Recombinant Treponema denticola 30S ribosomal protein S21 (rpsU)

What is the optimal expression system for recombinant Treponema denticola 30S ribosomal protein S21 (rpsU)?

For most basic structural studies and initial functional characterization, the E. coli expression system offers the best balance of yield, cost, and time efficiency. When selecting an expression system, consider:

• Research objectives (structural vs. functional studies)

• Required protein yield

• Importance of post-translational modifications

• Available laboratory resources and expertise

• Timeline constraints

How does the 30S ribosomal protein S21 (rpsU) contribute to bacterial ribosome assembly and function?

The 30S ribosomal protein S21 is crucial for proper bacterial ribosome assembly and translation efficacy . This protein plays a significant role in the formation of intersubunit bridges, particularly Bridge B4, which is essential for connecting the small (30S) and large (50S) ribosomal subunits .

Functionally, S21 facilitates the binding of other ribosomal proteins, including S6, S18, S11, and S21 in vitro . While some bacteria may have alternative assembly pathways in the absence of certain ribosomal proteins under optimal conditions, the absence of these proteins often results in weaker 70S ribosomal structures and cold-sensitive phenotypes . Under non-optimal or stress conditions, proteins like S21 become essential for bacterial survival .

Ribosomal proteins of the small subunit are particularly significant as targets for antibacterial agents, as certain antibiotics (such as gentamicin and kanamycin) exert their antimicrobial effects by specifically targeting the small ribosomal subunit of bacteria .

What purification techniques yield the highest purity of recombinant Treponema denticola 30S ribosomal protein S21?

For optimal purification of recombinant T. denticola 30S ribosomal protein S21, a multi-step chromatographic approach is recommended. While specific information about purifying this particular protein isn't provided in the search results, general approaches for ribosomal proteins can be applied:

• Affinity Chromatography: Using a histidine (His) tag is particularly effective for initial capture. The His-tagged protein can be purified using a nickel or cobalt resin column.

• Ion Exchange Chromatography: As a second step to remove contaminants with similar affinity but different charge properties.

• Size Exclusion Chromatography: As a final polishing step to separate the target protein based on molecular size.

For ribosomal proteins specifically, consider:

• Using high salt concentrations (300-500 mM NaCl) during initial binding steps to reduce non-specific interactions

• Adding RNase treatment during lysis to remove bound RNA

• Including reducing agents to prevent oxidation of cysteine residues

These approaches should yield protein with >95% purity suitable for functional and structural studies.

How can researchers design experimental frameworks to investigate the role of T. denticola 30S ribosomal protein S21 in bacterial pathogenesis?

Designing robust experimental frameworks to investigate the role of T. denticola 30S ribosomal protein S21 in pathogenesis requires a multi-factorial approach. Based on experimental design principles from factorial studies, researchers should consider:

• Gene Deletion/Complementation Studies:

  • Create a ΔrpsU mutant strain using optimized transformation protocols similar to those used for other T. denticola motility genes

  • Verify deletions through PCR and whole genome sequencing to confirm specificity

  • Complement the deletion with wild-type and modified versions of the rpsU gene

• Comparative Phenotypic Analysis: Compare wild-type and mutant strains using:

  • Growth kinetics under various stress conditions

  • Biofilm formation capacity (mono-species and polymicrobial with P. gingivalis)

  • Motility assays (as ribosomal proteins may indirectly affect motility proteins)

• Quantitative Proteomic Analysis:

  • Perform comprehensive proteomic profiling of wild-type vs. ΔrpsU mutants

  • Look for changes in abundance of proteins beyond the immediate ribosomal network

  • Analyze changes in stress response proteins and motility-associated proteins

• Experimental Design Considerations:

  • Use factorial design approaches to efficiently assess multiple variables simultaneously

  • Consider fractional factorial designs when resource constraints prevent testing all combinations

  • Balance scientific objectives with research economy

This framework allows for systematic analysis of both direct effects (on translation) and indirect effects (on virulence factors and stress responses) of S21 in T. denticola pathogenesis.

What are the comparative differences in structure and function between T. denticola 30S ribosomal protein S21 and homologs in other bacterial species?

While the search results don't provide specific comparative data for T. denticola S21 versus other bacterial homologs, we can establish a methodological approach to investigate these differences:

• Sequence Analysis:

  • Perform multiple sequence alignments of S21 proteins from various bacterial species

  • Identify conserved domains and species-specific variations

  • Calculate sequence identity and similarity percentages

• Structural Comparison:

  • Generate structural models through X-ray crystallography, cryo-EM, or computational modeling

  • Compare binding sites, especially those involved in RNA interactions

  • Analyze differences in secondary and tertiary structures

• Functional Divergence Assessment:

  • Test cross-species complementation (can S21 from another species restore function in T. denticola?)

