Recombinant Vibrio vulnificus 50S ribosomal protein L18 (rplR)

What is the 50S ribosomal protein L18 (rplR) in Vibrio vulnificus and its primary function?

The 50S ribosomal protein L18 (rplR) is a component of the large subunit (50S) of bacterial ribosomes in V. vulnificus. It plays a crucial role in ribosome assembly and stability, interacting with both ribosomal RNA and other ribosomal proteins. This protein specifically helps facilitate the binding of 5S rRNA to the large ribosomal subunit, making it essential for proper protein synthesis.

The protein is encoded by the rplR gene, which is typically found in ribosomal protein operons in the V. vulnificus genome. Unlike virulence factors such as RTX toxins or capsular polysaccharides that directly contribute to pathogenicity , rplR functions primarily as a housekeeping gene essential for bacterial survival through its role in protein translation.

How does rplR differ between clinical and environmental isolates of V. vulnificus?

Analysis of 21 clinical V. vulnificus isolates from patients in Ningbo, China (2013-2020) revealed conservation patterns in housekeeping genes, including ribosomal proteins . While virulence factors and antibiotic resistance genes show notable variation between isolates, essential ribosomal proteins like rplR typically display high sequence conservation.

This conservation contrasts sharply with the variable presence of virulence-associated genes observed across isolates. For example, clinical isolates consistently exhibited RTX gene clusters (rtxABCD), hemolysins, and various secretion systems , while housekeeping genes showed less variation, reflecting their essential functions.

What expression patterns does rplR exhibit during various growth phases of V. vulnificus?

Similar to other essential cellular components, rplR expression patterns can be compared to those observed in outer membrane proteins like TolCV1 and TolCV2. While TolCV1 expression in V. vulnificus increases time-dependently, TolCV2 shows decreased expression over time . Essential genes like rplR typically show expression patterns regulated by global stress responses, possibly mediated by sigma factors like RpoS, which accumulates during stationary phase .

How can rplR be used as a molecular target for studying V. vulnificus phylogenetic relationships?

The conserved nature of ribosomal proteins makes rplR a valuable molecular marker for phylogenetic analysis of V. vulnificus strains. Unlike variable virulence genes, rplR sequences can help establish core evolutionary relationships between isolates.

Table 1: Comparison of Molecular Markers for V. vulnificus Phylogenetic Analysis

When used in conjunction with variable markers like vcg genotyping (clinical-type vcgC or environment-type vcgE) and 16S rRNA typing (A-type or B-type) , rplR sequences provide complementary data for comprehensive evolutionary analyses of V. vulnificus strains.

What structural modifications occur in rplR during post-translational processing, and how do they affect function?

Post-translational modifications (PTMs) of ribosomal proteins, including rplR, can significantly impact ribosome assembly, stability, and function. Common PTMs in bacterial ribosomal proteins include methylation, acetylation, and phosphorylation.

For recombinant rplR production, researchers must consider whether proper PTMs will occur in heterologous expression systems. E. coli-expressed V. vulnificus rplR may lack certain modifications present in the native protein, potentially affecting structural studies and functional analyses.

Research approaches for studying PTMs include:

• Mass spectrometry to identify modification sites

• Site-directed mutagenesis to assess the functional importance of specific modified residues

• Comparative proteomics between native and recombinant protein forms

• Structural analysis using X-ray crystallography or cryo-EM to visualize modifications

How does temperature affect rplR expression and function in V. vulnificus during host infection?

V. vulnificus is known to respond to environmental changes, with climate warming likely expanding its geographical range . Temperature shifts between marine environments (typically cooler) and human hosts (37°C) trigger significant transcriptional reprogramming in this pathogen.

Ribosomal proteins including rplR likely undergo expression changes during temperature-induced stress responses. While specific data on rplR temperature response is limited, research on V. vulnificus outer membrane proteins shows that gene expression can be time-dependent and regulated by stress sigma factors like RpoS .

To investigate this phenomenon, researchers could employ:

• qRT-PCR to measure rplR transcript levels at different temperatures

• Ribosome profiling to assess translational efficiency

• Protein stability assays to determine if temperature affects protein half-life

• In vitro translation systems to measure functional impacts on protein synthesis rates

What are the optimal conditions for expressing recombinant V. vulnificus rplR in E. coli systems?

