Recombinant Chromobacterium violaceum 50S ribosomal protein L35 (rpmI)

What is Chromobacterium violaceum and why is it significant for ribosomal protein research?

Chromobacterium violaceum is a gram-negative, facultative anaerobic bacillus widely distributed in tropical and subtropical regions, particularly in soil and water environments . It produces a characteristic purple pigment called violacein that has antimicrobial properties and is thought to contribute to its virulence . Despite being primarily environmental, C. violaceum can occasionally cause severe infections in humans with high mortality rates .

The bacterium is significant for ribosomal protein research due to its remarkable adaptability to diverse environmental conditions and stress responses, making it an excellent model organism for studying ribosomal protein function and regulation under various physiological states . Additionally, the complete genome sequencing of C. violaceum has enabled detailed molecular and structural studies of its ribosomal components, including the 50S ribosomal protein L35.

What is the basic structure and function of the 50S ribosomal protein L35 (rpmI) in C. violaceum?

The 50S ribosomal protein L35 (encoded by the rpmI gene) in C. violaceum is a small protein component of the large ribosomal subunit that plays a crucial role in protein synthesis. While the search results don't provide specific structural details for L35 in C. violaceum, ribosomal proteins generally interact extensively with rRNA to maintain ribosome integrity and facilitate protein synthesis.

How does C. violaceum rpmI compare structurally with homologous proteins from other bacterial species?

While the search results don't provide direct comparative information about rpmI across species, we can infer some insights based on what is known about ribosomal protein conservation and variability. Typically, ribosomal proteins show high sequence conservation in functionally critical regions while displaying variability in peripheral regions.

Similar to how C. violaceum ribosomal proteins L1, L3, L5, and L6 show distinct expression patterns under stress conditions , the L35 protein likely maintains core structural features across bacterial species while potentially exhibiting C. violaceum-specific adaptations. These adaptations might relate to the bacterium's ability to thrive in diverse environmental conditions, including its response to pH and nutrient stress as observed in proteomic analyses .

What are the optimal conditions for heterologous expression of recombinant C. violaceum 50S ribosomal protein L35?

Based on general principles for recombinant protein expression and the specific growth conditions favorable for C. violaceum, the following methodological approach is recommended:

Expression System Selection:

• E. coli is typically the preferred host for heterologous expression of bacterial ribosomal proteins due to its well-established genetic tools and rapid growth

• For challenging expressions, consider using BL21(DE3) strains or derivatives that are optimized for recombinant protein production

Culture Conditions:

• Growth temperature: While C. violaceum grows optimally at 35°C , recombinant expression in E. coli often benefits from lower temperatures (18-25°C) to enhance protein folding

• Medium: Start with standard LB medium, but for higher yields, consider richer media like 2xYT or Terrific Broth

• Induction parameters: Lower IPTG concentrations (0.1-0.5 mM) combined with longer expression times (16-20 hours) at reduced temperatures often improve soluble protein yield for ribosomal proteins

Considerations for C. violaceum-specific characteristics:

• Since C. violaceum undergoes significant protein expression changes under stress , examining the native expression conditions of L35 may provide insights for optimizing recombinant production

• The loss of violacein pigmentation observed under stress conditions suggests significant metabolic shifts that might impact recombinant protein expression strategies

What purification strategy is most effective for obtaining high-purity recombinant C. violaceum L35 protein?

An effective purification strategy would involve:

Initial Capture:

• Affinity chromatography using a His6-tag is recommended as the first purification step

• Lysis buffer composition: 50 mM Tris-HCl pH 8.0, 300 mM NaCl, 10 mM imidazole, with protease inhibitors

• Consider including low concentrations of non-ionic detergents (0.1% Triton X-100) during lysis to minimize potential aggregation

Intermediate Purification:

• Ion exchange chromatography (typically cation exchange as ribosomal proteins are generally basic)

• Buffer conditions: 20 mM MES pH 6.0-6.5 with a gradient of 0-1 M NaCl

Polishing Step:

• Size exclusion chromatography using a Superdex 75 column

• Running buffer: 20 mM Tris-HCl pH 7.5, 150 mM NaCl

Quality Control:

• SDS-PAGE analysis to confirm protein purity

• Mass spectrometry to verify protein identity and integrity

• Circular dichroism to assess proper folding

How can isotopically labeled C. violaceum L35 protein be efficiently produced for NMR studies?

