Recombinant Chlamydophila caviae Phenylalanine--tRNA ligase alpha subunit (pheS)

What is the primary function of Phenylalanine-tRNA ligase alpha subunit in Chlamydophila caviae?

The alpha subunit of Phenylalanine-tRNA ligase (PheS) in C. caviae forms the catalytic core of the enzyme responsible for synthesizing Phe-tRNA(Phe) during protein synthesis. This enzyme belongs to the aminoacyl-tRNA synthetase family and plays a critical role in the translation process by ensuring the accurate charging of tRNA(Phe) with phenylalanine. The alpha subunit specifically contains the active site for amino acid activation and transfer to the tRNA molecule . Unlike the beta subunit which handles tRNA anticodon binding and editing functions, the alpha subunit primarily catalyzes the aminoacylation reaction itself .

How does the structure of C. caviae PheS compare to PheRS alpha subunits in other bacterial species?

The PheRS alpha subunit maintains high conservation across bacterial species, reflecting its essential catalytic function. While complete C. caviae PheS structural data isn't explicitly detailed in current literature, comparative genomic analyses indicate that C. caviae, like other Chlamydiaceae, maintains the core structural features of bacterial PheS proteins. The conservation is particularly evident in the N-terminal catalytic domain which contains the active site for aminoacylation . The C. caviae genome (1,173,390 nt) contains the pheS gene as part of its 1009 annotated genes .

Analysis of protein alignments across bacterial species shows that critical catalytic residues, particularly the catalytic histidine involved in amino acid activation, are highly conserved . Comparison with well-characterized PheRS structures from model organisms suggests the C. caviae PheS likely adopts the canonical alpha subunit fold with distinct domains for amino acid activation and tRNA interaction.

What are the optimal expression systems for recombinant production of C. caviae PheS?

Based on protocols developed for similar bacterial aminoacyl-tRNA synthetases, E. coli-based expression systems represent the most effective platform for recombinant production of C. caviae PheS. The recommended expression strategy involves:

• Cloning the C. caviae pheS gene into an expression vector with a strong inducible promoter (T7 or tac)

• Including an N-terminal or C-terminal affinity tag (His6 or GST) for purification

• Transforming into an E. coli expression strain optimized for protein production (BL21(DE3) or derivatives)

• Inducing expression at lower temperatures (16-20°C) to enhance proper folding

• Supplementing growth media with additional amino acids to support high-level protein production

Expression yields can be optimized by adjusting induction conditions (IPTG concentration 0.1-1.0 mM) and harvesting cells during mid to late log phase . For structural studies requiring isotopic labeling, minimal media supplemented with 15N-ammonium chloride and/or 13C-glucose would be necessary.

What purification challenges are specific to C. caviae PheS, and how can they be overcome?

Purification of recombinant C. caviae PheS presents several challenges:

• Solubility issues: PheS may form inclusion bodies when overexpressed. This can be addressed by:

  • Reducing induction temperature to 16°C

  • Co-expressing with molecular chaperones (GroEL/GroES)

  • Using solubility-enhancing fusion tags (MBP or SUMO)

• Maintaining enzyme activity: PheS activity is sensitive to oxidation of cysteine residues. Purification buffers should contain:

  • Reducing agents (1-5 mM DTT or β-mercaptoethanol)

  • 10-20% glycerol for stability

  • Appropriate pH buffering (typically pH 7.5-8.0)

• Separation from endogenous E. coli PheRS: Contamination can be minimized by:

  • Implementing a multi-step purification strategy combining affinity chromatography with ion exchange and size exclusion chromatography

  • Using stringent washing conditions during affinity purification

  • Incorporating tag removal and a second affinity step

Typical purification yields range from 2-5 mg of purified protein per liter of bacterial culture with >90% purity achievable following optimized protocols .

Does C. caviae PheS exhibit the growth-promoting and proliferation activities observed in other α-PheRS proteins?

Based on comparative analyses, C. caviae PheS likely possesses similar non-canonical functions to those observed in other systems. The N-terminal region of α-PheRS, which has been implicated in these alternative functions, shows sequence conservation patterns suggesting similar capabilities. Experimental investigation of C. caviae PheS would require:

• Expression of both full-length and N-terminal truncated versions

• Development of aminoacylation-deficient mutants (similar to the α-PheRSCys mutant described in Drosophila studies)

• Cell-based assays to measure proliferation effects

• Interaction studies with potential signaling partners

Notably, the N-terminal domain of α-PheRS has been shown to localize to the nucleus and potentially interfere with signaling pathways such as Notch , suggesting a possible regulatory role for C. caviae PheS beyond translation.

How can C. caviae PheS be used as a tool in antimicrobial research?

C. caviae PheS represents a valuable target for antimicrobial research due to several factors:

• Essential enzyme: As a critical component of the translation machinery, inhibition of PheS would effectively block protein synthesis in C. caviae

• Structural differences from human ortholog: Bacterial PheRS differs significantly from the human counterpart, offering potential for selective targeting

• Applications in drug discovery:

  • High-throughput screening platforms using purified recombinant C. caviae PheS to identify novel inhibitors

  • Structure-based drug design targeting unique pockets in the active site

  • Whole-cell assays with C. caviae to validate compound efficacy

• Development of reporter systems:

  • Construction of C. caviae strains with modified pheS genes encoding conditionally lethal mutations

  • These systems can serve as counterselectable markers in genetic studies

Research approaches could include comparative analyses of inhibition profiles across different Chlamydiaceae species to identify broad-spectrum candidates with activity against multiple pathogens. Preliminary data suggests that compounds targeting the aminoacylation active site show promise as selective inhibitors of bacterial growth .

What genetic systems are available for manipulating the pheS gene in C. caviae?

Recent advances in genetic manipulation of obligate intracellular bacteria have expanded the toolkit available for C. caviae gene modification. For pheS manipulation, several approaches can be employed:

• Transformation-based methods:

  • Electroporation of plasmid DNA containing modified pheS sequences

  • Selection using appropriate antibiotic markers (spectinomycin, ampicillin)

  • Monitoring transformants using fluorescent reporters

• Allelic exchange techniques:

  • Fluorescence-Reported Allelic Exchange Mutagenesis (FRAEM) can be adapted for C. caviae

  • FIoxed cassette allelic exchange mutagenesis (FLAEM) for creating markerless mutations

  • These approaches use conditional suicide vectors with regulated pgp6 expression

• Vector systems for C. caviae:

  • pSU6-based vectors modified for Chlamydiaceae

  • Shuttle vectors containing both E. coli and chlamydial origins of replication

  • Inducible promoter systems regulated by tetracycline

The transformation efficiency for C. caviae typically ranges from 10^-6 to 10^-7 transformants per input DNA molecule, with selection typically applied 8-24 hours post-infection to allow for bacterial establishment while preventing contamination with non-transformed organisms .

What site-directed mutagenesis approaches can be used to study structure-function relationships in C. caviae PheS?

Site-directed mutagenesis of C. caviae PheS enables detailed structure-function analyses of this essential enzyme. Based on established protocols for aminoacyl-tRNA synthetases, the following approaches are recommended:

• PCR-based mutagenesis:

  • QuikChange or overlap extension PCR methods

  • Targeting conserved catalytic residues identified through sequence alignment

  • Focus on residues in the aminoacylation active site and tRNA binding interface

• Key residues for targeted mutation:

  • Catalytic histidine residue essential for GTP hydrolysis (corresponding to H81 in other bacterial species)

  • ATP-binding pocket residues to modulate aminoacylation activity

  • Interface residues involved in alpha-beta subunit interactions

  • N-terminal domain residues implicated in non-canonical functions

• Functional analysis of mutants:

  • In vitro aminoacylation assays measuring tRNA charging efficiency

  • GTP hydrolysis assays to assess catalytic activity

  • Binding studies with nucleotides and tRNA substrates

  • Protein-protein interaction analyses for alpha-beta assembly

A systematic alanine-scanning approach targeting conserved regions would provide comprehensive functional mapping. Additionally, construction of chimeric proteins combining domains from different species could identify species-specific functional determinants .