Based on protein expression protocols used for other V. vulnificus proteins, the following approach is recommended for recombinant rplR expression:

Expression Protocol:

• Clone the rplR gene into a vector containing an N-terminal histidine tag (e.g., pET-28a)

• Transform into an expression strain like E. coli BL21(DE3)

• Grow cultures at 37°C until OD600 reaches 0.6-0.8

• Induce with IPTG (0.1-0.5 mM)

• Continue expression at 28°C for 4-6 hours to minimize inclusion body formation

• Harvest cells by centrifugation and proceed with purification

Purification Strategy:

• Lyse cells in buffer containing 50 mM Tris-HCl (pH 8.0), 300 mM NaCl, 10 mM imidazole, and protease inhibitors

• Purify using Ni-NTA affinity chromatography with an imidazole gradient (20-250 mM)

• Further purify by size exclusion chromatography if necessary

• Confirm purity by SDS-PAGE and Western blotting

This approach has been successful for related ribosomal proteins and can be adapted based on specific experimental requirements.

How can researchers effectively use cryo-EM to study the integration of rplR into the V. vulnificus ribosome?

Cryo-electron microscopy (cryo-EM) has revolutionized structural studies of ribosomes, allowing visualization of ribosomal proteins like rplR in their native context. When studying V. vulnificus rplR incorporation, researchers should consider:

• Sample Preparation:

  • Isolate intact 70S ribosomes from V. vulnificus cultures

  • Purify using sucrose gradient ultracentrifugation

  • Verify sample quality by negative stain EM before proceeding to cryo-EM

• Data Collection Parameters:

  • Use direct electron detectors with motion correction

  • Collect at 300 kV with pixel size ~1.0 Å

  • Collect 3,000-5,000 micrographs for high-resolution structure determination

• Data Processing Workflow:

  • Particle picking (automated with manual inspection)

  • 2D and 3D classification to identify complete ribosomes

  • Focused refinement on the L18 (rplR) binding region

  • Local resolution assessment to determine quality of rplR density

• Structural Analysis:

  • Compare rplR positioning with structures from model organisms

  • Analyze interactions with neighboring proteins and rRNA

  • Identify potential V. vulnificus-specific features

The resulting structures can provide insights into species-specific aspects of ribosome assembly and potential antimicrobial target sites.

What approaches can be used to study the interaction between rplR and other ribosomal components in V. vulnificus?

Multiple complementary techniques can investigate interactions between rplR and other ribosomal components:

Table 2: Methods for Studying rplR Interactions in V. vulnificus

When implementing these methods, researchers should consider using both recombinant components and native ribosomes purified from V. vulnificus to validate findings across different experimental systems.

Could rplR mutations affect antibiotic resistance profiles in clinical V. vulnificus strains?

Ribosomal proteins are common targets for antibiotics, making rplR potentially relevant to antibiotic resistance. In clinical V. vulnificus isolates from Ningbo, China, resistance to multiple antibiotics was observed, including 100% resistance to imipenem and 80.95% resistance to vancomycin .

While specific antibiotic resistance genes (ARGs) like varG, PBP3, parE, adeF, and CRP were identified in these isolates , mutations in ribosomal proteins like rplR could contribute to resistance phenotypes, particularly for antibiotics targeting the 50S subunit.

Research approaches to investigate this connection include:

• Sequence analysis of rplR genes from resistant and susceptible isolates to identify potential mutations

• Introduction of identified mutations into susceptible strains using site-directed mutagenesis

• Minimum inhibitory concentration (MIC) testing of mutant strains against various antibiotics

• Structural modeling to predict how mutations might affect antibiotic binding sites

• Ribosome functional assays to determine if mutations affect translation fidelity or rate

How does rplR expression change during different stages of V. vulnificus infection?

During infection, V. vulnificus undergoes transcriptional reprogramming to adapt to host environments. While specific data on rplR expression changes is limited, patterns may be inferred from related proteins.

Studies on V. vulnificus outer membrane proteins show that expression can be time-dependent and regulated by stress factors . Similarly, ribosomal proteins likely undergo regulation during infection stages as the bacterium shifts between growth and survival modes.

Experimental approaches to study rplR expression during infection include:

• Animal infection models:

  • Infect mice with V. vulnificus (similar to approaches in )

  • Harvest bacteria from different tissues/timepoints

  • Measure rplR expression by qRT-PCR or proteomics

• Cell culture models:

  • Infect human cell lines (e.g., HeLa cells as used in )

  • Isolate bacteria at various timepoints

  • Analyze expression changes using RNA-seq

• Ex vivo tissue models:

  • Use human tissue explants to mimic complex host environments

  • Track bacterial gene expression changes

  • Correlate with virulence factor expression

Could rplR be a potential target for novel antimicrobials against V. vulnificus?

