Recombinant Photorhabdus luminescens subsp. laumondii 50S ribosomal protein L18 (rplR)

What is the biological role of the L18 ribosomal protein in Photorhabdus luminescens?

L18 is one of three ribosomal proteins (alongside L5 and L25) that interact with 5S rRNA in bacterial ribosomes. In P. luminescens, as in other bacteria, it binds to and mediates the attachment of 5S rRNA into the large 50S ribosomal subunit, where it forms part of the central protuberance . This interaction is crucial for proper ribosome assembly and function. The protein helps stabilize the structure of the ribosome and is essential for cellular viability, as demonstrated by gene replacement studies in related bacteria . The gene encoding L18, rplR, is part of a polycistronic spc-operon and is regulated at the translation level by ribosomal protein S8 .

How does L18 differ between P. luminescens and model organisms like E. coli?

While both P. luminescens and E. coli L18 proteins serve similar functions in ribosome assembly, there are notable differences in their genetic context and regulation. In P. luminescens, the rplR gene is part of a genomic landscape that includes numerous additional genes not found in E. coli. Sample sequencing of the P. luminescens W14 genome revealed that approximately 53% of its genome is clearly distinct from that of E. coli K12 . These differences may reflect adaptations to P. luminescens' lifestyle as both a symbiont of entomopathogenic nematodes and a pathogen of insects. The larger genome size (~5.5 Mb compared to E. coli's 4.6 Mb) suggests functional redundancy that may be important for its complex life cycle .

What is the structure and functional domains of L18 in P. luminescens?

The L18 protein in P. luminescens contains domains specialized for:

• 5S rRNA binding

• Interactions with other ribosomal proteins (particularly those in the central protuberance)

• Structural stabilization of the large ribosomal subunit

What purification strategies yield the highest purity and activity for recombinant L18?

For optimal purification of recombinant L18 from P. luminescens, a multi-step approach is recommended:

• Initial capture: Affinity chromatography using His-tag or other fusion tags

• Intermediate purification: Ion exchange chromatography to separate based on charge properties

• Polishing: Size exclusion chromatography to remove aggregates and ensure homogeneity

The typical purification workflow is:

For maintaining protein stability, add protease inhibitors during lysis and keep the protein at 4°C throughout purification. For long-term storage, add 50% glycerol and store at -80°C .

How can I verify the correct folding and activity of purified recombinant L18?

To verify correct folding and activity of purified recombinant L18 from P. luminescens:

• Structural integrity assessment:

  • Circular dichroism (CD) spectroscopy to analyze secondary structure

  • Thermal shift assays to evaluate stability

  • Limited proteolysis to confirm proper folding

• Functional assays:

  • 5S rRNA binding assays using electrophoretic mobility shift assays (EMSA)

  • Filter binding assays to quantify RNA-protein interactions

  • Ribosome assembly assays using purified ribosomal components

• Activity validation:

  • In vitro translation assays using reconstituted ribosomes

  • Complementation of rplR deficient bacterial strains

A critical test is the ability of the recombinant protein to bind 5S rRNA and participate in ribosome assembly. For this, incubate the purified L18 with 5S rRNA and analyze the complex formation using native PAGE or size exclusion chromatography .

How does L18 interact with other ribosomal proteins and rRNA in P. luminescens?

L18 in P. luminescens interacts extensively with both ribosomal RNA and other proteins:

• RNA interactions:

  • Primarily binds to 5S rRNA through specific recognition of its structure

  • May also have contacts with domains of the 23S rRNA

• Protein interactions:

  • Forms part of a protein network including L5 and L25, which collectively bind 5S rRNA

  • Interacts with proteins L5, L25, L27, and other components of the central protuberance

  • May participate in bridges between the large and small ribosomal subunits

These interactions can be studied using:

• Cryo-EM to visualize the entire ribosome structure

• Cross-linking studies to identify specific contact points

• Co-immunoprecipitation to identify binding partners

• Two-hybrid systems to map protein-protein interactions

The interaction network surrounding L18 is highly conserved across bacterial species, indicating its fundamental importance to ribosome structure and function .

How can recombinant L18 be used to study ribosome biogenesis defects?

