Recombinant Chlamydophila caviae 30S ribosomal protein S10 (rpsJ)

What is the structure and function of the 30S ribosomal protein S10 in Chlamydophila caviae?

The 30S ribosomal protein S10 (rpsJ) in C. caviae is a component of the small ribosomal subunit involved in protein synthesis. The protein contains a flexible loop region that is critical for its function. The 30S ribosomal subunit has two primary functions: discriminating against aminoacyl transfer RNAs that don't match the mRNA codon (decoding) and working with the 50S subunit to move tRNAs and associated mRNA by precisely one codon (translocation) . The S10 protein is particularly important for these processes, as mutations in this protein can significantly affect ribosomal function.

What are the key structural domains of the S10 protein and their significance?

The S10 protein contains a flexible loop region that is critical for its function. The flexible loop tip has been particularly well-studied, with residues like V57 (based on E. coli numbering) playing crucial roles in both protein function and antibiotic interactions. This loop interacts with helix h31 of the 16S rRNA, and alterations in this region can affect both ribosome function and antibiotic susceptibility . The S10 protein in prokaryotes is incorporated during the late stages of ribosomal subunit biogenesis, making it important for proper ribosome assembly and function.

What are the optimal expression systems for producing recombinant C. caviae rpsJ?

Recombinant C. caviae S10 protein can be expressed in several systems including:

• Mammalian cell expression systems: Offers proper folding and post-translational modifications

• E. coli expression systems: Provides high yield but may require optimization of codon usage

• Baculovirus expression systems: Suitable for proteins requiring eukaryotic processing machinery

For most structural and functional studies, E. coli expression systems provide sufficient yields, though mammalian systems may be preferred when studying protein-protein interactions that might depend on specific folding characteristics.

What purification strategies yield the highest purity and activity for recombinant C. caviae S10?

Optimal purification of recombinant C. caviae S10 typically involves:

• Affinity chromatography (using His-tag or other fusion tags)

• Size exclusion chromatography to separate monomeric protein from aggregates

• Ion-exchange chromatography for removing contaminants with different charge properties

Purification should aim for >85% purity as assessed by SDS-PAGE . For functional studies, it's crucial to verify that the purification process doesn't affect the protein's secondary structure or activity. Buffer optimization is essential, with most protocols recommending storage in buffers containing 20 mM Tris-HCl (pH 7.0), 100-150 mM salt (NaCl or KCl), and stabilizers like glycerol (5-50%) .

How can researchers assess the proper folding and functionality of purified recombinant S10?

Functionality assessment should include:

• Structural integrity analysis: Circular dichroism spectroscopy to confirm secondary structure

• Binding assays: Measuring interaction with 16S rRNA or other ribosomal components

• Integration into partial ribosomal assemblies: Testing whether the protein can be incorporated into partial 30S subunit reconstitutions

• Thermal stability assays: Differential scanning fluorimetry to assess protein stability

Additionally, researchers should verify the absence of significant aggregation through dynamic light scattering or size exclusion chromatography profiles.

How can recombinant C. caviae S10 be used to study ribosomal assembly and function?

Recombinant C. caviae S10 can be employed in several experimental approaches:

• In vitro reconstitution studies: Using purified components to study 30S subunit assembly

• Binding assays with 16S rRNA: Determining interaction parameters and critical binding residues

• Cryo-EM structural studies: Incorporating labeled S10 to track its position during ribosomal assembly

• RNA-protein crosslinking experiments: Identifying S10 contact points with rRNA and mRNA

Research has shown that RNA polymerase can interact directly with the 30S ribosomal subunit, with a measured Kd of approximately 2.1 × 10^-8 M . This interaction may involve S10 and could be studied using the recombinant protein in reconstitution experiments.

What methodologies are most effective for studying S10 interactions with antibiotics?

To study S10-antibiotic interactions, researchers should consider:

• Isothermal titration calorimetry (ITC): For measuring binding constants between purified S10 and antibiotics

• Fluorescence-based assays: Using labeled antibiotics to track binding to S10 or S10-containing ribosomal complexes

• Mutagenesis studies: Creating specific mutations in the S10 flexible loop to assess their impact on antibiotic binding

• In vitro translation assays: Measuring how wild-type vs. mutant S10 affects antibiotic inhibition of translation

Studies with tetracycline derivatives show that mutations in S10, particularly at position V57, can significantly alter antibiotic susceptibility . For instance, the V57L mutation in E. coli increased the MIC to tigecycline from 0.125 μg/ml to 0.5 μg/ml .

