Recombinant Treponema denticola 30S ribosomal protein S11 (rpsK)
Description
Overview of Treponema denticola
Treponema denticola is a bacterium belonging to the oral microbiota and is associated with periodontal disease . Its genetic tractability makes it a model organism for studying Treponema physiology and host-microbe interactions .
Recombinant 30S Ribosomal Protein S11 (rpsK)
Recombinant Treponema denticola 30S ribosomal protein S11 (rpsK) is a synthetically produced version of the S11 protein, which is a component of the 30S ribosomal subunit in T. denticola . Ribosomal proteins like S11 are essential for protein synthesis within bacterial cells .
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Function: Ribosomal protein S11 is located on the platform of the 30S subunit and helps bridge RNA helices of the 16S rRNA . It also forms part of the Shine-Dalgarno cleft in the 70S ribosome .
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Interaction with S7: Studies indicate that S7 and S11 interact, contributing to the dynamics of the ribosome and influencing translational fidelity . Mutations in either S7 or S11 can affect the control of translational fidelity, increasing frameshifting, readthrough of nonsense codons, and codon misreading .
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Role in mRNA Binding: Research has demonstrated that mutated S7 or S11 enhances the capacity of 30S subunits to bind mRNA .
Characteristics of T. denticola 30S Ribosomal Protein S11
Recombinant Production and Applications
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Expression: The gene encoding the major outer sheath protein (Msp) of T. denticola can be expressed in E. coli to produce recombinant proteins .
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Applications: Recombinant T. denticola proteins are useful in studying protein function, interactions, and potential roles in pathogenesis . For example, recombinant Msp has been shown to adhere to laminin and fibronectin, suggesting its involvement in the extracellular matrix binding activity of T. denticola .
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Detection: PCR methods targeting the 16S rDNA can detect T. denticola in various infections, indicating its presence and potential role in disease .
Genetic Studies and Transformations
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Transformation Efficiency: The genetic transformation efficiency in T. denticola can be enhanced using SyngenicDNA-based plasmids that lack restriction-modification target motifs .
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R-M Systems: T. denticola possesses multiple restriction-modification (R-M) systems, which have implications for genetic manipulation and transformation efficiency .
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Mutagenesis: High-efficiency transposon mutagenesis of T. denticola is achievable using R-M-silent, codon-optimized transposase-based plasmids .
Immunological Studies
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Surface Epitopes: Immunological studies have identified surface epitopes on T. denticola proteins, aiding in understanding their surface topology and potential interactions with the host immune system .
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Msp Protein: The Msp protein of T. denticola has been investigated for its surface localization and variability among different strains using immunological techniques .
Disease Association
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T. denticola and Periodontal Disease: T. denticola is closely associated with periodontal disease, contributing to the dysregulation of tissue homeostasis and the breakdown of tooth-supporting tissues .
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Endodontic Infections: T. denticola has been detected in a significant percentage of endodontic infections, suggesting its involvement in the pathogenesis of periradicular lesions .
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Neuronal Apoptosis: Oral infection with T. denticola can induce neuronal apoptosis by promoting Aβ accumulation, indicating a potential link between oral bacteria and neurodegenerative diseases .
Product Specs
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Target Background
KEGG: tde:TDE0790
STRING: 243275.TDE0790
Q&A
What is the genomic organization of the rpsK gene in Treponema denticola?
The rpsK gene in Treponema denticola is part of the highly conserved ribosomal protein operons found in prokaryotes. Similar to other oral spirochetes, T. denticola demonstrates a genomic organization where ribosomal protein genes are clustered in operons. The rpsK gene encodes the 30S ribosomal protein S11, a critical component of the small ribosomal subunit involved in mRNA binding and translational accuracy. In T. denticola, this gene is typically located within a conserved gene cluster that includes other ribosomal proteins and often exhibits co-regulation with adjacent genes.
Based on comparative analysis with related spirochetes, researchers have determined that the gene expression is likely regulated through mechanisms similar to those observed in the dentilisin operon (prcB-prcA-prtP), which demonstrates coordinated expression of multiple gene products . Expression studies would typically employ RT-PCR and Northern blot analyses to confirm operon structure and transcriptional boundaries.
How does recombinant rpsK compare structurally to native protein from T. denticola?
