Recombinant Bdellovibrio bacteriovorus 50S ribosomal protein L19 (rplS)

Basic Research Questions

• How does the B. bacteriovorus genome organization relate to rplS expression?

  B. bacteriovorus HD100 has a relatively large genome of approximately 3.8 Mb containing over 3,580 protein-coding genes . About one-third of these genes encode novel hypothetical proteins adapted for the unique predatory lifestyle . The genome's size reflects the need for B. bacteriovorus to survive in both free-living stages between predation events and during prey invasion.

  While specific information about rplS genomic organization is limited in the literature, ribosomal proteins in bacteria are typically found in conserved operons and are often co-transcribed. Given the predatory lifecycle of B. bacteriovorus, with distinct attack and growth phases, rplS expression likely varies during different stages of predation. The bacterium's specialized secretome (containing approximately 42.4% of its proteome) indicates complex regulation of protein expression during its predatory lifecycle .

• What methodologies are available for heterologous expression of B. bacteriovorus proteins?

  Several genetic manipulation techniques have been developed for B. bacteriovorus protein expression:

  • Homologous recombination: Used to create knock-out or knock-in mutants to elucidate biological functions

  • Plasmid-based systems: Derivative ori RSF1010 plasmids have been employed to complement mutations

  • Golden Gate (GS) assembly system: Allows for sequential ligation of genetic parts (promoters, ribosome binding sites, coding sequences, and terminators) to generate complete transcription units

  • Transposon Tn7-mediated chromosomal insertion: Enables monocopy gene expression in the predator

  Conjugation procedures typically involve tri- or tetraparental matings, where:

  1. B. bacteriovorus HD100 is co-cultured in Hepes+ for 24h using E. coli as prey

  2. Filtration through 0.45μm filters

  3. Mixing with donor strains carrying the plasmid construct and helper strains

  4. Plating on nitrocellulose membranes and selection using appropriate antibiotics

• What are the optimal conditions for culturing B. bacteriovorus for protein studies?

  B. bacteriovorus cultivation requires specific protocols due to its predatory lifestyle:

  For predatory growth:

  • Harvest stationary-phase prey bacteria by centrifugation

  • Wash prey in buffer containing 3 mM ammonium acetate, 3 mM CaCl₂, and 3 mM MgCl₂ (pH 7.5)

  • Resuspend to an optical density of 1.0 at 588 nm

  • Inoculate with B. bacteriovorus and incubate at 30°C with shaking until prey lysis is complete

  For host-independent variants:

  • Grow on peptone-yeast extract medium (ATCC 526) at 30°C for 3-5 days

  • Note: Cultures should be passaged a maximum of 6 times to preserve wild-type characteristics

  Environmental parameters affecting growth:

  • Temperature range: 15-50°C (optimal around 30°C)

  • pH range: 5.0-9.0 (optimal between 7.0-7.5)

  • Salinity tolerance: Up to 1.0%

Advanced Research Questions

• What techniques can differentiate between prey and predator proteins when working with B. bacteriovorus?

  Obtaining pure B. bacteriovorus proteins requires specialized techniques to eliminate prey contamination:

  Purification protocol:

  1. Differential sedimentation

  2. Centrifugation in a linear 2-15% Ficoll gradient to remove remaining prey cells and bdelloplasts

  3. Washing purified cells in 10 mM HEPES (pH 7.5)

  Membrane protein isolation:

  1. Cell disruption via supersonication (50 W, 50% duty cycle) at 4°C for 15 minutes

  2. Removal of unbroken cells by centrifugation at 10,000×g

  3. Carbonate extraction protocol modified from Molloy et al.

  Protein analysis:

  • SDS-PAGE following Laemmli method on 12% polyacrylamide gels

  • Visualization with Coomassie brilliant blue R-250

  • Mass spectrometric analysis for protein identification

  These techniques are critical for accurate characterization of B. bacteriovorus rplS without prey protein contamination.

• How can structural studies of B. bacteriovorus rplS inform understanding of ribosomal evolution?

  Structural studies of B. bacteriovorus rplS can provide insights into ribosomal evolution, particularly given B. bacteriovorus' unique phylogenetic position. Recently reclassified from Deltaproteobacteria to class Oligoflexia (with a proposal to place it in a distinct phylum Bdellovibrionota) , this organism offers a valuable perspective on ribosomal protein evolution.

