Recombinant Protochlamydia amoebophila 50S ribosomal protein L35 (rpmI)

Basic Research Questions

• What is Protochlamydia amoebophila 50S ribosomal protein L35 (rpmI) and why is it significant for research?

  Protochlamydia amoebophila 50S ribosomal protein L35 (rpmI) is a component of the large ribosomal subunit in this amoebal endosymbiont. The significance of this protein lies in its role within the central protuberance (CP) structure of bacterial ribosomes. While not as extensively studied as other ribosomal proteins like L5, L35 is part of the protein cluster that includes L5, L16, L18, L25, L27, L31, and L33, which collectively contribute to the functionality of the 50S subunit . As a member of the Chlamydiae group, P. amoebophila represents an evolutionary intermediate between pathogenic chlamydiae and free-living bacteria, making its ribosomal components valuable for comparative studies of bacterial translation machinery.

• How is recombinant P. amoebophila 50S ribosomal protein L35 typically expressed and purified?

  Recombinant P. amoebophila rpmI can be expressed in multiple heterologous systems:

  Expression Host	Advantages	Considerations
  E. coli	High yield, rapid growth, cost-effective	May form inclusion bodies requiring refolding
  Yeast	Post-translational modifications	Longer cultivation time, more complex protocols
  Baculovirus	Higher eukaryotic modifications	Higher cost, specialized equipment needed
  Mammalian cells	Most complex modifications	Highest cost, lowest yield

  Purification typically involves affinity chromatography utilizing tagged constructs (His-tag being common), followed by size exclusion chromatography to achieve ≥85% purity as verified by SDS-PAGE . For functional studies, additional purification steps may be necessary to remove potential endotoxin contamination, particularly when expressed in bacterial systems.

• What structural features characterize P. amoebophila 50S ribosomal protein L35?

  While the specific structural details of P. amoebophila L35 are not extensively documented in the search results, ribosomal protein L35 typically:

  • Contains approximately 60-70 amino acids

  • Adopts a primarily α-helical structure

  • Interacts directly with rRNA in the 50S subunit

  • Is located near the central protuberance region

  The protein's structure would be expected to contain conserved residues that mediate RNA binding and interactions with neighboring ribosomal proteins such as L27, L31, and L33, which are known to cluster together in the assembled ribosome . Comparative analysis with L35 from other bacterial species would reveal evolutionary conservation patterns reflecting functional constraints on this ribosomal component.

Intermediate Research Questions

• How does P. amoebophila L35 contribute to ribosome assembly and what methods can assess this function?

  Based on studies of related ribosomal proteins, L35 likely contributes to the final stages of 50S subunit assembly. From research on E. coli ribosomes, we know that central protuberance proteins (including L35) are incorporated during later assembly phases . To assess L35's role in assembly:

  Methodology:

  • In vitro reconstitution assays: Ribosomes can be reconstituted from purified components with or without L35, followed by functional testing

  • Pulse-chase experiments: Track L35 incorporation timing during ribosome biogenesis

  • Cryo-EM analysis: Visualize assembly intermediates with and without L35

  • In vivo depletion studies: Similar to L5 depletion studies , conditional expression systems can be used to deplete L35 and observe effects on ribosome assembly

  L35 absence would likely affect the stability of neighboring components in the central protuberance structure, potentially creating assembly defects similar to those observed with L5 depletion, where several CP proteins (L16, L18, L25, L27, L31, L33) were also missing from resulting particles .

• What techniques are optimal for assessing the purity and activity of recombinant P. amoebophila L35?

  Purity Assessment:

  • SDS-PAGE (standard for ≥85% purity verification)

  • Western blotting with anti-L35 antibodies

  • Mass spectrometry (particularly LC-MS/MS for identity confirmation)

  • Size exclusion chromatography profiles

  Activity/Functionality Assessment:

  • Ribosome reconstitution assays (measuring incorporation into 50S subunits)

  • RNA binding assays (electrophoretic mobility shift assays with rRNA fragments)

  • Thermal shift assays to evaluate protein folding and stability

  • Circular dichroism to assess secondary structure integrity

  The combination of these techniques provides comprehensive characterization of both purity and functional integrity of the recombinant protein, critical for ensuring experimental reproducibility in downstream applications.

• How can recombinant P. amoebophila L35 be used to study host-pathogen interactions?

  Protochlamydia amoebophila has been studied for its interactions with amoebae and potentially with human cells. As demonstrated in studies with other Protochlamydia components, the bacterium can induce apoptosis in human immortal HEp-2 cells . Recombinant L35 could be used to:

  • Investigate whether ribosomal proteins like L35 are recognized by host immune systems

  • Study potential moonlighting functions outside of the ribosome

  • Develop antibodies for tracking bacterial protein expression during infection cycles

  • Examine interactions with host ribosomal machinery

  Experimental approach:

  • Production of fluorescently labeled L35 to track localization in host cells

  • Pull-down assays to identify host interaction partners

  • ELISA assays to measure antibody responses to L35 in infection models

  • Microarray analysis to detect host transcriptional responses to L35 exposure

  These approaches could reveal whether bacterial ribosomal proteins play roles beyond protein synthesis during host-pathogen interactions .

Advanced Research Questions

• What are the methodological considerations for studying L35 in the context of P. amoebophila metabolic activity?