What are the most effective assays for measuring C. caviae PheS aminoacylation activity?

Several robust assays can be employed to measure the aminoacylation activity of recombinant C. caviae PheS:

• Radioactive assays:

  • Incorporation of [³H]-phenylalanine or [¹⁴C]-phenylalanine into tRNA

  • Precipitation of charged tRNA followed by scintillation counting

  • Time-course measurements to determine initial velocities

  • Typical reaction conditions: 30°C, pH 7.5, with 4-10 mM MgCl₂, 2-4 mM ATP, 50-200 μM phenylalanine

• Pyrophosphate release assays:

  • Continuous monitoring using coupled enzyme systems

  • Detection of inorganic pyrophosphate released during aminoacylation

  • Advantages include real-time kinetics and avoidance of radioactivity

• tRNA charging efficiency measurement:

  • Acid gel electrophoresis to separate charged from uncharged tRNA

  • Northern blot analysis with tRNA^Phe-specific probes

  • Quantification of charging levels under various conditions

• Steady-state kinetic analysis:

  • Determination of Km values for phenylalanine, ATP and tRNA^Phe substrates

  • Analysis of kcat and catalytic efficiency (kcat/Km)

  • Investigation of competitive inhibitors

Michaelis-Menten parameters for bacterial PheRS typically show Km values of 20-60 μM for phenylalanine, 0.2-0.6 μM for tRNA^Phe, and 0.1-0.4 mM for ATP, with kcat values ranging from 2-8 s⁻¹ .

How can researchers distinguish between canonical and non-canonical functions of C. caviae PheS in experimental settings?

Distinguishing between the canonical aminoacylation function and potential non-canonical activities of C. caviae PheS requires specialized experimental approaches:

• Generation of activity-specific mutants:

  • Construction of aminoacylation-deficient PheS mutants (by mutating catalytic residues)

  • Development of PheS variants with intact aminoacylation but disrupted non-canonical functions

  • Domain deletion constructs isolating specific functional regions

• Cellular localization studies:

  • Fluorescent tagging of full-length and truncated PheS variants

  • Immunofluorescence detection of nuclear versus cytoplasmic distribution

  • Co-localization with markers for different cellular compartments

• Interaction analysis:

  • Yeast two-hybrid or pull-down assays to identify interaction partners

  • Focus on potential signaling proteins (e.g., Notch pathway components)

  • Verification of interactions using co-immunoprecipitation or FRET techniques

• Cell-based functional assays:

  • Cell proliferation measurements with wild-type versus mutant PheS

  • Gene expression profiling to detect pathways affected by PheS overexpression

  • In vitro differentiation assays to monitor cell fate decisions

A key approach is the use of aminoacylation-dead variants (similar to the α-PheRSCys mutant described in Drosophila) which can help isolate growth and proliferation effects from the canonical translation function .

How is the pheS gene conserved across different Chlamydiaceae species, and what does this reveal about evolutionary constraints?

Comparative genomic analysis reveals significant conservation patterns for the pheS gene across Chlamydiaceae species, providing insights into evolutionary constraints on this essential enzyme:

• Sequence conservation:

  • Core catalytic domains show >80% amino acid identity across Chlamydiaceae

  • The ATP-binding pocket and aminoacylation active site exhibit near-complete conservation

  • Greater sequence divergence is observed in peripheral regions less critical for catalysis

• Genomic context:

  • The pheS gene maintains syntenic relationships across Chlamydiaceae genomes

  • It is typically positioned outside the plasticity zone (PZ), indicating strong selective pressure against genomic rearrangements affecting this locus

  • Unlike many genes that show evidence of horizontal transfer in the Chlamydiaceae plasticity zone, pheS evolution appears primarily vertical

• Comparative evolutionary rates:

  • pheS shows slower evolutionary rates compared to membrane proteins and secreted factors

  • Non-synonymous to synonymous substitution ratios (dN/dS) are consistently <1, indicating purifying selection

  • Conservation patterns suggest functional constraints beyond simple aminoacylation activity

• Interspecies comparison table:

This conservation pattern suggests that PheS functionality is under strong selective pressure even as these species have adapted to different hosts and tissues .

