Recombinant Bdellovibrio bacteriovorus 30S ribosomal protein S20 (rpsT)

What is Bdellovibrio bacteriovorus and why is its ribosomal protein S20 significant?

Bdellovibrio bacteriovorus is a predatory deltaproteobacterium that invades and kills a broad spectrum of Gram-negative bacteria, including human pathogens such as Acinetobacter baumannii, Klebsiella pneumoniae, and Escherichia coli. This predatory bacterium has gained significant attention as a potential alternative to conventional antibiotics, especially in combating antimicrobial resistance. The 30S ribosomal protein S20, encoded by the rpsT gene, is a critical component of the small ribosomal subunit in B. bacteriovorus. Based on studies in related bacteria, S20 plays an essential role in ribosome assembly and function, specifically in mRNA binding and 30S-50S subunit association during translation initiation. Understanding this protein's structure and function in B. bacteriovorus provides valuable insights into the predator's protein synthesis machinery, which is crucial for its unique predatory lifecycle.

What expression systems are most effective for producing recombinant B. bacteriovorus S20 protein?

For recombinant expression of B. bacteriovorus S20 protein, E. coli-based expression systems typically offer the most efficient platform due to their well-established protocols and high yield potential. The BL21(DE3) strain or its derivatives are particularly suitable for ribosomal protein expression due to their reduced protease activity and tight control of induction. When designing expression constructs, researchers should consider codon optimization for E. coli, as B. bacteriovorus may have different codon usage patterns. Furthermore, fusion tags such as 6xHis or GST can facilitate purification, though their impact on protein folding should be carefully assessed. For optimal expression, culture conditions including temperature (typically reduced to 16-20°C post-induction), IPTG concentration (0.1-0.5 mM), and induction timing (mid-log phase) should be systematically optimized. Alternative expression systems, including cell-free protein synthesis platforms, may be considered if E. coli-based expression yields insoluble protein or leads to toxicity issues.

What purification strategies yield the highest purity of recombinant B. bacteriovorus S20 protein?

A multi-step purification strategy is recommended to achieve high purity of recombinant B. bacteriovorus S20 protein. The initial capture step typically employs affinity chromatography, with immobilized metal affinity chromatography (IMAC) being particularly effective if the protein contains a polyhistidine tag. Following affinity purification, ion exchange chromatography (typically using a strong cation exchanger due to the basic nature of most ribosomal proteins) effectively removes residual contaminants. Size exclusion chromatography as a polishing step helps eliminate aggregates and ensures monodispersity of the final preparation. Throughout the purification process, buffer optimization is crucial—typically maintaining pH between 7.0-8.0 with moderate ionic strength (150-300 mM NaCl) to prevent aggregation. Addition of reducing agents (1-5 mM DTT or β-mercaptoethanol) helps maintain protein stability by preventing oxidation of cysteine residues. Rigorous quality control using SDS-PAGE, Western blotting, and mass spectrometry is essential to confirm protein identity and purity. For functional studies, researchers should verify proper folding through circular dichroism or fluorescence spectroscopy.

What techniques can be used to assess the RNA-binding properties of B. bacteriovorus S20?

Multiple complementary techniques should be employed to comprehensively assess the RNA-binding properties of B. bacteriovorus S20 protein. Electrophoretic mobility shift assays (EMSAs) provide a straightforward initial approach to detect protein-RNA interactions, using labeled 16S rRNA fragments, particularly those corresponding to helix 44 which is known to interact with S20 in other bacteria. Surface plasmon resonance (SPR) or bio-layer interferometry (BLI) can be used to determine binding kinetics and affinity constants. Isothermal titration calorimetry (ITC) offers insights into the thermodynamic parameters of the binding interaction. For structural characterization of the protein-RNA complex, nuclear magnetic resonance (NMR) spectroscopy is valuable, particularly for mapping the binding interface. RNA footprinting techniques, including hydroxyl radical probing or SHAPE (Selective 2′-hydroxyl acylation analyzed by primer extension), can identify the specific nucleotides involved in the interaction. Additionally, fluorescence anisotropy provides information about the dynamics of the complex formation. These methodologies should be complemented with computational approaches such as molecular docking and molecular dynamics simulations to predict and validate binding models.

How does S20 deletion affect the predatory lifecycle of B. bacteriovorus?

