pfkA fusion proteins represent a promising approach for mucosal vaccine delivery systems. This application leverages several advantageous properties of S. gordonii:

Rationale for using S. gordonii as a vaccine vector:
S. gordonii is a human oral commensal bacterium that has been successfully developed as a vector for delivery of vaccines against various pathogens . The bacterium can colonize mucosal surfaces including oral, gut, and vaginal mucosa, enabling sustained antigen presentation to the host immune system. Studies have demonstrated that S. gordonii recombinants can induce both systemic and local immune responses against heterologous antigens expressed on their surface .

Methodological approach for pfkA-based vaccine constructs:

• Design of fusion constructs: pfkA can be genetically fused with antigenic epitopes from target pathogens, creating chimeric proteins that retain both metabolic function and immunogenicity.

• Surface display technology: Using cell wall anchoring domains (such as those from M protein or protein G), the pfkA-antigen fusion can be displayed on the S. gordonii cell surface, enhancing accessibility to immune cells.

• Strain development: Similar to established approaches using S. gordonii strain GP251 as a carrier strain , genetically modified S. gordonii expressing pfkA-antigen fusions can be developed through homologous recombination or plasmid-based expression systems.

• Immunization protocol: Oral administration of the recombinant strains allows for colonization of mucosal surfaces, providing continuous antigenic stimulation. Studies with similar S. gordonii recombinants have shown that this approach can effectively induce specific antibodies in serum and saliva .

Advantages and considerations:

• Mucosal delivery bypasses needles and cold-chain requirements

• The metabolic activity of pfkA may provide adjuvant-like effects

• Safety profile of S. gordonii as a commensal organism reduces concerns compared to attenuated pathogens

• Potential for developing multivalent vaccines by co-expressing multiple antigens

This approach represents an innovative intersection between metabolic engineering and vaccine development, utilizing recombinant S. gordonii pfkA as both a metabolically relevant component and an immunogenic carrier.

What role does pfkA play in S. gordonii biofilm formation and interspecies interactions?

Phosphofructokinase (pfkA) plays a multifaceted role in S. gordonii biofilm formation and interspecies interactions within the oral microbiome:

Metabolic contribution to biofilm development:
As a key glycolytic enzyme, pfkA influences the energetics of biofilm formation by regulating carbon flux and ATP generation. This metabolic activity is particularly important during the transition from planktonic to biofilm growth, where energy demands shift. Altered pfkA activity affects exopolysaccharide production, a critical component of biofilm matrix, by influencing the availability of metabolic intermediates.

Interspecies metabolic networking:
Within multispecies biofilms, S. gordonii serves as a primary colonizer that facilitates the attachment of secondary colonizers. Research has shown that S. gordonii frequently associates with other microorganisms such as Fusobacterium nucleatum, with these co-aggregation events triggering transcriptional changes in both bacteria . The metabolic activity governed by pfkA likely contributes to these interactions by:

• Influencing production of metabolites that serve as cross-feeding substrates for other species

• Affecting local microenvironment conditions (pH, oxygen consumption)

• Contributing to the generation of signaling molecules that mediate interbacterial communication

Immunomodulatory effects:
The metabolic activity of S. gordonii, partially regulated by pfkA, influences how the bacterium interacts with host immunity. Co-aggregation of S. gordonii with other bacteria affects their survival within macrophages and modulates the expression of pro-inflammatory cytokines . Similarly to how commensal S. cristatus dampens the response to F. nucleatum infection by inhibiting NF-κB and IL-8 production in oral epithelial cells , the metabolic state of S. gordonii likely influences its immunomodulatory properties.

Methodological approaches to study pfkA in biofilms:
Researchers investigating pfkA's role in biofilm formation typically employ:

• Comparison of wild-type and pfkA-mutant strains in static and flow biofilm systems

• Metabolomic analysis of biofilm exudates under varying conditions

• Transcriptomic profiling to identify co-regulated genes during biofilm development

• Confocal microscopy with fluorescent reporters to visualize metabolic activity within biofilm architecture

Understanding these interactions is crucial for developing strategies to modulate oral biofilms for health applications.

How might structural analysis of S. gordonii pfkA inform antimicrobial development?

