Recombinant Bdellovibrio bacteriovorus 30S ribosomal protein S15 (rpsO)

What is Bdellovibrio bacteriovorus and how does it relate to ribosomal protein studies?

Bdellovibrio bacteriovorus is a small predatory deltaproteobacterium that parasitizes other Gram-negative bacteria through a complex lifecycle. Initially, motile B. bacteriovorus cells collide with host bacteria, attach at the point of contact opposite to their flagellum, and penetrate the host cell wall . The parasite then multiplies intracellularly between the cell wall and plasma membrane of the host, eventually leading to host cell lysis . While primarily known for parasitizing Gram-negative bacteria, certain strains can also attack Gram-positive bacteria such as Streptococcus faecalis and Staphylococcus aureus through an "epibiotic" foraging strategy .

Studying ribosomal proteins like S15 (encoded by the rpsO gene) in B. bacteriovorus is particularly interesting due to the bacterium's unique predatory lifestyle, which may have influenced the evolution of its translation machinery compared to non-predatory bacteria.

How can researchers identify and confirm B. bacteriovorus isolates for rpsO studies?

Proper identification of B. bacteriovorus strains is essential before conducting any studies on specific genes like rpsO. Multiple complementary approaches should be used:

• Plaque assay observation: B. bacteriovorus forms distinctive transparent, round plaques (0.5-0.7 cm diameter) on lawns of prey bacteria that expand after prolonged incubation, differentiating them from bacteriophage plaques .

• PCR amplification: Use B. bacteriovorus-specific primers targeting:

  • 16S rRNA gene (~800 bp fragment for general Bdellovibrio detection)

  • Species-specific 16S rRNA partial sequences (~400 bp for B. bacteriovorus)

  • hit gene (~950 bp), which is relatively specific to B. bacteriovorus

• Sequence analysis: Perform phylogenetic analysis of 16S rRNA sequences using software like MEGA with bootstrap analysis (1,000 replicates) to confirm taxonomic placement .

• Microscopic examination: Gram staining and transmission electron microscopy (TEM) to confirm morphological characteristics - B. bacteriovorus appears as rod-shaped bacteria approximately 0.4-0.5 μm wide and 0.8-0.9 μm long with a single polar flagellum .

What is the comparative structure of ribosomal protein S15 across bacterial species?

The S15 ribosomal protein is relatively conserved across bacterial species, though with important variations that may reflect evolutionary adaptation. While the search results don't provide specific structural information about B. bacteriovorus S15, we can infer key characteristics based on ribosomal protein conservation patterns and available E. coli S15 data.

S15 typically functions in the assembly of the 30S ribosomal subunit and binds directly to 16S rRNA. When comparing S15 proteins between species, researchers should examine:

• Primary sequence conservation: Alignment analysis with homologous S15 proteins from related species

• Secondary structure elements: Alpha helices and beta sheets that contribute to RNA binding

• Key residues: Conservation of amino acids involved in rRNA interactions

• Species-specific variations: Unique residues that may reflect adaptation to B. bacteriovorus' predatory lifestyle

The ability of E. coli S15 to be incorporated into Serratia marcescens ribosomes suggests some functional conservation across species boundaries , which may extend to B. bacteriovorus as well.

What are the optimal expression systems for recombinant B. bacteriovorus rpsO?

Based on established protocols for expressing ribosomal proteins and the specific information about rpsO gene cloning in E. coli , researchers can employ several expression systems:

When choosing an expression system, consider that E. coli rpsO was successfully cloned using pBR322 and incorporated into functional ribosomes in a different species (S. marcescens) . The pRF3 vector, which changes copy number depending on growth temperature in temperature-sensitive polA hosts, has also proven effective for studying ribosomal protein expression dynamics .

How should researchers optimize codon usage for heterologous expression of B. bacteriovorus rpsO?

Codon optimization is critical when expressing B. bacteriovorus genes in E. coli due to potential differences in codon bias between these bacteria. A methodological approach includes:

• Analyze native codon usage: Calculate the codon adaptation index (CAI) of native B. bacteriovorus rpsO sequence.

• Identify rare codons: Map rare E. coli codons in the B. bacteriovorus sequence that might cause translational pausing or premature termination.

• Optimize expression strategy:

  • Option A: Synthesize a codon-optimized gene for E. coli

  • Option B: Co-express rare tRNAs using specialized E. coli strains (e.g., Rosetta™, CodonPlus®)

• Evaluate expression with pilot studies: Compare expression levels between native and optimized sequences.