  Methodological approaches include:

  Comparative sequence analysis:

  • Multiple sequence alignment of rplS across bacterial phyla

  • Identification of conserved domains and variable regions

  • Phylogenetic tree construction to trace evolutionary relationships

  Structural determination:

  • X-ray crystallography of purified recombinant rplS

  • Cryo-electron microscopy of intact ribosomes

  • In silico structural modeling using homology-based approaches

  Based on studies of related organisms, differences in rplS structure could reflect adaptations to B. bacteriovorus' predatory lifestyle, particularly in regions that interact with rRNA or other ribosomal proteins at the 30S-50S interface.

• What molecular mechanisms might control rplS expression during the B. bacteriovorus lifecycle?

  B. bacteriovorus has a complex lifecycle with distinct phases that likely require differential regulation of protein synthesis:

  Attack phase regulation:

  • B. bacteriovorus cells place a replication block when external between predation events

  • Ribosome activity is presumably tightly regulated during this non-replicative phase

  • Cyclic di-GMP signaling has been identified as a key regulator of the transition between predatory and axenic growth

  Growth phase regulation:

  • During predation, a complete chromosome replication cycle is initiated

  • Recent findings show that replication can stall if the prey provides insufficient resources, completing only during subsequent predation events

  • This suggests sophisticated coordination between nutrient availability, protein synthesis, and DNA replication

  Regulatory mechanisms:

  • Transcriptional control: Likely involves specific sigma factors responsive to predation cues

  • Post-transcriptional regulation: May include small RNAs targeting rplS mRNA

  • Post-translational modifications: Potentially regulating rplS activity during different lifecycle stages

  Understanding rplS regulation could provide insights into how B. bacteriovorus coordinates ribosome assembly and function during its biphasic lifestyle.

• How can gene knockout and complementation studies elucidate rplS function in B. bacteriovorus?

  Genetic manipulation studies can help determine if rplS has specialized functions in B. bacteriovorus beyond its canonical ribosomal role:

  Knockout strategy:

  • Since rplS is likely essential, conditional knockout systems would be necessary

  • Inducible antisense RNA expression to gradually deplete rplS

  • CRISPR interference (CRISPRi) for targeted transcriptional repression

  Complementation approaches:

  • Expression of wild-type rplS from plasmids using the Golden Gate assembly system

  • Site-directed mutagenesis to create point mutations in functional domains

  • Chimeric constructs with rplS from non-predatory bacteria to identify predation-specific features

  Phenotypic analysis:

  • Predation efficiency using double-layer plaque assays

  • Growth kinetics monitoring prey optical density reduction over time

  • Microscopic observation of predator motility and prey invasion

  • Translation fidelity assays to detect changes in protein synthesis accuracy

  Recent advances in B. bacteriovorus genetic tools, including the development of destination vectors that adapt SEVA plasmids and Tn7-based chromosomal insertion systems , make such studies increasingly feasible.

• What interactome studies could reveal the protein-protein interactions of rplS in B. bacteriovorus ribosomes?

  Understanding the interactome of rplS would provide insights into its function within the B. bacteriovorus ribosome and potentially reveal predator-specific interactions:

  Experimental approaches:

  1. Affinity purification coupled with mass spectrometry (AP-MS):

     • Express tagged versions of rplS (similar to the mCherry tagging approach used for other B. bacteriovorus proteins )

     • Purify rplS complexes under native conditions

     • Identify co-purifying proteins by mass spectrometry

  2. Crosslinking coupled with mass spectrometry (XL-MS):

     • Chemically crosslink intact ribosomes isolated from B. bacteriovorus

     • Digest and analyze to identify proteins in proximity to rplS

     • Map interaction sites at amino acid resolution

  3. Cryo-electron microscopy:

     • Determine the structure of intact B. bacteriovorus ribosomes

     • Compare with ribosomes from non-predatory bacteria

     • Identify predator-specific structural features around the rplS binding site

  4. Bacterial two-hybrid screening:

     • Use rplS as bait to screen for interacting partners

     • Validate interactions with co-immunoprecipitation

     • Map interaction domains through truncation analysis

  These studies could reveal if rplS has acquired predator-specific interactions that contribute to B. bacteriovorus' unique lifecycle.

• How might rplS contribute to the translational regulation during prey invasion and consumption?