Structural analysis of S. gordonii pfkA offers significant potential for informing novel antimicrobial development strategies, particularly those targeting oral streptococci. This approach leverages several key concepts:

Structural uniqueness as a basis for selectivity:
Detailed structural characterization of S. gordonii pfkA can reveal unique structural features that differ from human phosphofructokinase, enabling the design of inhibitors with selectivity for bacterial enzymes. Particular attention should focus on:

• The ATP-binding pocket architecture, which demonstrates different properties between bacterial and human PFKs

• Allosteric regulatory sites that may be unique to bacterial enzymes

• Oligomerization interfaces that could be targeted to disrupt quaternary structure

Targeting the dual functionality of ATP binding:
The complex relationship between ATP as both substrate and inhibitor of pfkA presents a unique opportunity for drug design. Compounds that mimic ATP but preferentially bind to the inhibitory site could amplify the natural substrate inhibition mechanism. Alternatively, molecules that prevent inhibitory ATP binding while permitting catalytic site binding could potentially hyperactivate the enzyme, disrupting metabolic homeostasis.

Rational design methodology:
A comprehensive approach to structure-based antimicrobial development would include:

• High-resolution structural determination of S. gordonii pfkA through X-ray crystallography or cryo-EM

• Computational screening of compound libraries against identified binding pockets

• Structure-activity relationship studies to optimize lead compounds

• Evaluation of species selectivity across oral streptococci and human PFK

• Assessment of effects on biofilm formation and interspecies interactions

Metabolic vulnerability exploitation:
S. gordonii relies on pfkA activity for energy production, particularly in the carbohydrate-rich environment of the oral cavity. Targeting this enzyme could disrupt the bacterium's ability to establish itself as a primary colonizer in dental plaque. Furthermore, as S. gordonii is associated with over 30% of native valve endocarditis cases along with other oral streptococci , developing antimicrobials targeting pfkA could have applications beyond oral health.

This structure-based approach represents a promising strategy for developing narrow-spectrum antimicrobials with reduced impact on beneficial microbiota compared to conventional broad-spectrum antibiotics.

How should researchers address inconsistent kinetic data when characterizing recombinant S. gordonii pfkA?

Inconsistent kinetic data is a common challenge when characterizing enzymes like recombinant S. gordonii pfkA. Researchers should implement a systematic troubleshooting approach:

Methodological approach to reconciling contradictory data:

• Evaluate assay conditions: Different buffer systems, pH values, and ionic strengths can significantly impact pfkA activity measurements. Standardize these parameters and test activity across a range of conditions to identify potential sources of variability.

• Compare direct and coupled assay methods: Discrepancies often arise between direct product measurement and coupled spectrophotometric assays. When possible, validate findings using both the initial-velocity method and modeling method approaches . The modeling method can separate ATP's dual roles as substrate and inhibitor, providing deeper insights into apparent contradictions.

• Consider enzyme preparation differences: Variations in expression systems, purification methods, and storage conditions can affect enzyme conformation and activity. Document and standardize preparation protocols, including:

  • Expression host and conditions

  • Purification strategy and buffer composition

  • Storage conditions and freeze-thaw cycles

• Analyze oligomeric state: PFK enzymes function as multimers, and different oligomeric forms may exhibit distinct kinetic properties. Use size exclusion chromatography or analytical ultracentrifugation to determine the oligomeric distribution in different preparations.

• Statistical approaches: When analyzing inhibition data, particularly for complex phenomena like substrate inhibition, apply appropriate statistical models:

  • Compare different inhibition models (competitive, noncompetitive, mixed)

  • Use global fitting approaches for multiple datasets

  • Implement bootstrap analysis to estimate parameter confidence intervals

• Control for batch-to-batch variability: Establish internal standards and reference preparations to normalize data across experimental sessions.

By systematically addressing these factors and implementing robust analytical approaches, researchers can reconcile apparently contradictory kinetic data and develop a more comprehensive understanding of S. gordonii pfkA behavior under diverse conditions.

What are effective strategies for optimizing codon usage in recombinant S. gordonii pfkA expression?

Optimizing codon usage is critical for achieving high-level expression of recombinant S. gordonii pfkA in heterologous hosts. The following strategies have proven effective:

Host-specific codon optimization:
Different expression hosts have distinct codon usage preferences that reflect their tRNA abundance profiles. For E. coli expression, the pfkA sequence should be optimized by:

• Replacing rare codons (AGA/AGG for arginine, CTA for leucine, ATA for isoleucine) with more frequent synonymous codons

• Balancing GC content to 40-60% to improve mRNA stability

• Avoiding consecutive rare codons that can cause ribosomal stalling

Strategic approach to codon optimization:

• Whole gene optimization: Tools like GenScript's OptimumGene™ or IDT's Codon Optimization Tool can analyze and optimize the entire pfkA sequence for your specific expression host.

• Targeted optimization: Focus optimization efforts on:

  • N-terminal codons (first 15-25 amino acids) that strongly influence translation initiation

  • Regions encoding structurally critical domains

  • Areas with predicted secondary structure in mRNA that might impede translation

• Harmonization rather than maximization: Rather than simply using the most frequent codons throughout, "harmonize" codon usage to mimic the natural translational rhythm, which can improve protein folding.

