Recombinant Chromobacterium violaceum 50S ribosomal protein L18 (rplR)

What is the functional role of 50S ribosomal protein L18 in Chromobacterium violaceum?

The 50S ribosomal protein L18 (rplR) in Chromobacterium violaceum is a critical component of the large ribosomal subunit involved in protein synthesis. Based on structural homology with bacterial L18 proteins, it plays a crucial role in ribosome assembly through specific interactions with 5S rRNA. This interaction causes a conformational change that promotes the binding of other ribosomal proteins to 5S rRNA in the final phase of ribosome assembly .

The binding of L18 to 5S rRNA is essential for the unification of 5S rRNA with 23S rRNA in the large subunit, which contains the peptidyl transferase center where proteins are synthesized . In the genomic analysis of C. violaceum, ORFs for all ribosomal proteins except S22 were found, confirming the presence of functional rplR in this organism .

How is recombinant C. violaceum rplR typically expressed and purified for research applications?

Expression and purification of recombinant C. violaceum 50S ribosomal protein L18 typically follows protocols similar to those used for other ribosomal proteins:

Expression Systems:

• E. coli expression systems are commonly used, similar to those employed for the rplF protein from C. violaceum

• Alternative systems include yeast, baculovirus, or mammalian cell lines depending on research requirements

Expression Procedure:

• Clone the rplR gene into an appropriate expression vector with N-terminal and/or C-terminal tags

• Transform into competent E. coli cells

• Induce expression with IPTG (for T7 promoter systems)

• Harvest cells and lyse using methods that preserve protein structure

Purification Strategy:

• Initial clarification by centrifugation

• Affinity chromatography using the protein tag (His-tag is common)

• Size exclusion chromatography for further purification

• Ion exchange chromatography for final polishing

• Concentrate to ≥85% purity as determined by SDS-PAGE

Storage recommendations include lyophilization or liquid formulation (determined during manufacturing), with long-term storage at -20°C or -80°C and working aliquots maintained at 4°C for up to one week .

How does C. violaceum rplR contribute to antibiotic sensitivity and resistance mechanisms?

The relationship between C. violaceum 50S ribosomal protein L18 and antibiotic mechanisms is significant, as this protein is part of the large ribosomal subunit that is targeted by several antibiotics:

Antibiotic Targeting:

• Ribosomal proteins, including those in the 50S subunit, are targets for antibiotics that inhibit the polypeptide elongation step of translation

• Sublethal doses of translation-inhibiting antibiotics can induce specific responses in C. violaceum, including violacein production, biofilm formation, and virulence factors

Research Findings:

• C. violaceum activates an antibiotic-induced response (air) two-component regulatory system when exposed to antibiotics targeting translation

• Exposure to translation inhibitors that target the polypeptide elongation step affects quorum sensing through connections with the Air system

• Modifications in ribosomal proteins, potentially including L18, might contribute to adaptations against antibiotics

To study the specific role of rplR in these mechanisms, researchers can employ site-directed mutagenesis of key residues in the protein and observe changes in antibiotic sensitivity profiles.

What structural characteristics define C. violaceum L18 and how do they compare to homologous proteins?

The structural characteristics of C. violaceum 50S ribosomal protein L18 can be inferred from homologous proteins and genomic data:

Key Structural Features:

• Located in the central protuberance of the large ribosomal subunit

• Contains an RL18/L5e superfamily domain

• Likely interacts with 5S rRNA through specific binding domains

• The ortholog of RL18/L5e protein in bacteria and archaea is known as L18

Comparative Structural Analysis:

• In E. coli, L18 is involved in forming the peptidyl transferase center

• The pattern of RNA-protein interactions appears conserved across species

To experimentally characterize the structure, researchers would typically employ X-ray crystallography or cryo-electron microscopy of purified recombinant protein or ribosomal complexes.

What experimental approaches can be used to study interactions between C. violaceum rplR and 5S rRNA?

To study the interactions between C. violaceum 50S ribosomal protein L18 and 5S rRNA, researchers can employ several complementary techniques:

For the MST analysis, protocols similar to those used to study CviR protein interactions could be adapted . This would involve:

• Fluorescent labeling of either the rplR protein or 5S rRNA

• Preparing serial dilutions of the unlabeled binding partner

• Measuring the thermophoretic movement in a temperature gradient

• Calculating binding affinity (Kd) from the resulting data

How can researchers design effective mutation studies to investigate functional domains in C. violaceum rplR?