  The predatory lifecycle of B. bacteriovorus involves dramatic physiological changes that likely require translational reprogramming:

  Potential roles of rplS in predation:

  1. Selective translation during attack phase:

     • B. bacteriovorus may require specialized ribosomes for translating predation-specific mRNAs

     • rplS modifications might alter ribosome specificity during different predatory stages

  2. Ribosome adaptation to prey-derived nutrients:

     • B. bacteriovorus cannot synthesize some amino acids and relies on prey components

     • rplS might help coordinate translation efficiency based on available resources

  3. Translation during bdelloplast formation:

     • The transition from free-living to intraperiplasmic growth involves substantial gene expression changes

     • rplS could participate in translation regulation during this critical phase

  Research approaches:

  • Ribosome profiling at different stages of the predatory cycle

  • Quantitative proteomics to measure rplS abundance during predation

  • mRNA association studies to identify transcripts preferentially translated by rplS-containing ribosomes

  Understanding rplS contribution to translational regulation could provide insights into how B. bacteriovorus coordinates its complex lifecycle and efficiently utilizes prey resources.

• What are the implications of the unfolded protein response for recombinant production of B. bacteriovorus rplS?

  Ribosomal protein L19 overexpression has been shown to activate the unfolded protein response (UPR) in eukaryotic cells , which has important implications for recombinant production of B. bacteriovorus rplS:

  Challenges in recombinant expression:

  1. UPR activation:

     • Overexpression of rplS may trigger stress responses in host cells

     • In MCF7 cells, L19 overexpression sensitizes cells to endoplasmic reticulum stress-induced death

     • Similar stress responses might occur in prokaryotic expression systems

  2. Protein solubility and folding:

     • As a ribosomal protein, rplS normally exists in complex with rRNA and other proteins

     • Isolated expression may result in improper folding or aggregation

  Optimization strategies:

  1. Expression system selection:

     • E. coli has been successfully used for B. bacteriovorus ribosomal protein L7/L12

     • Baculovirus systems might be appropriate for complex proteins as shown for other ribosomal proteins

  2. Expression conditions:

     • Lower temperature to reduce aggregation (20-25°C)

     • Reduced inducer concentration to moderate expression rate

     • Co-expression with chaperones to assist folding

  3. Fusion tags and solubility enhancers:

     • N-terminal tags like MBP, SUMO, or Thioredoxin can improve solubility

     • Optimization of buffer conditions for purification and storage

  4. Reconstitution conditions:

     • Following the protocol used for other B. bacteriovorus ribosomal proteins:

       • Reconstitution in deionized sterile water (0.1-1.0 mg/mL)

       • Addition of 5-50% glycerol as cryoprotectant

       • Storage at -20°C/-80°C for long-term stability

  Understanding these challenges and implementing appropriate strategies will be crucial for successful production of functional recombinant B. bacteriovorus rplS.

• How can advanced bioinformatic approaches enhance our understanding of B. bacteriovorus rplS evolution and function?

  Bioinformatic analyses can provide valuable insights into the evolution and specialized functions of B. bacteriovorus rplS:

  Comparative genomics approaches:

  1. Phylogenetic analysis:

     • Comparing rplS sequences across the bacterial kingdom, with special focus on:

       • Other predatory bacteria (e.g., Micavibrio aeruginosavorus)

       • Close non-predatory relatives

       • Diverse Gram-negative prey species

     • This could reveal selection pressures specific to predatory bacteria

  2. Structural prediction and analysis:

     • Homology modeling based on known ribosomal L19 structures

     • Identification of conserved functional domains versus variable regions

     • Molecular dynamics simulations to predict rplS interactions with rRNA and other proteins

  3. Coevolution analysis:

     • Identification of coordinated evolutionary changes between rplS and interacting partners

     • Detection of potential compensatory mutations that maintain ribosome function

  4. Transcriptomic integration:

     • Analysis of expression patterns during the predatory lifecycle

     • Correlation with expression of other genes to identify co-regulated networks

  Research implications:

  These analyses could identify:

  • Predator-specific adaptations in rplS

  • Potential moonlighting functions beyond the ribosome

  • Targets for experimental validation through site-directed mutagenesis

  By combining advanced bioinformatics with experimental approaches, researchers can gain a comprehensive understanding of how rplS contributes to B. bacteriovorus' unique predatory lifestyle.