Recombinant Photorhabdus luminescens subsp. laumondii 30S ribosomal protein S4 (rpsD)

What is the functional role of ribosomal protein S4 in Photorhabdus luminescens?

Ribosomal protein S4 serves multiple critical functions in P. luminescens:

• Assembly nucleation: S4 functions as one of two assembly initiator proteins for the 30S ribosomal subunit, binding directly to 16S rRNA where it nucleates assembly of the body of the 30S subunit .

• Translational regulation: S4 acts as a translational repressor protein, controlling the translation of the alpha-operon (which codes for S13, S11, S4, RNA polymerase alpha subunit, and L17) by binding to its mRNA .

• Transcriptional regulation: S4 functions as a rho-dependent antiterminator of rRNA transcription, increasing rRNA synthesis under conditions of excess protein .

• Translational accuracy: Together with S5 and S12, S4 plays an important role in translational accuracy .

The protein contributes to the processive rRNA transcription and antitermination complex (rrnTAC), forming an RNA-chaperone ring around the RNA exit tunnel of RNA polymerase, supporting rapid transcription and antitermination of rRNA operons .

How can I design a basic experiment to express recombinant P. luminescens S4 protein?

A methodological approach to express recombinant P. luminescens S4 protein involves:

• Gene selection and vector design:

  • Identify and amplify the rpsD gene from P. luminescens genomic DNA using PCR

  • Include appropriate restriction sites for cloning into an expression vector (e.g., pET system)

  • Consider adding an N- or C-terminal His-tag for purification purposes

• Expression system selection:

  • E. coli is the preferred expression system (typically BL21(DE3) strain)

  • Expression can be regulated by IPTG induction (if using T7 promoter-based vectors)

• Culture conditions:

  • Standard LB media with appropriate antibiotics

  • Induction at OD600 = 0.6-0.8

  • Typical expression at 30°C for 4-6 hours (to balance yield and solubility)

• Purification strategy:

  • Lyse cells using sonication or French press in buffer containing 50 mM Tris-HCl pH 8.0, 300 mM NaCl

  • Purify using Ni-NTA affinity chromatography for His-tagged protein

  • Further purify by ion-exchange chromatography if needed

• Quality control:

  • Verify purity by SDS-PAGE (expect ~20-25 kDa band)

  • Confirm identity by western blot or mass spectrometry

The expressed protein should achieve >85% purity as determined by SDS-PAGE, consistent with commercial standards .

What are the key structural features of the 30S ribosomal protein S4?

The 30S ribosomal protein S4 from P. luminescens exhibits several key structural features important for its function:

• Domain organization: Contains stable C-terminal domains that include a winged-helix motif which directly contacts the center of the five-way junction (5WJ) in the rRNA

• RNA binding site: Recognizes the five-way junction (5WJ) between helices (H) 3, 4, 16, 17 and 18, which flank the 5′ and 3′ ends of the 16S 5′ domain

• Interaction mode: Primarily interacts with the rRNA backbone with a few base-specific contacts in the 5WJ

• Size and composition: Typically ranges from 200-206 amino acids (similar to the E. coli homolog)

The binding mechanism induces structural changes in the 5′ and 3′ domains of the 16S rRNA, facilitating further steps in 30S assembly . These conformational changes help create a platform for binding of subsequent ribosomal proteins and proper folding of the ribosomal RNA.

How does the experimental design differ when studying S4 protein interactions with rRNA versus its role in translational accuracy?

When investigating different functional aspects of S4 protein, experimental approaches must be tailored to the specific research questions:

For S4-rRNA interaction studies:

• In vitro binding assays:

  • RNA electrophoretic mobility shift assays (EMSA)

  • Filter binding assays with labeled rRNA fragments

  • Isothermal titration calorimetry for binding kinetics

• Structural analysis:

  • Hydroxyl radical footprinting to map binding sites

  • Crystallography of S4-rRNA complexes

  • Cryo-EM of assembly intermediates

• Experimental controls:

  • Use isolated 5WJ RNA construct as positive control

  • Include non-specific RNA as negative control

  • Validate with known binding mutants

For translational accuracy studies:

• Reporter systems:

  • Dual luciferase reporters with programmed frameshifts

  • β-galactosidase readthrough assays

  • In vitro translation systems with defined components

• Mutation analysis:

  • Focus on streptomycin-dependent mutants and suppressors

  • Study ribosomal ambiguity mutations (ram) in S4

  • Analyze error rates with different S4 variants

• Complementation experiments:

  • Express wild-type or mutant S4 in appropriate genetic backgrounds

  • Quantify miscoding events and stop codon readthrough

This comparative experimental approach allows researchers to isolate specific S4 functions while controlling for variables that might confound results in more complex systems.

