Recombinant Bdellovibrio bacteriovorus 50S ribosomal protein L23 (rplW)

What is the function of ribosomal protein L23 in Bdellovibrio bacteriovorus?

L23 in B. bacteriovorus functions as a critical component of the 50S ribosomal subunit. Based on studies in other bacterial species, L23 serves as a chaperone docking site on the ribosome, directly linking protein biosynthesis with chaperone-assisted protein folding . It's positioned at the exit of the peptide tunnel, where nascent polypeptides emerge from the ribosome.

Methodological approach for functional characterization:

• Cross-linking studies to identify interactions between L23 and potential binding partners

• Mutational analysis of exposed residues to determine critical regions for chaperone binding

• Comparative genomic analysis with L23 proteins from non-predatory bacteria

What is the structure and location of L23 within the ribosome?

L23 is one of the proteins that surrounds the polypeptide exit tunnel on the outside of the ribosome . It belongs to the universal ribosomal protein uL23 family, which is highly conserved across bacterial species. Though the specific structure of B. bacteriovorus L23 has not been fully determined, structural studies of bacterial ribosomes show that L23 forms a critical part of the nascent peptide exit site.

Methodological approaches for structural determination:

• Cryo-electron microscopy of intact B. bacteriovorus ribosomes

• X-ray crystallography of purified recombinant L23

• Homology modeling based on structurally characterized L23 proteins from related bacteria

How does L23 contribute to ribosome assembly?

L23 is classified as one of the early assembly proteins during ribosome biogenesis . It binds directly to 23S rRNA and plays a crucial role in the hierarchical assembly of the 50S ribosomal subunit. Based on studies of ribosome assembly intermediates , L23 likely binds during the initial stages of assembly, creating binding sites or promoting proper conformations for later-binding proteins.

Assembly pathway analysis methods:

• In vitro reconstitution assays with purified components

• Time-resolved structural analysis of assembly intermediates

• Depletion studies to observe accumulation of specific precursors

What is the role of L23 in the context of B. bacteriovorus predation?

While the specific role of L23 in predation has not been directly studied, ribosomal proteins are essential for the predatory lifecycle of B. bacteriovorus. During the invasion and replication within prey bacteria, B. bacteriovorus must rapidly synthesize proteins, making functional ribosomes critical for successful predation . L23, as a core ribosomal component, would be essential for this process.

Methodological approaches to study L23 in predation:

• Gene expression analysis during different stages of the predatory cycle

• Conditional mutants to examine effects on predation efficiency

• Transposon sequencing to assess gene essentiality during predation

What is the molecular weight and sequence characteristics of B. bacteriovorus L23?

The specific molecular weight of B. bacteriovorus L23 is not directly reported in the literature, but based on homologous proteins, it likely has a molecular weight of approximately 10-15 kDa . The protein belongs to the uL23 family and would be expected to share the conserved structural features of this family while potentially having sequence adaptations specific to B. bacteriovorus.

How does the sequence and structure of L23 from B. bacteriovorus compare to L23 from other bacteria?

Methodological approach for comparative analysis:

• Multiple sequence alignment of L23 proteins from diverse bacterial species

• Phylogenetic analysis to assess evolutionary relationships

• Structure-based comparison of conserved versus variable regions

• Functional complementation studies between different bacterial species

What methods are optimal for expression and purification of recombinant B. bacteriovorus L23?

Based on approaches used for other B. bacteriovorus ribosomal proteins , recombinant L23 would typically be expressed in E. coli systems with appropriate affinity tags. Ribosomal proteins often present purification challenges due to their high isoelectric points and RNA-binding properties.

Recommended purification protocol:

• Clone the rplW gene into an expression vector with an N-terminal His-tag

• Transform into E. coli BL21(DE3) or similar expression strain

• Induce expression at low temperature (16-18°C) to enhance solubility

• Lyse cells in high-salt buffer to disrupt RNA-protein interactions

• Purify using nickel affinity chromatography followed by size exclusion chromatography

• Include RNase treatment if RNA contamination is observed

• Verify purity by SDS-PAGE and functionality through in vitro binding assays

Common challenges and solutions:

How can advanced structural studies of L23 inform our understanding of B. bacteriovorus ribosome function?

Structural studies of L23 can provide insights into ribosome evolution and function specific to this predatory bacterium. Based on cryo-EM studies of ribosomal assembly intermediates , the structure of L23 and its interactions with other components can reveal crucial information about ribosome biogenesis and function.

Advanced structural characterization methods:

• High-resolution cryo-EM of B. bacteriovorus ribosomes at different functional states

• Hydrogen-deuterium exchange mass spectrometry to map dynamic regions

• Cross-linking mass spectrometry to identify interaction partners

• NMR studies of L23 dynamics and binding interactions

What experimental approaches can be used to study the interaction of L23 with chaperones specific to B. bacteriovorus?

Building on studies from E. coli where L23 interacts with Trigger Factor , similar approaches can be applied to identify and characterize potential chaperone interactions in B. bacteriovorus.

Methodological approaches:

• Protein-protein interaction analysis:

  • Co-immunoprecipitation coupled with mass spectrometry

  • Surface plasmon resonance to measure binding kinetics

  • Bacterial two-hybrid screening to identify interaction partners

• Structural characterization:

  • Cryo-EM of ribosome-chaperone complexes

  • X-ray crystallography of L23 with bound chaperone domains

  • NMR titration experiments to map binding interfaces

• Functional validation:

  • Site-directed mutagenesis of predicted interaction sites

  • In vitro translation assays with purified components

  • In vivo complementation studies with L23 variants

How can genetic manipulation of the rplW gene provide insights into B. bacteriovorus predatory mechanisms?

