Recombinant Vibrio vulnificus 30S ribosomal protein S4 (rpsD)

What is the role of 30S ribosomal protein S4 in Vibrio vulnificus?

The 30S ribosomal protein S4 (rpsD) in Vibrio vulnificus plays multiple critical roles in ribosome assembly and function. It serves as a primary binding protein that initiates the assembly of the 30S ribosomal subunit by binding directly to 16S rRNA. This protein acts as an RNA chaperone, ensuring proper folding of ribosomal RNA during assembly. Additionally, S4 contributes to translational fidelity by participating in mRNA decoding and helps maintain the structural integrity of the small ribosomal subunit. Similar to other bacterial species, V. vulnificus S4 likely functions in translation regulation and may participate in various stress responses .

How does the structure of V. vulnificus S4 compare to that of other bacterial species?

V. vulnificus S4 shares structural similarities with other bacterial ribosomal S4 proteins, particularly with those from related Vibrio species and E. coli. The protein typically features an N-terminal domain containing multiple alpha-helices involved in RNA binding and a C-terminal domain with a mixed alpha/beta structure. Based on comparative analysis with E. coli S4, the V. vulnificus variant likely maintains the characteristic fold while exhibiting sequence variations that reflect its adaptation to the V. vulnificus cellular environment. The E. coli S4 protein spans amino acids 2-206 with a specific sequence pattern that contributes to its RNA binding properties . Structural studies of other V. vulnificus ribosomal proteins, such as S1, have revealed unique domain architectures that play specific roles in mRNA binding and RNA chaperoning activities .

What expression systems are typically used for producing recombinant V. vulnificus ribosomal proteins?

Recombinant V. vulnificus ribosomal proteins, including S4, are commonly expressed using prokaryotic expression systems, with E. coli being the predominant host. The selection of an appropriate expression vector is crucial for achieving optimal protein yield and solubility. Based on experiences with other recombinant proteins, pET vectors (particularly pET-28a and pET-SUMO) often provide better results than pAE vectors for ribosomal protein expression. The pET-SUMO vector system has been demonstrated to overcome common problems of low expression and poor solubility in recombinant protein production by adding a SUMO tag at the N-terminal of the target protein . The expression conditions typically involve culture optimization at lower temperatures (16-25°C) to enhance protein solubility and proper folding .

What purification strategies work best for recombinant V. vulnificus S4 protein?

The most effective purification strategy for recombinant V. vulnificus S4 protein typically involves a multi-step approach. Based on successful purification of other recombinant proteins, the recommended method includes:

• Affinity chromatography using nickel or cobalt resin (for His-tagged constructs)

• Protein tag cleavage (if using systems like SUMO-fusion)

• Buffer exchange to remove imidazole and other contaminants

• Secondary purification steps such as ion-exchange or size-exclusion chromatography

This approach has shown success in producing highly pure and soluble recombinant proteins from Vibrio species and other bacteria. The single-step purification method involving affinity chromatography, protein cleavage, buffer exchange, and protein elution has been particularly effective for obtaining soluble proteins with preserved biological activity .

How can researchers overcome solubility issues when expressing recombinant V. vulnificus S4 protein?

Overcoming solubility challenges when expressing recombinant V. vulnificus S4 protein requires a multi-faceted approach. Based on similar challenges encountered with other recombinant proteins, researchers should consider:

• Fusion tag selection: The SUMO fusion system has demonstrated particular effectiveness in improving protein solubility. For example, when expressing LipL21 (an outer membrane protein), using the pET-SUMO vector significantly improved solubility compared to pET-28a and pAE vectors .

• Expression temperature optimization: Lowering the expression temperature to 16-20°C often reduces inclusion body formation by slowing protein synthesis and allowing proper folding.

• Media and induction conditions: Using enriched media (e.g., TB or 2xYT) and lower IPTG concentrations (0.1-0.5 mM) can improve soluble protein yield.

• Co-expression with molecular chaperones: Systems incorporating GroEL/GroES or DnaK/DnaJ/GrpE chaperones can assist with proper protein folding.

• Buffer optimization: The addition of specific stabilizing agents (e.g., arginine, trehalose, or specific salt concentrations) during lysis and purification can maintain protein solubility.