Recombinant Treponema denticola 30S ribosomal protein S11 (rpsK) typically retains most structural features of the native protein, though differences can arise based on expression systems and purification methods. Structural analyses comparing native and recombinant forms generally reveal:
| Feature | Native rpsK | Recombinant rpsK | Analytical Method |
|---|---|---|---|
| Secondary Structure | α-helical content: ~35-40% β-sheet content: ~25-30% | Similar α-helical content Possible variations in β-sheet organization | Circular Dichroism (CD) spectroscopy |
| Tertiary Structure | Compact folding with RNA-binding domains | Generally conserved, potential differences in flexible regions | X-ray crystallography, NMR |
| Post-translational Modifications | Potential methylation and acetylation | Typically absent unless engineered | Mass spectrometry |
| Solubility | Naturally integrated into ribosome complex | Often more soluble as recombinant protein | Size-exclusion chromatography |
Methodological approaches for comparing these proteins typically involve biophysical characterization using circular dichroism spectroscopy, fluorescence spectroscopy, and limited proteolysis to assess structural integrity. Research has demonstrated that most recombinant ribosomal proteins maintain functional domains required for RNA interactions and ribosome assembly, though subtle differences can impact activity assays.
What expression systems are most effective for producing recombinant T. denticola rpsK protein?
The choice of expression system for recombinant T. denticola 30S ribosomal protein S11 (rpsK) production depends on research objectives and downstream applications. Comparative analysis of expression systems reveals:
| Expression System | Advantages | Limitations | Yield (approx.) | Purification Method |
|---|---|---|---|---|
| E. coli BL21(DE3) | High yield, simple protocol, cost-effective | Potential codon bias issues, inclusion body formation | 15-25 mg/L culture | IMAC, followed by ion exchange |
| E. coli Rosetta™ | Better handling of rare codons found in T. denticola | Increased cost, additional antibiotic selection | 12-20 mg/L culture | IMAC, followed by ion exchange |
| Cell-free systems | Rapid production, avoids toxicity issues | Higher cost, lower yield | 0.5-2 mg/mL reaction | Affinity chromatography |
| Baculovirus/insect cells | Better folding, potential for PTMs | Complex protocol, higher cost | 5-15 mg/L culture | Multiple chromatography steps |
When designing expression protocols, researchers should consider codon optimization to account for differences between T. denticola and the expression host. This approach is similar to methodologies used for other T. denticola proteins, where expression optimization has been achieved through careful vector design and growth condition adjustment .
The experimental design should incorporate randomized block designs to account for batch-to-batch variation in protein expression, as demonstrated in statistical approaches for experimental optimization . Expression conditions should be systematically tested using factorial design to determine optimal temperature, induction timing, and media composition.
How can researchers effectively study interactions between rpsK and other ribosomal components?
Studying interactions between recombinant T. denticola rpsK and other ribosomal components requires multiple complementary approaches:
| Technique | Application | Data Output | Limitations |
|---|---|---|---|
| Pull-down assays | Identifying direct binding partners | Protein-protein interaction maps | May miss transient interactions |
| Surface Plasmon Resonance (SPR) | Quantifying binding kinetics | Association/dissociation constants | Requires protein immobilization |
| Isothermal Titration Calorimetry (ITC) | Thermodynamic parameters of binding | ΔH, ΔS, Kd values | Requires significant protein amounts |
| Cryo-EM | Structural context within ribosome | 3D structural models | Complex sample preparation |
| Cross-linking Mass Spectrometry | Proximity mapping | Identification of interaction sites | Potential artifacts from cross-linking |
When designing interaction studies, researchers should consider incorporation of response surface methodology (RSM) to optimize experimental conditions, particularly for complex multi-component systems . This approach enables systematic exploration of factors affecting binding interactions, similar to approaches used in studying dentilisin complex formation in T. denticola .
For in vitro ribosome assembly studies, researchers typically employ a staggered nested design to distinguish between variability in the experimental method versus true biological variation in assembly kinetics . Analysis of results should incorporate statistical approaches similar to those used in studies of T. denticola virulence factors, where interrelated expression has been observed between different protein complexes .
What experimental contradictions exist in the current literature regarding T. denticola ribosomal protein function?
Current research on T. denticola ribosomal proteins reveals several experimental contradictions and knowledge gaps:
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Extraribosomal Functions: There are conflicting reports regarding potential extraribosomal functions of rpsK and other ribosomal proteins in T. denticola. Some studies suggest involvement in gene regulation, while others indicate these are experimental artifacts.
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Post-translational Modifications: Various studies report different patterns of post-translational modifications on ribosomal proteins from oral spirochetes, with inconsistent findings regarding their functional significance.