An effective mutation study to investigate functional domains in C. violaceum rplR should follow these methodological steps:

1. Domain Identification:

• Perform sequence alignment with homologous L18 proteins from well-characterized organisms

• Use bioinformatics tools to predict functional domains (RNA-binding regions, protein-protein interaction sites)

• Identify conserved residues likely to be functionally important

2. Mutagenesis Strategy:

• Design both alanine-scanning mutations (replacing residues with alanine) and conservative substitutions

• Create point mutations in suspected 5S rRNA binding domains

• Engineer deletion mutants for predicted functional domains

3. Expression and Analysis:

• Express wild-type and mutant proteins in E. coli using protocols similar to those for rplF

• Purify proteins using affinity chromatography

• Assess structural integrity using circular dichroism

• Measure RNA binding using techniques listed in Question 5

• Evaluate effects on translation using in vitro translation assays

4. In vivo Functional Studies:

• Complement L18-deficient strains with mutant variants

• Assess growth rates, ribosome assembly, and translation efficiency

• Measure antibiotic sensitivity profiles

This approach parallels methods used for studying other C. violaceum proteins, such as the site-directed mutagenesis of the CviR DNA binding site, which helped define the DNA sequence required for promoter recognition .

What is the role of C. violaceum rplR in translation, particularly during environmental stress conditions?

The role of 50S ribosomal protein L18 from C. violaceum in translation during stress conditions is particularly relevant given the organism's adaptability to diverse environments:

Normal Translation Function:

• Essential component of the peptidyl transferase center where proteins are synthesized

• Contributes to the structural integrity of the 50S ribosomal subunit

• Helps maintain proper rRNA folding during translation

Under Environmental Stress:

• May contribute to stress-responsive translation regulation

• Potentially involved in selective mRNA translation during stress

• Could mediate ribosome hibernation or stabilization under adverse conditions

C. violaceum demonstrates remarkable versatility in environmental adaptation, with its genome revealing extensive systems for stress adaptation, which likely involve translation regulation mechanisms . The bacterium can thrive under diverse environmental conditions, including nutrient limitation and oxygen deprivation .

To study this role experimentally, researchers could:

• Compare ribosome composition and L18 modifications under normal and stress conditions

• Analyze translation efficiency of stress-responsive mRNAs in wild-type vs. L18-mutant strains

• Investigate potential post-translational modifications of L18 during stress response

How does the genomic organization of rplR in C. violaceum compare to other bacterial species?

The genomic organization of rplR in Chromobacterium violaceum reflects important evolutionary aspects of ribosomal protein genes in bacteria:

Genomic Context in C. violaceum:

• The complete genome sequence of C. violaceum ATCC 12472 contains 4,751,080 bp with 4,431 predicted protein-coding ORFs

• Ribosomal proteins, including rplR, are among the 168 genes categorized under "translation, ribosomal structure, and biogenesis" (COG category J)

• Like many bacteria, ribosomal protein genes in C. violaceum are likely organized in operons

Comparative Analysis:

• The typical organization in bacteria includes rplR in the S10 operon along with other ribosomal proteins

• Conservation of this organization provides insights into evolutionary relationships

• Specific genomic context may influence expression regulation under different conditions

To experimentally study the genomic organization, researchers could:

• Use RT-PCR to confirm operon structure and co-transcription of adjacent genes

• Employ genome walking techniques to characterize regulatory elements

• Perform comparative genomic analysis with other bacteria, particularly closely related species like Neisseria meningitidis and Ralstonia solanacearum, which share significant genomic similarity with C. violaceum

What methods are most effective for studying the role of C. violaceum rplR in ribosome assembly?