Recombinant L18 from P. luminescens can be used as a powerful tool to study ribosome biogenesis defects through several approaches:

• Complementation studies:

  • Introducing recombinant L18 into cells with defective endogenous L18

  • Analyzing whether wild-type or mutant versions can rescue biogenesis defects

• In vitro assembly assays:

  • Using recombinant L18 to reconstitute ribosomes from cells with assembly defects

  • Identifying which steps of assembly require functional L18

• Structure-function analysis:

  • Creating point mutations in recombinant L18 to disrupt specific interactions

  • Analyzing how these mutations affect ribosome assembly and function

For example, research on other ribosomal proteins shows that defects in late-stage assembly of 50S subunits can be analyzed using cryo-EM to visualize intermediate structures. In the case of the 45S particles (precursors to 50S), researchers identified two major intermediate states that differed in the stability of functional centers . Similar approaches could be applied to study L18's role by creating L18 variants and analyzing their effects on ribosome structure.

These studies are particularly valuable for understanding fundamental aspects of bacterial ribosome assembly and could potentially lead to new antibiotic targets .

How is L18 expression regulated in P. luminescens, and how does this compare to other bacteria?

In P. luminescens and related bacteria, L18 expression is tightly regulated within the context of ribosome biogenesis:

• Transcriptional regulation:

  • The rplR gene encoding L18 is part of the polycistronic spc-operon in enterobacteria

  • Expression is coordinated with other ribosomal proteins to ensure stoichiometric production

• Translational regulation:

  • Similar to E. coli, L18 synthesis in P. luminescens is likely regulated at the translation level by the ribosomal protein S8

  • This represents a feedback mechanism ensuring balanced ribosomal protein production

• Comparative regulation:

  • Unlike some other ribosomal proteins (like L25/rplY), which have independent transcription units and autoregulatory mechanisms, L18 is regulated as part of an operon

  • The regulatory mechanisms appear to be conserved across enterobacteria, including P. luminescens

Research on related ribosomal proteins like L25 has shown sophisticated autoregulatory mechanisms involving structured regions in the mRNA that form regulatory elements. For example, in E. coli, L25 binds to specific hairpin structures in its own mRNA to repress translation . Whether similar mechanisms exist for L18 within the context of the spc-operon remains an area for investigation.

To study regulation experimentally, approaches such as reporter gene fusions (e.g., rplR-lacZ) can be used to monitor expression under different conditions and in response to mutations in potential regulatory factors .

What role does L18 play in antibiotic resistance and bacterial stress responses?

L18's role in antibiotic resistance and stress responses is multifaceted:

• Antibiotic interactions:

  • The ribosome is a major target for antibiotics, and structural changes in ribosomal proteins like L18 can affect drug binding

  • Mutations in L18 may potentially confer resistance to certain classes of antibiotics that target the large ribosomal subunit

• Stress response mechanisms:

  • During stress conditions, alterations in ribosome composition and structure may occur

  • L18 may participate in specialized ribosomes that preferentially translate stress-response mRNAs

• Comparative analysis with stress proteins:

  • Interestingly, in Bacillus subtilis, a general stress protein CTC is homologous to the ribosomal protein L25 (which, like L18, binds 5S rRNA)

  • CTC can partially complement the growth defect of L25-defective cells in E. coli

  • This suggests potential overlap between ribosomal proteins and stress-response systems

The role of L18 in stress responses can be studied by:

• Analyzing changes in L18 expression under various stress conditions

• Examining phenotypes of strains with L18 mutations during stress

• Comparing translation profiles between wild-type and L18-mutant strains under stress

This research area connects ribosome biology to bacterial adaptation and may reveal new insights into how bacteria respond to environmental challenges .

How can site-directed mutagenesis of recombinant L18 be used to probe specific structure-function relationships?

Site-directed mutagenesis of recombinant L18 from P. luminescens provides powerful insights into structure-function relationships:

• Key residue identification and mutation strategy:

  • Identify conserved residues involved in 5S rRNA binding, protein-protein interactions, or structural stability

  • Design mutations that specifically disrupt these interactions

  • Create a library of single and multiple mutations targeting different functional aspects

• Experimental approaches:

  • Express and purify mutant versions using the methods outlined in section 2

  • Assess RNA binding using electrophoretic mobility shift assays

  • Evaluate ribosome assembly using reconstitution experiments

  • Test translation activity in ribosome function assays

• Structural analysis:

  • Use cryo-EM or X-ray crystallography to determine structural changes caused by mutations

  • Map the effects of mutations on the three-dimensional arrangement of the ribosome

For example, a mutagenesis study might target:

• Positively charged residues likely involved in RNA binding

• Residues at interfaces with other ribosomal proteins

• Residues in highly conserved regions

The results can be analyzed in a table format:

This approach has been successfully used with other ribosomal proteins and can reveal the precise molecular contributions of L18 to ribosome structure and function .

