Recombinant Bordetella petrii Phosphoserine aminotransferase (serC)

What is Phosphoserine aminotransferase (serC) in Bordetella petrii and what is its functional role?

Phosphoserine aminotransferase (serC) in Bordetella petrii is an enzyme belonging to the class-V pyridoxal-phosphate-dependent aminotransferase family, specifically the SerC subfamily . The enzyme catalyzes two primary reversible reactions:

• The conversion of 3-phosphohydroxypyruvate to phosphoserine

• The conversion of 3-hydroxy-2-oxo-4-phosphonooxybutanoate to phosphohydroxythreonine

In B. petrii, serC is a 376 amino acid protein with a molecular mass of approximately 40.7 kDa . These reactions represent key steps in amino acid biosynthesis pathways, particularly for serine and threonine production. The enzyme likely plays a critical role in B. petrii's metabolic flexibility, potentially contributing to its remarkable adaptability across diverse environmental conditions.

How does Bordetella petrii differ from other Bordetella species?

Bordetella petrii exhibits several distinctive characteristics compared to other members of the genus:

B. petrii has been isolated from clinical samples associated with jaw, ear bone, cystic fibrosis, and chronic pulmonary disease, demonstrating its emergence as an opportunistic human pathogen .

How does genomic plasticity in B. petrii affect serC expression and function?

B. petrii undergoes "massive genomic rearrangements" both in vitro and likely in vivo, which has significant implications for gene expression and protein function . While the search results don't specifically address serC regulation within this context, several research considerations emerge:

• Genomic rearrangements may affect serC expression through:

  • Promoter modifications altering transcription efficiency

  • Changes in global regulators that control amino acid biosynthesis pathways

  • Alterations in operon structure affecting co-transcription with neighboring genes

• Studies with sequential clinical isolates have demonstrated phenotypic variations in:

  • Growth characteristics

  • Antibiotic susceptibility

  • Antigenic profiles and immune recognition

These variations suggest that genomic rearrangements impact multiple cellular processes, potentially including metabolic pathways where serC functions. Researchers should consider analyzing serC sequence, expression, and enzymatic activity across sequential isolates to determine if this enzyme undergoes adaptation during persistent infection.

What role might serC play in B. petrii adaptation to diverse environmental niches?

SerC likely contributes to B. petrii's remarkable environmental adaptability through several mechanisms:

• Metabolic flexibility: As an enzyme involved in amino acid biosynthesis, serC may enable adaptation to environments with varying nutrient availability.

• Stress response integration: In many bacteria, amino acid biosynthesis pathways interact with stress response systems. The stringent response, mediated by ppGpp (as mentioned in relation to other bacteria), often regulates metabolic pathways during adaptation .

• Host-pathogen interaction: Amino acid metabolism can influence virulence factor production and bacterial survival within host environments.

• Biofilm formation: Metabolic enzymes often play roles in biofilm development, which represents an important survival strategy in both environmental and host settings.

Experimental approaches to investigate these hypotheses include:

• Comparative transcriptomics of serC expression under various environmental conditions

• Creation of serC mutants to assess fitness across different niches

• Metabolomics analysis to trace carbon flux through serC-dependent pathways under different conditions

How does the immune recognition of B. petrii correlate with bacterial adaptation during infection?

Research has revealed fascinating insights into B. petrii's immune evasion capabilities:

• Sequential B. petrii isolates from the same patient exhibited different recognition patterns by the patient's antibodies .

• Mouse studies confirmed this pattern, with antibodies from mice inoculated with different B. petrii strains recapitulating the same strain-specific recognition observed with patient serum .

• One immune evasion mechanism involved modifications to the lipopolysaccharide O-antigen, with a specific mutation identified that affected antibody recognition .

This evidence strongly suggests that B. petrii undergoes adaptive changes during infection that affect antigenic presentation and immune recognition. These adaptations may contribute to the bacterium's ability to establish persistent infections lasting more than one year in patients with chronic pulmonary conditions .

This immune evasion capability appears to be an important aspect of B. petrii's pathogenic potential and distinguishes it from environmental bacteria lacking such adaptive mechanisms.

What are optimal expression systems and conditions for producing recombinant B. petrii serC?