• Consider selective pressure: The expression of S15 is likely tightly regulated in nature, as demonstrated by the observation that even when plasmid copy number increased more than 20-fold, the relative synthesis rate of S15 only doubled , suggesting sophisticated feedback mechanisms.

What purification strategies yield high-purity B. bacteriovorus ribosomal proteins?

Purifying recombinant S15 protein requires a multi-step approach:

• Cell lysis optimization:

  • Sonication or French press for mechanical disruption

  • Lysozyme treatment (0.1-1 mg/ml) in appropriate buffer systems

  • Addition of nucleases to reduce contamination with nucleic acids

• Initial capture:

  • Immobilized metal affinity chromatography (IMAC) if using His-tagged constructs

  • Ion exchange chromatography based on S15's predicted isoelectric point

• Intermediate purification:

  • Ammonium sulfate precipitation to separate protein fractions

  • Size exclusion chromatography to separate monomeric S15 from aggregates

• Polishing step:

  • Reverse-phase HPLC for highest purity

  • Removal of affinity tags via specific proteases if needed

• Quality control:

  • SDS-PAGE and western blotting to confirm identity and purity

  • Mass spectrometry to verify molecular weight and post-translational modifications

  • Circular dichroism to assess secondary structure integrity

Given that S15 interacts with RNA in its native context, researchers should verify that the purified recombinant protein retains RNA-binding capability through electrophoretic mobility shift assays (EMSA).

How might the predatory lifestyle of B. bacteriovorus influence its ribosomal protein function?

B. bacteriovorus exhibits a unique biphasic lifecycle alternating between free-living attack phase and intraperiplasmic growth phase within its prey. This lifestyle may have placed selective pressures on its translation machinery:

• Rapid protein synthesis requirements: During the intraperiplasmic growth phase, B. bacteriovorus rapidly synthesizes proteins using resources from its host . This may have led to adaptations in its ribosomal proteins, potentially including S15, to optimize translation efficiency under these conditions.

• Host-derived translation components: Research should investigate whether B. bacteriovorus incorporates host-derived ribosomal components or must rely entirely on its own translation machinery during intracellular growth.

• Regulatory adaptations: The observation that S15 synthesis increases only 2-fold despite a 20-fold increase in gene copy number suggests sophisticated regulation mechanisms. In B. bacteriovorus, these regulatory mechanisms may be specially adapted to coordinate rapid growth during the predatory cycle.

• Structural adaptations: Comparative structural analysis of B. bacteriovorus S15 with homologs from non-predatory bacteria might reveal specific adaptations related to its predatory lifestyle.

What experimental approaches can determine S15's role in B. bacteriovorus predation?

Understanding S15's potential role in predation requires targeted experimental designs:

• Gene knockout/knockdown studies:

  • CRISPR-Cas9 genome editing to create conditional S15 mutants

  • Evaluation of mutant phenotypes in predation efficiency assays

  • Complementation studies with native and modified S15 variants

• Localization studies:

  • Fluorescent protein tagging of S15 to track localization during predatory cycle

  • Immunogold labeling for electron microscopy to achieve higher resolution localization

  • FRAP (Fluorescence Recovery After Photobleaching) to study protein dynamics

• Interaction studies:

  • Pull-down assays to identify prey proteins that might interact with B. bacteriovorus S15

  • Two-hybrid screening to detect potential protein-protein interactions

  • Crosslinking mass spectrometry to capture transient interactions

• Comparative expression analysis:

  • RNA-seq to measure rpsO expression changes during different stages of predation

  • Ribosome profiling to assess translation efficiency of rpsO mRNA

  • Quantitative proteomics to measure S15 protein levels throughout the predatory cycle

How can researchers study the potential influence of B. bacteriovorus S15 on prey ribosomes?

Given B. bacteriovorus' predatory nature, researchers might investigate whether its ribosomal proteins interact with prey translation machinery:

• Hybrid ribosome assembly assays:

  • In vitro reconstitution of ribosomes using components from both predator and prey

  • Functional testing of hybrid ribosomes through in vitro translation assays

  • Structural characterization of hybrid ribosomes using cryo-electron microscopy

• Translation interference studies:

  • Introduction of recombinant B. bacteriovorus S15 into prey cells to observe effects

  • Cell-free translation systems to test direct effects on prey ribosomes

  • Competition assays between native prey S15 and B. bacteriovorus S15

• Structural comparison:

  • Comparative modeling of S15 proteins from B. bacteriovorus and common prey species

  • Identification of structural differences that might enable competitive binding

• Evolution analysis:

  • Phylogenetic analysis of S15 sequences across predatory and non-predatory bacteria

  • Selection pressure analysis to identify positively selected residues potentially involved in predation

What methods are appropriate for determining the structure of B. bacteriovorus S15?