Deletion of the rpsT gene encoding S20 in B. bacteriovorus would likely result in profound effects on the predatory lifecycle due to compromised ribosomal function. Based on studies in other bacteria, S20 deletion significantly impairs 30S subunit function, reducing mRNA binding rates and inhibiting association with the 50S subunit to form functional 70S ribosomes. In B. bacteriovorus, these defects would manifest as growth rate reduction and potentially altered predatory efficiency. To investigate these effects experimentally, researchers should generate a conditional knockout strain using techniques adapted for B. bacteriovorus, such as the conjugation procedures described for transferring plasmids from E. coli. Once established, the mutant strain should be systematically evaluated across all stages of the predatory lifecycle (attack phase, transition phase, and growth phase). Key parameters to measure include:

The predicted outcomes would include significantly reduced predatory capability, potentially similar to the effects observed when type IV pili were disrupted, which abolished predatory capabilities while still allowing prey-independent growth. Analysis should also include RNA-seq to determine transcriptome-wide effects and potential compensatory mechanisms that the predator might employ.

What role does S20 play in the regulation of prey-specific gene expression in B. bacteriovorus?

S20 may participate in prey-specific gene regulation in B. bacteriovorus beyond its canonical role in ribosome function. While primarily recognized as a structural component of the 30S ribosomal subunit, emerging research in bacteria suggests certain ribosomal proteins can moonlight as regulators of gene expression, particularly under stress conditions. To investigate this hypothesis, researchers should employ a comprehensive strategy combining genomics, transcriptomics, and biochemical approaches. The research methodology should include:

• Chromatin immunoprecipitation sequencing (ChIP-seq) using tagged S20 to identify any direct DNA binding sites in the B. bacteriovorus genome

• RNA-seq comparison between wild-type and S20-depleted strains during predation on different prey species to identify differentially expressed genes

• Ribosome profiling to analyze translational efficiency of mRNAs in the presence and absence of S20

• Protein-protein interaction studies using pull-down assays followed by mass spectrometry to identify potential regulatory partners of S20 outside the ribosome

Special attention should be given to genes encoding predation-related proteins, particularly the secreted nucleases (Bd0934 and Bd3507) and type IV pili components that are critical for predatory function. The timing of expression during the predatory cycle should be carefully analyzed, as B. bacteriovorus exhibits sequential expression of different factors throughout its life cycle. If S20 does play a regulatory role, it may function as part of a feedback mechanism coordinating ribosome assembly with predatory gene expression, particularly during the transition between attack phase and intraperiplasmic growth.

How do post-translational modifications of S20 affect ribosome assembly and function in B. bacteriovorus?

Post-translational modifications (PTMs) of ribosomal proteins, including S20, can significantly impact ribosome assembly, structure, and function. While PTMs of B. bacteriovorus S20 have not been specifically characterized in the available literature, this represents a promising avenue for advanced research. To comprehensively investigate this question, researchers should:

• Employ high-resolution mass spectrometry techniques, particularly LC-MS/MS with electron transfer dissociation (ETD) fragmentation, to identify and map PTMs on purified native S20 from different stages of the B. bacteriovorus lifecycle

• Generate site-directed mutants where potential modification sites are replaced with non-modifiable amino acids

• Develop in vitro ribosome assembly assays specifically for B. bacteriovorus components to assess the impact of S20 modifications on assembly kinetics and efficiency

• Conduct comparative ribosome profiling between wild-type and mutant strains to evaluate translational impacts

Potential PTMs to investigate include phosphorylation, methylation, and acetylation, which have been documented in ribosomal proteins from other species. The dynamic nature of these modifications should be analyzed across different growth conditions, particularly comparing host-dependent and host-independent growth modes. Special attention should be paid to modifications that might occur during the transition between predatory phases or in response to different prey species. Functional consequences of these modifications could include altered RNA binding affinity, changed intersubunit interactions, or modified interactions with translation factors that might be particularly relevant during the rapid growth phase inside the bdelloplast.

What mechanisms explain the interaction between B. bacteriovorus S20 and secreted nucleases during prey DNA degradation?