What methods are optimal for analyzing the interaction between P. luminescens S4 protein and the bacterial Type VI secretion system?

Analyzing interactions between P. luminescens S4 protein and the Type VI secretion system (T6SS) requires specialized methods to capture these complex molecular relationships:

• Protein-protein interaction analysis:

  • Bacterial two-hybrid screening to identify potential binding partners within the T6SS

  • Co-immunoprecipitation using anti-S4 antibodies followed by mass spectrometry

  • Pull-down assays with His-tagged S4 to isolate interacting T6SS components

• Localization studies:

  • Immunofluorescence microscopy using fluorescently labeled antibodies

  • GFP fusion proteins to track S4 localization relative to T6SS structures

  • Cell fractionation to determine subcellular distribution

• Functional assays:

  • Antibacterial competition assays using wild-type and S4-deficient strains

  • T6SS secretion assays to measure effector export efficiency

  • ADP-ribosylation assays to assess modification of target 23S rRNA

• Genetic approaches:

  • Construct deletion mutants of S4 and T6SS components

  • Complementation studies with various S4 domains

  • Site-directed mutagenesis of key S4 residues

Based on research with related Photorhabdus proteins, the T6SS appears to deliver Rhs-linked toxins that ADP-ribosylate the 23S ribosomal RNA in target cells . While S4 itself may not directly interact with T6SS, understanding these pathways provides context for ribosomal protein functions in bacterial competition and pathogenicity.

What are the key differences between P. luminescens S4 protein and E. coli S4 protein in terms of structure and function?

Comparing P. luminescens S4 protein with its E. coli homolog reveals both conservation and divergence:

While core functions are conserved, P. luminescens S4 may have evolved unique properties related to the bacterium's dual lifestyle as both an insect pathogen and nematode symbiont . The P. luminescens genome contains large genomic islands that differ from the Escherichia/Yersinia-like backbone, which could influence ribosomal protein function in species-specific contexts .

What methodological approaches would be most effective for studying the role of S4 protein in a research-practice partnership framework?

Implementing research on P. luminescens S4 protein within a research-practice partnership (RPP) framework requires careful consideration of both scientific rigor and practical application:

Methodological Framework:

• Partnership establishment:

  • Identify stakeholders from research institutions and agricultural/pest management sectors

  • Establish shared goals connecting basic S4 protein research to practical applications

  • Create protocols for bidirectional knowledge exchange

• Research design considerations:

  • Instrumental use approach: Apply existing research on S4 structure-function directly to develop biocontrol applications

  • Conceptual use approach: Use S4 research to extend understanding of bacterial-nematode symbiosis

  • Process use approach: Employ S4 research methods to increase practitioners' capacity for improving pest management

• Implementation strategies:

  • Establish experimental field sites with controlled variables

  • Develop laboratory-to-field transition protocols

  • Create practitioner-friendly assessment tools for biological control efficacy

• Research questions categorization :

  • Diagnostic questions: Why does S4 function differently across Photorhabdus strains?

  • Impact questions: How does S4 modification affect insecticidal activity?

  • Implementation questions: What resources and conditions are required for S4-based applications?

• Evaluation framework:

  • Measure knowledge transfer using the R3I Method (Relevance, Inference, Impact, Importance)

  • Document practitioner engagement using structured observation protocols

  • Track changes in practice resulting from research findings

This approach acknowledges that academic understanding of S4 protein should inform practical applications in biological control, while ensuring research questions remain relevant to end-users .

How might the PDX pathway interact with the function of S4 protein in P. luminescens, and what experimental approaches would best reveal these interactions?