Based on the challenges reported with recombinant Bordetella protein expression in E. coli and the properties of serC, researchers should consider:

Optimization parameters:

• Temperature: 15-25°C often improves solubility for challenging proteins

• Inducer concentration: Lower concentrations with extended expression times

• Media supplementation: Consider pyridoxal phosphate addition as it's a required cofactor

• Fusion partners: Solubility-enhancing tags (MBP, SUMO, GST) if folding issues arise

Expression verification:

• SDS-PAGE analysis (expected size: 40.7 kDa plus any fusion tags)

• Western blotting with anti-His (or relevant tag) antibodies

• Enzymatic activity assays to confirm functional folding

What purification strategies yield functional recombinant B. petrii serC?

A multi-step purification strategy is recommended for obtaining high-purity, functional serC:

• Initial capture:

  • Immobilized metal affinity chromatography (IMAC) if His-tagged

  • Alternative affinity methods based on fusion partner (amylose resin for MBP-tagged constructs, etc.)

• Intermediate purification:

  • Ion exchange chromatography based on calculated pI of serC

  • Heparin affinity chromatography (effective for many nucleic acid-binding proteins)

• Polishing step:

  • Size exclusion chromatography to remove aggregates and verify oligomeric state

  • Potentially identify bound cofactors (pyridoxal phosphate should appear as A388 absorption)

Critical buffer considerations:

• Include pyridoxal phosphate (50-100 μM) in all buffers to maintain enzyme structure

• Evaluate protein stability across pH range (typically pH 7.0-8.0)

• Include reducing agents if cysteine residues are present in the sequence

• Determine glycerol requirements for long-term storage stability

Quality assessment methods:

• Specific activity determination compared to literature values for related SerC enzymes

• Thermal shift assays to evaluate stability under different buffer conditions

• Dynamic light scattering to assess homogeneity and aggregation state

How can researchers effectively assay serC enzymatic activity?

Several complementary approaches can be employed to characterize B. petrii serC activity:

• Spectrophotometric coupled assays:

  • Forward reaction: Couple phosphoserine formation to NADH oxidation via auxiliary enzymes

  • Reverse reaction: Monitor 3-phosphohydroxypyruvate formation through coupled reactions

  • Standard conditions: 50 mM Tris-HCl pH 7.5, 100 μM pyridoxal phosphate, 5 mM MgCl₂, 37°C

• HPLC-based direct product quantification:

  • Separate and quantify phosphoserine formation using ion-exchange or reverse-phase HPLC

  • Include internal standards for accurate quantification

  • Useful for kinetic parameter determination (Km, Vmax, kcat)

• Isothermal titration calorimetry:

  • Directly measure heat released/absorbed during enzymatic reaction

  • Allows detailed thermodynamic characterization

  • Particularly useful for determining binding affinities for substrates and inhibitors

Example assay protocol:

• Reaction mixture: 50 mM HEPES pH 7.5, 100 μM pyridoxal phosphate, 5 mM MgCl₂, 2 mM 3-phosphohydroxypyruvate, 10 mM glutamate, purified serC (0.1-1 μg)

• Incubate at 37°C for 5-30 minutes

• Terminate reaction with equal volume of 10% trichloroacetic acid

• Analyze phosphoserine formation by appropriate detection method

Controls and validations:

• Heat-inactivated enzyme as negative control

• Commercial aminotransferase as positive control if available

• Substrate and cofactor titrations to establish optimal assay conditions

What experimental approaches can elucidate serC's role in B. petrii pathogenesis?

To investigate serC's potential contributions to B. petrii pathogenesis, researchers should consider this multi-faceted approach:

• Genetic manipulation studies:

  • Generate precise serC deletion mutants using allelic exchange techniques

  • Create complemented strains to confirm phenotypes are directly attributable to serC

  • Develop conditional expression systems for essential genes

• Phenotypic characterization:

  • Growth kinetics in minimal versus rich media

  • Stress tolerance assays (oxidative, temperature, pH, nutrient limitation)

  • Biofilm formation capacity

  • Antibiotic susceptibility profiles

• Host-pathogen interaction models:

  • Macrophage infection assays comparing wild-type and ΔserC strains

  • Measurement of intracellular survival and replication

  • Analysis of host cell cytokine/chemokine responses

  • Small animal infection models if appropriate biosafety facilities are available

• Systems biology approaches:

  • Transcriptomics to identify serC-dependent gene expression networks

  • Metabolomics to characterize pathway alterations in ΔserC mutants

  • Proteomics to detect changes in virulence factor production

• Comparative analysis of clinical isolates:

  • Sequence serC across sequential isolates from persistent infections

  • Compare enzymatic activity of serC from different clinical strains

  • Correlate serC variations with changes in antibiotic susceptibility or immune recognition

This integrated approach would provide comprehensive insights into serC's role in B. petrii pathogenesis and potentially identify novel therapeutic targets for persistent Bordetella infections.