  • Compare binding affinities to ribosomal RNA and other ribosomal proteins

  • Assess differences in temperature sensitivity and response to antibiotics

• Evolutionary Analysis:

  • Construct phylogenetic trees to understand evolutionary relationships

  • Identify positively selected sites that may relate to species-specific adaptations

  • Correlate structural/functional differences with evolutionary distance

The comparative analysis should focus on differences that may influence pathogenicity, antibiotic susceptibility, and species-specific adaptation mechanisms.

How does post-translational modification of recombinant T. denticola 30S ribosomal protein S21 affect its functionality in experimental systems?

Post-translational modifications (PTMs) of recombinant T. denticola 30S ribosomal protein S21 can significantly impact its functionality in experimental systems. When investigating PTMs:

• Expression System Selection:

  • E. coli and yeast systems provide high yields but limited PTMs

  • Insect cells with baculovirus or mammalian cell expression systems provide many of the post-translational modifications necessary for correct protein folding and activity retention

• Identification of Critical PTMs:

  • Employ mass spectrometry to identify specific PTMs (methylation, acetylation, phosphorylation)

  • Compare PTM profiles between native and recombinant proteins from different expression systems

  • Correlate specific PTMs with functional properties

• Functional Assays to Assess PTM Impact:

  • In vitro translation assays comparing differently modified versions

  • RNA binding assays to measure affinity changes

  • Structural stability assessments under various conditions

• Engineering Specific PTMs:

  • Site-directed mutagenesis to remove or mimic PTM sites

  • Co-expression with relevant modification enzymes

  • Chemical modification approaches for specific PTMs

A comprehensive approach would include creating variants with different PTM profiles and testing them in functional assays that measure ribosomal assembly, translation efficiency, and response to stress conditions.

What methodological approaches can be used to investigate the interaction between T. denticola 30S ribosomal protein S21 and bacterial stress response mechanisms?

To investigate interactions between T. denticola 30S ribosomal protein S21 and stress response mechanisms, researchers should consider:

• Stress Exposure Experimental Design:

  • Subject wild-type and S21-modified strains to various stressors (oxidative, pH, temperature, nutrient limitation)

  • Measure growth kinetics, survival rates, and recovery times

  • Implement factorial experimental designs to efficiently assess multiple stress variables simultaneously

• Transcriptomic and Proteomic Analyses:

  • Perform RNA-seq to identify differentially expressed genes under stress conditions

  • Conduct quantitative proteomics similar to approaches used for T. denticola motility mutants

  • Look for changes in established stress response proteins like desulfoferrodoxin/neelaredoxin, RecA, DNA topoisomerase I (TopA), and transcription termination factor Rho

• tRNA and Ribosome Function Assessment:

  • Examine changes in tRNA modification and processing enzymes similar to those reported in P. aeruginosa stress responses

  • Monitor aminoacyl-tRNA synthetase activity, particularly those involved in the editing of misacylated tRNAs

  • Assess ribosomal assembly integrity under stress conditions

• Protein-Protein Interaction Studies:

  • Perform co-immunoprecipitation to identify stress-specific interaction partners

  • Use bacterial two-hybrid systems to verify direct interactions

  • Employ proximity labeling approaches to capture transient interactions during stress response

This methodological framework enables researchers to systematically characterize how S21 contributes to stress adaptation in T. denticola, potentially revealing new targets for antimicrobial development.

How can researchers assess the impact of T. denticola 30S ribosomal protein S21 on polymicrobial biofilm formation?

Based on studies of T. denticola motility mutants and their effects on biofilm formation, researchers can develop a methodology to assess S21's impact on polymicrobial biofilms:

• Biofilm Assay Development:

  • Create an rpsU knockout mutant using optimized transformation protocols similar to those developed for flgE and motB genes

  • Establish mono-species and dual-species biofilm models with P. gingivalis (a known synergistic partner)

  • Use confocal laser scanning microscopy to visualize biofilm architecture and composition

• Quantitative Biofilm Analysis:

  • Measure biofilm biomass using crystal violet staining

  • Determine species ratios within polymicrobial biofilms using species-specific qPCR

  • Calculate synergy ratios (dual-species biofilm biomass divided by the sum of monospecies biofilms)

• Mechanistic Investigation:

  • Analyze expression of biofilm-related genes in the presence/absence of S21

  • Investigate potential cross-species signaling affected by ribosomal protein function

  • Examine extracellular matrix composition for differences in wild-type versus mutant strains

• Complementation Studies:

  • Reintroduce wild-type rpsU to confirm phenotype restoration

  • Create point mutations in functional domains to identify critical regions

  • Test heterologous complementation with S21 from other species

This approach allows for comprehensive characterization of how ribosomal protein S21 influences the complex process of polymicrobial biofilm formation between T. denticola and other oral pathogens.