The relationship between the vitamin B6 (PDX) biosynthetic pathway and S4 ribosomal protein function represents a complex but potentially significant intersection in P. luminescens biology:

Theoretical Background:
P. luminescens requires vitamin B6 for pathogenicity, as demonstrated by pdxB mutants showing attenuated virulence against C. elegans and other insects . Given S4's critical role in ribosome assembly and translation regulation, there may be functional interactions between these systems that affect pathogenicity and symbiosis.

Proposed Experimental Approaches:

• Transcriptomic analysis:

  • Compare gene expression profiles between wild-type, pdxB mutants, and S4 partial knockdowns

  • Analyze differential expression patterns during insect infection versus nematode symbiosis

  • Identify potential gene regulatory networks connecting these pathways

• Proteomic interaction studies:

  • Perform immunoprecipitation of S4 protein followed by mass spectrometry

  • Identify PDX pathway enzymes that co-precipitate with S4 or ribosomes

  • Validate interactions using yeast two-hybrid or proximity labeling approaches

• Metabolomic profiling:

  • Quantify vitamin B6 vitamers in wild-type versus S4-depleted conditions

  • Measure translation rates and fidelity under vitamin B6 limitation

  • Assess changes in the bacterial metabolome during host infection

• Genetic interaction analysis:

  • Construct double mutants with partial S4 depletion and pdxB mutations

  • Perform synthetic genetic array analysis to identify genetic interactions

  • Measure epistatic effects on growth and virulence phenotypes

• Structural biology approaches:

  • Investigate potential binding of PLP (pyridoxal 5'-phosphate) to S4 or assembled ribosomes

  • Perform structural studies of ribosomes from PLP-depleted cells

  • Assess changes in rRNA modification patterns in pdxB mutants

A proposed experimental workflow would involve:

• Initial screens for genetic interactions between pdxB and S4

• Transcriptomic/proteomic profiling to identify mechanistic connections

• Focused biochemical studies on specific interactions

• In vivo validation using insect and nematode models

This research direction could reveal how nutritional status affects ribosome function and translation fidelity during P. luminescens life cycle transitions between pathogenicity and symbiosis.

What are the most appropriate controls when studying the effects of recombinant S4 protein on ribosome assembly?

Designing rigorous controls is critical when investigating the effects of recombinant S4 protein on ribosome assembly:

Essential Control Types:

• Negative controls:

  • Buffer-only conditions to establish baseline measurement parameters

  • Irrelevant protein (similar size/charge but non-ribosomal) to control for non-specific effects

  • Heat-denatured S4 protein to control for non-functional protein effects

• Positive controls:

  • Native S4 protein purified from P. luminescens (not recombinant)

  • Well-characterized E. coli S4 protein as reference standard

  • Reconstitution with complete 30S assembly factors to verify system functionality

• Experimental validation controls:

  • Concentration series to establish dose-dependent effects

  • Time-course experiments to capture assembly kinetics

  • Temperature variations to assess thermodynamic parameters

• Specificity controls:

  • S4 mutants with known assembly defects

  • Alternative ribosomal proteins (e.g., S7) to test protein-specific effects

  • Heterologous rRNA to test species-specificity

• Technical validation:

  • Multiple preparation batches of recombinant protein

  • Different purification tags (His, GST, etc.) to control for tag effects

  • Endotoxin removal to eliminate contamination effects

Implementation of these controls should be documented in a systematic experimental matrix to ensure all variables are appropriately controlled across experimental conditions.

How can researchers effectively isolate and characterize protein-protein interactions between S4 and other ribosomal proteins in P. luminescens?

Characterizing protein-protein interactions between S4 and other ribosomal proteins requires a multi-method approach to capture both stable and transient interactions:

Methodological Workflow:

• In vivo crosslinking approaches:

  • Formaldehyde crosslinking followed by immunoprecipitation

  • Photo-activatable crosslinkers for increased specificity

  • In vivo biotinylation using proximity-dependent methods (BioID, APEX)

  Protocol focus: Optimize crosslinker concentration (0.1-1%) and reaction time (5-15 minutes) to capture physiologically relevant interactions without artificial aggregation.