What are the experimental considerations for using L18 to study the evolution of the translation machinery?

When using L18 from P. luminescens for evolutionary studies of translation machinery:

• Sequence comparison framework:

  • Collect L18 sequences from diverse bacterial species, including free-living and symbiotic bacteria

  • Align sequences to identify conserved and variable regions

  • Construct phylogenetic trees to map the evolutionary history of L18

• Functional conservation testing:

  • Express recombinant L18 from different bacterial species

  • Test cross-species complementation by introducing various L18 proteins into L18-deficient strains

  • Evaluate if L18 from P. luminescens can function in distantly related bacteria

• Structural comparative analysis:

  • Compare L18 positioning within ribosomes from different bacteria using cryo-EM structures

  • Identify structural adaptations that might relate to bacterial lifestyle

• Experimental design considerations:

  • Use codon-optimized sequences when expressing L18 from different species

  • Control for differences in regulatory elements that might affect expression

  • Consider using chimeric proteins to map functional domains across species

This approach can reveal insights into how essential ribosomal components like L18 have evolved while maintaining their critical functions in protein synthesis. Of particular interest would be comparing L18 from P. luminescens with homologs from free-living relatives to understand adaptations related to its symbiotic and pathogenic lifestyle .

What are common challenges when working with recombinant L18, and how can they be addressed?

When working with recombinant L18 from P. luminescens, researchers commonly encounter several challenges:

• Solubility issues:

  • Problem: L18 may form inclusion bodies when overexpressed

  • Solution: Lower induction temperature (16-20°C), reduce IPTG concentration, use solubility-enhancing fusion tags (SUMO, MBP), or co-express with chaperones

• RNA contamination:

  • Problem: Purified L18 may contain bound RNA from expression host

  • Solution: Include high salt washes (500 mM NaCl) during purification, add RNase treatment steps, use ion exchange chromatography

• Protein instability:

  • Problem: L18 may aggregate or degrade during purification

  • Solution: Maintain reducing conditions (add DTT or TCEP), keep samples cold, add protease inhibitors, optimize buffer conditions

• Binding partner requirements:

  • Problem: L18 may be unstable without binding partners (5S rRNA or other ribosomal proteins)

  • Solution: Purify as complexes with binding partners, or stabilize with appropriate buffer conditions

• Activity verification:

  • Problem: Difficult to confirm biological activity of purified L18

  • Solution: Develop 5S rRNA binding assays, use complementation of knockout strains, or incorporate into in vitro ribosome assembly systems

Each of these challenges can be systematically addressed through optimization of expression conditions, purification protocols, and functional assays .

How can cryo-EM be used to study L18's role in ribosome assembly intermediates?

Cryo-electron microscopy (cryo-EM) offers powerful approaches to investigate L18's role in ribosome assembly:

• Sample preparation for assembly intermediates:

  • Isolate ribosome assembly intermediates using sucrose gradient centrifugation

  • Prepare samples with and without recombinant L18 to observe structural differences

  • Use strain-specific depletion systems to generate L18-deficient assembly intermediates

• Data collection and processing strategy:

  • Collect high-resolution cryo-EM data (ideally <3Å resolution)

  • Use 3D classification to identify different assembly states

  • Focus refinement on the central protuberance region where L18 resides

• Analysis approaches:

  • Generate temperature maps showing regions of structural flexibility

  • Compare with mature ribosomes to identify conformational differences

  • Use flexible fitting of atomic models into cryo-EM maps

• Visualization and interpretation:

  • Map the position of L18 relative to 5S rRNA and other proteins

  • Identify structural rearrangements dependent on L18 incorporation

  • Analyze the state of functional centers in the presence/absence of L18

What analytical techniques are most informative for studying L18-RNA interactions at the molecular level?