What role does T. denticola 30S ribosomal protein S21 play in antibiotic resistance mechanisms?

While the search results don't specifically address S21's role in antibiotic resistance in T. denticola, a methodological framework to investigate this question includes:

• Minimum Inhibitory Concentration (MIC) Determination:

  • Compare susceptibility profiles of wild-type and ΔrpsU mutant strains

  • Test various antibiotic classes, particularly those targeting protein synthesis

  • Focus on antibiotics known to target the small ribosomal subunit (like aminoglycosides)

• Ribosome Binding Studies:

  • Perform in vitro binding assays with purified ribosomes from wild-type and mutant strains

  • Use radiolabeled or fluorescently labeled antibiotics to measure binding kinetics

  • Compare structural changes in ribosomes upon antibiotic binding

• Genetic Approaches:

  • Generate spontaneous antibiotic-resistant mutants and sequence rpsU

  • Create site-directed mutants based on known resistance mutations in homologous proteins

  • Perform complementation studies with mutant versions of rpsU in knockout strains

• Translation Fidelity Assessment:

  • Measure translation error rates in the presence of subinhibitory antibiotic concentrations

  • Compare error rates between wild-type and ΔrpsU strains

  • Assess stop codon readthrough and frameshifting frequencies

These methodological approaches would provide valuable insights into whether S21 contributes to intrinsic antibiotic resistance in T. denticola and potential mechanisms involved.

How can researchers optimize expression and purification protocols for structural studies of T. denticola 30S ribosomal protein S21?

For structural studies of T. denticola 30S ribosomal protein S21, optimization of expression and purification is critical. A comprehensive approach includes:

• Expression System Optimization:

  • Compare yields and purity from E. coli and yeast expression systems

  • Test different E. coli strains designed for problematic protein expression (BL21(DE3), Rosetta, C41/C43)

  • Optimize induction conditions (temperature, inducer concentration, duration)

• Construct Design for Structural Studies:

  • Create constructs with removable affinity tags (His6, GST, MBP)

  • Consider testing multiple constructs with different boundaries

  • Introduce surface entropy reduction mutations to promote crystallization

• Purification Protocol Refinement:

  • Develop a multi-step purification strategy combining:

    • Affinity chromatography (IMAC, GST)

    • Ion exchange chromatography

    • Size exclusion chromatography

  • Optimize buffer conditions to enhance stability (pH, salt concentration, additives)

  • Implement quality control checks at each purification step (SDS-PAGE, Western blot)

• Structural Integrity Assessment:

  • Employ circular dichroism to verify secondary structure

  • Use thermal shift assays to identify stabilizing conditions

  • Perform dynamic light scattering to assess homogeneity

For X-ray crystallography specifically, incorporate sparse matrix screening of crystallization conditions followed by optimization of promising hits. For NMR studies, establish isotope labeling protocols using minimal media supplemented with 15N-ammonium chloride and 13C-glucose.

How does T. denticola 30S ribosomal protein S21 function compare to its role in P. aeruginosa and other pathogenic bacteria?

To establish a comprehensive comparison between T. denticola S21 and its counterparts in other pathogenic bacteria:

• Functional Conservation Analysis:

  • Compare phenotypic effects of S21 deletion across species

  • In P. aeruginosa, the absence of certain ribosomal proteins can lead to significant changes in stress responses and bacterial adaptation

  • Examine whether T. denticola S21 shows similar essentiality patterns as observed in E. coli, where some ribosomal proteins become critical under non-optimal conditions

• Structural Comparison Methodology:

  • Align sequences to identify conserved motifs across species

  • Create homology models based on available structures

  • Compare binding sites and interaction surfaces

• Species-Specific Interaction Networks:

  • Map protein-protein interactions unique to each species

  • In P. aeruginosa, ribosomal protein S18 shows strong interactions with S15

  • Determine if T. denticola has similar compensatory mechanisms

• Differential Response to Environmental Stressors:

  • Cold sensitivity is a known phenotype of ribosomal protein mutations in E. coli

  • Test whether T. denticola S21 mutants show similar temperature-dependent phenotypes

  • Compare responses to other stressors (oxidative stress, pH changes, nutrient limitation)

Through this comparative approach, researchers can identify both conserved functions essential across bacterial species and specialized roles that may have evolved in T. denticola to support its unique ecological niche and pathogenic potential.

