Recombinant Pseudomonas putida 30S ribosomal protein S3 (rpsC)

What is the structure and function of 30S ribosomal protein S3 in Pseudomonas putida?

The 30S ribosomal protein S3 in P. putida S16 is a small ribosomal protein encoded by the PPS_0456 locus, located on the positive strand of the chromosome at position 541957-542643 . It is characterized by:

Functionally, S3 is essential for ribosome assembly and protein synthesis, forming part of the 30S ribosomal subunit. Structural evidence from electron microscopy studies of related Pseudomonas species indicates high conservation, with P. aeruginosa S3 showing 95-96% identity to P. putida S3 .

How does P. putida ribosomal protein S3 compare with homologous proteins in other bacterial species?

P. putida S3 shares significant sequence homology with other bacterial S3 proteins, particularly those from related Pseudomonas species. Electron microscopy studies reveal that P. putida S3 is structurally similar to P. aeruginosa S3, with approximately 96.1% sequence identity . This high conservation reflects the essential nature of this protein in ribosomal function.

The protein falls within the UniRef90_A4XZ84 and UniRef50_A0LIJ6 clusters, indicating substantial similarity with S3 proteins across bacterial species . Comparative analysis methodology typically involves:

• Multiple sequence alignment using MUSCLE or CLUSTAL

• Phylogenetic analysis using maximum likelihood methods

• Structural comparison using homology modeling and superimposition

• Functional domain conservation analysis

What genomic context surrounds the rpsC gene in P. putida?

The rpsC gene in P. putida S16 (PPS_0456) is positioned within the ribosomal protein operon, characteristic of bacterial genomes. To analyze this genomic context:

• Utilize genome browsers specific for P. putida strains

• Perform promoter analysis using BPROM or similar software

• Identify transcription factor binding sites using MEME Suite

• Characterize operon structure using RNA-seq data

The gene is located on the positive strand at chromosome position 541957-542643, within the context of other ribosomal protein genes . This clustering facilitates coordinated expression of ribosomal components, essential for maintaining proper stoichiometry during ribosome assembly.

What expression systems are optimal for recombinant production of P. putida 30S ribosomal protein S3?

For recombinant expression of P. putida S3 protein, consider the following expression systems based on their advantages:

Methodology recommendations:

• Clone the PPS_0456 gene with its native ribosome binding site

• Use inducible promoters (T7 or Pm/XylS systems) for controlled expression

• Include a C-terminal His-tag for purification while minimizing functional interference

• Express at lower temperatures (16-25°C) to enhance proper folding

• Supplement growth media with essential cofactors if needed

P. putida itself has emerged as an excellent platform for recombinant protein production, offering advantages in certain applications compared to common hosts like E. coli .

How can codon optimization improve recombinant expression of P. putida rpsC?

Codon optimization strategies for P. putida rpsC expression should be tailored to the chosen host organism:

• Analyze codon usage bias in the target expression host using the Codon Adaptation Index (CAI)

• Eliminate rare codons that may cause translational pausing

• Adjust GC content to match the expression host (particularly important as P. putida has a high GC content)

• Remove potential RNA secondary structures in the 5' region

• Eliminate internal Shine-Dalgarno-like sequences that may cause translational stalling

When expressing in E. coli, optimize for E. coli codon preferences while maintaining secondary structure elements critical for proper folding. For expression in other Pseudomonas species, less extensive optimization may be needed due to similar codon preferences.

What purification strategies yield highest purity recombinant P. putida S3 protein?

A robust purification strategy for recombinant P. putida S3 protein involves:

• Initial capture:

  • Immobilized metal affinity chromatography (IMAC) using Ni-NTA for His-tagged protein

  • Cation exchange chromatography leveraging the high pI (10.66) of the protein

• Intermediate purification:

  • Heparin affinity chromatography (exploiting the nucleic acid binding properties of S3)

  • Ammonium sulfate fractionation (40-60% saturation range)

• Polishing:

  • Size exclusion chromatography using Superdex 75 or similar matrix

  • Hydroxyapatite chromatography

• Quality control:

  • SDS-PAGE to confirm MW (25.7 kDa)

  • Western blotting with anti-S3 antibodies

  • Mass spectrometry for accurate mass determination and PTM identification

  • Dynamic light scattering for aggregation assessment

To minimize degradation, maintain buffers at pH 7.5-8.5 and include protease inhibitors throughout purification.

What analytical methods best characterize the structural integrity of recombinant P. putida S3?

Multiple complementary techniques should be employed to comprehensively characterize recombinant P. putida S3 structural integrity:

• Spectroscopic methods:

  • Circular dichroism (CD) to assess secondary structure content

  • Fluorescence spectroscopy to evaluate tertiary structure (intrinsic tryptophan fluorescence)

  • FTIR spectroscopy for complementary secondary structure analysis

• Hydrodynamic techniques:

  • Size exclusion chromatography with multi-angle light scattering (SEC-MALS)

  • Analytical ultracentrifugation to determine oligomeric state

  • Dynamic light scattering for polydispersity assessment

• Thermal stability:

  • Differential scanning calorimetry (DSC)

  • Thermal shift assays (DSF/Thermofluor)

• Functional assays:

  • RNA binding assays (electrophoretic mobility shift assays)

  • 30S subunit reconstitution assays

  • Translation efficiency in reconstituted systems

Comparison with structural data from P. aeruginosa S3 protein (96.1% identity) can provide valuable reference points for validation .

