Recombinant Chromobacterium violaceum 30S ribosomal protein S16 (rpsP)

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

Chromobacterium violaceum is a Gram-negative bacterium commonly found in soil and water in tropical and subtropical regions worldwide . While primarily environmental, it can act as an opportunistic pathogen causing severe infections in humans with high mortality rates of 60-80% in disseminated infections . The bacterium produces a characteristic purple pigment called violacein, which contributes to its virulence and has antibiotic-inhibiting properties .

The significance of C. violaceum for ribosomal protein research stems from several factors:

• It represents an important model of an environmental opportunistic pathogen with unique pathogenicity mechanisms

• The bacterium possesses distinct virulence factors, including two type III secretion systems (T3SSs)

• Its genome has been fully sequenced (strain ATCC 12472), providing essential reference data for protein studies

• The bacterium demonstrates resistance to multiple antibiotics, making its ribosomal proteins potential targets for novel antimicrobial development

What is the structure and function of the 30S ribosomal protein S16 (rpsP) in bacteria?

The 30S ribosomal protein S16 is a component of the small subunit (30S) of the bacterial ribosome. While the search results don't provide specific information about rpsP in C. violaceum, general bacterial ribosomal protein knowledge indicates:

• The S16 protein plays a critical role in the assembly and stability of the 30S ribosomal subunit

• It contributes to the proper folding of 16S rRNA during ribosome assembly

• The protein typically consists of approximately 80-90 amino acids with a conserved RNA binding domain

• In many bacteria, S16 is essential for cell viability, making it a potential antimicrobial target

For C. violaceum specifically, the rpsP gene encoding S16 would be expected to share considerable homology with other bacterial species while potentially presenting unique characteristics that could be exploited for specific targeting.

How is recombinant Chromobacterium violaceum 30S ribosomal protein S16 expressed and purified?

Based on general recombinant protein methodologies and limited information from the search results, the expression and purification of recombinant C. violaceum 30S ribosomal protein S16 would typically follow these procedures:

• Gene cloning:

  • Isolation of genomic DNA from C. violaceum ATCC 12472 (or other characterized strain)

  • PCR amplification of the rpsP gene using primers designed from the known genome sequence

  • Cloning into an appropriate expression vector (pET system vectors are commonly used)

• Expression in host system:

  • Transformation of expression vectors into E. coli BL21(DE3) or similar expression strains

  • Induction of protein expression using IPTG or auto-induction media

  • Optimization of expression conditions (temperature, induction time, media composition)

• Purification strategy:

  • Cell lysis using sonication or mechanical disruption

  • Initial purification using affinity chromatography (His-tag is commonly employed)

  • Further purification using ion exchange chromatography

  • Final polishing step using size exclusion chromatography

• Verification of purity and identity:

  • SDS-PAGE analysis

  • Western blotting

  • Mass spectrometry confirmation

  • Activity assessment

Similar recombinant enzymes from C. violaceum have been successfully purified to homogeneity and crystallized using PEG 4000 via the microbatch method , suggesting this approach could be applicable to rpsP as well.

What are the distinctive structural and functional characteristics of C. violaceum 30S ribosomal protein S16 compared to other bacterial species?

The structural and functional characteristics that distinguish C. violaceum 30S ribosomal protein S16 from those of other bacterial species would require comprehensive comparative analysis. A methodological approach would include:

• Structural analysis:

  • X-ray crystallography or cryo-electron microscopy of the purified protein

  • NMR spectroscopy for dynamic analysis

  • In silico structural prediction and comparison with S16 proteins from other bacterial species

  • Analysis of protein-RNA interfaces specific to C. violaceum

• Sequence analysis:

  • Multiple sequence alignment with S16 proteins from related and distant bacterial species

  • Identification of conserved domains versus unique regions

  • Evolutionary analysis to determine phylogenetic relationships

• Functional characterization:

  • In vitro ribosome assembly assays

  • RNA binding studies using techniques such as EMSA, filter binding assays, or SPR

  • Effect of mutations on ribosome assembly and function

  • Complementation studies in S16-deficient strains

Since C. violaceum has pathogenicity mechanisms distinct from many other bacteria, including its specialized T3SS encoded by Chromobacterium pathogenicity islands , its ribosomal proteins may have evolved unique characteristics that could be exploited for targeted interventions.

How does the expression of rpsP correlate with virulence in C. violaceum infections?

The correlation between rpsP expression and virulence in C. violaceum would require investigation through several methodological approaches:

• Expression analysis during infection:

  • qRT-PCR analysis of rpsP expression during different stages of infection

  • RNA-seq to compare transcriptomic profiles between virulent and avirulent strains

  • Proteomics to quantify S16 protein levels during infection processes

• Genetic manipulation studies:

  • Construction of rpsP conditional mutants (as complete deletion may be lethal)

  • Analysis of virulence phenotypes in the mouse infection model

  • Complementation studies to confirm phenotypic changes

• Host-pathogen interaction analysis:

  • Investigation of host immune responses to wild-type versus rpsP-modified strains

  • Assessment of bacterial survival in host cells and tissues

  • Examination of the role of rpsP in resistance to host defense mechanisms

Given that C. violaceum causes fulminant hepatitis in mouse infection models and the high mortality rate in human infections (62.1%) , understanding the relationship between ribosomal proteins and virulence could provide valuable insights into pathogenesis.

What experimental approaches are most effective for studying the interaction between C. violaceum rpsP and antibiotics?

Investigating interactions between C. violaceum rpsP and antibiotics requires multiple experimental strategies:

• In vitro binding and inhibition studies:

  • Surface plasmon resonance (SPR) to measure direct binding of antibiotics to purified rpsP

  • Isothermal titration calorimetry (ITC) for thermodynamic analysis of interactions

  • Fluorescence-based assays to monitor structural changes upon antibiotic binding

• Structural studies of antibiotic-protein complexes:

  • Co-crystallization of rpsP with various antibiotics

  • Cryo-EM analysis of ribosome-antibiotic complexes focusing on S16 interactions

  • In silico molecular docking and dynamics simulations

• Resistance development and mechanisms:

  • Selection of antibiotic-resistant mutants and sequencing of rpsP

  • Introduction of specific mutations in rpsP to confirm their role in resistance

  • Transcriptomic and proteomic analysis of resistant strains

• Therapeutic potential assessment:

  • Evaluation of synergistic effects between S16-targeting compounds and conventional antibiotics

  • Development of S16-specific inhibitors based on structural data

  • In vivo efficacy testing in infection models

This approach is particularly relevant given that C. violaceum demonstrates resistance to multiple antibiotics, including penicillin, beta-lactams, and clindamycin, while showing sensitivity to carbapenems, aminoglycosides, chloramphenicol, quinolones, tetracyclines, and trimethoprim-sulfamethoxazole .

What are the challenges and solutions in developing recombinant C. violaceum rpsP as a potential therapeutic target?

Developing recombinant C. violaceum rpsP as a therapeutic target presents several challenges with corresponding methodological solutions:

The therapeutic potential is significant given the high mortality rate of C. violaceum infections (62.1%) and the challenge of treating abscesses that can persist for extended periods (clinical course duration median of 18 days, range 2–264 days) .

How can rpsP be utilized in genotyping and evolutionary studies of Chromobacterium species?

The utilization of rpsP in genotyping and evolutionary studies of Chromobacterium species could follow methodological approaches similar to those applied with other genes like recA :

• Sequence-based analysis:

  • PCR amplification and sequencing of rpsP from multiple Chromobacterium isolates

  • Comparative sequence analysis to identify conserved and variable regions

  • Phylogenetic tree construction to establish evolutionary relationships

• PCR-RFLP methodology:

  • Design of rpsP-specific primers for Chromobacterium species

  • Selection of appropriate restriction enzymes based on sequence analysis

  • Development of a standardized rpsP PCR-RFLP protocol similar to the recA PCR-RFLP analysis described for C. violaceum

• Multi-locus sequence typing (MLST) integration:

  • Inclusion of rpsP as one of several housekeeping genes in an MLST scheme

  • Analysis of sequence types and clonal complexes

  • Correlation of sequence types with geographical distribution and virulence characteristics

• Whole genome context analysis:

  • Examination of rpsP genomic context across different Chromobacterium species

  • Analysis of selection pressures acting on ribosomal protein genes

  • Comparative genomics to identify species-specific signatures

This approach could provide valuable insights into the evolution and diversification of Chromobacterium species, similar to how recA PCR-RFLP analysis identified at least three different genospecies among C. violaceum strains .

What are the optimal conditions for expression and crystallization of recombinant C. violaceum rpsP?

Based on information from similar recombinant proteins from C. violaceum , the optimization of expression and crystallization conditions for rpsP would follow this methodology:

• Expression optimization:

  • Testing multiple expression vectors (pET series, pGEX, pMAL) with different fusion tags

  • Screening expression temperatures (16°C, 25°C, 30°C, 37°C)

  • Evaluating induction conditions (IPTG concentration, induction time)

  • Testing different E. coli expression strains (BL21(DE3), BL21(DE3)pLysS, Rosetta, Arctic Express)

  • Optimizing media composition (LB, TB, auto-induction media)

• Solubility enhancement:

  • Addition of solubility-enhancing tags (MBP, SUMO, TRX)

  • Co-expression with chaperones

  • Addition of stabilizing agents during lysis (glycerol, specific ions)

  • Testing detergents for membrane-associated fractions

• Purification refinement:

  • Sequential chromatography steps (IMAC, ion exchange, size exclusion)

  • On-column refolding if inclusion bodies form

  • Buffer optimization for stability (pH, salt concentration, additives)

• Crystallization screening:

  • Initial screening using commercial sparse matrix screens

  • Focused optimization around successful conditions

  • Specific testing of PEG 4000 conditions using the microbatch method, which has been successful for other C. violaceum proteins

  • Seeding techniques to improve crystal quality

  • Co-crystallization with RNA fragments or antibiotics

A systematic approach to these parameters would increase the likelihood of obtaining high-quality crystals suitable for structural determination, similar to what has been achieved with other recombinant enzymes from C. violaceum .

How can RNA-protein interactions between C. violaceum 16S rRNA and rpsP be effectively studied?

Studying RNA-protein interactions between C. violaceum 16S rRNA and rpsP requires a multi-faceted experimental approach:

• In vitro binding assays:

  • Electrophoretic mobility shift assays (EMSA) with purified rpsP and 16S rRNA fragments

  • Filter binding assays to determine binding affinities

  • Surface plasmon resonance (SPR) for real-time interaction analysis

  • Isothermal titration calorimetry (ITC) for thermodynamic parameters

• Structural characterization of complexes:

  • X-ray crystallography of rpsP-RNA complexes

  • Cryo-electron microscopy of reconstituted 30S subunits

  • NMR spectroscopy for dynamic interaction studies

  • Chemical crosslinking followed by mass spectrometry to identify interaction sites

• Functional assays:

  • In vitro reconstitution of 30S subunits with wild-type or mutant rpsP

  • Translation assays to assess the impact of rpsP mutations on protein synthesis

  • Ribosome profiling to identify translation changes in vivo

• Computational approaches:

  • Molecular dynamics simulations of rpsP-RNA interactions

  • RNA structure prediction and docking studies

  • Sequence conservation analysis across related bacterial species

Understanding these interactions could provide insights into the unique aspects of C. violaceum ribosome assembly and function, potentially revealing targetable differences from host ribosomes.

How does the structure and function of rpsP contribute to the virulence and antibiotic resistance of C. violaceum?

Investigating the relationship between rpsP and C. violaceum virulence/antibiotic resistance requires methodological approaches that integrate structural, functional, and clinical data:

• Structural analysis in pathogenicity context:

  • Comparison of rpsP structure between virulent and avirulent strains

  • Identification of structural domains that interact with virulence-related factors

  • Analysis of potential post-translational modifications during infection

• Functional studies in pathogenicity models:

  • Construction of rpsP point mutants affecting specific structural domains

  • Assessment of mutant strains in mouse infection models

  • Evaluation of hepatocyte invasion and abscess formation capacity

• Antibiotic resistance mechanisms:

  • Identification of rpsP mutations in naturally resistant isolates

  • Introduction of these mutations into sensitive strains to confirm their role

  • Structural analysis of how mutations affect antibiotic binding

• Translation regulation of virulence factors:

  • Ribosome profiling to assess translation of virulence factors with wild-type versus mutant rpsP

  • Investigation of potential regulatory roles of rpsP in stress response during infection

  • Analysis of translation efficiency of T3SS components in different rpsP backgrounds

This integrated approach could reveal whether rpsP contributes to the characteristic properties of C. violaceum infections, such as abscess formation in internal organs (36.4% of cases) and high mortality rates despite antimicrobial therapy.

What are the implications of C. violaceum rpsP research for developing new diagnostic tools for infection?

Developing diagnostic tools based on C. violaceum rpsP research involves several methodological approaches:

• Molecular detection methods:

  • Design of rpsP-specific PCR primers for rapid identification

  • Development of loop-mediated isothermal amplification (LAMP) assays targeting rpsP sequences

  • Digital PCR approaches for quantification in clinical samples

• Immunological detection systems:

  • Production of monoclonal antibodies against recombinant rpsP

  • Development of lateral flow immunoassays for rapid diagnosis

  • ELISA-based detection systems for clinical laboratory use

• Mass spectrometry applications:

  • Identification of rpsP-specific peptide markers for MALDI-TOF diagnosis

  • Development of multiple reaction monitoring (MRM) assays for targeted detection

  • Integration with clinical mass spectrometry platforms

• Point-of-care diagnostic development:

  • Miniaturized biosensor platforms incorporating rpsP-specific detection elements

  • Smartphone-based diagnostic applications using portable detection devices

  • Field-deployable systems for use in resource-limited settings

Early detection is critical given the rapid progression of C. violaceum infection, with a median incubation period of 4.0 days (IQR 2.0–8.0 days) and high mortality without prompt and appropriate treatment.

What are the most promising research directions for C. violaceum rpsP studies in the next decade?

The future of C. violaceum rpsP research holds significant promise in several key areas:

• Structural biology advancements:

  • Complete structural characterization using cryo-EM and AI-assisted structure prediction

  • Dynamic studies of ribosome assembly and function specific to C. violaceum

  • Integration of structural data into drug discovery pipelines

• Therapeutic development:

  • Design of rpsP-specific inhibitors based on structural differences from host ribosomes

  • Development of combination therapies targeting multiple ribosomal components

  • Creation of delivery systems capable of penetrating abscesses and biofilms

• Diagnostic applications:

  • Implementation of rapid molecular and immunological detection methods

  • Integration with point-of-care systems for resource-limited settings

  • Development of prognostic markers based on rpsP variants

• Ecological and evolutionary research:

  • Comprehensive analysis of rpsP across the expanding number of identified Chromobacterium species

  • Investigation of horizontal gene transfer and evolution of ribosomal components

  • Understanding the role of rpsP in environmental adaptation across different habitats

These research directions align with the growing recognition of C. violaceum as an important model of an environmental opportunistic pathogen and the need for better diagnostic and therapeutic approaches given its high mortality rate.

How can collaborative research approaches enhance our understanding of C. violaceum ribosomal proteins?

Effective collaborative approaches to enhance understanding of C. violaceum ribosomal proteins would include:

• Interdisciplinary research teams:

  • Integration of structural biologists, microbiologists, clinicians, and bioinformaticians

  • Combination of experimental and computational approaches

  • Collaboration between academic institutions and healthcare facilities

• Technology sharing platforms:

  • Development of standardized protocols for rpsP expression and purification

  • Creation of repositories for C. violaceum strains and mutants

  • Sharing of crystallization conditions and structural data

• Clinical research networks:

  • Establishment of surveillance systems for C. violaceum infections

  • Collection and characterization of clinical isolates

  • Correlation of microbial characteristics with clinical outcomes

• Data integration frameworks:

  • Creation of databases integrating genomic, structural, and clinical data

  • Development of predictive models for virulence and antibiotic resistance

  • Implementation of machine learning approaches for pattern recognition

Such collaborative approaches would accelerate progress in understanding the role of ribosomal proteins in C. violaceum pathogenicity and potentially lead to improved management of this severe infection, which currently has a 62.1% mortality rate .