Genetic studies of the rplW gene can help determine its importance in the predatory lifecycle. Using techniques such as those described for high-throughput genetic analysis in B. bacteriovorus , researchers can assess the impact of L23 modifications on predation efficiency.

Experimental approaches:

• Creation of conditional L23 mutants using inducible promoters

• Site-directed mutagenesis to alter specific functional domains

• CRISPR-Cas9 genome editing to introduce specific modifications

• Complementation studies with L23 variants to restore predatory function

What role might L23 play in the late-stage assembly of the B. bacteriovorus 50S ribosomal subunit?

While L23 is considered an early assembly protein , understanding its potential role in the later stages of ribosome assembly is also important. Studies on late-stage assembly intermediates of the bacterial 50S ribosomal subunit can inform research on B. bacteriovorus-specific assembly processes.

Research methodology:

• Isolation and characterization of assembly intermediates

• Pulse-chase experiments to track L23 incorporation into ribosomal particles

• Quantitative mass spectrometry of ribosome assembly intermediates

• Cryo-EM structural analysis of assembly states

The assembly of late-stage proteins (L28, L16, L33, L36, L35) has been identified as a bottleneck in ribosome assembly , as shown in this table of underrepresented proteins in 45S particles:

How might L23 contribute to antibiotic resistance or sensitivity in B. bacteriovorus?

Given that B. bacteriovorus is being studied as a potential "living antibiotic" , understanding how its own ribosomal components interact with antibiotics is important. L23, positioned near the peptide exit tunnel, could play a role in antibiotic sensitivity or resistance.

Experimental approaches:

• Comparative sequence analysis of L23 from antibiotic-resistant versus sensitive strains

• Structural studies of B. bacteriovorus ribosomes with bound antibiotics

• Site-directed mutagenesis of key residues to alter antibiotic binding

• Minimum inhibitory concentration (MIC) testing of B. bacteriovorus strains with L23 variants

• In vitro translation assays with purified components to assess drug effects

What are the best methods for formulating research questions regarding L23 in B. bacteriovorus?

Following principles of effective research question formulation , studies on L23 should consider:

• Feasibility: Ensure questions can be answered with available technology and resources

• Interest: Address gaps in current knowledge about predatory bacteria

• Novelty: Focus on unique aspects of L23 in the context of bacterial predation

• Relevance: Connect to broader understanding of ribosome function or antibiotic resistance

Recommended approach for developing L23 research questions:

• Start by identifying specific aspects of interest (structure, function, interactions)

• Conduct preliminary research on that subject to identify knowledge gaps

• Define what still needs to be known

• Assess the implied questions for FINER criteria (Feasible, Interesting, Novel, Ethical, Relevant)

What qualitative research methods are applicable to studying L23 function?

In addition to quantitative approaches, qualitative methods can provide valuable insights into L23 function . These approaches help researchers understand the "how" and "why" of biological processes.

Applicable qualitative methods:

• Interpretative phenomenological analysis to understand the meaning of observed phenomena

• Standpoint theory approaches to examine L23 from different theoretical perspectives

• Reflexive analysis of experimental results to account for researcher biases

• Mixed methods research combining qualitative and quantitative approaches

What are optimal protocols for studying L23 in the context of predatory lifecycle?

Building on high-throughput methods used to study B. bacteriovorus , specific protocols can be developed to investigate L23 during predation:

• Temporal expression analysis:

  • qRT-PCR of rplW gene expression during predatory cycle

  • Ribosome profiling at different stages of predation

  • Proteomics analysis of L23 abundance and modifications

• Predation efficiency assays:

  • Co-culture experiments with L23 variants and prey bacteria

  • Microscopy-based tracking of predation stages

  • Viability assays measuring predator and prey populations over time

• Ribosome functionality tests:

  • In vitro translation assays with ribosomes from different predation stages

  • Structural analysis of ribosomes during predation

  • Antibiotic susceptibility testing during predatory growth

How can L23 research contribute to understanding ribosome evolution in predatory bacteria?

The study of L23 in B. bacteriovorus offers opportunities to understand ribosomal adaptation in the context of a predatory lifestyle. Comparative analyses with non-predatory bacteria can reveal evolutionary adaptations specific to predation.

Research approaches:

• Phylogenetic analysis of L23 sequences across bacterial predators

• Structural comparisons to identify predator-specific adaptations

• Functional studies to determine if L23 has acquired predator-specific roles

• Computational modeling of evolutionary constraints on L23 in predatory contexts

What potential applications exist for engineered B. bacteriovorus L23 variants?

Engineered L23 variants could potentially enhance B. bacteriovorus predation efficiency or specificity, which has implications for its use as a "living antibiotic" .

Potential applications:

• Enhanced predation efficiency through optimized ribosome function

• Altered prey specificity by modifying protein synthesis capabilities

• Increased resistance to antibiotics that might be present in treatment environments

• Improved stability for therapeutic formulations

How might systems biology approaches enhance our understanding of L23 in the context of the B. bacteriovorus proteome?

Systems biology approaches can place L23 function in the broader context of cellular networks within B. bacteriovorus.

Recommended approaches:

• Network analysis of L23 interactions with other cellular components

• Integration of transcriptomic, proteomic, and metabolomic data

• Mathematical modeling of ribosome assembly pathways

• Constraint-based modeling of protein synthesis during predation