Recombinant Vibrio vulnificus Ribosomal RNA small subunit methyltransferase C (rsmC)

What is the biological function of RsmC in Vibrio vulnificus?

RsmC in Vibrio vulnificus functions as a methyltransferase that catalyzes the transfer of a methyl group from S-adenosyl-L-methionine (SAM) to G1207 of 16S rRNA. This post-transcriptional modification is critical for proper ribosome assembly and function. Mutations affecting G1207 methylation have dominant lethal phenotypes in bacterial models, underscoring the significance of this modification for ribosome function and bacterial viability . Like its homolog in E. coli, the V. vulnificus RsmC likely contains two homologous domains tandemly duplicated within a single polypeptide, with specialized functions for substrate binding and catalysis.

How does RsmC structure relate to its function in pathogenic Vibrio species?

The RsmC protein contains two homologous domains that have undergone subfunctionalization through complementary degeneration of redundant functions. Crystal structure analysis at 2.1 Å resolution reveals that while both domains share structural similarity, they have specialized for different aspects of the methylation reaction . One domain primarily mediates substrate recognition and binding to the 16S rRNA, while the other domain maintains the catalytic function for methyl transfer. This domain specialization represents an evolutionary strategy that enhances the efficiency and specificity of the methylation process. In pathogenic Vibrio species, proper ribosome function supported by RsmC activity is essential for expression of virulence factors and adaptation to host environments.

How is RsmC expression regulated in Vibrio vulnificus during infection?

RsmC expression in V. vulnificus appears to be regulated as part of the pathogen's response to host environments. During infection, V. vulnificus encounters various stresses including changes in temperature, pH, osmolarity, and nutrient availability. Research indicates that stress-sensing protein complexes (stressosomes) respond to environmental cues such as oxygen levels , potentially affecting the expression of ribosome-associated factors including RsmC. Additionally, quorum sensing regulators like SmcR (a LuxR homolog) control the expression of numerous genes during infection . While direct regulation of RsmC by SmcR has not been definitively established, the interconnection between ribosome modification and virulence gene expression suggests coordinated regulation mechanisms that optimize bacterial fitness during host colonization.

What expression systems are most suitable for producing recombinant V. vulnificus RsmC?

For recombinant V. vulnificus RsmC production, E. coli-based expression systems are generally most suitable due to their high yield, ease of genetic manipulation, and cost-effectiveness. The selection criteria should consider the following factors:

For most research applications, BL21(DE3) or Rosetta strains with pET-based vectors containing T7 promoters provide optimal expression levels. Codon optimization may be necessary to address potential rare codon issues between V. vulnificus and E. coli .

What are the critical factors for successful cloning of V. vulnificus rsmC into expression vectors?

Successful cloning of V. vulnificus rsmC requires careful consideration of several critical factors:

• Gene sequence verification: The complete coding sequence should be confirmed by sequencing prior to cloning, with attention to potential strain-specific variations. V. vulnificus strains show significant genetic polymorphism, including in ribosomal genes .

• Vector selection: Choose vectors with appropriate promoters (T7 for high expression), affinity tags (His6, GST, or MBP), and fusion partners that may enhance solubility.

• Restriction site strategy: Design primers with:

  • Compatible restriction sites absent in the gene sequence

  • Appropriate reading frame alignment

  • Kozak sequence or ribosome binding site optimization

  • 6-base overhangs for efficient restriction enzyme digestion

• Codon optimization: While maintaining critical functional regions, optimize codons for E. coli expression, especially if the GC content differs significantly from the expression host.

• Signal sequence consideration: For periplasmic expression, include an appropriate signal sequence (e.g., pelB) to enhance proper folding and reduce inclusion body formation .

Careful PCR conditions with high-fidelity polymerases minimize the risk of mutations during amplification, and sequence verification of the final construct is essential before proceeding to expression.

How can Design of Experiments (DoE) approaches be applied to optimize recombinant V. vulnificus RsmC expression?

DoE approaches provide a systematic framework for optimizing RsmC expression with fewer experiments than traditional one-factor-at-a-time methods. A robust optimization strategy would follow these steps:

• Factor screening using fractional factorial design:

  • Identify 5-7 potential factors influencing expression (temperature, inducer concentration, media composition, etc.)

  • Use a 2^(k-p) design to screen significant factors

  • Analyze main effects to identify critical variables

• Response surface methodology (RSM) for optimization:

  • Apply central composite design (CCD) with the significant factors

  • Develop a polynomial model relating factors to protein yield

  • Identify optimal operating conditions

For RsmC expression, a typical DoE workflow might include:

This approach typically reduces the number of experiments by 30-50% compared to one-factor-at-a-time methods, while revealing interaction effects that might otherwise be missed .