A potential functional relationship between ribosomal protein S20 and secreted nucleases in B. bacteriovorus presents an intriguing research question. The predator's life cycle involves the coordinated expression of various proteins, including secreted nucleases like Bd0934 and Bd3507, which are crucial for degrading prey DNA during the intraperiplasmic growth phase. Investigating potential interactions between S20 and these nucleases requires a multi-faceted approach:

• Co-immunoprecipitation studies using tagged versions of both S20 and the secreted nucleases, followed by mass spectrometry, to detect direct protein-protein interactions

• Fluorescence microscopy with differentially labeled proteins to track co-localization patterns during the predatory cycle

• Transcriptome analysis comparing wild-type and S20-depleted strains to identify changes in nuclease gene expression

• Biochemical assays measuring nuclease activity in the presence and absence of purified S20 protein

The research should be structured around investigating whether S20 might have extra-ribosomal functions in B. bacteriovorus, potentially serving as a regulatory factor for nuclease expression or activity. This hypothesis is supported by the observation that both nucleases (Bd0934 and Bd3507) are secreted into the bdelloplast milieu during predation, and their expression patterns could be coordinated with ribosome function via S20. The investigation should specifically examine whether S20 plays a role in the sequential and orchestrated release of nucleases observed during the intraperiplasmic growth phase, potentially linking translation efficiency with nuclease activity to optimize predatory efficiency.

How does the structure and function of S20 differ between host-dependent and host-independent B. bacteriovorus strains?

Host-independent (HI) B. bacteriovorus strains represent a fascinating adaptation where the predator can grow axenically without requiring prey invasion. Comparing S20 structure and function between host-dependent (HD) and HI strains could reveal important insights into ribosomal adaptations during this lifestyle switch. This research question should be approached through:

• Comparative genomics analysis of the rpsT gene sequences between multiple HD and HI strains to identify any consistent mutations

• Transcriptome and proteome profiling to quantify S20 expression levels in both lifestyles

• Structural biology approaches (X-ray crystallography or cryo-EM) to identify any conformational differences in S20 between the two growth modes

• Ribosome profiling to assess translational efficiency and mRNA selection differences

• Complementation studies where S20 from HD strains is expressed in HI strains and vice versa

Research should focus particularly on whether any modifications to S20 contribute to the altered growth characteristics observed in HI strains, which typically display diminished intraperiplasmic-growth capabilities and form smaller, more turbid plaques than wild-type strains. The potential role of S20 in adapting the translation machinery to different nutrient acquisition strategies (predatory versus saprophytic) should be investigated, potentially revealing how ribosomal components evolve to accommodate major lifestyle shifts. Special attention should be paid to whether S20 contributes to the regulation of genes involved in predation versus axenic growth, potentially serving as a molecular switch that helps coordinate the transition between these distinct physiological states.

What are the optimal conditions for expressing recombinant B. bacteriovorus S20 in heterologous systems?

Successful expression of recombinant B. bacteriovorus S20 protein requires careful optimization of expression conditions. Based on experiences with other ribosomal proteins, researchers should consider the following parameters:

To validate successful expression, Western blotting with anti-His antibodies should be performed, followed by small-scale purification tests. If expression in E. coli proves challenging, alternative expression systems such as insect cells (baculovirus expression system) or cell-free protein synthesis should be considered. For structural studies requiring isotope labeling, minimal media with 15N-ammonium sulfate and/or 13C-glucose should be employed with modified induction protocols to accommodate slower growth in minimal media.

How can researchers effectively study S20-16S rRNA interactions in B. bacteriovorus?

Investigating the interactions between S20 and 16S rRNA in B. bacteriovorus requires a combination of in vitro and in vivo approaches. Researchers should implement the following methodological strategy:

• RNA fragment identification: Based on structural knowledge from other bacteria, design and synthesize B. bacteriovorus 16S rRNA fragments corresponding to helix 44 and other potential binding regions.

• Binding assays:

  • Electrophoretic mobility shift assays (EMSAs) with purified S20 and labeled RNA fragments

  • Filter binding assays to quantify interaction strengths

  • Isothermal titration calorimetry to determine thermodynamic parameters

  • Surface plasmon resonance to measure association and dissociation kinetics

• Structural characterization:

  • Chemical and enzymatic footprinting to identify protected nucleotides

  • SHAPE (Selective 2′-hydroxyl acylation analyzed by primer extension) analysis to detect structural changes upon S20 binding

  • Cryo-electron microscopy of reconstituted 30S subunits with and without S20

  • Nuclear magnetic resonance (NMR) studies if feasible with isotopically labeled components

• In vivo validation:

  • UV crosslinking and immunoprecipitation (CLIP) to capture interactions in living cells

  • Ribosome assembly mapping using sucrose gradient fractionation in wild-type versus S20-depleted strains

  • Point mutations in both S20 and 16S rRNA to validate specific interaction sites

Special attention should be paid to comparing these interactions between attack phase and intraperiplasmic growth phase B. bacteriovorus cells, as ribosome function may be differentially regulated during these distinct physiological states. The data should be integrated to generate a comprehensive model of S20-16S rRNA interactions specific to B. bacteriovorus, highlighting any unique features that may relate to its predatory lifestyle.