• Affinity purification strategies:

  • Tandem affinity purification using dual-tagged S4

  • Quantitative SILAC to distinguish specific from non-specific interactions

  • On-bead digestion to minimize contamination

  Statistical validation: Compare prey abundance across at least three biological replicates versus controls to calculate significance scores.

• Structural characterization methods:

  • Hydrogen-deuterium exchange mass spectrometry to map interaction surfaces

  • Cryo-electron microscopy of assembly intermediates

  • Integrative modeling combining different structural data

• Functional validation:

  • Mutational analysis of predicted interaction interfaces

  • In vitro reconstitution with purified components

  • Complementation assays in S4-depleted backgrounds

• Data analysis and integration:

  • Apply appropriate statistical filters (p < 0.05, fold change > 2)

  • Generate interaction network maps

  • Cross-reference with existing ribosomal assembly models

This comprehensive approach should yield a high-confidence interaction map of S4 with other ribosomal proteins, providing insight into P. luminescens-specific aspects of ribosome assembly.

What experimental design would be most appropriate for investigating how environmental factors affect the expression and function of S4 protein during P. luminescens lifecycle transitions?

Investigating environmental effects on S4 protein during P. luminescens lifecycle transitions requires a factorial design approach to capture complex interactions:

Proposed Randomized Block Design (RBD):

• Environmental factors (treatments):

  • Temperature (15°C, 25°C, 37°C)

  • pH (5.5, 7.0, 8.5)

  • Nutrient availability (minimal, intermediate, rich media)

  • Oxygen levels (anaerobic, microaerobic, aerobic)

  • Host-derived signals (insect hemolymph, nematode extracts, none)

• Lifecycle stages (blocks):

  • Free-living bacteria

  • Nematode colonization phase

  • Early insect infection

  • Late insect infection

• Measured outcomes:

  • S4 protein expression levels (quantitative Western blot)

  • S4 localization (fluorescent microscopy)

  • Global translation rates (35S-methionine incorporation)

  • Ribosome profile analysis (polysome profiling)

  • S4-dependent gene expression (RT-qPCR of target genes)

• Experimental implementation:

  • Each treatment combination tested across all blocks

  • Minimum of 4 biological replicates per condition

  • Randomization within blocks to control for batch effects

  • Include appropriate controls for each block and treatment

• Statistical analysis:

  • Two-way ANOVA with interaction terms

  • Post-hoc comparisons with appropriate correction for multiple testing

  • Multivariate analysis to identify patterns across outcome measures

This RBD approach removes block-to-block variation from the experimental error, making it more sensitive in detecting treatment effects than a completely randomized design . The factorial nature also enables identification of interaction effects between environmental factors, which is crucial for understanding the complex regulation of S4 during lifecycle transitions.

What are the most common technical challenges when purifying recombinant P. luminescens S4 protein, and how can they be addressed?

Purification of recombinant P. luminescens S4 protein presents several technical challenges that require specific troubleshooting approaches:

Challenge 1: Poor expression levels

• Solution: Optimize codon usage for expression host; test multiple expression strains (BL21(DE3), Rosetta, Arctic Express); evaluate different promoters (T7, tac, araBAD)

• Technical details: Expression at lower temperatures (16-25°C) often increases yield of correctly folded protein

Challenge 2: Protein insolubility/inclusion bodies

• Solution: Reduce induction temperature to 18°C; decrease IPTG concentration to 0.1-0.3 mM; co-express with chaperones (GroEL/ES, DnaK)

• Protocol adjustment: Add 5-10% glycerol and 0.1% Triton X-100 to lysis buffer to improve solubility

Challenge 3: Co-purification of RNA

• Solution: Include high salt wash steps (500 mM-1 M NaCl); treat with RNase A before purification; add polyethyleneimine precipitation step

• Quality control: Monitor A260/A280 ratio to verify RNA removal (target ratio ~0.6-0.8)

Challenge 4: Protein instability during purification

• Solution: Include protease inhibitors in all buffers; maintain samples at 4°C; add reducing agents (5 mM DTT or 2 mM β-mercaptoethanol)