The methodological approach should be systematic, testing each parameter individually while monitoring expression levels and solubility through SDS-PAGE and Western blot analysis .

What are the key considerations for designing structural studies of V. vulnificus S4 protein?

Designing effective structural studies for V. vulnificus S4 protein requires careful consideration of several factors:

• Sample preparation: Based on successful NMR studies of other V. vulnificus ribosomal proteins (e.g., S1 domains), researchers should prepare uniformly labeled (15N, 13C) protein samples at concentrations of 0.5-1.0 mM in buffers optimized for structural stability .

• Methodology selection: While X-ray crystallography provides high-resolution structures, NMR spectroscopy has been successfully applied to other V. vulnificus ribosomal proteins and offers insights into dynamics and RNA interactions. For example, NMR has been used to determine the solution structure of the minimal mRNA-binding fragment of V. vulnificus S1 protein .

• Construct design: Consider analyzing both full-length S4 and specific functional domains separately. Domain boundary determination based on sequence analysis and limited proteolysis is crucial.

• RNA-binding studies: Design experiments to characterize S4-RNA interactions using techniques such as EMSA, filter-binding assays, and NMR titration experiments.

• Comparative analysis: Integrate structural data with existing structures from related organisms (e.g., E. coli S4) to identify unique features of V. vulnificus S4.

The approach should be iterative, with initial low-resolution structural characterization guiding more detailed studies of specific protein domains or complexes .

How does the sequence and function of rpsD correlate with V. vulnificus virulence and adaptation?

The relationship between rpsD (S4 protein) and V. vulnificus virulence represents a complex interplay between ribosomal function and pathogenicity. Several lines of evidence suggest important connections:

• Transcriptional regulation: Similar to the regulation observed with rpoS in V. vulnificus, rpsD expression may be modulated by environmental signals relevant to pathogenesis. The rpoS gene in V. vulnificus is regulated through two distinct transcriptional initiation sites (proximal and distal promoters), with expression levels inversely correlated with intracellular cAMP levels . Such sophisticated regulatory mechanisms might also apply to rpsD, influencing its expression during infection.

• Genomic context: V. vulnificus isolates exhibit diverse virulence factors, including outer membrane components, flagella, RTX toxins, and various secretion systems. Recent genomic analyses have identified specialized secretion systems (T6SS1) in specific phylogenetic lineages associated with increased numbers of genomic islands and virulence factors . The genomic context of rpsD and its co-expression patterns with these virulence factors could provide insights into its role in pathogenicity.

• Adaptive evolution: Multilocus sequence analysis of Vibrio species has revealed significant evolutionary relationships within the central clade. While studies have focused on genes like 16S rRNA, recA, pyrH, rpoD, gyrB, rctB, and toxR , extending such analyses to include rpsD could elucidate its evolutionary history and potential adaptive significance.

• Stress response: As ribosomal proteins often participate in stress responses, rpsD may contribute to V. vulnificus adaptation to host environments through mechanisms similar to those observed with RpoS, which confers resistance against various stresses and regulates virulence factor expression .

Investigating these connections requires integrated genomic, transcriptomic, and functional approaches to map the role of rpsD in V. vulnificus pathogenicity networks .

What experimental approaches can be used to characterize the RNA-binding properties of V. vulnificus S4?

Characterizing the RNA-binding properties of V. vulnificus S4 requires a multi-technique approach focusing on both qualitative and quantitative aspects of RNA-protein interactions:

• Electrophoretic Mobility Shift Assays (EMSA): This fundamental technique can establish the basic binding capability of S4 to various RNA substrates, including 16S rRNA fragments and potential mRNA targets. Careful titration of protein concentrations against fixed RNA amounts enables determination of apparent Kd values.

• Filter-binding assays: These provide a quantitative measurement of binding affinity between S4 and radiolabeled RNA substrates, complementing EMSA data with more precise Kd calculations.

• Fluorescence-based approaches: Techniques such as fluorescence anisotropy or FRET can monitor binding interactions in solution, providing real-time kinetic data on association and dissociation rates.

• NMR spectroscopy: Drawing from approaches used to study other V. vulnificus ribosomal proteins, NMR can map specific residues involved in RNA binding. For example, NMR has been successfully applied to characterize the minimal mRNA-binding fragment of V. vulnificus S1 protein, revealing key interaction interfaces .