What genetic manipulation techniques are most effective for studying S20 function in B. bacteriovorus?

Genetic manipulation of B. bacteriovorus presents unique challenges due to its predatory lifestyle and distinctive biology. For studying S20 function, researchers should consider the following approaches:

• Knockout strategies:

  • Since complete deletion of S20 may be lethal, conditional knockout systems should be employed

  • Tetracycline-inducible expression systems where the native gene is deleted and complemented with an inducible copy

  • CRISPR interference (CRISPRi) for tunable repression of rpsT expression

• Gene replacement approaches:

  • Allelic exchange using suicide vectors carrying homologous recombination regions

  • Markerless deletion systems based on counter-selection markers

  • Introduction of point mutations to study specific functional domains

• Expression systems:

  • IncQ-type plasmids which have been demonstrated to replicate autonomously in B. bacteriovorus

  • Integration of expression constructs into the chromosome for stable expression

• Conjugation protocols:

  • Established conjugation procedures for transferring plasmids from E. coli to B. bacteriovorus

  • Optimization of recipient:donor ratios and media conditions for maximum conjugation efficiency

• Verification methods:

  • PCR verification of genetic modifications

  • Whole-genome sequencing to confirm genetic integrity

  • RT-qPCR to verify expression levels

  • Western blotting to confirm protein production

When manipulating S20, researchers should be prepared for potential growth defects based on observations in other bacterial systems where S20 deletion significantly impairs growth. Working with host-independent strains may facilitate some genetic manipulations, though results should be validated in host-dependent strains when possible. For functional studies, complementation with wild-type S20 should always be performed to verify that phenotypes are specifically due to S20 loss rather than polar effects or secondary mutations.

How should researchers optimize in vitro translation systems to study B. bacteriovorus S20 function?

Developing and optimizing an in vitro translation system specific for B. bacteriovorus would provide a powerful tool for studying S20 function. The following methodological approach is recommended:

• Component preparation:

  • Purification of B. bacteriovorus ribosomes using sucrose gradient ultracentrifugation

  • Isolation of translation factors (initiation factors, elongation factors, release factors) from B. bacteriovorus or closely related species

  • Preparation of aminoacyl-tRNAs specific to B. bacteriovorus codon usage

  • Synthesis of model mRNAs containing B. bacteriovorus gene sequences

• Reconstitution experiments:

  • Development of 30S subunit reconstitution protocols with purified B. bacteriovorus ribosomal proteins and 16S rRNA

  • Comparative reconstitution with and without S20 to assess assembly effects

  • Assembly kinetics measurements using light scattering or fluorescence-based assays

• Functional assays:

  • mRNA binding assays using fluorescently labeled mRNAs and filter binding techniques

  • 50S subunit association assays using light scattering or sucrose gradient analysis

  • Complete translation assays measuring incorporation of labeled amino acids into peptides

  • Toe-printing assays to analyze initiation complex formation

• Comparative analysis:

  • Side-by-side comparison of wild-type versus S20-depleted 30S subunits

  • Complementation experiments adding back purified S20 to depleted subunits

  • Analysis of translation efficiency across different mRNA templates, particularly those derived from predation-specific genes

The system should be optimized for buffer conditions (pH, ionic strength, magnesium concentration) that best reflect the B. bacteriovorus intracellular environment. Special attention should be paid to temperature optimization, as predatory activity and potentially translation efficiency may vary with temperature. The development of such a system would not only enable detailed studies of S20 function but would also provide a valuable tool for investigating the unique features of translation in this predatory bacterium.

What approaches are recommended for investigating the potential role of S20 in B. bacteriovorus stress response?