Experimental validation of optimization strategies:
Different optimization approaches should be empirically tested, as theoretical predictions don't always translate to improved expression. Compare:

• Expression levels (via Western blot or activity assays)

• Solubility profiles

• Enzymatic activity of the purified protein

Additional considerations beyond codons:

• Remove internal Shine-Dalgarno-like sequences that could cause translational pausing

• Eliminate cryptic splice sites if expressing in eukaryotic systems

• Consider the impact of codon changes on mRNA secondary structure

By implementing these strategies, researchers can significantly improve recombinant S. gordonii pfkA expression levels, potentially increasing yields by 5-10 fold compared to non-optimized sequences.

How can researchers accurately interpret substrate inhibition patterns in S. gordonii pfkA kinetic studies?

Accurate interpretation of substrate inhibition patterns in S. gordonii pfkA kinetic studies requires sophisticated analytical approaches that account for the enzyme's complex regulatory mechanisms. Researchers should adopt the following methodological framework:

Dual analysis approach:
Employ both initial-velocity methods and comprehensive modeling approaches when analyzing pfkA activity data . While the initial-velocity method (calculating time slope from the first datapoints) provides a qualitative picture of inhibition, it cannot fully separate ATP's dual roles. In contrast, the modeling method using differential equations that explicitly represent ATP's effects can quantify the degree of inhibition and provide mechanistic insights .

Mathematical models for substrate inhibition:
Several models can be applied to interpret substrate inhibition data:

• Basic substrate inhibition equation:
  $v = \frac{V_{max}[S]}{K_m + [S] + \frac{[S]^2}{K_i}}$

• Expanded model for ATP's dual role:
  $v = \frac{V_{max}[ATP][F6P]}{(K_m^{ATP} + [ATP])(K_m^{F6P} + [F6P])(1 + \frac{[ATP]}{K_i^{ATP}})}$

These models should be fitted to experimental data using non-linear regression, ideally with global fitting across multiple substrate concentrations.

Interpretation guidelines:
When analyzing fitted parameters, consider these key aspects:

• Affinity comparison: Compare ATP affinity for the catalytic site (Km) versus the inhibitory site (Ki). For S. gordonii pfkA, ATP affinity is typically much greater for the catalytic site than the inhibitory site, yet inhibition still occurs because the inhibited complex is significantly slower in product generation .

• pH dependence of inhibition: Substrate inhibition patterns often vary with pH. Analyze how Ki changes across pH values to understand the role of protonation states in the inhibition mechanism.

• Allosteric effector interactions: Test how known PFK allosteric effectors (AMP, ADP, PEP, citrate) modify the substrate inhibition pattern, which can reveal insights into the regulatory network.

• Temperature effects: Examine how inhibition patterns change with temperature, as this can differentiate between entropy and enthalpy-driven inhibition mechanisms.

By implementing these sophisticated analytical approaches, researchers can move beyond simply documenting substrate inhibition to understanding its mechanistic basis and physiological significance in S. gordonii metabolism.

What emerging technologies could advance the study of S. gordonii pfkA structure-function relationships?

Several cutting-edge technologies are poised to significantly advance our understanding of S. gordonii pfkA structure-function relationships:

Cryo-electron microscopy (Cryo-EM) has revolutionized structural biology by enabling visualization of proteins in near-native states without crystallization. For S. gordonii pfkA, cryo-EM offers:

• Visualization of different conformational states, particularly capturing the transition between active and inhibited forms

• Structural insights into oligomerization interfaces that are critical for function

• Ability to visualize the enzyme with multiple bound ligands, illuminating ATP's dual binding modes

Hydrogen-deuterium exchange mass spectrometry (HDX-MS) provides dynamic information about protein structure by measuring the rate at which backbone hydrogens exchange with deuterium. This technique can:

• Map conformational changes induced by substrate binding

• Identify regions with altered dynamics upon ATP inhibition

• Detect allosteric communication networks connecting distant binding sites

AlphaFold2 and other AI-based structural prediction tools offer unprecedented accuracy in predicting protein structures and can:

• Generate reliable models of S. gordonii pfkA variants without experimental structures

• Predict effects of mutations on structure and dynamics

• Model protein-protein interactions with metabolic partners

Single-molecule enzymology techniques allow observation of individual enzyme molecules rather than ensemble averages:

• Single-molecule FRET can track conformational changes in real-time

• Optical tweezers can measure mechanical forces during conformational transitions

• These approaches could reveal heterogeneity in pfkA behavior and identify rare but functionally important states

Microfluidic enzyme assays enable high-throughput screening under precisely controlled conditions:

• Rapid testing of hundreds of conditions simultaneously

• Minimal sample consumption

• Integration with imaging for real-time activity monitoring

These technologies, particularly when used in combination, promise to provide unprecedented insights into the molecular mechanisms underlying S. gordonii pfkA's complex regulation and its role in bacterial metabolism.

How might gene editing technologies be used to study pfkA function in S. gordonii in vivo?

Advanced gene editing technologies offer powerful approaches for investigating pfkA function in S. gordonii within its natural biological context:

CRISPR-Cas9 based approaches provide precise genetic manipulation capabilities:

• Generation of clean knockouts: Creating pfkA deletion mutants without antibiotic resistance markers or other genomic scars allows for clean interpretation of phenotypic effects.

• Point mutation introduction: Engineering specific amino acid substitutions at catalytic or regulatory sites can probe structure-function relationships in vivo without completely eliminating the enzyme.

• Promoter modifications: Replacing the native pfkA promoter with inducible or repressible elements enables controlled expression to study dose-dependent effects of pfkA activity on cellular physiology.

• Fluorescent tagging: Fusing fluorescent reporters to pfkA while maintaining its native genomic context allows real-time visualization of expression patterns and protein localization during biofilm formation.

Recombineering approaches using phage-derived recombination systems:

• Lambda Red recombineering adapted for streptococci allows efficient chromosomal modifications

• Can be combined with CRISPR-Cas9 for enhanced efficiency and specificity

In vivo applications of these technologies:

• Biofilm studies: Comparing wild-type and pfkA-modified S. gordonii strains in both monospecies and polymicrobial biofilm models to assess metabolic interactions .

• Host-pathogen interaction models: Using engineered strains to study how pfkA activity affects S. gordonii's interactions with host cells, particularly in the context of platelet adhesion and aggregation which are relevant to endocarditis pathogenesis .

• Competitive fitness assays: Co-culturing wild-type and pfkA-modified strains to assess how alterations in glycolytic efficiency affect fitness in various nutritional environments.

• Mucosal colonization models: Testing colonization efficiency of engineered strains in animal models to understand how pfkA contributes to S. gordonii's commensal lifestyle and potential as a vaccine vector .

These gene editing approaches, when combined with appropriate in vivo models, can provide insights into pfkA function that would be impossible to obtain through in vitro studies alone, advancing our understanding of S. gordonii metabolism in its natural ecological context.

What cross-disciplinary approaches could enhance applications of recombinant S. gordonii pfkA research?

Integrating knowledge and methodologies from multiple scientific disciplines can substantially enhance the applications and impact of recombinant S. gordonii pfkA research:

Synthetic biology and metabolic engineering:

• Designing synthetic metabolic circuits incorporating modified pfkA variants to control glycolytic flux

• Engineering S. gordonii strains with altered pfkA regulation for enhanced production of valuable metabolites

• Creating "kill switches" based on pfkA activity to control recombinant S. gordonii persistence in applications as live biotherapeutics

Immunology and vaccine development:

• Exploiting S. gordonii's proven capability as a vaccine vector to develop recombinant strains expressing pfkA-antigen fusion proteins

• Investigating how metabolically active S. gordonii influences mucosal immunity

• Developing multi-antigen presentation systems using pfkA as one component of a more complex immunogenic construct

Computational biology and systems biology:

• Developing genome-scale metabolic models of S. gordonii with accurate representation of pfkA regulation

• Simulating how pfkA modifications affect metabolic flux under various environmental conditions

• Predicting emergent properties of multi-species communities containing engineered S. gordonii strains

Materials science and biofilm engineering:

• Creating programmable biofilms with engineered S. gordonii that respond to specific metabolic signals

• Developing biosensors based on pfkA activity to detect environmental conditions

• Incorporating S. gordonii with modified pfkA into structured biofilm communities for biocatalysis applications

Translational medicine:

• Investigating modified S. gordonii as a probiotic for oral health, with pfkA engineering to optimize colonization without pathogenic potential

• Exploring potential of pfkA-targeted inhibitors as narrow-spectrum antimicrobials against viridans group streptococci involved in endocarditis

• Developing diagnostic tools based on pfkA activity to assess oral microbiome metabolic function

These cross-disciplinary approaches not only enhance the fundamental understanding of S. gordonii pfkA but also expand its potential applications in biotechnology, medicine, and environmental science. The integration of these diverse perspectives could lead to innovative solutions for challenges in oral health, infectious disease, and beyond.