• Cross-linking coupled with mass spectrometry: This approach can identify specific amino acid residues that directly contact RNA, providing detailed molecular insights into the binding interface.

• Mutagenesis studies: Systematic mutation of conserved residues predicted to be involved in RNA binding, followed by functional assays, can validate the importance of specific amino acids for RNA recognition.

The data obtained should be integrated to develop a comprehensive model of S4-RNA interaction dynamics, comparing the findings with known characteristics of S4 proteins from E. coli and other bacterial species .

How can transcriptional regulation of V. vulnificus rpsD be studied in relation to environmental stressors?

Investigating the transcriptional regulation of V. vulnificus rpsD in response to environmental stressors requires a systematic approach integrating molecular genetics, transcriptomics, and biochemical analyses:

• Promoter mapping: Similar to studies on V. vulnificus rpoS, which identified two distinct transcriptional initiation sites (Pp and Pd) , primer extension experiments can define the transcriptional start sites of rpsD. This mapping would form the foundation for understanding transcriptional regulation.

• Reporter fusion constructs: Developing rpsD::luxAB transcriptional fusions can quantitatively measure promoter activity under various environmental conditions (temperature, pH, osmolarity, nutrient limitation) that mimic different stages of infection or environmental survival.

• Regulatory protein identification: Based on insights from rpoS regulation, where the cAMP-CRP complex acts as a direct repressor , chromatin immunoprecipitation (ChIP) followed by sequencing can identify potential transcription factors that bind the rpsD promoter region.

• In vitro transcription assays: Reconstituted systems using purified RNA polymerase, potential regulatory proteins (e.g., CRP), and rpsD promoter templates can directly assess transcriptional regulation mechanisms.

• Global regulon analysis: RNA-seq comparing wild-type and regulatory mutant strains under different stress conditions can position rpsD within broader regulatory networks.

The experimental design should include the following key variables:

This integrated approach would reveal how V. vulnificus modulates rpsD expression during pathogenesis and environmental adaptation, potentially uncovering new regulatory mechanisms that could be targeted for therapeutic intervention .

What are the optimal expression conditions for producing recombinant V. vulnificus S4 in E. coli?

Achieving optimal expression of recombinant V. vulnificus S4 protein in E. coli requires careful optimization of multiple parameters:

• Expression vector selection: Comparative analysis with other recombinant proteins suggests that pET-SUMO vectors often yield better results than pAE or pET-28a vectors for challenging proteins. The SUMO fusion tag has been shown to significantly improve protein solubility and expression levels .

• Host strain selection: BL21(DE3) and its derivatives (Rosetta, Arctic Express, or SHuffle) should be evaluated, with Rosetta strains being particularly suitable if V. vulnificus S4 contains rare codons that may limit expression in standard E. coli strains.

• Temperature optimization: A systematic comparison of expression at 37°C, 30°C, 25°C, and 16°C is essential, with lower temperatures often favoring soluble protein production. For instance, studies with other recombinant proteins have shown that reduced temperature can significantly decrease inclusion body formation .

• Induction parameters: The optimal combination of IPTG concentration (0.1-1.0 mM) and induction duration (3-24 hours) should be established through small-scale expression trials evaluated by SDS-PAGE analysis.

• Media composition: Rich media (LB, TB, 2xYT) versus minimal media supplemented with specific nutrients should be compared for their effects on protein yield and solubility.

The recommended protocol based on successful expression of other recombinant proteins includes:

• Transformation into BL21(DE3) or Rosetta(DE3) cells

• Culture growth in TB medium at 37°C until OD600 reaches 0.6-0.8

• Temperature reduction to 18-20°C

• Induction with 0.5 mM IPTG

• Continued growth for 16-18 hours

• Cell harvesting and lysis under native conditions

• Initial assessment of protein expression and solubility by SDS-PAGE and Western blot analysis

What analytical methods are most effective for assessing the structural integrity of purified V. vulnificus S4?

Comprehensive assessment of the structural integrity of purified V. vulnificus S4 protein requires a multi-technique approach:

• Circular Dichroism (CD) spectroscopy: This provides a rapid evaluation of secondary structure content and thermal stability. Far-UV CD spectra (190-260 nm) can determine the proportion of alpha-helices and beta-sheets, which should be compared to predicted structural elements based on homology with E. coli S4 .