As an essential protein for bacterial survival, rplR represents a potential antimicrobial target. The high conservation of ribosomal proteins between bacterial species presents both advantages and challenges:

• Target validation:

  • Generate conditional knockdown strains to confirm essentiality

  • Assess growth defects when rplR expression is reduced

  • Determine minimum expression levels required for viability

• Screening strategies:

  • Structure-based virtual screening using crystallographic data

  • High-throughput screens using translation inhibition assays

  • Fragment-based approaches to identify binding molecules

• Selectivity considerations:

  • Compare V. vulnificus rplR to human ribosomal proteins to identify differences

  • Target V. vulnificus-specific structural features

  • Design assays to measure selectivity ratios

• Evaluation in infection models:

  • Test candidate molecules in mouse infection models similar to those used in previous V. vulnificus studies

  • Measure survival rates and bacterial burden

  • Compare efficacy to established antibiotics

What structural features distinguish V. vulnificus rplR from homologs in other bacterial species?

While specific structural data for V. vulnificus rplR is limited, comparative sequence analysis suggests both conserved core elements and species-specific features. Based on ribosomal protein evolution patterns:

Table 3: Predicted Structural Features of V. vulnificus rplR

Researchers could verify these predictions through:

• X-ray crystallography of purified recombinant V. vulnificus rplR

• Cryo-EM of intact V. vulnificus ribosomes

• Homology modeling using closely related species' structures

• Molecular dynamics simulations to identify functional motions

How do post-translational modifications of rplR affect ribosome assembly and function in V. vulnificus?

Post-translational modifications (PTMs) can significantly impact ribosomal protein function, affecting ribosome assembly, stability, and translational activity. Common bacterial ribosomal protein PTMs include methylation, acetylation, and hydroxylation.

Research approaches to study PTMs in V. vulnificus rplR:

• PTM identification:

  • Mass spectrometry analysis of native rplR purified from V. vulnificus

  • Comparison with recombinant protein expressed in E. coli

  • Mapping modifications to structural models

• Functional impact assessment:

  • In vitro ribosome assembly assays with modified vs. unmodified rplR

  • Translation efficiency measurements using reconstituted ribosomes

  • Site-directed mutagenesis to mimic or prevent specific modifications

• Regulation under stress conditions:

  • Analysis of PTM patterns under different growth conditions

  • Correlation with environmental stressors relevant to infection

  • Identification of enzymes responsible for specific modifications

How has rplR evolved among different Vibrio species, and what does this tell us about V. vulnificus pathogenicity?

Comparative analysis of rplR sequences across Vibrio species can provide insights into evolutionary history and potential functional adaptations. Unlike virulence factors that show high variability among clinical isolates , ribosomal proteins generally show high conservation due to their essential functions.

• Sequence conservation patterns between pathogenic (V. vulnificus, V. cholerae) and non-pathogenic Vibrio species

• Selection pressure analysis to identify residues under positive or negative selection

• Correlation between specific rplR variants and clinical vs. environmental isolates

• Potential horizontal gene transfer events affecting ribosomal protein genes

Such analyses could complement typing methods currently used for V. vulnificus, including vcg typing (clinical-type vcgC or environment-type vcgE) and 16S rRNA typing (A-type or B-type) .

What can functional genomics approaches reveal about rplR's role in the V. vulnificus stress response?

Functional genomics approaches can elucidate how rplR contributes to V. vulnificus adaptation to various stressors encountered during infection. Similar to observations with outer membrane proteins like TolCV1, whose expression is regulated by the stress sigma factor RpoS , ribosomal proteins likely play roles in stress adaptation.

Experimental approaches include:

• Transcriptomics:

  • RNA-seq analysis under various stress conditions (temperature shift, osmotic stress, nutrient limitation)

  • Correlation of rplR expression with global stress response patterns

  • Identification of co-regulated genes suggesting functional relationships

• Proteomics:

  • Quantitative proteomics to measure protein abundance changes

  • Ribosome profiling to assess translational efficiency

  • Protein-protein interaction networks under stress conditions

• Genetic approaches:

  • Construction of rplR reporter strains to monitor expression in real-time

  • CRISPR interference to modulate rplR expression levels

  • Suppressor mutant screens to identify genetic interactions