How can researchers study the role of S10 in Chlamydial species-specific ribosomal functions?

To investigate species-specific ribosomal functions:

• Comparative binding studies: Using recombinant S10 from different Chlamydia species to identify differences in binding to conserved or species-specific rRNAs

• Chimeric protein construction: Creating hybrid S10 proteins with domains from different species to map functional regions

• Heterologous complementation: Testing whether C. caviae S10 can functionally replace S10 in other bacterial systems

• Genomic recombination experiments: Analyzing natural recombination events involving rpsJ between Chlamydia species

Interspecies genetic exchange can occur in Chlamydia, though the exchanged fragments tend to be smaller in interspecies crosses (averaging considerably less than the 181,000 bp seen in intraspecies exchanges) .

How conserved is the rpsJ gene across Chlamydial species and what does this reveal about evolutionary pressures?

The rpsJ gene shows high conservation across Chlamydial species, reflecting its essential role in protein synthesis. Analysis of genomic data shows:

Despite differences in genome size and host tropism, core ribosomal proteins including S10 remain highly conserved . This conservation suggests strong purifying selection on ribosomal components, though specific regions like the flexible loop may show greater variability related to antibiotic resistance or host adaptation.

What genetic recombination events involving rpsJ have been documented in Chlamydia, and what are their implications?

While specific recombination events involving rpsJ weren't detailed in the search results, studies have documented that interspecies genetic exchange can occur in Chlamydia. Fragments encompassing 79% of the C. muridarum chromosome have been introduced into a C. trachomatis background, with total coverage contained on 142 independent recombinant clones . These recombination events potentially include ribosomal genes like rpsJ and could impact ribosome function or antibiotic susceptibility. The largest exchanged fragment in interspecies crosses was approximately 124,000 bp, significantly smaller than those observed in intraspecies crosses .

How does S10 interact with RNA polymerase in Chlamydial transcription-translation coupling?

The interaction between S10 and RNA polymerase is critical for transcription-translation coupling. Research in E. coli has shown that RNA polymerase (RNAP) binds directly to the 30S ribosomal subunit with high affinity (Kd ≈ 2.1 × 10^-8 M) . This interaction likely involves S10, as studies have shown that RNAP co-purifies with ribosomal proteins S1 and S2 .

In Chlamydia, which have reduced genomes and streamlined cellular processes, this coupling may be even more critical. The interaction could be studied using:

• Co-immunoprecipitation with purified recombinant S10 and chlamydial RNAP

• Crosslinking studies followed by mass spectrometry to identify interaction surfaces

• Cryo-EM studies of the 30S- RNAP complex with tagged S10 to visualize the interaction

What role might S10 play in regulating Chlamydial gene expression during different developmental stages?

Chlamydia species have a biphasic developmental cycle, transitioning between infectious elementary bodies (EBs) and replicative reticulate bodies (RBs). S10 may play different roles during these stages:

• In EBs, where protein synthesis is minimal, S10 might be involved in maintaining ribosome integrity

• During the transition to RBs, changes in S10 activity could help activate translation machinery

• In RBs, S10 likely functions in normal translation and possibly in regulatory feedback mechanisms

Recent findings suggest that gene expression in Chlamydia can be regulated by metabolic signals. For example, the enolase-RsbU pathway connects glycolytic flux to gene regulation . While not directly involving S10, these findings suggest that translation machinery components like S10 might also respond to metabolic cues during the developmental cycle.

How does the interaction between S10 and antibiotics affect Chlamydial susceptibility profiles?

Studies in E. coli have shown that mutations in the S10 flexible loop, particularly at position V57, can significantly alter susceptibility to tetracycline derivatives . While direct studies in Chlamydia weren't reported, the mechanism likely applies across bacterial species:

• The S10 flexible loop interacts with helix h31 of the 16S rRNA, which is also a binding site for tetracyclines

• Mutations in this loop can alter the positioning of h31, affecting both antibiotic binding and S10 function

• Different amino acid substitutions have varying effects - some increase resistance while others can actually increase sensitivity

For example, in E. coli, the V57L mutation increased the MIC to tigecycline from 0.125 μg/ml to 0.5 μg/ml, while V57K actually decreased resistance to tetracycline . These findings suggest that the interaction between S10 and antibiotics is complex and could be exploited for developing species-specific antibiotics.

