Recombinant Chlamydophila caviae Serine--tRNA ligase (serS)

What is the genomic context of serS in Chlamydophila caviae?

SerS is encoded within the 1.17 Mb genome of Chlamydophila caviae (formerly Chlamydia psittaci GPIC isolate). The complete genome sequencing revealed 1009 annotated genes, with serS being part of the core genome shared across Chlamydiaceae species . The gene is likely part of the 798 core genes that represent the "minimal gene content" required for the developmental cycle and intracellular survival of Chlamydiaceae . As an essential enzyme for protein synthesis, serS is highly conserved across chlamydial species, including C. trachomatis, C. pneumoniae, and C. muridarum.

How does C. caviae serS compare structurally to other bacterial tRNA synthetases?

While the specific crystal structure of C. caviae serS has not been determined according to the provided search results, inferences can be made from related serS proteins. Based on data from Chlamydia abortus serS (a closely related species), the protein consists of 424 amino acids with distinct functional domains . The catalytic core contains the ATP binding site and serine recognition domains. Like other bacterial SerRS enzymes, C. caviae serS likely adopts a dimeric structure with each monomer comprising an N-terminal domain and a catalytic C-terminal domain responsible for aminoacylation activity.

What expression systems are recommended for producing recombinant C. caviae serS?

Based on information from similar recombinant proteins, mammalian expression systems have been successfully used to produce recombinant Chlamydial proteins . For optimal expression of C. caviae serS:

• Expression vector: Vectors containing strong promoters (e.g., CMV) are recommended

• Host system: Mammalian cell lines have proven effective for chlamydial protein expression

• Purification: His-tag purification methods with >85% purity achievable by SDS-PAGE

• Storage conditions: Store at -20°C/-80°C with 5-50% glycerol (50% is optimal) to prevent activity loss

• Stability: Liquid form maintains stability for approximately 6 months, while lyophilized form remains stable for up to 12 months at -20°C/-80°C

What is the role of serS in the C. caviae developmental cycle?

SerS plays a critical role in the biphasic developmental cycle of C. caviae, which transitions between elementary bodies (EBs) and reticulate bodies (RBs). As a tRNA synthetase, serS is essential for protein synthesis during both developmental stages. The transition between EBs and RBs involves extensive regulation of protein synthesis and function , and serS likely plays a crucial role in this process by ensuring the fidelity of serine incorporation into proteins needed for developmental transitions. Phosphoproteomic studies have identified stage-specific phosphorylated proteins in C. caviae EBs and RBs, suggesting that protein synthesis machinery (potentially including serS) may be regulated during the developmental cycle .

How can tRNA synthetase inhibitors be used to study serS function in C. caviae?

tRNA synthetase inhibitors represent powerful tools for studying serS function in C. caviae. Research has shown that prokaryotic-specific tRNA synthetase inhibitors like indolmycin (targeting tryptophanyl-tRNA synthetase) and AN3365 can induce persistence in Chlamydia species . A similar approach can be applied to study serS:

• Experimental design: Treat C. caviae-infected cells with serS-specific inhibitors at different time points post-infection

• Expected outcomes: Growth inhibition, changes in morphology, and transcriptional alterations consistent with persistence

• Reversibility: Growth inhibition should be reversible upon removal of the inhibitor

• Applications: This approach allows for mechanistic study of persistence using genetic tools and enables amino acid starvation to be induced at specific times

The table below summarizes the effects of tRNA synthetase inhibitors on Chlamydia:

What experimental approaches can be used to detect post-translational modifications of serS in C. caviae?

Post-translational modifications (PTMs) of serS may regulate its activity during the C. caviae developmental cycle. Based on phosphoproteomic studies of C. caviae , the following experimental approaches are recommended:

• Two-dimensional gel electrophoresis coupled with phosphoprotein staining

• MALDI-TOF/TOF analysis for protein identification

• Thin-layer chromatography to identify phosphorylated amino acid residues

• Isolation of both EB and RB forms at different time points (e.g., 15h for RBs and 43h for EBs) using density differential centrifugation

• Western blot analysis with anti-phospho antibodies

Considerations for experimental design:

• Use multiple biological replicates to ensure reproducibility

• Include appropriate controls for phosphorylation detection

• Be aware that low-abundance phosphoproteins may be missed (Pro-Q Diamond stain detection limit: 1-2 ng for multiphosphorylated proteins and 8 ng for singly phosphorylated proteins)

How might Surface Enhanced Raman Spectroscopy (SERS) be optimized for studying C. caviae serS structure and function?