How can site-directed mutagenesis of P. putida S3 inform ribosomal assembly mechanisms?

Site-directed mutagenesis of P. putida S3 can elucidate critical residues involved in ribosome assembly and function. Implement the following methodological approach:

• Target selection:

  • Conserved residues identified through multiple sequence alignment

  • Residues at RNA-binding interfaces based on structural homology models

  • Charged residues (given the high positive charge of +17.67 at pH 7)

• Mutagenesis protocol:

  • QuikChange or Q5 site-directed mutagenesis for single mutations

  • Gibson Assembly for multiple simultaneous mutations

  • Golden Gate Assembly for systematic domain swapping

• Functional characterization:

  • In vitro reconstitution assays with purified 30S components

  • In vivo complementation of S3-depleted strains

  • Ribosome profiling to assess translation efficiency

• Structural impact assessment:

  • Hydrogen-deuterium exchange mass spectrometry

  • Cryo-EM of reconstituted ribosomes containing mutant S3

  • SHAPE-MaP RNA structure probing of associated rRNA regions

How does P. putida S3 contribute to antibiotic resistance mechanisms?

To investigate the role of P. putida S3 in antibiotic resistance:

• Experimental design:

  • Generate strains with mutations in conserved S3 residues

  • Create strains with modified S3 expression levels

  • Develop S3-rRNA crosslinking assays

• Resistance profiling:

  • Minimum inhibitory concentration (MIC) determination against various antibiotics

  • Time-kill kinetics with ribosome-targeting antibiotics

  • Ribosome binding studies with labeled antibiotics

• Mechanistic investigations:

  • Ribosome footprinting in the presence of antibiotics

  • Cryo-EM structural studies of the ribosome-antibiotic complex

  • Molecular dynamics simulations of S3-antibiotic interactions

Historical context: Mutations in ribosomal proteins, including S12 in P. putida (which has been shown to confer streptomycin resistance through the K43T mutation), demonstrate how alterations in ribosomal components can affect antibiotic susceptibility .

What role does P. putida S3 play in stress response during adaptive laboratory evolution?

Investigate the role of S3 in stress response during adaptive laboratory evolution using this methodology:

• Experimental design:

  • Utilize automated DIY frameworks for P. putida evolution experiments

  • Implement semi-continuous log-phase bioreactor systems with anti-biofilm measures

  • Apply selection pressure through various stressors (antibiotics, temperature, pH)

• Genomic analysis:

  • Whole genome sequencing of evolved strains

  • Targeted sequencing of the rpsC gene region

  • Transcriptomic analysis under stress conditions

• Comparative framework:

  • Monitor for mutations similar to those observed in RNA polymerase genes (rpoC) during P. putida evolution

  • Investigate potential epistatic interactions between S3 and RNA polymerase mutations

  • Analyze fitness effects through competition assays

RNA polymerase mutations have been identified as significant adaptations during laboratory evolution of P. putida strains, suggesting potential interplay between transcription and translation machinery under stress .

How can CRISPR-Cas9 genome editing be optimized for modification of the rpsC gene in P. putida?

For CRISPR-Cas9 editing of the P. putida rpsC gene, implement this methodological framework:

• sgRNA design considerations:

  • Select target sites with minimal off-target effects using algorithms specific for P. putida's genome

  • Avoid targeting regions with secondary structures that may impede Cas9 access

  • Design sgRNAs with appropriate PAM sequences for the chosen Cas9 variant

• Delivery method optimization:

  • Electroporation of ribonucleoprotein (RNP) complexes

  • Plasmid-based delivery with inducible Cas9 expression

  • Conjugation-based transfer of CRISPR components

• Repair template design:

  • Include ~1 kb homology arms flanking the edit site

  • Introduce silent mutations in the PAM or seed region to prevent re-cutting

  • Consider codon optimization while maintaining RNA structural elements

• Screening strategies:

  • HRMA (High Resolution Melt Analysis) for mutation detection

  • Sanger sequencing of PCR amplicons spanning the target region

  • Phenotypic screening if edits confer selectable traits

• Validation approaches:

  • RT-qPCR to confirm transcript expression

  • Western blotting to verify protein production

  • Ribosome profiling to assess functional integration

What methodologies enable structural studies of recombinant P. putida S3 protein?

For structural characterization of recombinant P. putida S3:

• X-ray crystallography approach:

  • Screen various buffer conditions (pH 7.0-8.5) with different precipitants

  • Test both free S3 and S3 bound to RNA fragments

  • Implement surface entropy reduction mutations to promote crystal contacts

  • Use microseed matrix screening to improve crystal quality

• Cryo-EM methodology:

  • Reconstitute S3 with other 30S components for ribosomal complex studies

  • Utilize Vitrobot or similar plunge-freezing devices

  • Implement motion correction and CTF estimation

  • Apply 3D classification to sort heterogeneous conformations

• NMR spectroscopy strategy:

  • Express isotopically labeled protein (13C, 15N) in minimal media

  • Record HSQC spectra to assess structural integrity

  • Collect triple-resonance spectra for backbone assignment

  • Analyze chemical shift perturbations upon RNA binding

Existing structural data from P. aeruginosa ribosomes, showing high sequence identity (95-96%) to P. putida S3, provides valuable reference models for molecular replacement or comparative modeling approaches .