What are the most common challenges in expressing and purifying functional recombinant C. caviae S10?

Researchers frequently encounter these challenges:

• Expression yield issues: As a ribosomal protein, S10 may interact with host ribosomes and affect expression

  • Solution: Use tunable expression systems and optimize induction conditions

• Protein solubility: Ribosomal proteins often aggregate when expressed without their binding partners

  • Solution: Express with solubility tags (MBP, SUMO) or use mild detergents in purification buffers

• Protein functionality: Ensuring the recombinant protein maintains its native structure

  • Solution: Verify structure using circular dichroism and functional assays

• Contaminant ribosomal RNA: Host rRNA may co-purify with S10

  • Solution: Include RNase treatment steps and high-salt washes during purification

How can researchers optimize storage conditions to maintain S10 stability and activity?

Optimal storage recommendations include:

• Store purified protein at high concentration (0.1-1.0 mg/mL)

• Add 5-50% glycerol as a cryoprotectant

• Store at -20°C/-80°C with stability up to 6 months (liquid form) or 12 months (lyophilized)

• Avoid repeated freeze-thaw cycles; store working aliquots at 4°C for up to one week

• Include reducing agents (DTT or β-mercaptoethanol) to prevent oxidation of cysteine residues

What quality control measures should be implemented when working with recombinant S10?

Essential quality control measures include:

• Purity assessment: SDS-PAGE analysis (target >85% purity)

• Identity confirmation: Mass spectrometry or western blotting with anti-S10 antibodies

• Structural integrity: Circular dichroism spectroscopy to verify secondary structure

• Oligomeric state analysis: Size-exclusion chromatography to check for aggregation

• Functional validation: RNA binding assays or incorporation into partial ribosome assemblies

How can C. caviae S10 be used to study transcription-translation coupling in obligate intracellular bacteria?

Transcription-translation coupling is particularly important in organisms with reduced genomes like Chlamydia. Research approaches could include:

• Reconstituted systems combining purified S10, RNA polymerase, and other components: These can reveal direct interactions and kinetic parameters

• Single-molecule approaches: Using fluorescently labeled S10 to track its dynamics during coupled transcription-translation

• Crosslinking mass spectrometry (XL-MS): To map the interaction surfaces between S10, RNA polymerase, and other factors

• Cryo-EM of native complexes: Similar to the approaches used to study the 30S- RNAP complex in E. coli, which revealed direct binding between RNAP and the 30S subunit

Such studies could help understand how Chlamydia, with their limited genetic resources, optimize protein synthesis efficiency through coupling of transcription and translation.

What insights can comparative studies of S10 across Chlamydial species provide about host adaptation mechanisms?

Comparative analysis of S10 from different Chlamydial species that infect diverse hosts (humans, guinea pigs, mice, etc.) could reveal:

• Sequence variations that correlate with host tropism: Particularly in regions that might interact with host factors

• Differential responses to host-derived signals: Such as metabolites or stress indicators

• Species-specific interactions with translation factors: Which might reflect adaptation to different host environments

The Chlamydiaceae family includes species with diverse host ranges and tissue tropisms, from C. trachomatis (human genital/ocular infections) to C. caviae (guinea pig conjunctivitis) . Comparing S10 function across these species could reveal adaptation mechanisms.

How might S10 mutations contribute to antimicrobial resistance mechanisms in Chlamydial pathogens?

Studies in E. coli have shown that S10 mutations, particularly in the flexible loop region, can significantly alter susceptibility to tetracyclines and other antibiotics . In Chlamydia, which are already difficult to treat due to their intracellular lifestyle, such mutations could contribute to treatment failures.

Research approaches should include:

• Genetic screening of clinical isolates: To identify naturally occurring S10 mutations in resistant strains

• Directed evolution experiments: To identify potential resistance mutations before they emerge clinically

• Structural studies of S10-antibiotic interactions: To understand the molecular basis of resistance

• Combination therapy testing: To identify approaches that prevent resistance development

Understanding these mechanisms could be critical for developing new treatment strategies for persistent Chlamydial infections.