SERS provides a non-destructive method for structural analysis of proteins like serS. To optimize SERS for studying recombinant C. caviae serS:

• Implement Design of Experiments (DoE) approach :

  • Identify critical factors affecting SERS enhancement (pH, concentration, aggregating agent)

  • Select appropriate experimental design (fractional factorial or full factorial)

  • Conduct targeted experiments based on the design matrix

  • Analyze results to identify optimal conditions

• Optimize key parameters :

  • Metal substrate selection (silver or gold)

  • Reducing agent and concentration

  • Aggregating agent and concentration

  • Acquisition time and laser power

• Data analysis methods :

  • Univariate analysis: Plot peak area of characteristic vibrations

  • Calculate mean, standard deviation, and full width at half maximum (FWHM)

  • Consider multivariate analysis of whole SERS spectrum

  • Apply principal component analysis for data comparison

This approach can reduce the total number of experiments needed while identifying optimal conditions for serS structural analysis .

What role might serS play in C. caviae persistence and host adaptation?

C. caviae serS likely plays a critical role in bacterial persistence and host adaptation mechanisms:

• Persistence induction: tRNA synthetase activity is linked to persistence in Chlamydia . When amino acids become limiting, the resulting stress response can trigger persistence. As SerS is responsible for serine incorporation into proteins, inhibition of its activity would disrupt protein synthesis, potentially triggering persistence.

• Host adaptation: C. caviae has been detected in guinea pigs with 2.7-8.9% occurrence , and has recently been identified as causing zoonotic infections in humans . The serS enzyme must function effectively across different host environments with potentially different serine availability.

• Developmental regulation: Phosphoproteomic analysis has revealed stage-specific phosphorylation patterns between EBs and RBs in C. caviae . Whether serS is among the differentially phosphorylated proteins is unknown, but such regulation could contribute to developmental transitions.

• Potential therapeutic target: As an essential enzyme, serS could be targeted by inhibitors to block C. caviae growth. The recent identification of C. caviae as a zoonotic pathogen causing severe community-acquired pneumonia in humans makes this a particularly relevant consideration.

How does genomic conservation and recombination affect serS in Chlamydia species?

Analysis of recombination and selection in Chlamydial genomes provides insights into serS evolution:

• Conservation: serS likely belongs to the 836 core genes identified in C. trachomatis genomes . The high conservation reflects its essential function in protein synthesis.

• Recombination rate: The ratio of recombination events to mutation (ρ/θ) in Chlamydia is approximately 0.07, but recombination significantly affects genetic diversification (r/m = 0.71) . It's unknown whether serS specifically has undergone recombination, but essential genes often show less recombination than virulence-associated genes.

• Selection pressure: 92 genes in Chlamydia show evidence of positive selection . As an essential housekeeping gene, serS likely experiences purifying selection to maintain function rather than diversifying selection.

• Adaptation implications: If serS has undergone recombination between Chlamydial species, this could potentially be associated with adaptation to different host conditions or developmental requirements.

What approaches can be used to study potential interactions between serS and other Chlamydial proteins?

To investigate protein-protein interactions involving C. caviae serS:

• Yeast two-hybrid screening to identify potential interacting partners

• Co-immunoprecipitation followed by mass spectrometry

• Bioluminescence resonance energy transfer (BRET) or fluorescence resonance energy transfer (FRET) for in vivo interaction monitoring

• Bacterial two-hybrid systems adaptable for obligate intracellular bacteria

Potential interacting partners may include:

• Other components of the translation machinery

• Regulatory proteins like those in the anti-anti-sigma factor system that respond to metabolic stress and regulate development

• Protein kinases that might phosphorylate serS, such as PknD, which has been shown to exhibit dual amino acid specificity in C. pneumoniae

When designing experiments to study these interactions, researchers should consider the challenges of working with an obligate intracellular pathogen and may need to rely on recombinant proteins or heterologous expression systems.