How might serC function contribute to B. petrii persistence in chronic respiratory conditions?

B. petrii has demonstrated remarkable persistence capabilities, with documented cases of infection lasting more than one year in patients with chronic pulmonary conditions . Several mechanisms may connect serC function to this persistence:

• Metabolic adaptation:

  • SerC's role in amino acid biosynthesis may enable adaptation to nutrient-limited environments within the respiratory tract

  • Metabolic flexibility could facilitate survival under varying conditions in chronically diseased lungs

• Stress response integration:

  • Amino acid metabolism often interfaces with bacterial stress responses

  • The SerC pathway may contribute to survival under host-imposed stresses (oxidative, nitrosative, pH)

• Biofilm contribution:

  • Metabolic enzymes frequently play secondary roles in biofilm development

  • Persistence in chronic respiratory conditions is often associated with biofilm formation

• Immune evasion support:

  • Metabolic adaptations can alter surface structure expression

  • B. petrii demonstrates strain-specific immune recognition patterns that evolve during infection

Experimental evidence from sequential clinical isolates shows that B. petrii undergoes significant adaptation during persistent infection, with changes in growth characteristics, antibiotic susceptibility, and immune recognition . Investigating serC's potential contributions to these adaptive processes represents an important research direction.

What are the most promising approaches for studying B. petrii genome plasticity and its relationship to serC function?

The remarkable genome plasticity of B. petrii presents unique research opportunities:

• Longitudinal genomic analysis:

  • Whole genome sequencing of sequential isolates from persistent infections

  • Identification of common rearrangement patterns and their functional consequences

  • Monitoring of serC sequence and surrounding genomic regions for stability/variation

• Experimental evolution studies:

  • Laboratory evolution under defined selective pressures

  • Tracking genome rearrangements and correlating with phenotypic changes

  • Specific focus on metabolic gene adaptations including serC

• Functional genomics approaches:

  • CRISPR interference to modulate serC expression without permanent genetic changes

  • Transposon sequencing (Tn-seq) to identify genetic interactions with serC

  • RNA-seq under various environmental conditions to map transcriptional networks involving serC

• Comparative genomics:

  • Analysis of serC conservation and surrounding genomic architecture across Bordetella species

  • Identification of horizontally acquired elements that may influence serC function

  • Comparison of environmental versus clinical B. petrii isolates

These approaches would provide valuable insights into how genome plasticity affects metabolic functions in B. petrii and potentially reveal adaptations that enable persistence in human hosts. Elucidating the relationship between genome rearrangements and serC function could enhance our understanding of bacterial adaptation and inform development of intervention strategies for persistent infections.

How might recombinant B. petrii serC be utilized in structural biology and drug discovery efforts?

Recombinant B. petrii serC offers several applications in structural biology and therapeutic development:

• Structural characterization:

  • X-ray crystallography to determine three-dimensional structure

  • Cryo-EM for analysis of conformational states

  • NMR studies for dynamic regions and ligand interactions

  • Comparison with SerC structures from other pathogens to identify unique features

• Inhibitor development:

  • High-throughput screening against purified recombinant serC

  • Structure-based design of selective inhibitors

  • Fragment-based approaches to identify binding pockets

  • Development of transition-state analogs as potential inhibitors

• Therapeutic applications:

  • Evaluation of serC as a potential drug target for persistent B. petrii infections

  • Cross-species inhibitor testing to develop broad-spectrum agents

  • Exploration of allosteric regulation sites for selective targeting

• Biochemical tool development:

  • SerC-based biosensors for metabolite detection

  • Engineered variants with altered substrate specificity

  • Development of activity-based probes for in vivo enzyme monitoring