Investigating the potential role of S20 in B. bacteriovorus stress response requires a systematic approach combining genetics, molecular biology, and physiological studies. The following methodology is recommended:

• Stress exposure experiments:

  • Subject wild-type and S20-depleted strains to various stressors (oxidative, pH, temperature, osmotic, nutrient limitation)

  • Compare survival rates, recovery times, and predatory efficiency post-stress

  • Analyze morphological changes using advanced microscopy techniques

• Transcriptomic and proteomic analysis:

  • Perform RNA-seq under various stress conditions comparing wild-type and S20-depleted strains

  • Conduct quantitative proteomics to identify differentially expressed proteins

  • Use ribosome profiling to assess translational efficiency under stress conditions

• Stress-specific reporter systems:

  • Develop fluorescent reporters for key stress response genes

  • Monitor expression patterns in real-time during stress exposure

  • Compare expression kinetics between wild-type and S20-depleted strains

• Biochemical characterization:

  • Examine post-translational modifications of S20 under stress conditions

  • Assess potential interactions between S20 and stress response regulators

  • Investigate changes in ribosome composition and integrity during stress

• In vivo visualization:

  • Use fluorescently tagged S20 to track localization changes during stress

  • Employ super-resolution microscopy to visualize potential redistribution

  • Combine with prey cell labeling to assess impact on predatory functions

Particular attention should be paid to starvation stress, which may be especially relevant to the predatory lifestyle of B. bacteriovorus, as well as stress conditions that might be encountered during the transition between free-swimming and intraperiplasmic phases. The potential role of S20 in facilitating selective translation of stress-response genes should be thoroughly investigated, as ribosomal proteins in other bacteria have been implicated in stress-specific translational regulation. This research could reveal novel adaptations in the translational machinery that contribute to the remarkable ecological versatility of this predatory bacterium.

What are the most promising future research directions for B. bacteriovorus S20?

The study of B. bacteriovorus S20 protein presents several promising research directions that could significantly advance our understanding of predatory bacteria and ribosomal function. The most compelling future research avenues include:

• Comparative genomics and evolution: Investigating how S20 structure and function have evolved across predatory and non-predatory bacteria could reveal adaptations specific to the predatory lifestyle. This should include analysis of selection pressures on the rpsT gene and identification of predator-specific features.

• Structural biology approaches: Determining the high-resolution structure of B. bacteriovorus S20 in complex with its binding partners, particularly 16S rRNA, would provide invaluable insights into its molecular function. Cryo-electron microscopy of complete B. bacteriovorus ribosomes would be particularly informative.

• Extra-ribosomal functions: Exploring potential moonlighting roles of S20 beyond its canonical function in translation could reveal novel regulatory mechanisms. This includes investigating potential interactions with predation-specific proteins and nucleic acids.

• Translational regulation during predation: Examining how S20 contributes to the reprogramming of translation during the transition between attack phase and intraperiplasmic growth would illuminate a critical aspect of the predatory lifecycle.

• Therapeutic applications: As B. bacteriovorus continues to be explored as a potential living antibiotic, understanding how S20 contributes to predatory efficiency could lead to engineered strains with enhanced therapeutic potential.

These research directions would benefit from the development of improved genetic tools for B. bacteriovorus and the establishment of standardized assays for measuring predatory efficiency. Collaborative approaches combining expertise in structural biology, microbial genetics, and systems biology would be particularly effective in advancing our understanding of this fascinating component of the predatory bacterial machinery.

How might understanding B. bacteriovorus S20 contribute to developing engineered predatory bacteria for therapeutic applications?

Understanding the structure, function, and regulation of B. bacteriovorus S20 could significantly contribute to the development of engineered predatory bacteria for therapeutic applications. The potential contributions include:

The safety profile of engineered B. bacteriovorus strains is paramount for therapeutic applications. The finding that B. bacteriovorus can be engulfed by human phagocytic cells and persist for 24 hours without affecting host cell viability is promising, but further research into long-term effects and potential immune responses is essential. Manipulating S20 and other ribosomal components represents one avenue for fine-tuning the balance between predatory efficiency and safety in these emerging living antibiotics.

Comparative Analysis of S20 Protein Across Different Bacterial Species

*Predicted length based on genomic analysis; exact characterization pending

Expression Profile of B. bacteriovorus Genes Related to Ribosomal Function During Predatory Cycle

Methodological Approaches for Studying S20 Function in B. bacteriovorus