• Fluorescence spectroscopy: Intrinsic tryptophan fluorescence measurements can assess tertiary structure integrity and conformational changes under different conditions (pH, temperature, ionic strength).

• Size-Exclusion Chromatography coupled with Multi-Angle Light Scattering (SEC-MALS): This provides information about the oligomeric state and homogeneity of the purified protein, revealing potential aggregation or degradation.

• Nuclear Magnetic Resonance (NMR) spectroscopy: 1H-15N HSQC spectra can serve as structural fingerprints to confirm proper folding. More detailed NMR analyses have been successfully applied to other V. vulnificus ribosomal proteins to determine solution structures .

• Limited proteolysis: Digestion with proteases (trypsin, chymotrypsin) followed by mass spectrometry analysis can identify stable domains and flexible regions, providing insights into the structural organization.

• Thermal shift assays (Thermofluor): These can assess protein stability under various buffer conditions, guiding optimization of storage conditions.

• Functional assays: RNA-binding assays using model substrates can confirm that the purified protein retains its biological activity, which is the ultimate measure of structural integrity.

The integration of these complementary approaches provides a comprehensive assessment of both the global folding and specific structural features of the recombinant S4 protein .

How can researchers effectively design site-directed mutagenesis experiments to study critical residues in V. vulnificus S4?

Designing effective site-directed mutagenesis experiments for V. vulnificus S4 protein research requires a strategic approach based on structural and functional insights:

• Target residue identification: Begin with sequence alignment of V. vulnificus S4 with well-characterized S4 proteins from E. coli and other bacteria to identify:

  • Highly conserved residues across bacterial species

  • Residues unique to Vibrio species that may confer specialized functions

  • Known RNA-binding residues based on structural studies of E. coli S4

  • Residues involved in protein-protein interactions within the ribosome

• Mutation strategy:

  • Conservative substitutions (e.g., Lys→Arg) to test the importance of specific chemical properties

  • Non-conservative substitutions (e.g., Lys→Ala) to completely abolish side-chain functionality

  • Charge-reversal mutations to test electrostatic interactions (e.g., Lys→Glu)

• Experimental design matrix:

• Mutagenesis method selection: Overlap extension PCR or commercial site-directed mutagenesis kits (e.g., QuikChange) are recommended for generating the desired mutations.

• Validation approach:

  • Confirm mutations by DNA sequencing

  • Verify expression and purification using the same protocols established for wild-type protein

  • Perform structural integrity checks using CD spectroscopy and thermal stability assays

  • Apply functional assays specific to the predicted role of the mutated residue

The systematic analysis of mutant phenotypes will provide insights into structure-function relationships and potentially identify novel regulatory mechanisms specific to V. vulnificus S4 .

How can V. vulnificus S4 protein be used in comparative studies of ribosome structure and function among Vibrio species?

V. vulnificus S4 protein represents a valuable tool for comparative studies of ribosome structure and function across Vibrio species, offering insights into both conserved ribosomal mechanisms and species-specific adaptations:

• Evolutionary analysis: Integration of rpsD sequences into multilocus sequence analysis (MLSA) frameworks that have previously utilized genes such as 16S rRNA, recA, pyrH, rpoD, gyrB, rctB, and toxR can provide a more comprehensive evolutionary picture of the Vibrio genus . Such analyses can reveal selection pressures on different ribosomal components across ecological niches.

• Structural comparisons: Recombinant S4 proteins from different Vibrio species can be studied using NMR spectroscopy or X-ray crystallography to identify structural variations that may correlate with ecological adaptations. Similar approaches have been successfully applied to characterize the domain architecture of V. vulnificus S1 protein .

• Functional conservation testing: Cross-species complementation experiments, where the rpsD gene from one Vibrio species is expressed in another species with its native rpsD deleted or conditionally repressed, can assess functional conservation and identify species-specific roles.

• Ribosome assembly comparative studies: In vitro reconstitution experiments using S4 proteins from different Vibrio species can reveal variations in assembly pathways, kinetics, and temperature dependencies that may reflect adaptation to different environmental conditions.