To study the role of C. violaceum 50S ribosomal protein L18 in ribosome assembly, researchers should employ a multi-faceted approach:

In vitro Reconstitution Studies:

• Purify all components of the 50S subunit including rRNAs and proteins

• Perform reconstitution with and without L18

• Analyze assembly intermediates using sucrose gradient ultracentrifugation

• Monitor incorporation of other proteins dependent on L18 (particularly those interacting with 5S rRNA)

In vivo Assembly Analysis:

• Create conditional L18 mutants in C. violaceum

• Monitor ribosome profiles under depleted conditions

• Identify accumulating assembly intermediates

• Use pulse-chase experiments with radiolabeled precursors

Structural Approaches:

• Use photolabile oligoRNAs complementary to 23S rRNA regions that interact with L18

• Analyze crosslinking patterns similar to methods used for studying 50S components neighboring 23S rRNA

• Employ cryo-EM to visualize assembly intermediates with and without L18

Real-time Assembly Monitoring:

• Develop fluorescently-tagged L18 variants

• Monitor incorporation into assembling ribosomes using fluorescence microscopy

• Track assembly kinetics using techniques like FRET

This research is particularly valuable because the bacterial L18 plays an important role in ribosome assembly through interaction with 5S rRNA, causing conformational changes that promote the binding of other proteins and ultimately ensuring proper formation of the peptidyl transferase center .

What post-translational modifications might affect C. violaceum rplR function?

While specific post-translational modifications (PTMs) of C. violaceum 50S ribosomal protein L18 have not been directly characterized in the search results, potential modifications can be inferred from studies of homologous proteins and general bacterial ribosomal protein biology:

Potential Post-translational Modifications:

Research Approaches:

• Proteomic Analysis:

  • Purify ribosomes from C. violaceum grown under different conditions

  • Perform mass spectrometry to identify PTMs on L18

  • Compare modification patterns between growth conditions

• Functional Studies:

  • Create site-directed mutants mimicking or preventing specific PTMs

  • Evaluate impacts on ribosome assembly and translation

  • Assess effects on antibiotic sensitivity

• Environmental Response:

  • Compare modification patterns during normal growth vs. stress conditions

  • Analyze PTMs in response to antibiotics targeting translation

Given C. violaceum's remarkable environmental adaptability , PTMs of ribosomal proteins could be part of the regulatory mechanisms that allow it to thrive in diverse conditions, including surviving in stagnant water, the natural habitat where it has been isolated from human infections .

How can researchers effectively express and purify C. violaceum rplR for structural studies?

For structural studies of C. violaceum 50S ribosomal protein L18, researchers require high-yield, high-purity protein preparations. The following comprehensive protocol is recommended:

Expression Vector Design:

• Amplify the rplR gene using PCR with primers containing appropriate restriction sites

• Clone into an expression vector with a strong, inducible promoter (T7 recommended)

• Include a cleavable affinity tag (His6 recommended for initial purification)

• Optimize codon usage for the expression host if necessary

Expression Optimization:

• Transform into E. coli expression strains (BL21(DE3) or Rosetta for rare codons)

• Test expression at different temperatures (18°C, 25°C, 37°C)

• Optimize induction conditions (IPTG concentration, induction time)

• Evaluate solubility in different growth media (LB, TB, minimal media)

Purification Strategy:

• Cell Lysis:

  • Resuspend cells in buffer containing protease inhibitors

  • Lyse using sonication or French press

  • Clarify lysate by centrifugation (20,000 × g, 30 min)

• Multi-step Purification:

  • Immobilized metal affinity chromatography (IMAC) using Ni-NTA

  • Tag cleavage with TEV protease

  • Ion exchange chromatography (IEX)

  • Size exclusion chromatography (SEC)

• Quality Control:

  • Assess purity by SDS-PAGE (target ≥95% for structural studies)

  • Verify identity by mass spectrometry

  • Check homogeneity by dynamic light scattering

Structural Study Preparation:

• Concentrate to 5-10 mg/mL for crystallization

• For NMR studies, express in minimal media with isotope labeling (15N, 13C)

• For cryo-EM, ensure sample homogeneity and proper buffer conditions

This protocol is adaptable from methods used for other C. violaceum proteins and incorporates general principles for ribosomal protein purification similar to those used for 50S ribosomal protein L6 (rplF) .

What is the significance of C. violaceum rplR in bacterial evolution and ribosomal conservation?