What strategies can enhance the solubility of recombinant V. vulnificus RsmC during expression?

Enhancing solubility of recombinant V. vulnificus RsmC requires multiple complementary strategies:

• Fusion partners: Implementing solubility-enhancing fusion tags such as:

  • MBP (Maltose Binding Protein): Provides up to 60% improvement in solubility

  • SUMO: Enhances folding and can be cleaved without leaving residual amino acids

  • Thioredoxin (Trx): Particularly effective for proteins with multiple cysteines

• Expression conditions optimization:

  • Lower temperatures (16-25°C): Reduces aggregation by slowing protein synthesis

  • Reduced inducer concentration: Lowers expression rate, allowing proper folding

  • Co-expression with chaperones (GroEL/ES, DnaK, trigger factor): Assists proper folding

  • Addition of compatible solutes (sorbitol, glycine betaine): Stabilizes folding intermediates

• Host strain selection:

  • E. coli strains with enhanced folding capacity (SHuffle, Origami)

  • Strains with reduced protease activity (BL21, BL21(DE3)pLysS)

• Periplasmic targeting:

  • Direct protein to periplasmic space using appropriate signal sequences

  • More oxidizing environment supports disulfide bond formation

  • Less crowded environment reduces aggregation tendency

• Media composition modifications:

  • Supplementation with cofactors (S-adenosylmethionine)

  • Addition of osmolytes (5-10% sorbitol, 0.5-1M NaCl)

  • Modified mineral composition to support proper folding

Combining these approaches has shown synergistic effects, with successful case studies demonstrating up to 80% improvement in soluble recombinant protein yield .

What is the most efficient purification strategy for recombinant V. vulnificus RsmC with minimal activity loss?

An efficient purification strategy for recombinant V. vulnificus RsmC should balance purity requirements with preservation of enzymatic activity. Based on the characteristics of methyltransferases, a multi-step approach is recommended:

• Initial capture step: Immobilized metal affinity chromatography (IMAC) using Ni-NTA or Co-based resins for His-tagged RsmC

  • Buffer recommendation: 50 mM Tris-HCl pH 8.0, 300 mM NaCl, 5% glycerol, 5 mM β-mercaptoethanol

  • Inclusion of 1 mM SAM (S-adenosylmethionine) in all buffers helps stabilize the enzyme structure

• Intermediate purification: Ion exchange chromatography

  • Buffer selection based on RsmC theoretical pI (typically pH 7.5)

  • Q-Sepharose (anion exchange) if pI < 7.0 or SP-Sepharose (cation exchange) if pI > 7.0

• Polishing step: Size exclusion chromatography (Superdex 75 or 200)

  • Buffer recommendation: 25 mM HEPES pH 7.5, 150 mM NaCl, 1 mM DTT, 0.5 mM EDTA, 5% glycerol

Throughout purification, incorporate these activity-preserving strategies:

• Maintain temperature at 4°C during all steps

• Include protease inhibitors in initial lysis buffer

• Add reducing agents (1-5 mM DTT or β-mercaptoethanol) to prevent oxidation

• Keep SAM concentration at 0.5-1 mM in all buffers

• Avoid freeze-thaw cycles by aliquoting final product

A typical purification table would show:

This approach typically yields 5-10 mg of highly pure, active enzyme per liter of bacterial culture .

What are the critical quality attributes that should be assessed for recombinant V. vulnificus RsmC?

Comprehensive characterization of recombinant V. vulnificus RsmC should include assessment of the following critical quality attributes:

• Purity and Identity:

  • SDS-PAGE analysis (≥95% purity)

  • Western blot with anti-His or anti-RsmC antibodies

  • Mass spectrometry (intact mass and peptide fingerprinting)

  • N-terminal sequencing to confirm correct processing

• Structural Integrity:

  • Circular dichroism (CD) spectroscopy to assess secondary structure

  • Thermal shift assay (Tm determination) for stability assessment

  • Dynamic light scattering for aggregation state analysis

  • Native PAGE to assess oligomeric state

• Functional Activity:

  • Methyltransferase activity using:

    • Radiometric assay (³H-SAM incorporation into 16S rRNA substrate)

    • Fluorescence-based methyltransferase assays

    • SAH (S-adenosylhomocysteine) detection assays

  • Binding studies:

    • Isothermal titration calorimetry (ITC) for SAM binding parameters

    • RNA electrophoretic mobility shift assay (EMSA) for 16S rRNA binding

• Homogeneity and Stability:

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

  • Accelerated stability studies (activity retention at 4°C, 25°C, 37°C)

  • Freeze-thaw stability (activity retention after multiple freeze-thaw cycles)

Typical specifications would include:

These characterization methods ensure that the recombinant RsmC is functionally equivalent to the native enzyme and suitable for downstream research applications .