Several analytical techniques provide valuable insights into L18-RNA interactions:

• Biophysical methods:

  • Isothermal titration calorimetry (ITC): Measures binding thermodynamics (ΔH, ΔS, Kd)

  • Surface plasmon resonance (SPR): Provides real-time binding kinetics (kon, koff)

  • Microscale thermophoresis (MST): Detects binding under near-native conditions

• Structural methods:

  • Nuclear magnetic resonance (NMR): Maps interaction sites at atomic resolution

  • X-ray crystallography: Provides high-resolution static structures of complexes

  • Small-angle X-ray scattering (SAXS): Analyzes complex formation in solution

• Biochemical approaches:

  • RNA footprinting: Identifies RNA regions protected by L18 binding

  • Cross-linking and immunoprecipitation (CLIP): Maps RNA-protein contacts in vivo

  • Hydroxyl radical probing: Detects structural changes in RNA upon protein binding

• Computational methods:

  • Molecular dynamics simulations: Model dynamic aspects of interactions

  • Docking algorithms: Predict binding modes and interaction energies

These techniques can be combined to build a comprehensive picture of how L18 recognizes and binds 5S rRNA. For example, crystallography might reveal the static structure of the complex, while NMR could identify dynamic aspects of the interaction. RNA footprinting can map the binding site, which can then be validated through mutagenesis studies .

How might L18 be targeted for antimicrobial development?

L18's essential nature and unique structural features make it a potential target for novel antimicrobials:

• Rationale for targeting L18:

  • The rplR gene is essential for bacterial viability as shown in knockout studies

  • L18's role in ribosome assembly represents a critical vulnerability

  • The protein's interactions with 5S rRNA offer specific targeting opportunities

• Potential targeting strategies:

  • Small molecules that disrupt L18-5S rRNA binding

  • Peptide mimetics that interfere with L18's incorporation into the ribosome

  • Compounds that prevent proper folding of L18

• Screening approaches:

  • Structure-based virtual screening against the L18-RNA interface

  • High-throughput biochemical assays measuring L18-RNA binding

  • Phenotypic screens for compounds that disrupt ribosome assembly

• Selectivity considerations:

  • Target structural differences between bacterial L18 and eukaryotic homologs

  • Focus on bacterial-specific interactions within the ribosome

  • Exploit differences in ribosome assembly pathways

An advantage of targeting L18 is that it could provide activity against a broad spectrum of bacteria, including P. luminescens and related pathogens. Furthermore, since L18 is part of a highly conserved cellular machinery, resistance development might be slower than for other antibiotic targets .

What potential applications exist for recombinant L18 in biotechnology and synthetic biology?

Recombinant L18 from P. luminescens offers several innovative applications:

• Engineered ribosomes:

  • Creating hybrid ribosomes with altered translation properties

  • Developing orthogonal translation systems for synthetic biology

  • Incorporating modified L18 to alter ribosome specificity or function

• Diagnostic and research tools:

  • Developing L18-based probes for studying ribosome biogenesis

  • Creating biosensors that respond to changes in translation capacity

  • Using L18 as a specific RNA-binding module in synthetic circuits

• Protein expression enhancement:

  • Engineering L18 to optimize translation of difficult proteins

  • Creating specialized ribosomes for biotechnology applications

  • Developing conditional translation systems based on modified L18

• Educational and research reagents:

  • Using recombinant L18 in educational kits for teaching ribosome assembly

  • Creating standardized reagents for ribosome research

  • Developing in vitro translation systems with defined components

These applications build on our understanding of L18's fundamental role in ribosome structure and function. By engineering this protein, researchers could create novel tools for both basic science and biotechnological applications .

How can systems biology approaches integrate L18 research into broader understanding of bacterial adaptation?

Systems biology offers powerful frameworks to integrate L18 research into comprehensive models of bacterial adaptation:

• Multi-omics integration:

  • Combine transcriptomics, proteomics, and ribosome profiling to trace L18's role across conditions

  • Map how L18 expression correlates with other cellular components

  • Develop predictive models of how ribosome composition changes affect bacterial physiology

• Network analysis approaches:

  • Place L18 within protein-protein interaction networks

  • Identify regulatory hubs that control L18 expression

  • Map the connections between ribosome assembly and other cellular processes

• Evolutionary systems biology:

  • Compare L18 function across bacterial species with different lifestyles

  • Identify co-evolving components in the translation machinery

  • Model how selective pressures shape ribosome composition

• Experimental system design:

  • Create reporter systems to monitor L18 expression and incorporation in vivo

  • Develop high-throughput phenotyping of L18 variants

  • Design synthetic circuits to probe how L18 alterations affect cellular responses

This integrated approach would connect L18 function to P. luminescens' unique lifestyle as both a symbiont of nematodes and a pathogen of insects. For example, researchers could investigate how L18 expression changes during the transition between these different ecological roles, and how such changes contribute to the bacterium's success in different environments .