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Antigenic Properties: Contradictory findings exist regarding the immunogenicity and surface exposure of ribosomal proteins, similar to controversies observed with TprK protein in T. pallidum .
To address these contradictions, researchers should employ multiple methodological approaches, including:
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Targeted gene deletion and complementation studies
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Comprehensive mass spectrometry analysis under different growth conditions
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Cell fractionation combined with immunological techniques
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Comparative analyses across related spirochete species
These approaches should be designed as factorial experiments with appropriate controls to systematically investigate the sources of experimental variation, as outlined in statistical design principles . When designing such experiments, randomized complete block designs may help control for batch effects and other experimental variables.
How can researchers distinguish between direct and indirect effects when studying rpsK knockout mutants?
Distinguishing between direct and indirect effects in T. denticola rpsK knockout studies presents significant challenges due to the essential nature of ribosomal proteins. Researchers should consider these methodological approaches:
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Conditional knockout systems: Employ tetracycline-regulated or similar systems to control expression levels rather than complete knockouts.
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Point mutations vs. gene deletion: Create specific point mutations affecting function rather than complete gene deletion to minimize global translation effects.
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Complementation strategies: Use ectopic expression of wild-type and mutant variants to confirm phenotype specificity.
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Time-course experiments: Monitor changes immediately following depletion to identify primary versus secondary effects.
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Global analysis approaches: Combine transcriptomics, proteomics, and metabolomics to map regulatory networks.
These approaches should be implemented within a carefully designed experimental framework that incorporates both generalized complete block design and, where appropriate, split-plot arrangements to account for multiple experimental factors . Statistical analysis should employ mixed-effects models to separate experimental variation from true biological effects.
When interpreting results, researchers should consider that ribosomal protein mutations may have pleiotropic effects similar to those observed in studies of T. denticola virulence factors, where interrelated expression patterns have been documented between seemingly unrelated gene products .
What are the methodological approaches to studying the role of rpsK in T. denticola stress response?
To investigate the potential role of rpsK in T. denticola stress response, researchers can implement these methodological approaches:
| Stress Condition | Experimental Approach | Measurement Parameters | Controls |
|---|---|---|---|
| Oxidative stress | H₂O₂ exposure gradient | Growth inhibition, protein carbonylation, gene expression | Wild-type and complemented strains |
| Antibiotic stress | Sub-MIC antibiotic exposure | Ribosome assembly, translation fidelity | Other ribosomal protein mutants |
| Nutrient limitation | Defined media with limiting components | Growth kinetics, ribosome profiles | Global transcription/translation inhibitors |
| pH stress | Controlled pH microenvironments | Protein stability, RNA binding capacity | pH-resistant mutants |
| Temperature stress | Heat/cold shock protocols | Ribosome assembly kinetics | Constitutive vs. inducible expression |
These stress response studies should employ factorial experimental designs to explore interaction effects between different stressors, as detailed in statistical design literature . This approach allows researchers to construct response surface models that can predict rpsK behavior under various stress combinations.
Validation experiments should incorporate approaches similar to those used in studying T. denticola virulence factors, where gene expression regulators (e.g., TDE_0127, TDE_0814, and TDE_0344) have been identified as playing critical roles in stress adaptation . Exploring potential links between rpsK and iron homeostasis may be particularly valuable, as connections between protein expression and iron regulation have been documented in T. denticola research .
What is the current understanding of rpsK's potential role in T. denticola virulence and host interaction?
The potential role of rpsK in T. denticola virulence remains an emerging area of research with several intriguing directions:
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Moonlighting functions: Beyond its canonical role in translation, rpsK may contribute to virulence through secondary functions, similar to ribosomal proteins in other pathogens.
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Host immune recognition: Ribosomal proteins can serve as pathogen-associated molecular patterns (PAMPs) that trigger host immune responses.
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Stress adaptation: rpsK may contribute to survival within the periodontal pocket through roles in stress response pathways.
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Translational regulation: Selective translation of virulence factors during infection may involve rpsK-mediated mechanisms.
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Biofilm formation: Potential contributions to community behavior and interspecies interactions.
Research approaches should employ multiple methodologies, including targeted mutagenesis, host-pathogen interaction models, and comparative genomics. These studies should integrate randomized block experimental designs to control for host variability in interaction studies, and where appropriate, split-plot designs to efficiently manage multiple experimental factors .
The relationship between rpsK and established virulence factors such as dentilisin and Msp should be examined, as research has demonstrated interrelated expression patterns between major virulence determinants in T. denticola . Understanding these relationships may provide insights into coordinated expression of virulence traits during infection.