How can isothermal titration calorimetry (ITC) be applied to study RsmC interactions with SAM and RNA substrates?

ITC provides a powerful methodology for quantitatively characterizing the thermodynamic parameters of RsmC interactions with its substrates. For effective application to RsmC studies:

• Experimental setup for SAM binding analysis:

  • Sample cell: RsmC protein (20-50 μM) in buffer (typically 50 mM HEPES pH 7.5, 150 mM NaCl)

  • Syringe: SAM solution (200-500 μM) in identical buffer

  • Control experiment: SAM titration into buffer alone (for heat of dilution)

  • Temperature: 25°C with stirring (300-400 rpm)

  • Injection protocol: 0.5 μL initial injection followed by 19-25 injections of 2 μL each

• Setup for RNA substrate binding:

  • Sample cell: RsmC protein (10-20 μM)

  • Syringe: 16S rRNA fragment containing G1207 (100-200 μM)

  • Special considerations: RNA must be properly folded and free of contaminants

  • For cooperative binding studies: Pre-incubate RsmC with SAM before RNA titration

• Data analysis and interpretation:

  • Fit using appropriate binding models (one-site, two-site, or sequential binding)

  • Extract thermodynamic parameters: K₀ (binding constant), ΔH (enthalpy change), ΔS (entropy change)

  • Calculate derived parameters: ΔG (Gibbs free energy) and stoichiometry

Based on studies with E. coli RsmC, expected parameters might include:

The thermodynamic signature can reveal the nature of binding interactions (hydrogen bonding, hydrophobic interactions) and conformational changes upon binding. For RsmC, the specialized functions of the two domains can be distinguished by strategic mutations and comparative ITC analyses .

What mutagenesis approaches can be used to investigate the functional roles of specific residues in V. vulnificus RsmC?

Strategic mutagenesis of V. vulnificus RsmC provides critical insights into structure-function relationships. A comprehensive approach includes:

• Site-directed mutagenesis strategies:

  • Alanine scanning of conserved residues in both domains

  • Conservative substitutions to probe specific interactions (e.g., D→E, K→R)

  • Domain swapping between the two homologous domains

  • Creation of single-domain variants to assess domain independence

• Target selection based on structural information:

  • SAM-binding motif residues (typically G-X-G-X-G motif)

  • Putative catalytic residues (based on homology to other methyltransferases)

  • Domain interface residues (to probe interdomain communication)

  • RNA recognition residues (based on conserved positively charged patches)

• Functional assessment of mutants:

  • Methyltransferase activity assays comparing wild-type vs mutant enzymes

  • Substrate binding studies using ITC or fluorescence spectroscopy

  • Thermal stability analysis to assess structural integrity

  • Crystal structure determination of critical mutants

A systematic mutagenesis approach might include the following targets and expected outcomes:

Based on studies with E. coli RsmC, mutations in the N-terminal domain typically affect catalytic function while C-terminal domain mutations more often impact substrate recognition, revealing the specialized roles each domain adopted through evolutionary subfunctionalization .

How can V. vulnificus RsmC be used as a model system to study domain duplication and subfunctionalization in enzyme evolution?

V. vulnificus RsmC provides an excellent model system for studying domain duplication and subfunctionalization in enzyme evolution due to its distinctive structural organization with two homologous domains that have developed specialized functions. A comprehensive research program would include:

• Comparative genomic analysis:

  • Sequence alignment of RsmC from diverse bacterial species to identify conserved and divergent regions

  • Phylogenetic reconstruction of domain evolution across bacterial lineages

  • Identification of single-domain RsmC homologs as potential evolutionary precursors

• Domain isolation experiments:

  • Expression of individual N and C domains as separate proteins

  • Functional characterization of isolated domains

  • Complementation studies with domain mixtures to assess cooperative function

  • Domain swapping across different bacterial species

• Evolution simulation through directed evolution:

  • Creation of libraries with random mutations in one or both domains

  • Selection for methyltransferase activity under varying conditions

  • Sequencing of successful variants to identify compensatory mutations

  • Tracking of mutational trajectories that lead to further specialization

• Structural dynamics studies:

  • Hydrogen-deuterium exchange mass spectrometry to assess domain flexibility

  • NMR studies of interdomain communication

  • Molecular dynamics simulations of domain movements during catalysis

Research using this approach has revealed fundamental principles of protein evolution:

This research not only illuminates RsmC function but provides broader insights into how protein domains evolve specialized functions while maintaining interdependence - a common theme in protein evolution .