Can C. caviae PheS be utilized as a phylogenetic marker for bacterial classification and evolutionary studies?

The PheS protein serves as an excellent phylogenetic marker for several reasons:

• Advantages of pheS for phylogenetic studies:

  • Essential gene present in all bacteria with minimal lateral gene transfer

  • Sufficient sequence variation to distinguish closely related species

  • Less prone to recombination than 16S rRNA

  • Consistent with genome-wide phylogenetic signals

• Application methodologies:

  • Multi-locus sequence typing (MLST) incorporating pheS with other housekeeping genes

  • Single-gene phylogenies using conserved pheS regions

  • Whole-protein structural phylogenetics comparing folding patterns

• Taxonomic resolution:

  • pheS sequences can differentiate between closely related Chlamydiaceae species

  • Particularly useful for resolving relationships within the C. psittaci complex

  • Can distinguish C. caviae from related species such as C. felis

• Technical considerations:

  • PCR amplification using degenerate primers targeting conserved regions

  • Sequence analysis focusing on informative variable regions

  • Phylogenetic reconstruction using maximum likelihood or Bayesian methods

Studies have demonstrated that pheS phylogenies typically agree with those based on other markers such as 16S rRNA, but often provide better resolution at the species and strain levels. Within Chlamydiaceae, pheS sequences have helped clarify relationships between C. caviae, C. felis, and C. psittaci, confirming the distinct species status of C. caviae despite their common classification as GPIC (guinea pig inclusion conjunctivitis) isolates .

How can structural studies of C. caviae PheS contribute to understanding species-specific aminoacylation mechanisms?

Structural studies of C. caviae PheS offer valuable insights into species-specific aminoacylation mechanisms that could advance both basic science and applied research:

• Crystallographic approaches:

  • X-ray crystallography of purified recombinant C. caviae PheS alone and in complex with substrates

  • Co-crystallization with tRNA^Phe, phenylalanine, and ATP analogues

  • Structural comparison with PheS from model organisms like E. coli

• Species-specific structural features:

  • Identification of unique binding pocket architectures

  • Analysis of interface regions involved in alpha-beta subunit assembly

  • Characterization of conformational changes during catalysis

• Mechanistic insights from structural biology:

  • Elucidation of the precise reaction mechanism for aminoacylation

  • Understanding the basis for tRNA recognition specificity

  • Identification of structural determinants for editing mischarged Tyr-tRNA^Phe

• Computational approaches:

  • Molecular dynamics simulations to model enzyme flexibility

  • Virtual screening for species-selective inhibitors

  • Quantum mechanical calculations of transition states

Preliminary structural analyses suggest that while the core catalytic domain architecture is conserved across bacterial species, subtle differences in surface loops and binding pocket geometry may contribute to species-specific substrate preferences and catalytic efficiencies . These differences could potentially be exploited for the development of selective inhibitors targeting C. caviae without affecting the human ortholog.

What are the challenges in developing a cell-free protein synthesis system using C. caviae translation components?

Developing a cell-free protein synthesis (CFPS) system incorporating C. caviae translation components presents several technical challenges and research opportunities:

• Component purification challenges:

  • Expression and purification of all C. caviae translation factors (including PheRS)

  • Ensuring proper folding and activity of recombinant proteins

  • Maintaining stable complexes for multi-subunit factors

• System optimization parameters:

  • Buffer composition (pH 7.5-8.0, Mg²⁺ concentration 3-5 mM)

  • Energy regeneration components (phosphoenolpyruvate or creatine phosphate)

  • Optimal concentrations of aminoacyl-tRNA synthetases (0.1-0.5 μM)

  • tRNA pool composition and concentration (0.5-2.0 mg/ml)

• Technical considerations:

  • Template design with appropriate regulatory elements for C. caviae

  • Ribosome isolation and stability maintenance

  • Prevention of RNase contamination

  • Development of appropriate activity assays

• Research applications:

  • Investigation of species-specific translation mechanisms

  • Incorporation of unnatural amino acids through engineered PheRS

  • Production of proteins toxic to living cells

  • High-throughput screening platform for translation inhibitors

What are the common pitfalls in working with recombinant C. caviae PheS, and how can they be addressed?