• Storage conditions: Add 10% glycerol to final preparation; flash-freeze in small aliquots; avoid repeated freeze-thaw cycles

Challenge 5: Low purity

• Solution: Implement multi-step purification strategy (IMAC followed by ion exchange and gel filtration); optimize imidazole concentrations for IMAC

• Target specifications: Final purity should exceed 85% as determined by SDS-PAGE, consistent with commercial standards

Decision flow chart for troubleshooting:

• Assess expression level by SDS-PAGE → If low, optimize expression conditions

• Check solubility in lysis buffer → If insoluble, adjust buffer composition and lysis method

• Evaluate initial IMAC purification → If co-purifying contaminants present, add secondary purification steps

• Measure A260/A280 ratio → If >1.0, implement RNA removal strategies

• Test protein activity → If low activity despite high purity, evaluate buffer conditions and storage

This systematic approach addresses the most common challenges while maintaining the goal of >85% purity for downstream applications.

How can researchers accurately quantify and assess the functional activity of purified recombinant S4 protein?

Comprehensive functional assessment of purified recombinant S4 protein requires multiple complementary assays:

Quantification Methods:

• Protein concentration determination:

  • Bradford assay: Linear range 0.1-1.4 mg/ml, minimize interference by using BSA standard curve

  • BCA assay: Linear range 0.02-2 mg/ml, more compatible with detergents

  • A280 measurement: Calculate extinction coefficient based on amino acid composition

• Purity assessment:

  • SDS-PAGE: Target >85% purity as determined by densitometry

  • Size-exclusion chromatography: Monitor for aggregates and degradation products

  • Mass spectrometry: Confirm exact mass and sequence coverage

Functional Activity Assays:

• RNA binding assays:

  • Electrophoretic mobility shift assay (EMSA): Titrate protein against labeled 16S rRNA fragments

  • Filter binding assay: Determine binding constants (typical Kd ~10-100 nM for specific binding)

  • Fluorescence polarization: Real-time binding kinetics with fluorescently labeled RNA

• Ribosome assembly assays:

  • In vitro reconstitution: Monitor 30S subunit assembly with purified components

  • Sucrose gradient analysis: Quantify formation of 30S particles

  • Light scattering: Real-time assembly kinetics

• Translational function tests:

  • In vitro translation: Measure protein synthesis rates in reconstituted systems

  • Translational fidelity: Assess error rates using reporter constructs

  • Anti-termination activity: Measure effect on transcription termination efficiency

• Thermal stability assessment:

  • Differential scanning fluorimetry: Determine melting temperature (Tm)

  • Circular dichroism: Monitor secondary structure stability

  • Limited proteolysis: Identify stable domains and flexible regions

Data integration and quality standards:

• Calculate specific activity (activity units per mg protein)

• Compare to reference standards (e.g., E. coli S4)

• Establish minimum performance criteria for each assay

• Document batch-to-batch variation

This multi-parameter approach provides comprehensive characterization of both quantity and functional quality of the purified S4 protein, ensuring reliability for downstream applications.

What are the key considerations when designing primers for cloning the P. luminescens rpsD gene, and how should sequencing validation be approached?

Designing effective primers and validation strategies for cloning the P. luminescens rpsD gene requires careful consideration of multiple factors:

Primer Design Considerations:

• Sequence verification sources:

  • Use complete genome sequences of P. luminescens subsp. laumondii

  • Verify gene boundaries using multiple database annotations

  • Check for strain-specific variations in the rpsD sequence

• Core primer design parameters:

  • Length: 18-30 nucleotides for gene-specific region

  • GC content: Target 40-60% for stable annealing

  • Tm matching: Design primer pairs with Tm within 3°C of each other (typically 58-62°C)

  • 3' stability: Ensure 1-2 G/C bases at 3' end for extension efficiency

  • Secondary structure: Avoid hairpins (ΔG > -3 kcal/mol) and self-complementarity

• Cloning-specific modifications:

  • Add appropriate restriction sites with 4-6 base 5' overhangs

  • Include Kozak sequence if needed for expression

  • Consider adding protease cleavage sites for tag removal

  • Optimize codon usage for expression host if synthesizing gene