The 50S ribosomal protein L18 from Chromobacterium violaceum represents an important case study in bacterial evolution and ribosomal conservation:

Evolutionary Conservation:

• Ribosomal proteins are among the most highly conserved proteins across all domains of life

• L18 serves as an evolutionary marker due to its essential role in ribosome structure and function

• The ortholog of RL18/L5e protein in bacteria and archaea is known as L18, while in mammals, the related protein MRP-L18 is localized in mitochondria

Genomic Analysis Findings:

• Comparative genomic analysis of C. violaceum reveals that 17.4% of its ORFs have closest similarity to Ralstonia solanacearum, 9.75% to Neisseria meningitidis, and 9.61% to Pseudomonas aeruginosa

• This suggests evolutionary relationships that can be further explored through ribosomal protein analysis

• The presence of 8 rRNA operons in C. violaceum indicates potential for rapid adaptation to environmental changes

Research Approaches:

• Perform phylogenetic analysis of L18 sequences across bacterial species

• Compare RNA binding domains for conservation patterns

• Analyze coevolution between L18 and interacting ribosomal components

• Examine selective pressure on different domains within the protein

Understanding the evolution of C. violaceum rplR provides insights into not only ribosomal evolution but also the adaptive mechanisms that enable this organism to thrive in diverse environmental niches, from soil and water ecosystems to occasional pathogenic infections in humans .

How does C. violaceum rplR interact with antibiotics that target the 50S ribosomal subunit?

The interaction between C. violaceum 50S ribosomal protein L18 and antibiotics targeting the 50S ribosomal subunit is a critical area for research, particularly given the organism's potential pathogenicity and the need for effective treatments:

Antibiotic Interactions:

• Several classes of antibiotics target the 50S ribosomal subunit, including macrolides, lincosamides, streptogramins, and chloramphenicol

• L18, as part of the central protuberance of the large subunit, may be directly or indirectly affected by these antibiotics

• Specific binding sites may exist near the L18-5S rRNA interface

Research Findings:

• Sublethal doses of antibiotics targeting translation in C. violaceum induce significant changes in gene expression and behavior

• C. violaceum activates an antibiotic-induced response (air) two-component regulatory system when exposed to translation-inhibiting antibiotics

• This response affects quorum sensing, biofilm formation, and virulence

Experimental Approach for Studying L18-Antibiotic Interactions:

• Binding Studies:

  • Direct binding assays using purified L18 and radiolabeled antibiotics

  • Competition assays with 5S rRNA to identify binding interference

• Structural Analysis:

  • Co-crystallization of L18 with antibiotics

  • Molecular docking simulations to predict binding sites

• Resistance Mechanisms:

  • Site-directed mutagenesis of L18 residues potentially involved in antibiotic binding

  • Selection and characterization of spontaneous antibiotic-resistant mutants

• Functional Impact:

  • In vitro translation assays to measure inhibition with wild-type vs. mutant L18

  • Ribosome assembly assays to determine if antibiotics disrupt L18-dependent steps

Understanding these interactions has significant implications for treating the rare but potentially fatal infections caused by C. violaceum , which often require complex antibiotic regimens due to the organism's intrinsic resistance patterns.

What role does C. violaceum rplR play in the organism's pathogenicity and virulence mechanisms?

While 50S ribosomal protein L18 is not typically classified as a virulence factor, its role in C. violaceum pathogenicity merits investigation:

Context of Pathogenicity:

• C. violaceum causes rare but severe infections that can progress to fulminating septicemia with multiple abscesses in liver, lung, spleen, skin, lymph nodes, and brain

• These infections have a high fatality rate, often reaching 60-80%

• The bacterium typically enters through broken skin after contact with contaminated soil or water

Potential Contributions of rplR to Virulence:

• Translation of Virulence Factors:

  • As a component of the ribosome, L18 is involved in the translation of all proteins, including virulence factors

  • Efficient translation is critical during infection and host adaptation

• Stress Response during Infection:

  • C. violaceum must adapt to various stresses within the host environment

  • Translation regulation, potentially involving L18, may be important for this adaptation

• Antibiotic Resistance:

  • L18 modifications might contribute to the bacterium's intrinsic resistance to certain antibiotics

  • This could impact treatment efficacy during infection

Research Approaches:

• Compare L18 sequence and modifications between virulent and avirulent strains

• Examine L18 expression levels during infection using qRT-PCR

• Create L18 variants with site-directed mutations and assess impacts on virulence in animal models

• Study the translation of specific virulence factors in wild-type vs. L18-mutant strains