Researchers working with recombinant C. caviae PheS commonly encounter several technical challenges:

• Expression and solubility issues:

  • Problem: Formation of inclusion bodies during overexpression

  • Solutions:

    • Lower induction temperature (16-18°C)

    • Reduce IPTG concentration (0.1-0.2 mM)

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

    • Test different E. coli expression strains (BL21, Rosetta, Arctic Express)

• Protein stability concerns:

  • Problem: Activity loss during purification and storage

  • Solutions:

    • Include stabilizing agents (10-15% glycerol, 1-5 mM DTT)

    • Maintain temperature at 4°C throughout purification

    • Store at -80°C in small aliquots to avoid freeze-thaw cycles

    • Use optimized buffer conditions (pH 7.5-8.0, 100-200 mM NaCl)

• Heterotetramer assembly challenges:

  • Problem: Difficulties in obtaining properly assembled α₂β₂ complex

  • Solutions:

    • Co-express alpha and beta subunits from a single vector

    • Implement a two-step purification strategy targeting different tags on each subunit

    • Include molecular chaperones during expression

    • Verify complex formation using size exclusion chromatography

• Activity assay troubleshooting:

  • Problem: Low or inconsistent aminoacylation activity

  • Solutions:

    • Verify tRNA quality (intact acceptor stem is critical)

    • Optimize Mg²⁺ concentration (typically 3-8 mM)

    • Ensure ATP freshness and appropriate concentration (2-4 mM)

    • Include pyrophosphatase to prevent product inhibition

    • Verify that phenylalanine is pure and correctly prepared

Implementing these strategies has been shown to improve both yield and functional quality of recombinant aminoacyl-tRNA synthetases, with potential yield improvements of 2-3 fold and activity enhancements of up to 5-fold in optimized systems .

How can researchers overcome the challenges of working with Chlamydophila caviae as an obligate intracellular pathogen when studying PheS function?

Studying PheS function in the context of C. caviae presents unique challenges due to the organism's obligate intracellular lifestyle:

• Cell culture and infection challenges:

  • Problem: Difficulty maintaining and propagating C. caviae

  • Solutions:

    • Use optimized cell lines (HeLa or McCoy cells) for propagation

    • Implement DEAE-dextran treatment (30 μg/ml) to enhance infection efficiency

    • Centrifuge inoculum at 700 × g for 60 minutes to promote bacterial entry

    • Culture in media supplemented with cycloheximide (1 μg/ml) to inhibit host protein synthesis

• Genetic manipulation approaches:

  • Problem: Limited genetic tools for obligate intracellular bacteria

  • Solutions:

    • Utilize transformation protocols with electroporation (conditions: 2.5 kV, 25 μF, 200 Ω)

    • Employ selection 8-24 hours post-infection using appropriate antibiotics

    • Use fluorescent reporters for tracking transformed bacteria

    • Implement conditional expression systems regulated by tetracycline

• Isolation of bacterial components:

  • Problem: Contamination with host cell material

  • Solutions:

    • Purify elementary bodies using density gradient centrifugation

    • Implement differential centrifugation protocols with sucrose gradients

    • Use selective lysis conditions to separate bacterial from host components

    • Verify purity using microscopy and Western blot analysis

• Functional studies in the native context:

  • Problem: Difficulty distinguishing bacterial PheS activity from host

  • Solutions:

    • Use species-specific antibodies for immunofluorescence studies

    • Develop reporter systems based on bacterial-specific promoters

    • Implement RNA-seq approaches with species-specific mapping

    • Create tagged versions of PheS for tracking within infected cells

These methodological adaptations enable researchers to investigate PheS function within its native context despite the challenges posed by the obligate intracellular lifestyle of C. caviae .