For NMR studies requiring isotopically labeled protein:

• Use minimal media supplemented with 15N-ammonium chloride and/or 13C-glucose as the sole nitrogen and carbon sources respectively

• Implement a two-stage culture protocol:

  • Grow cells in rich medium to achieve high cell density

  • Transfer cells to isotopically enriched minimal medium prior to induction

• Extend expression time (20-24 hours) while reducing temperature (16-18°C) to maximize incorporation of labeled nutrients

• Utilize high cell density fermentation techniques to improve yield while minimizing isotope costs

• Consider deuteration for larger proteins or complex analyses by growing in D2O-based media

This approach balances the need for high protein yield with the efficient incorporation of expensive isotopically labeled precursors.

How does C. violaceum regulate L35 expression under different environmental stresses?

While specific information about L35 regulation is not directly provided in the search results, insights can be drawn from the documented behavior of other ribosomal proteins in C. violaceum:

Research has shown that C. violaceum undergoes significant proteomic changes in response to environmental stresses such as pH variation and nutrient starvation . Most ribosomal proteins examined (including L1, L5, and L6) showed reduced expression under stress conditions, suggesting a general downregulation of the translation machinery .

Interestingly, the ribosomal protein L3, which plays a role in protein folding, was an exception and maintained higher expression under stress conditions . This differential regulation suggests a specialized adaptive response where certain ribosomal components are selectively preserved or enhanced to support cellular survival under adverse conditions.

For L35 specifically, researchers should consider:

• Examining expression patterns across growth phases using quantitative PCR or ribosome profiling

• Comparing expression under reference conditions (nutrient-rich, pH 7.0) versus stress conditions (nutrient starvation, acidic or alkaline pH)

• Investigating potential interactions with stress-response regulators or alternative sigma factors

What techniques are most effective for studying L35 protein interactions within the ribosome complex?

Several complementary approaches are recommended:

Structural Analysis:

• Cryo-electron microscopy (cryo-EM) to visualize L35 within the intact ribosome

• X-ray crystallography of the 50S subunit with focus on the L35 interaction network

• Hydrogen-deuterium exchange mass spectrometry (HDX-MS) to map interaction surfaces

Biochemical Approaches:

• Site-directed mutagenesis followed by in vitro reconstitution assays

• Crosslinking studies to identify neighboring proteins and rRNA contacts

• Pull-down assays using tagged L35 to identify strong interaction partners

Biophysical Methods:

• Fluorescence resonance energy transfer (FRET) to measure distances between L35 and other ribosomal components

• Surface plasmon resonance (SPR) to determine binding kinetics with rRNA segments

• Nuclear magnetic resonance (NMR) to identify specific residues involved in interactions

Computational Approaches:

• Molecular dynamics simulations to predict dynamic interactions

• Sequence co-evolution analysis to identify functionally coupled residues

How does L35 contribute to ribosome assembly and stability in C. violaceum?

Based on general ribosomal assembly principles and the available information about C. violaceum's adaptability:

L35 likely plays a critical role in the late stages of 50S subunit assembly, serving as a bridge between rRNA helices and neighboring proteins. Its presence might be essential for proper folding of specific rRNA domains and the recruitment of other ribosomal proteins.

The contribution of L35 to ribosome stability could be particularly important under stress conditions. In C. violaceum, which shows remarkable adaptability to environmental changes , L35 may provide structural support that maintains ribosome integrity during stress exposure. This is supported by observations that C. violaceum exhibits distinct proteomic profiles under different growth conditions , suggesting specialized ribosomal configurations adapted to various environmental challenges.

To experimentally characterize L35's contribution to ribosome assembly:

• Create conditional knockdowns or depletions of L35 in C. violaceum

• Analyze resulting partially assembled ribosomes using sucrose gradient centrifugation

• Perform complementation studies with mutant variants to identify critical residues

• Compare assembly kinetics under optimal versus stress conditions

How has the C. violaceum L35 protein evolved compared to homologs in other bacterial species?

The evolutionary context of C. violaceum L35 would be instructive to analyze through:

• Phylogenetic analysis: Constructing a phylogenetic tree of L35 sequences across diverse bacterial phyla to identify conserved and variable regions.

• Selection pressure analysis: Calculating dN/dS ratios to determine which regions of the protein have been under purifying selection (highly conserved functional domains) versus positive selection (potentially species-specific adaptations).

• Structural conservation mapping: Identifying structurally conserved regions that likely perform core functions versus variable regions that might contribute to species-specific adaptations.

• Comparative genomics: Analyzing the genomic context of the rpmI gene across species to identify conservation or divergence in operon structure and regulatory elements.

Given C. violaceum's remarkable adaptability to different environmental conditions , its L35 protein might contain unique adaptations that contribute to ribosome stability under stress conditions common in its natural habitat. These adaptations could be particularly relevant in the context of the bacterium's ability to maintain protein synthesis under challenging conditions such as pH stress and nutrient limitation .

What functional differences exist between C. violaceum L35 and similar proteins in pathogenic versus non-pathogenic bacteria?

Though the search results don't directly address this question for L35, we can propose a research framework based on what's known about C. violaceum:

C. violaceum exists primarily as an environmental saprophyte but can cause severe infections under certain conditions . This dual lifestyle suggests potential functional adaptations in its ribosomal proteins, including L35, that might contribute to its pathogenic potential.

Recommended comparative analysis approach:

• Compare L35 sequences between C. violaceum and closely related pathogenic and non-pathogenic species

• Identify sequence or structural features unique to pathogenic strains

• Examine expression patterns of L35 under host-mimicking conditions versus environmental conditions

• Investigate whether L35 is differentially regulated during transition to pathogenic state

The analysis should account for C. violaceum's ability to adapt its protein expression under stress , which might be relevant to its transition between environmental and pathogenic lifestyles. Particular attention should be paid to any structural features that might influence ribosome function under the stress conditions encountered during infection.

How can C. violaceum L35 protein be utilized in structural studies of antibiotic binding and resistance mechanisms?

Ribosomal proteins are frequent targets for antibiotics, making structural studies of L35 potentially valuable for understanding antimicrobial resistance. Research approaches could include:

Structural Analysis of Antibiotic Interactions:

• Co-crystallization of the C. violaceum 50S subunit with various antibiotics to identify binding sites near L35

• Cryo-EM studies of intact ribosomes with bound antibiotics to visualize conformational changes

• NMR studies with isotopically labeled L35 to detect chemical shift perturbations upon antibiotic binding

Resistance Mechanism Investigation:

• Compare L35 sequences between antibiotic-sensitive and resistant C. violaceum strains

• Create site-directed mutants based on identified variations to test their impact on antibiotic sensitivity

• Perform functional assays to assess translation efficiency in the presence of antibiotics

Translation Dynamics:

• Single-molecule FRET studies to monitor conformational changes in ribosomes containing modified L35 variants

• Ribosome profiling to detect changes in translation patterns when L35 is altered

This is particularly relevant given that C. violaceum shows resistance to multiple antibiotics, and the violacein pigment itself has antibiotic properties . Understanding how L35 contributes to ribosome function in this context could provide insights into novel resistance mechanisms.

What role might L35 play in C. violaceum's adaptation to extreme environmental conditions?

C. violaceum demonstrates remarkable adaptability to various environmental stresses, including pH extremes and nutrient limitation . The role of L35 in this adaptation could be investigated through:

• Stress-Specific Expression Analysis:

  • Quantify L35 expression levels under different stress conditions using RT-qPCR

  • Compare with expression patterns of other ribosomal proteins like L3, which shows stress-specific regulation

  • Determine if L35 follows the general pattern of reduced expression under stress or shows specialized regulation

• Functional Characterization Under Stress:

  • Create conditional L35 mutants and assess their growth under various stress conditions

  • Perform ribosome profiling to determine if L35 alterations affect the translation of stress-response genes

  • Analyze ribosome stability under extreme conditions with wild-type versus modified L35

• Comparative Analysis Between Strains:

  • Compare L35 sequence and expression between C. violaceum strains isolated from different environmental niches

  • Identify potential adaptations that correlate with habitat-specific stressors

Table 1: Predicted L35 Expression Patterns Based on C. violaceum Ribosomal Protein Behavior Under Stress

How might gene knockout or mutation studies of C. violaceum L35 inform our understanding of ribosome assembly pathways?

Systematic genetic manipulation of L35 in C. violaceum could provide valuable insights into ribosome assembly:

Experimental Design Approach:

• Create conditional depletion systems for L35 using regulatable promoters

• Develop CRISPR-Cas9 based approaches for precise genetic manipulation

• Engineer strains with tagged L35 variants to track assembly intermediates

Analysis Methods:

• Sucrose gradient centrifugation to isolate and characterize partially assembled ribosomal particles

• Mass spectrometry to identify the composition of assembly intermediates

• Electron microscopy to visualize structural abnormalities in ribosomes when L35 is altered

Expected Research Outcomes:

This research would be particularly informative in C. violaceum due to its adaptability to different growth conditions , potentially revealing condition-specific assembly pathways that contribute to bacterial survival under stress.

How can heterologous expression systems for C. violaceum L35 be optimized for structural biology applications?

Based on the unique characteristics of C. violaceum and its proteins, optimization strategies should include:

• Expression Vector Design:

  • Incorporate C. violaceum-specific codon optimization

  • Design fusion constructs with solubility-enhancing partners like MBP or SUMO

  • Include precision protease sites for tag removal that leave minimal or no residual amino acids

• Host Selection and Modification:

  • Test specialized E. coli strains designed for expression of proteins with rare codons

  • Consider Pseudomonas-based expression systems for proteins that are challenging in E. coli

  • Evaluate co-expression with C. violaceum-specific chaperones if folding issues are observed

• Culture Condition Optimization:

  • Implement statistical design of experiments to simultaneously optimize multiple parameters

  • Test auto-induction media formulations to avoid the toxicity sometimes associated with IPTG

  • Explore the impact of mimicking C. violaceum's natural growth conditions on protein quality

• Purification Enhancements:

  • Develop on-column refolding protocols for cases where the protein forms inclusion bodies

  • Implement multi-parametric screening to identify optimal buffer conditions

  • Consider the impact of violacein or other C. violaceum metabolites on protein stability

The optimization process should take into account C. violaceum's responsiveness to environmental conditions , potentially incorporating stress elements that might improve protein expression or folding based on the bacterium's natural adaptability mechanisms.

What insights can comparative ribosomal studies between C. violaceum and pathogenic bacteria provide for antibiotic development?

Comparative studies offer several valuable research directions:

• Structural Comparisons:

  • Identify unique structural features in C. violaceum L35 compared to pathogenic bacteria

  • Map conservation patterns across species to identify potential selective targets

  • Analyze binding pockets near L35 that might be exploited for species-specific targeting

• Functional Divergence:

  • Characterize differences in L35 contributions to ribosome assembly between species

  • Identify functions that are essential in pathogens but not in C. violaceum (or vice versa)

  • Explore whether L35 interacts differently with translation factors across species

• Resistance Mechanism Analysis:

  • Compare natural resistance mechanisms involving L35 across species

  • Evaluate how mutations in L35 affect antibiotic sensitivity differently between species

  • Study ribosome protection mechanisms that might involve L35

• Drug Development Implications:

  • Screen for compounds that bind differentially to L35 from different species

  • Exploit structural differences to design selective inhibitors

  • Use C. violaceum as a model system for testing ribosome-targeting compounds

The unique characteristics of C. violaceum, including its violacein pigment with natural antibiotic properties , may provide additional context for understanding ribosome-targeting antibiotic mechanisms and resistance development.