What experimental approaches can be used to investigate the impact of T. denticola 30S ribosomal protein S21 modifications on translation accuracy?

To investigate how modifications to T. denticola 30S ribosomal protein S21 affect translation accuracy, researchers should consider:

• Reporter System Implementation:

  • Develop dual-luciferase reporter systems containing programmed errors

  • Measure readthrough of premature stop codons

  • Quantify frameshifting frequency at slippery sequences

  • Compare results between wild-type and S21-modified strains

• In Vitro Translation Assays:

  • Reconstitute ribosomes with wild-type or modified S21

  • Use mRNA templates with known error-prone sequences

  • Quantify amino acid misincorporation rates using mass spectrometry

  • Measure translation speed and processivity

• tRNA Selection and Accommodation Analysis:

  • Examine how S21 modifications affect tRNA selection

  • Similar to P. aeruginosa studies, analyze changes in tRNA ligase profiles

  • Focus on glutamine-tRNA ligase and cysteine-tRNA ligase, which have been implicated in bacterial translation accuracy

• Ribosome Structural Analysis:

  • Use cryo-EM to determine structural changes in ribosomes with modified S21

  • Analyze changes in intersubunit bridge formation

  • Examine effects on mRNA and tRNA binding sites

These approaches provide complementary data on how S21 modifications impact translation at the molecular level, potentially revealing mechanisms by which ribosomal proteins contribute to both translation accuracy and stress adaptation in T. denticola.

What are the most promising future research directions for T. denticola 30S ribosomal protein S21 in periodontal disease treatment?

Based on current understanding of ribosomal proteins and their roles in bacterial pathogenesis, promising future research directions include:

• Therapeutic Target Validation:

  • Assess whether S21 can be specifically targeted without affecting human ribosomal function

  • Examine whether S21 inhibition affects T. denticola virulence in animal models

  • Investigate synergistic effects of targeting S21 alongside other virulence factors

• Biofilm Disruption Strategies:

  • Develop peptides or small molecules targeting S21 to disrupt polymicrobial biofilms

  • Test combination approaches targeting both motility and translation machinery

  • Leverage the knowledge that T. denticola motility is essential for synergistic biofilm formation with P. gingivalis

• Cross-Species Communication Mechanisms:

  • Investigate how translation regulation through S21 affects production of signaling molecules

  • Examine potential roles in quorum sensing and interspecies communication

  • Develop intervention strategies targeting these communication pathways

• Ribosome-Targeting Antimicrobial Development:

  • Design species-specific inhibitors of T. denticola ribosomes

  • Test repurposed antibiotics that target the small ribosomal subunit

  • Develop delivery systems for targeting biofilm-embedded T. denticola

These research directions build on our current understanding while pushing toward translational applications that could impact periodontal disease treatment strategies.

How can multi-factorial experimental designs be optimized for studying the interactions between T. denticola 30S ribosomal protein S21 and other virulence factors?

Optimizing multi-factorial experimental designs for studying complex interactions involving T. denticola S21 requires:

• Resource-Efficient Design Selection:

  • Implement fractional factorial designs when resource constraints prevent testing all combinations

  • Prioritize scientific objectives based on their potential impact

  • Balance between service to scientific objectives and research economy

• Factor Prioritization Framework:

  • Consider whether research questions are framed as main effects or simple effects

  • Identify which effects might be aliased (confounded) in particular designs

  • Calculate the number of experimental conditions required for statistical power

• Integration of Molecular and Phenotypic Approaches:

  • Combine transcriptomic, proteomic, and phenotypic assays

  • Develop quantitative readouts for virulence factor expression

  • Implement systems biology approaches to model complex interactions

• Standardized Reporting Protocols:

  • Establish minimum information standards for experimental reporting

  • Include detailed methodology for reproduction

  • Implement data sharing practices to facilitate meta-analyses

This optimized approach allows researchers to efficiently investigate the complex interactions between ribosomal function and virulence while maintaining scientific rigor and making effective use of limited research resources.