Multiple complementary approaches can be used to determine S15 structure:

• X-ray crystallography:

  • Expression and purification of sufficient quantities of recombinant S15 protein

  • Crystallization screening to identify optimal conditions

  • Data collection and structural determination at high resolution

• Nuclear Magnetic Resonance (NMR) spectroscopy:

  • Isotopic labeling (15N, 13C) of recombinant S15

  • Collection of multidimensional NMR data

  • Structure calculation based on distance and angular constraints

• Cryo-electron microscopy:

  • Particularly useful for visualizing S15 in the context of the whole ribosome

  • Single-particle analysis to achieve near-atomic resolution

  • Focused classification to improve resolution of the S15 region

• In silico approaches:

  • Homology modeling based on structures of S15 from related species

  • Molecular dynamics simulations to study conformational flexibility

  • Protein-RNA docking to predict interactions with 16S rRNA

How can researchers assess RNA-binding properties of recombinant B. bacteriovorus S15?

S15 primarily functions through RNA interactions, necessitating specialized techniques to characterize these interactions:

• Electrophoretic Mobility Shift Assays (EMSA):

  • Titration of labeled RNA fragments with increasing concentrations of S15

  • Determination of binding constants and cooperativity

  • Competition assays to assess binding specificity

• Surface Plasmon Resonance (SPR):

  • Real-time monitoring of S15-RNA interactions

  • Determination of association and dissociation rates

  • Analysis of the effect of buffer conditions on binding kinetics

• Isothermal Titration Calorimetry (ITC):

  • Measurement of thermodynamic parameters of binding

  • Elucidation of the enthalpy-entropy balance of the interaction

  • Assessment of binding stoichiometry

• Fluorescence-based methods:

  • Fluorescence anisotropy measurements with labeled RNA

  • FRET experiments to measure distances between labeled S15 and RNA

  • Microscale thermophoresis for binding affinity determination

• Hydroxyl radical footprinting:

  • Identification of RNA regions protected by S15 binding

  • Mapping of the interaction interface at nucleotide resolution

How does B. bacteriovorus S15 compare to its homologs in prey bacteria?

Comparative analysis between predator and prey S15 proteins can reveal evolutionary adaptations:

A thorough comparative analysis would involve examining S15 proteins from a range of bacteria including B. bacteriovorus, common prey species (E. coli, Pseudomonas aeruginosa), and other predatory bacteria to identify convergent evolutionary adaptations.

What insights can be gained from studying ribosomal proteins in predatory bacteria?

Ribosomal proteins from predatory bacteria like B. bacteriovorus offer unique perspectives on bacterial evolution:

• Adaptation to predatory lifestyle:

  • Comparative genomics to identify signatures of selection in ribosomal genes

  • Investigation of translation efficiency during rapid growth inside prey

  • Assessment of potential dual functions of ribosomal proteins in predation

• Host-predator coevolution:

  • Analysis of ribosomal protein evolution rates in predator-prey pairs

  • Investigation of whether predatory bacteria target prey ribosomes during attack

  • Identification of potential resistance mechanisms in prey ribosomes

• Biotechnological applications:

  • Engineering of B. bacteriovorus ribosomes for specialized translation

  • Development of inhibitors targeting prey-specific ribosomal features

  • Exploration of B. bacteriovorus as a "living antibiotic"

• Cross-species compatibility:

  • Testing whether B. bacteriovorus S15 can function in heterologous systems

  • Investigation of the minimal set of species-specific features required for function

  • Development of hybrid ribosomes with enhanced properties

What are the most promising research avenues for B. bacteriovorus ribosomal proteins?

Several high-priority research directions emerge from current knowledge:

• Structural biology approaches to determine high-resolution structures of B. bacteriovorus ribosomal proteins and complete ribosomes, particularly comparing attack phase and growth phase configurations.

• Systems biology studies examining the dynamics of ribosome assembly and function during the predatory lifecycle, potentially revealing phase-specific adaptations.

• Synthetic biology applications exploring whether unique features of B. bacteriovorus ribosomes can be harnessed for biotechnological purposes, such as specialized translation systems.

• Comparative genomics across predatory bacteria to identify convergent adaptations in translation machinery related to predation.

• Therapeutic applications investigating whether B. bacteriovorus ribosomal components could serve as targets for enhancing or regulating its activity as a "living antibiotic" .