What approaches can be used to identify small molecule inhibitors of V. vulnificus RsmC and evaluate their potential as antimicrobial agents?

Developing small molecule inhibitors of V. vulnificus RsmC requires a multi-faceted approach spanning computational, biochemical, and microbiological methods:

• Target-based virtual screening:

  • Structure-based pharmacophore modeling based on SAM binding pocket

  • Molecular docking of compound libraries against RsmC crystal structure

  • Molecular dynamics simulations to account for protein flexibility

  • Consensus scoring to prioritize compounds for experimental testing

• Biochemical screening cascade:

  • Primary screening: SAH-Glo methyltransferase assay for inhibition potency

  • Secondary assays: ITC for direct binding assessment

  • Counter-screening against human methyltransferases to assess selectivity

  • Mechanism of action studies (competitive vs. allosteric inhibition)

• Structure-activity relationship (SAR) development:

  • Medicinal chemistry optimization of hit compounds

  • X-ray crystallography of RsmC-inhibitor complexes

  • Fragment-based approaches to identify novel chemical scaffolds

• Antimicrobial evaluation:

  • Determination of minimum inhibitory concentration (MIC) against V. vulnificus

  • Activity testing against antibiotic-resistant clinical isolates

  • Assessment of resistance development frequency

  • Cytotoxicity evaluation against human cell lines

Promising compound classes might include:

The most successful RsmC inhibitors would target unique features of the bacterial enzyme not shared with human methyltransferases. Given the essential nature of rRNA methylation for bacterial viability and the rising antibiotic resistance in V. vulnificus, RsmC inhibitors represent a promising avenue for novel antimicrobial development .

How does the methyltransferase activity of RsmC contribute to antibiotic resistance mechanisms in Vibrio vulnificus?

The RsmC methyltransferase contributes to antibiotic resistance through several mechanisms related to ribosome modification and function:

• Direct effects on antibiotic binding sites:

  • Methylation at G1207 in 16S rRNA alters ribosome structure in regions that overlap with binding sites for aminoglycoside antibiotics

  • Structural changes may reduce binding affinity of antibiotics that target the 30S ribosomal subunit

• Impact on translation accuracy and efficiency:

  • RsmC-mediated methylation ensures proper ribosome assembly and function

  • Properly modified ribosomes maintain translation accuracy despite antibiotic stress

  • This contributes to bacterial survival under antibiotic pressure

• Relationship with other resistance mechanisms:

  • V. vulnificus clinical isolates show varying degrees of antibiotic resistance with 66.7% resistant to multiple antibiotics

  • The interplay between ribosome modifications and expression of resistance genes creates complex resistance profiles

  • RsmC activity may influence the expression of other resistance factors

Research findings on the relationship between V. vulnificus rRNA modifications and antibiotic resistance profiles:

Advanced studies suggest that targeting RsmC function could potentially re-sensitize resistant strains to certain antibiotics, particularly those that target the ribosome. This creates potential for combination therapies that target both the resistance mechanisms and the underlying translational machinery .

What role does RsmC play in Vibrio vulnificus virulence, and how might it interact with other known virulence factors?

While direct evidence linking RsmC specifically to V. vulnificus virulence is limited, several lines of evidence suggest important interconnections between ribosomal modification and virulence mechanisms:

• Coordination with translational regulation of virulence factors:

  • Proper ribosome function mediated by RsmC is essential for efficient translation of virulence factor mRNAs

  • Key virulence proteins in V. vulnificus include:

    • RtxA1 toxin: Primary cytotoxin responsible for cellular damage

    • VvhA: Cytolysin/hemolysin with hemolytic activity

    • VvpE: Extracellular protease involved in tissue damage and biofilm detachment

  • Expression timing and levels of these factors depend on optimal translation efficiency

• Relationship with stress response and environmental adaptation:

  • RsmC may contribute to survival under stress conditions encountered during infection

  • Stressosome-mediated responses to oxygen and other stressors likely depend on proper ribosome function

  • Adaptation to host iron limitation, temperature shifts, and immune responses requires coordinated protein synthesis

• Potential interaction with regulatory networks:

  • Quorum sensing regulator SmcR controls numerous virulence factors

  • RsmC-mediated ribosome modification may influence the translation efficiency of regulatory proteins

  • This creates a potential feedback loop between ribosome modification and virulence regulation

The interconnection between RsmC and virulence factors is summarized in this relationship network: