Recombinant Escherichia fergusonii Phosphoserine aminotransferase (serC)

What is Escherichia fergusonii and how does it relate to other Escherichia species?

Escherichia fergusonii is an emerging pathogen within the genus Escherichia that has gained increased attention due to its prevalence in both human and animal infections globally. E. fergusonii is closely related to E. coli but represents a distinct species with unique genomic characteristics. Initially, E. fergusonii was often misidentified as E. coli using conventional biochemical methods like the API 20E identification system, highlighting the challenges in accurate species differentiation .

Molecular identification has revealed that E. fergusonii is increasingly observed in clinical settings and environmental samples. Core genome analysis demonstrates that E. fergusonii has evolved as a significant pathogen capable of acquiring multiple resistance determinants, including β-lactamases and carbapenemases, contributing to its clinical importance . Phylogenetic analyses have positioned E. fergusonii as an important member of Enterobacteriaceae requiring increased surveillance, particularly as it has been associated with antimicrobial resistance transmission.

What is the function of phosphoserine aminotransferase (serC) in bacterial metabolism?

Phosphoserine aminotransferase (PSAT), encoded by the serC gene, catalyzes a critical step in the phosphorylated pathway of serine biosynthesis. This enzyme specifically converts 3-phosphohydroxypyruvate to 3-phosphoserine using glutamate as an amino group donor. The reaction represents the second step in the three-step phosphorylated serine biosynthesis pathway.

The enzyme functions as part of the larger serine metabolic network that intersects with multiple metabolic pathways, including:

• Amino acid metabolism (particularly glycine and cysteine)

• One-carbon metabolism via tetrahydrofolate derivatives

• Phospholipid biosynthesis through serine incorporation

• Cellular redox balance maintenance

In bacteria, the phosphorylated pathway of serine biosynthesis is particularly important under conditions where direct uptake of serine from the environment is limited. The serC gene product is therefore essential for bacterial growth in minimal media lacking serine supplementation and plays a crucial role in bacterial adaptation to diverse environmental conditions .

How are E. fergusonii strains typically isolated and identified in research settings?

The isolation and identification of E. fergusonii require specialized techniques due to its phenotypic similarity to E. coli. Researchers employ a multi-step approach:

Isolation Protocol:

• Initial cultivation on enrichment media such as MacConkey agar, which allows for the visualization of lactose-fermenting colonies

• Selection on specialized media containing antimicrobials (such as colistin at 2 mg/L) for screening resistant strains

• Preservation of isolates at -80°C in appropriate storage media for subsequent analysis

Identification Methods:
Traditional biochemical methods like API 20E have limitations, frequently misidentifying E. fergusonii as E. coli. More reliable identification requires molecular techniques:

For definitive identification, a duplex PCR approach using EFER 13- and EFER YP-specific primers has proven highly effective. This molecular method can reduce identification time from six days to three days compared to traditional biochemical methods . This approach targets conserved genes specific to E. fergusonii including genes encoding conserved hypothetical cellulose synthase protein and putative transcriptional activator for multiple antibiotic resistance.

What expression systems are most effective for recombinant E. fergusonii serC?

The choice of expression system for recombinant E. fergusonii phosphoserine aminotransferase depends on research objectives and downstream applications. Based on studies with related proteins, the following systems have demonstrated effectiveness:

E. coli-Based Expression Systems:
E. coli BL21(DE3) remains the workhorse for recombinant protein expression, particularly with the following vectors:

• pET expression system: Offers tight regulation of expression through T7 promoter and provides high yields of recombinant protein

• pGEX vectors: Allow expression of serC as a GST-fusion protein, which can enhance solubility and facilitate purification

The GST-fusion approach has been particularly successful with phosphoserine aminotransferase enzymes, as demonstrated in human PSAT studies where the relative enzyme activity of GST-PSAT beta expressed in E. coli appeared to be 6.8 times higher than that of GST-PSAT alpha .

Optimal Expression Conditions:

• Induction: 0.1-0.5 mM IPTG at OD600 of 0.6-0.8

• Temperature: Lowering to 16-25°C post-induction often improves solubility

• Media supplementation: Addition of pyridoxal phosphate (PLP) as a cofactor can improve folding and stability

• Expression duration: 4-16 hours depending on temperature and construct design

Alternative Systems:

• Cell-free protein synthesis systems for proteins that may be toxic to host cells

• Specialized E. coli strains like Rosetta or Origami for proteins with rare codons or disulfide bonds

• Bacillus subtilis expression systems for secreted production with native N-terminus

What purification strategies yield highest activity for recombinant serC protein?

Purification of recombinant phosphoserine aminotransferase requires careful consideration of the enzyme's biochemical properties. The following multi-step purification strategy has been empirically determined to maintain high enzymatic activity:

Recommended Purification Protocol:

• Cell Lysis Optimization:

  • Buffer composition: 50 mM Tris-HCl (pH 8.0), 150 mM NaCl, 1 mM EDTA, 1 mM DTT

  • Addition of protease inhibitors (PMSF, leupeptin, pepstatin)

  • Gentle lysis methods (sonication with cooling intervals or enzymatic lysis)

• Initial Capture:

  • For GST-tagged constructs: Glutathione Sepharose affinity chromatography

  • For His-tagged constructs: Immobilized metal affinity chromatography (IMAC)

  • Wash stringency balancing: Sufficient to remove contaminants without protein loss

• Tag Removal and Secondary Purification:

  • Site-specific protease cleavage (PreScission, TEV, or thrombin depending on construct)

  • Ion exchange chromatography (typically Q Sepharose at pH 8.0)

  • Size exclusion chromatography as a polishing step

• Activity Preservation Measures:

  • Include pyridoxal phosphate (10-50 μM) in all buffers

  • Maintain reducing conditions with 1-5 mM DTT or 2-mercaptoethanol

  • Consider addition of 5-10% glycerol for storage stability

  • Flash freeze in liquid nitrogen and store at -80°C in small aliquots

This approach typically yields >95% pure protein with specific activity comparable to native enzyme. The purified enzyme should be characterized by SDS-PAGE, Western blotting, and enzymatic assays to confirm identity and activity.

How can the enzymatic activity of recombinant phosphoserine aminotransferase be accurately measured?

Accurate measurement of phosphoserine aminotransferase activity requires careful consideration of reaction conditions and detection methods. The following approaches provide reliable quantification:

Spectrophotometric Coupled Enzyme Assays:

The most common method couples PSAT activity to another enzymatic reaction that produces a measurable spectrophotometric change:

• Forward Reaction (3-phosphohydroxypyruvate → 3-phosphoserine):

  • Couple to glutamate dehydrogenase, which consumes NADH when converting α-ketoglutarate to glutamate

  • Monitor decrease in NADH absorbance at 340 nm

  • Reaction conditions: 50 mM HEPES (pH 7.5), 100 mM KCl, 5 mM MgCl2, 0.2 mM NADH, 5 mM α-ketoglutarate, 0.5-5 mM 3-phosphohydroxypyruvate, 2-5 units glutamate dehydrogenase

• Reverse Reaction (3-phosphoserine → 3-phosphohydroxypyruvate):

  • Couple to lactate dehydrogenase, which consumes NADH when converting pyruvate to lactate

  • Monitor decrease in NADH absorbance at 340 nm

  • Reaction conditions: 50 mM HEPES (pH 7.5), 100 mM KCl, 5 mM MgCl2, 0.2 mM NADH, 5 mM α-ketoglutarate, 0.5-5 mM 3-phosphoserine, 2-5 units lactate dehydrogenase

Direct Detection Methods:

• HPLC-based quantification of reaction products

• LC-MS/MS for highly sensitive detection of 3-phosphoserine formation

• Radiometric assays using 14C-labeled substrates

Kinetic Parameter Determination:
For comprehensive characterization, determine the following parameters:

Comparing these parameters between E. fergusonii PSAT and other bacterial PSATs provides valuable insights into evolutionary adaptations and catalytic efficiency differences.

How does serC expression correlate with bacterial stress responses and environmental adaptation?

Phosphoserine aminotransferase plays a crucial role in bacterial stress responses and environmental adaptation through its central position in serine metabolism. Analysis of serC expression patterns reveals complex regulatory networks:

Stress Response Correlation:
Phosphoserine aminotransferase expression is modulated in response to several environmental stressors:

Environmental Adaptation Mechanisms:
Environmental factors that influence serC expression include:

Understanding these regulatory patterns provides insight into the ecological versatility of E. fergusonii and its ability to colonize diverse niches, from environmental reservoirs to mammalian hosts.

What is the relationship between serC function and antimicrobial resistance in E. fergusonii?

While serC itself is not directly associated with antimicrobial resistance, emerging research suggests several indirect connections between phosphoserine aminotransferase function and resistance mechanisms in E. fergusonii:

Metabolic Support for Resistance Mechanisms:

• Cell wall modification: Serine is a key component of bacterial peptidoglycan, and alterations in serine metabolism may influence cell wall composition, potentially affecting susceptibility to cell wall-targeting antimicrobials.

• Efflux pump energetics: Active efflux systems that expel antimicrobials require significant energy. As a central metabolic enzyme, serC function may influence the cell's energy balance and thus the efficiency of efflux-mediated resistance.

• Stress response coordination: The metabolic pathways involving serC intersect with general stress response networks that can increase bacterial survival during antimicrobial exposure.

Genomic Context and Co-occurrence Patterns:
Analysis of E. fergusonii genomes has revealed interesting patterns in the genomic neighborhood of serC:

• E. fergusonii strains carrying mobile colistin resistance (mcr-1) gene show distinct metabolic profiles, suggesting possible interactions between resistance determinants and core metabolic functions .

• The genomic organization of serC in relation to resistance elements varies among strains, with some evidence suggesting co-selection of certain metabolic gene variants with resistance determinants.

• Multidrug-resistant E. fergusonii isolates often show alterations in metabolic pathway regulation, including pathways intersecting with serC function.

Experimental Evidence Table:

These observations suggest that while serC may not directly confer resistance, its function is integrated into the broader metabolic and physiological adaptations that support antimicrobial resistance phenotypes in E. fergusonii.

How do sequence variations in serC affect enzyme function across different E. fergusonii strains?

Comparative genomic analysis of serC across E. fergusonii isolates reveals sequence variations that may influence enzyme function, substrate specificity, and regulatory mechanisms:

Structural and Functional Implications of Sequence Variation:

• Active site residues: Conservative substitutions in residues coordinating the PLP cofactor can subtly alter catalytic efficiency or substrate specificity.

• Substrate binding pocket: Variations in amino acids lining the substrate binding pocket may influence substrate affinity (Km) and turnover rate (kcat).

• Oligomerization interfaces: PSAT typically functions as a homodimer. Mutations at subunit interfaces can affect quaternary structure stability and allosteric regulation.

• Surface-exposed loops: Sequence diversity is highest in surface-exposed loops, which may influence protein-protein interactions without directly affecting catalytic function.

Strain-Specific Variations and Their Consequences:

Analysis of serC sequences from clinical and environmental E. fergusonii isolates reveals three main patterns:

• Core conserved residues: Catalytic residues directly involved in PLP binding and catalysis show near-complete conservation across all strains, highlighting functional constraints.

• Lineage-specific polymorphisms: Certain amino acid substitutions correlate with specific phylogenetic lineages, suggesting potential adaptive significance.

• Host-associated variations: Strains isolated from different host species (human vs. animal) show characteristic sequence patterns, potentially reflecting host adaptation.

Structure-Function Relationship Model:

Based on structural homology to crystal structures of E. coli phosphoserine aminotransferase (PDB: 1BJN) and other bacterial PSATs , the following structure-function relationships can be proposed:

These variations likely contribute to fine-tuning of enzyme kinetics rather than dramatic functional changes, consistent with the essential metabolic role of phosphoserine aminotransferase.

What are the optimal methods for studying serC gene regulation in E. fergusonii?

Investigating serC gene regulation in E. fergusonii requires a multi-faceted approach combining molecular genetics, transcriptomics, and reporter systems:

Promoter Analysis and Transcriptional Regulation:

• 5' RACE (Rapid Amplification of cDNA Ends):

  • Identifies transcription start sites and maps the serC promoter architecture

  • Reveals potential alternative promoters or transcription initiation sites

  • Protocol modification: Use specialized RNA extraction methods to preserve primary transcripts with 5' triphosphate

• Reporter Fusion Constructs:

  • Create serC promoter fusions to reporter genes (GFP, luciferase, lacZ)

  • Enable real-time monitoring of promoter activity under various conditions

  • Design consideration: Include sufficient upstream sequence (1-2 kb) to capture distal regulatory elements

• ChIP-seq (Chromatin Immunoprecipitation Sequencing):

  • Identifies transcription factors binding to the serC promoter region

  • Reveals genome-wide binding patterns of regulators affecting serC

  • Technical challenge: Requires antibodies against E. fergusonii transcription factors or epitope-tagged constructs

Transcriptional Profiling:

• RNA-seq under Various Conditions:

  • Provides comprehensive view of serC expression across environmental conditions

  • Reveals co-regulated genes in the same metabolic or stress response pathways

  • Experimental design: Include replicate samples and appropriate reference conditions

• Quantitative RT-PCR:

  • For targeted validation of expression changes under specific conditions

  • Higher sensitivity than RNA-seq for detecting subtle expression changes

  • Critical control: Careful selection of reference genes stable under test conditions

Regulatory Network Mapping:

• Transcription Factor Binding Site (TFBS) Analysis:

  • In silico prediction of regulatory motifs in the serC promoter region

  • Cross-species comparison to identify conserved regulatory elements

  • Validation: Confirm predicted sites by site-directed mutagenesis of reporter constructs

• Global Regulator Mutant Analysis:

  • Examine serC expression in strains with mutations in global regulators (e.g., CRP, H-NS, Lrp)

  • Identifies key regulators controlling serC expression

  • Approach: Generate clean deletion mutants using lambda Red recombination system

Metabolic Regulation Analysis:

• Metabolomic Profiling:

  • Correlate serC expression with intracellular metabolite levels

  • Identifies potential feedback regulation mechanisms

  • Method: LC-MS/MS analysis of key metabolites in serine biosynthesis pathway

• Riboswitch and Small RNA Investigations:

  • Examine potential post-transcriptional regulation of serC

  • Northern blotting and structure probing of the 5' UTR region

  • Look for conservation of RNA structural elements across related species

This comprehensive approach enables mapping of the complex regulatory network controlling serC expression in response to environmental and metabolic signals, providing insight into the integration of serine biosynthesis with broader cellular processes.

What are common challenges in recombinant serC protein expression and how can they be overcome?

Recombinant expression of E. fergusonii phosphoserine aminotransferase presents several challenges that can be addressed through optimization strategies:

Challenge 1: Protein Solubility Issues

Phosphoserine aminotransferase may form inclusion bodies during overexpression, particularly at high induction levels or elevated temperatures.

Solutions:

• Lower induction temperature to 16-20°C post-induction

• Reduce IPTG concentration to 0.1-0.2 mM

• Co-express with chaperones (GroEL/GroES, DnaK/DnaJ)

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

• Add stabilizing additives to lysis buffer (10% glycerol, 50-100 mM arginine)

Analogous to human PSAT expression, where GST-fusion significantly enhanced solubility and activity, GST-tagged constructs have shown particular promise with E. fergusonii PSAT .

Challenge 2: Cofactor Incorporation and Enzyme Activity

As a PLP-dependent enzyme, proper cofactor incorporation is essential for obtaining active phosphoserine aminotransferase.

Solutions:

• Supplement expression media with pyridoxine (50-100 μM)

• Add PLP (50-100 μM) to all purification buffers

• Include a reconstitution step with excess PLP followed by dialysis

• Monitor spectroscopic signature of PLP incorporation (peak at 412-420 nm)

• Avoid prolonged exposure to light during purification

Challenge 3: Protein Stability During Purification

Phosphoserine aminotransferase may show activity loss during purification due to cofactor loss or oligomeric state disruption.

Solutions:

Challenge 4: Oligomeric State Verification

Ensuring the correct oligomeric state (typically dimeric) is critical for full enzymatic activity.

Solutions:

• Assess oligomeric state by size exclusion chromatography

• Confirm by native PAGE or analytical ultracentrifugation

• Include mild detergents (0.01-0.05% Triton X-100) to prevent non-specific aggregation

• Optimize salt concentration to maintain proper quaternary structure

Challenge 5: Heterologous Expression Bias

Codon usage differences between E. fergusonii and expression host may limit expression levels.

Solutions:

• Use codon-optimized synthetic gene constructs

• Express in Rosetta strains carrying rare tRNA genes

• Adjust expression conditions to allow slower, more accurate translation

By systematically addressing these challenges, researchers can achieve high-yield expression of active recombinant E. fergusonii phosphoserine aminotransferase suitable for structural and functional studies.

What methods are most effective for studying protein-protein interactions involving serC in E. fergusonii?

Understanding the interaction partners of phosphoserine aminotransferase provides insight into its integration within metabolic networks and potential regulatory mechanisms. Several complementary approaches can effectively characterize these interactions:

In Vivo Interaction Methods:

• Bacterial Two-Hybrid (B2H) Systems:

  • Based on reconstitution of adenylate cyclase or split transcription factors

  • Allows screening of interaction partners in a bacterial cellular context

  • Advantages: Conducted in prokaryotic environment; suitable for membrane proteins

  • Limitations: May detect indirect interactions within complexes

• Protein-Fragment Complementation Assays (PCA):

  • Split reporter proteins (GFP, luciferase) reconstitute upon interaction

  • Enables visualization of interactions in living bacterial cells

  • Can detect spatial and temporal dynamics of interactions

  • Implementation: Construct genomic fusion libraries to screen for interaction partners

• In vivo Crosslinking and Co-Immunoprecipitation:

  • Chemical crosslinkers (formaldehyde, DSP) capture transient interactions

  • Epitope-tagged serC allows specific pulldown of complexes

  • Mass spectrometry identifies interaction partners

  • Critical control: Compare crosslinked samples with non-crosslinked controls

In Vitro Biochemical Approaches:

• Pull-down Assays with Recombinant Proteins:

  • Immobilize purified tagged serC as bait

  • Incubate with E. fergusonii lysate or purified candidate proteins

  • Identify bound proteins by Western blotting or mass spectrometry

  • Quantitative analysis: Calculate apparent KD values for validated interactions

• Surface Plasmon Resonance (SPR) or Bio-Layer Interferometry (BLI):

  • Provides real-time kinetic analysis of interactions

  • Determines association and dissociation rates and affinity constants

  • Requires highly purified proteins and specialized instrumentation

  • Particularly valuable for characterizing regulatory interactions

• Isothermal Titration Calorimetry (ITC):

  • Measures thermodynamic parameters of interactions

  • Determines stoichiometry, enthalpy, and binding constants

  • Provides detailed energetic profile of interactions

  • No protein modification or immobilization required

Structural Approaches:

• X-ray Crystallography of Complexes:

  • Provides atomic-level details of interaction interfaces

  • Reveals conformational changes upon complex formation

  • Challenge: Obtaining diffracting crystals of protein complexes

  • Strategy: Use crosslinking or fusion constructs to stabilize transient complexes

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

  • Maps interaction interfaces through differential solvent exposure

  • Detects conformational changes upon binding

  • Does not require crystal formation

  • Particularly useful for dynamic or transient interactions

Network Analysis Methods:

• Proximity-Dependent Biotin Identification (BioID):

  • Fusion of serC to a promiscuous biotin ligase

  • Biotinylates proteins in close proximity in vivo

  • Identifies the spatial "neighborhood" of serC in the cell

  • Adaptation for bacteria: Use shorter linkers and optimize expression

• Integrative Multi-Omics Approaches:

  • Correlate protein interaction data with transcriptomics and metabolomics

  • Construct pathway models integrating different data types

  • Identifies functional consequences of protein-protein interactions

  • Provides systems-level understanding of serC function

These methods provide complementary information about serC interactions, from binary partner identification to detailed structural characterization of complexes, enabling comprehensive mapping of its interaction network within E. fergusonii.

How might E. fergusonii serC be explored as a potential antimicrobial target?

Phosphoserine aminotransferase represents a potential antimicrobial target due to its essential role in serine biosynthesis, particularly in environments where serine availability is limited. Several research avenues could explore this potential:

Target Validation Approaches:

• Essentiality Assessment:

  • Conditional knockout systems (e.g., CRISPR interference)

  • Depletion studies using regulatable promoters

  • Transposon sequencing (Tn-seq) under various growth conditions

  • Critical experiments: Demonstrate serC essentiality in infection-relevant conditions

• Chemical Genetics:

  • Screen for small molecule inhibitors of serC

  • Validate on-target effects through resistance mutations mapping to serC

  • Assess cross-species activity against other pathogenic bacteria

  • Advantage: Simultaneously validates target and provides lead compounds

Structure-Based Drug Design Strategy:

• Structural Characterization:

  • Determine high-resolution crystal structures of E. fergusonii serC

  • Compare with human PSAT to identify structural differences

  • Map catalytic residues and substrate binding pockets

  • Focus on unique structural features absent in mammalian orthologs

• Virtual Screening and Fragment-Based Approaches:

  • Computational docking of compound libraries targeting the active site

  • Fragment screening to identify chemical starting points

  • Structure-activity relationship studies of promising leads

  • Particular focus on compounds that exploit differences from human PSAT

Specificity and Selectivity Considerations:

The key challenge in targeting serC is achieving selectivity over human PSAT. Approaches to address this include:

• Differential inhibition strategy: Target amino acid residues unique to bacterial serC proteins

• Allosteric inhibition: Identify bacterial-specific regulatory sites distinct from the catalytic center

• Prodrug approach: Design compounds activated by bacterial-specific enzymes

Potential Impact and Challenges:

Research targeting E. fergusonii serC could yield insights applicable to a broader range of pathogens, as the serine biosynthesis pathway is conserved across many bacterial species, potentially addressing the critical need for novel antimicrobial targets in this era of increasing resistance .

How do environmental factors influence serC expression and evolution in E. fergusonii?

The expression and evolution of phosphoserine aminotransferase in E. fergusonii are shaped by environmental pressures that influence both regulatory mechanisms and sequence conservation:

Environmental Regulation of serC Expression:

E. fergusonii inhabits diverse environments, from the mammalian intestinal tract to environmental reservoirs such as soil and water. These distinct niches impose different selective pressures:

• Host-Associated Environments:

  • Nutrient availability fluctuations influence serC regulation

  • Competition with the host for serine and other amino acids

  • Response to host-derived antimicrobial compounds

  • Integration with virulence factor expression during colonization

• Environmental Reservoirs:

  • Adaptation to nutrient-limited conditions increases reliance on de novo synthesis

  • Temperature fluctuations drive expression pattern changes

  • Soil chemistry influences metabolic pathway utilization

  • Biofilm formation in environmental settings alters metabolic priorities

Evolutionary Patterns and Selection Pressures:

Comparative genomic analysis across E. fergusonii strains from different sources reveals:

• Conservation Patterns:

  • Catalytic core residues show highest conservation

  • Surface-exposed regions display greater sequence diversity

  • Lineage-specific polymorphisms correlate with ecological niches

  • Horizontal gene transfer events may introduce variant alleles

• Selection Signatures:

  • Evidence of purifying selection on catalytic residues

  • Positive selection on regions involved in protein-protein interactions

  • Balancing selection maintaining polymorphisms in certain populations

  • Convergent evolution in strains adapting to similar niches

Research Approaches to Study Environmental Adaptation:

• Experimental Evolution Studies:

  • Long-term cultivation under defined selective pressures

  • Monitor serC sequence changes and expression patterns

  • Correlate with fitness measurements and metabolic phenotypes

  • Particularly valuable for understanding adaptation to new niches

• Phylogenomic Analysis:

  • Compare serC sequences across E. fergusonii strains with known provenance

  • Identify environment-specific sequence signatures

  • Calculate dN/dS ratios to detect selection patterns

  • Reconstruct ancestral sequences to map evolutionary trajectories

• Transcriptional Response Profiling:

  • RNA-seq under conditions mimicking different environments

  • Identify environment-specific regulatory patterns

  • Map transcriptional responses to metabolic network models

  • Compare with other species to identify conserved response patterns

Understanding these environmental influences provides insight into the ecological versatility of E. fergusonii and its ability to adapt to diverse niches, which may contribute to its emergence as a pathogen of increasing clinical significance .

What emerging technologies are advancing our understanding of serC function?

Recent technological advances are revolutionizing our understanding of phosphoserine aminotransferase function and its integration within bacterial metabolic networks:

Structural Biology Innovations:

• Cryo-Electron Microscopy (Cryo-EM):

  • Enables visualization of large macromolecular complexes

  • Captures conformational dynamics not accessible by crystallography

  • Particularly valuable for studying serC in the context of metabolic complexes

  • Application: Visualizing potential "metabolons" involving serine biosynthesis enzymes

• Time-Resolved X-ray Crystallography:

  • Captures enzyme reaction intermediates

  • Provides insight into catalytic mechanism at atomic resolution

  • Requires specialized synchrotron beamlines or XFEL facilities

  • Could resolve longstanding questions about the PLP-dependent transamination mechanism

Systems Biology Approaches:

Genetic Technology Applications:

• CRISPR-Based Technologies:

  • CRISPRi for precise transcriptional control of serC

  • Base editing for introducing specific mutations without selection markers

  • In vivo tracking of serC dynamics using Cas13-based RNA detection

  • Advantage: Enables manipulation in previously genetically intractable strains

• Genome-Wide Interaction Mapping:

  • Synthetic genetic arrays identify genetic interactions with serC

  • Transposon sequencing under selective conditions reveals functional relationships

  • Double-knockout libraries identify compensatory pathways

  • Provides insight into genetic buffering and pathway redundancy

Single-Cell Technologies:

• Single-Cell RNA-seq:

  • Reveals population heterogeneity in serC expression

  • Identifies distinct metabolic states within bacterial populations

  • Particularly relevant for understanding bacterial persistence phenotypes

  • Technical challenge: Adapting protocols for bacterial cells with tough cell walls

• Microfluidics-Based Approaches:

  • Tracks single-cell growth and gene expression in controlled environments

  • Enables precise manipulation of environmental conditions

  • High-throughput screening of mutant libraries

  • Application: Understanding stochastic variation in metabolic states

In Situ Visualization Methods:

• Expansion Microscopy for Bacteria:

  • Physical expansion of cells enables super-resolution imaging with standard equipment

  • Visualizes protein localization patterns with nanoscale precision

  • Can be combined with multiplexed protein labeling

  • Application: Mapping subcellular distribution of serC and interaction partners

• Proximity Labeling In Vivo:

  • APEX2 or TurboID fusions for in situ protein interaction mapping

  • Spatial mapping of serC within the bacterial proteome

  • Identifies transient or weak interactions missed by traditional methods

  • Provides context-dependent interaction data in native cellular environment

These emerging technologies promise to transform our understanding of serC from a simple metabolic enzyme to a dynamic component integrated within complex regulatory and metabolic networks in E. fergusonii.