• Translational fidelity analysis: Systems comparing translational error rates in ribosomes containing S4 proteins from different Vibrio species can identify variations in decoding accuracy that may contribute to stress adaptation.

The methodological approach should include:

• Construction of a comprehensive phylogenetic framework based on rpsD sequences

• Production of recombinant S4 proteins from multiple Vibrio species using standardized expression and purification protocols

• Parallel structural and functional characterization

• Correlation of molecular differences with ecological and pathogenic characteristics of each species

This comparative approach would significantly advance our understanding of ribosomal adaptation in the Vibrio genus and potentially identify novel targets for species-specific antimicrobial development .

What approaches can be used to study interactions between V. vulnificus S4 and other ribosomal components?

Investigating the interactions between V. vulnificus S4 and other ribosomal components requires a multi-faceted approach combining biochemical, biophysical, and structural methods:

• Co-purification studies: Pull-down assays using tagged recombinant S4 as bait can identify interacting ribosomal proteins and rRNA fragments from V. vulnificus lysates. Mass spectrometry analysis of co-purified components can reveal the composition of S4-associated complexes.

• Yeast two-hybrid or bacterial two-hybrid screening: These methods can systematically identify protein-protein interactions between S4 and other ribosomal proteins, potentially revealing interactions unique to V. vulnificus compared to model organisms.

• Chemical cross-linking coupled with mass spectrometry (XL-MS): This approach can capture transient interactions and identify specific residues involved in protein-protein contacts within the ribosomal context.

• Reconstitution of partial ribosomal complexes: In vitro assembly of defined subassemblies containing S4 and selected rRNA domains or proteins can elucidate the hierarchy and cooperativity of interactions during ribosome assembly.

• Cryo-electron microscopy: While challenging, this technique could potentially resolve the structure of V. vulnificus ribosomes, allowing direct visualization of S4 interactions within the native context.

• Hydroxyl radical footprinting: This technique can map the RNA binding sites of S4 on 16S rRNA with nucleotide resolution, identifying potential differences from E. coli S4-rRNA interactions.

• Surface plasmon resonance (SPR) or isothermal titration calorimetry (ITC): These methods provide quantitative measurements of binding affinities and thermodynamic parameters for S4 interactions with specific ribosomal components.

The integration of these complementary approaches would generate a comprehensive interaction map of V. vulnificus S4 within the ribosome, potentially identifying novel structural or functional features specific to this pathogen .

How might the study of V. vulnificus S4 contribute to understanding antimicrobial resistance mechanisms?

The study of V. vulnificus S4 protein offers unique perspectives on antimicrobial resistance mechanisms, particularly those involving ribosome-targeting antibiotics:

• Structural basis of antibiotic resistance: Comparative structural analysis of V. vulnificus S4 with counterparts from antibiotic-resistant bacteria could reveal amino acid substitutions or conformational differences that contribute to altered antibiotic binding. Techniques such as NMR spectroscopy, which has been successfully applied to other V. vulnificus ribosomal proteins , can provide detailed structural information at the atomic level.

• Antibiotic binding studies: Direct measurement of antibiotic binding to reconstituted ribosomal subunits containing wild-type or mutant V. vulnificus S4 can identify specific contributions of this protein to antibiotic sensitivity or resistance.

• Ribosome heterogeneity investigation: Analysis of potential post-translational modifications of S4 under stress conditions might reveal adaptive mechanisms that contribute to transient antibiotic tolerance without genetic changes.

• Regulatory roles exploration: Similar to the complex regulation observed with rpoS in V. vulnificus , S4 might be subject to sophisticated regulatory mechanisms that influence its expression under antibiotic stress. Investigation of transcriptional and translational regulation of rpsD under antibiotic exposure could reveal novel resistance strategies.

• Evolution of resistance tracking: Integration of rpsD sequences into multilocus sequence analysis frameworks from clinical isolates with varying antibiotic susceptibility profiles could identify evolutionary patterns associated with resistance development.

Experimental approaches should include:

• Site-directed mutagenesis of specific residues predicted to influence antibiotic binding

• In vitro translation assays measuring antibiotic inhibition in systems with wild-type versus mutant S4

• Transcriptomic and proteomic analyses of V. vulnificus under antibiotic stress, focusing on ribosomal protein expression

• Structural studies of S4-containing ribosomal subassemblies in complex with various antibiotics

These studies would contribute to the broader understanding of ribosome-targeted antibiotic action and resistance mechanisms, potentially guiding the development of new antimicrobial strategies specific to Vibrio pathogens .