  P. amoebophila elementary bodies (EBs) demonstrate unexpected metabolic activity that challenges traditional views of this developmental stage . Studying L35 in this context requires specialized approaches:

  Methodology:

  1. Developmental stage-specific analysis: Isolate EBs and reticulate bodies (RBs) using density gradient ultracentrifugation

  2. Activity monitoring: Use CTC reduction assays to correlate ribosomal activity with respiratory function

  3. In situ protein labeling: Apply techniques like BONCAT (bio-orthogonal non-canonical amino acid tagging) to detect newly synthesized L35 during host-free periods

  4. Isotope ratio mass spectrometry (IRMS): Measure protein synthesis activity when supplemented with labeled amino acids

  Technical challenges include:

  • Maintaining viability of P. amoebophila during experimentation

  • Distinguishing bacterial proteins from host contaminants

  • Working with the fastidious growth requirements of this obligate intracellular organism

  These approaches can connect L35 synthesis and incorporation to the surprising metabolic capabilities of P. amoebophila EBs, potentially revealing specialized translational regulation during different developmental stages .

• How can molecular genetic techniques be applied to study L35 function in P. amoebophila despite its obligate intracellular lifestyle?

  Studying gene function in obligate intracellular bacteria presents significant challenges. For P. amoebophila L35 research, several sophisticated approaches can overcome these limitations:

  Technique	Application to L35 Research	Limitations
  RNA interference in host cells	Disrupt host factors required for L35 function	Indirect approach, may affect multiple pathways
  Antisense RNA expression	Targeted inhibition of rpmI expression	Requires efficient delivery system
  Host-free systems	Study L35 function during extracellular phase	Limited timeframe of bacterial viability
  Heterologous expression systems	Express L35 variants in E. coli to assess function	May not replicate natural environment
  Fluorescence in situ hybridization (FISH)	Localize L35 mRNA during developmental cycle	Requires optimization for intracellular detection

  A particularly promising approach involves adapting techniques from transposon-insertion sequencing studies developed for other pathogens , potentially allowing identification of genes that interact functionally with rpmI despite the challenges of working with an intracellular organism.

• What is the relationship between L35 and other central protuberance proteins in the context of ribosomal evolution?

  Advanced evolutionary analysis of L35 can provide insights into ribosomal evolution across bacterial phyla:

  Research approaches:

  1. Comparative genomics: Analyze L35 sequence conservation across Chlamydiae and related bacteria

  2. Structural bioinformatics: Model co-evolution networks between L35 and other CP proteins

  3. Ancestral sequence reconstruction: Infer evolutionary trajectories of L35 within bacterial lineages

  The interdependence between L35 and other CP components (L5, L16, L18, L25, L27, L31, L33) observed in E. coli studies suggests a coordinated evolutionary module. Analysis of L5-depleted ribosomes revealed that L35 absence correlates with missing CP structure, indicating these proteins evolved as a functional unit .

  This evolutionary perspective on L35 can potentially illuminate how P. amoebophila, positioned between environmental bacteria and obligate pathogens, represents an intermediate evolutionary stage in the transition to obligate intracellular lifestyle.

• How might recombinant P. amoebophila L35 be engineered for specialized research applications?

  Advanced protein engineering approaches can enhance the utility of recombinant L35:

  Engineering strategies:

  • Site-directed mutagenesis: Create L35 variants to map functional domains

  • Fusion constructs: Generate L35 fusions with fluorescent proteins or affinity tags for specialized applications

  • Protein complementation assays: Design split-reporter systems to study L35 interactions

  • Thermostability engineering: Modify L35 for enhanced stability during experimental manipulation

  Applications in experimental systems:

  Engineering Approach	Research Application	Expected Outcome
  Cysteine incorporation	Site-specific labeling for FRET studies	Dynamic information on L35 interactions during assembly
  Chimeric L35 constructs	Domain swapping with other bacterial L35 proteins	Functional conservation mapping
  Conditionally stable L35	Temperature-sensitive variants	Temporal control of L35 function
  Biotinylated L35	Pull-down experiments	Identification of novel interaction partners

  These engineered variants could overcome limitations in studying this protein from an obligate intracellular bacterium while providing molecular tools for broader ribosomal research applications.

• What role might P. amoebophila L35 play in the developmental cycle of this organism in amoebal hosts?

  P. amoebophila undergoes a biphasic developmental cycle similar to other Chlamydiae, transitioning between elementary bodies (EBs) and reticulate bodies (RBs). Recent studies have shown that EBs maintain unexpected metabolic activity .

  Research considerations:

  • During the transition from EB to RB (the metabolically active form), ribosome activation is critical

  • L35 synthesis and incorporation likely occurs during early stages of RB development

  • Respiratory activity detected in EBs suggests potential translational activity involving L35-containing ribosomes

  • Studies using CTC reduction assays have shown that respiratory activity in EB fractions cannot be solely attributed to contamination with other developmental stages

  Methodological approach:

  1. Stage-specific proteomics to quantify L35 abundance across the developmental cycle

  2. Ribosomal profiling during EB-to-RB transition

  3. Fluorescence microscopy with L35-specific antibodies to track ribosome distribution

  4. Host-free incubation studies to evaluate translation in isolated developmental forms

  These approaches could reveal how L35-containing ribosomes contribute to the surprising metabolic capabilities observed in P. amoebophila developmental forms .