What are common challenges in expressing and purifying V. vulnificus S4, and how can they be addressed?

Recombinant expression and purification of V. vulnificus S4 protein present several challenges that can be systematically addressed through optimized protocols:

• Low expression levels:

  • Challenge: Insufficient protein production despite confirmed correct sequence

  • Solution: Test multiple expression vectors, as demonstrated in comparative studies where pET-SUMO vectors significantly outperformed pAE vectors for recombinant protein expression

  • Implementation: Compare expression levels using pET-28a, pET-SUMO, and other specialized vectors through small-scale expression trials

• Inclusion body formation:

  • Challenge: Expressed protein aggregates in insoluble fraction

  • Solution: Reduce expression temperature to 16-20°C, as lower temperatures have been shown to improve protein solubility for challenging recombinant proteins

  • Implementation: Conduct parallel expressions at multiple temperatures (37°C, 30°C, 25°C, 18°C) with consistent induction parameters

• Protein instability:

  • Challenge: Purified protein shows degradation or aggregation during storage

  • Solution: Optimize buffer composition through systematic screening of pH, salt concentration, and stabilizing additives

  • Implementation: Perform thermal shift assays to identify optimal buffer conditions that maximize protein stability

• Co-purification of contaminants:

  • Challenge: RNA or other bacterial proteins co-purify with the target protein

  • Solution: Implement sequential purification strategies including both affinity and size-exclusion chromatography

  • Implementation: Add RNase treatment steps and high-salt washes during purification to remove non-specifically bound nucleic acids

• Low yield after tag removal:

  • Challenge: Significant protein loss during protease cleavage of fusion tags

  • Solution: Optimize cleavage conditions and implement on-column cleavage protocols

  • Implementation: Compare different proteases (e.g., TEV, SUMO protease) and reaction conditions to maximize recovery of cleaved protein

The table below summarizes a systematic troubleshooting approach:

This systematic approach has proven successful for improving the expression and purification of challenging recombinant proteins from various bacterial species .

How can researchers distinguish between functional and non-functional recombinant V. vulnificus S4 protein preparations?

Establishing functional validation criteria for recombinant V. vulnificus S4 protein preparations is essential for ensuring meaningful experimental outcomes:

• RNA binding assays:

  • Primary functional test: Electrophoretic mobility shift assays (EMSA) using specific 16S rRNA fragments known to interact with S4

  • Validation criteria: Concentration-dependent binding with Kd values comparable to those reported for E. coli S4 (typically in the nanomolar range)

  • Controls: Heat-denatured S4 protein as negative control; E. coli S4 as positive control if available

• Ribosome incorporation tests:

  • Functional assay: In vitro reconstitution of 30S ribosomal subunits using recombinant S4 and either native or recombinant ribosomal components

  • Validation method: Sucrose gradient centrifugation to confirm incorporation into 30S particles

  • Analysis: Comparison of reconstitution efficiency with native S4 protein

• Translation activity assessment:

  • Functional test: In vitro translation assays using reconstituted ribosomes containing recombinant S4

  • Measurement: Production of reporter protein (e.g., luciferase) as indicator of translation efficiency

  • Benchmarking: Comparison with activity of ribosomes containing native S4

• Structural integrity verification:

  • Biophysical characterization: Circular dichroism (CD) spectroscopy to confirm secondary structure content

  • Thermal stability: Monitoring unfolding transitions using CD or differential scanning fluorimetry

  • Comparison: Correlation with functional activity across different protein preparations

• Binding partner interaction validation:

  • Co-precipitation assays: Pull-down experiments with known S4 interaction partners (other ribosomal proteins)

  • Analytical ultracentrifugation: Characterization of complex formation with specific rRNA fragments

  • Surface plasmon resonance: Quantitative measurement of binding kinetics with interaction partners

Decision matrix for functional validation:

Implementing this comprehensive validation approach ensures that only functionally competent S4 protein preparations are used for subsequent mechanistic studies, improving experimental reliability and reproducibility .