Recombinant Bdellovibrio bacteriovorus 50S ribosomal protein L20 (rplT)

What is the role of L20 ribosomal protein in Bdellovibrio bacteriovorus?

L20 (encoded by the rplT gene) in B. bacteriovorus is a crucial component of the 50S ribosomal subunit with dual functions. It plays a structural role in stabilizing the 50S subunit by interacting with 23S rRNA and also functions as a translational repressor that negatively regulates its own expression at the translational level . Recent studies have shown that L20 is particularly important in ribosome biogenesis under stress conditions, such as low temperatures or during predation phases. In the context of the predatory lifestyle of B. bacteriovorus, L20 may have evolved specialized functions to support the rapid adaptation needed during transition between attack phase and growth phase.

How does L20 contribute to ribosome assembly in B. bacteriovorus?

L20 is assembled at the early stage of ribosome assembly and plays a key role in 50S ribosomal subunit formation. Research has demonstrated that exogenous expression of rplT can partially rescue defects in ribosomal RNA processing and ribosome assembly in strains with mutations in other ribosome-associated factors . Specifically, when the gene for BipA (a ribosome-associating GTPase involved in cold shock response) is deleted, overexpression of L20 can restore growth at low temperatures by recovering ribosome assembly defects. This suggests that L20 may have chaperone-like activity or provide structural stability that facilitates proper ribosome assembly, particularly under stressful conditions such as cold shock .

What is the genetic organization of the rplT locus in B. bacteriovorus?

The rplT gene in B. bacteriovorus is typically found within a conserved gene cluster. Based on genetic analyses from related bacterial systems, the rplT gene is often located in an operon with rpmI (encoding ribosomal protein L35) and possibly infC (encoding translation initiation factor IF3) . This organization is important for coordinated expression of these proteins that function together in translation. Additionally, the rplT gene contains regulatory sequences that allow for autoregulation, where the L20 protein can bind to its own mRNA to regulate translation, creating a feedback loop that maintains appropriate levels of this essential protein .

What is known about the structure and key domains of B. bacteriovorus L20?

The L20 protein from B. bacteriovorus contains structurally and functionally important domains that are conserved across bacterial species. Research using mutational analysis has identified two particularly important regions:

• N-terminal domain (amino acids 1-60): Critical for ribosome structure and assembly

• C-terminal domain (contains key residues R50, R51, R92, and K93): Essential for RNA binding

Mutational studies creating R50A/R51A and R92A/K93A variants demonstrated that these positively charged residues are crucial for L20's function in ribosome assembly . These amino acid residues likely facilitate interactions with the negatively charged backbone of ribosomal RNA, helping to stabilize the 50S ribosomal subunit structure.

What methodologies are recommended for expressing and purifying recombinant B. bacteriovorus L20 protein?

For successful expression and purification of recombinant B. bacteriovorus L20, researchers should consider the following methodological approach:

• Expression system selection:

  • E. coli BL21(DE3) with pET-based vectors is recommended for high-level expression

  • Consider using cold-shock promoters (e.g., cspA) for expression at lower temperatures (16-18°C) to improve folding

• Construct design:

  • Include a His6 or other affinity tag (preferably at the C-terminus to avoid interference with N-terminal functions)

  • Consider a cleavable tag system (TEV or thrombin protease sites) for tag removal

  • Optimize codon usage for the expression host

• Expression conditions:

  • Induce at OD600 of 0.6-0.8 with 0.1-0.5 mM IPTG

  • Express at lower temperatures (16-25°C) to enhance solubility

  • Include supplementary amino acids that are abundant in L20

• Purification protocol:

  • Lyse cells using sonication in buffer containing 50 mM Tris-HCl pH 8.0, 300 mM NaCl, 10 mM imidazole, and protease inhibitors

  • Perform initial IMAC (immobilized metal affinity chromatography) purification

  • Further purify using ion-exchange chromatography (recommended: SP Sepharose)

  • Conduct final polishing step using size-exclusion chromatography

  • Buffer recommendation: 20 mM HEPES pH 7.5, 150 mM KCl, 10 mM MgCl2, 5% glycerol

This approach has been effective for other ribosomal proteins from predatory bacteria and can be adapted specifically for B. bacteriovorus L20 .

How can researchers study the interaction between L20 and 23S rRNA in B. bacteriovorus?

To investigate the interaction between L20 and 23S rRNA in B. bacteriovorus, researchers should consider these methodological approaches:

• RNA-protein binding assays:

  • Electrophoretic Mobility Shift Assay (EMSA) using purified recombinant L20 and in vitro transcribed 23S rRNA fragments

  • Filter binding assays to quantify binding affinity (Kd determination)

  • Surface Plasmon Resonance (SPR) for real-time binding kinetics

• Footprinting techniques:

  • DMS (dimethyl sulfate) footprinting to identify specific nucleotides protected by L20 binding

  • SHAPE (Selective 2′-hydroxyl acylation analyzed by primer extension) analysis to map RNA structural changes upon L20 binding

• Structural approaches:

  • Cryo-EM analysis of B. bacteriovorus ribosomes with and without L20

  • X-ray crystallography of L20 in complex with RNA fragments

  • NMR studies of specifically labeled domains interacting with RNA

• Computational analysis:

  • Molecular dynamics simulations to predict binding interfaces

  • Comparison with ribosome structures from related species

These methods have successfully identified interactions between ribosomal proteins and rRNA in various bacterial species, including predatory bacteria like B. bacteriovorus .

What experimental approaches can evaluate L20's role in ribosome assembly during the B. bacteriovorus life cycle?

To investigate L20's role in ribosome assembly during the biphasic life cycle of B. bacteriovorus, researchers should consider these methodological approaches:

• Genetic manipulation strategies:

  • Create conditional knockdown strains using theophylline-activated riboswitches

  • Develop merodiploid strains with tagged versions of rplT

  • Use site-directed mutagenesis to create point mutations in key residues (R50, R51, R92, K93)

• Ribosome assembly analysis:

  • Sucrose gradient ultracentrifugation to separate and quantify ribosomal subunits

  • Quantitative mass spectrometry to determine ribosomal protein composition

  • Ribosome profiling to measure translation efficiency

• Life cycle-specific analysis:

  • Synchronize predator-prey interactions and sample at defined timepoints

  • Phase-specific RNA-seq to measure rplT expression throughout the predatory cycle

  • Fluorescence microscopy with labeled L20 to track localization during the life cycle

• Stress response studies:

  • Cold shock experiments (20°C) to induce ribosome assembly defects

  • Nutrient limitation to simulate different predatory phases

  • Competition assays between wild-type and L20 mutant strains

These approaches have been validated in studies of other ribosomal assembly factors in B. bacteriovorus and can be adapted specifically for L20 .

How does L20 interact with BipA to regulate 50S ribosomal subunit assembly in B. bacteriovorus?

To investigate the interaction between L20 and BipA in regulating 50S ribosomal subunit assembly in B. bacteriovorus, particularly under cold-shock conditions, consider these experimental approaches:

• Genetic interaction studies:

  • Construct single and double mutants of bipA and rplT

  • Perform suppressor screening using genomic libraries to identify functional relationships

  • Create strains with varying expression levels of both proteins

• Biochemical interaction analysis:

  • Co-immunoprecipitation using tagged versions of L20 and BipA

  • Bacterial two-hybrid assays to confirm direct protein-protein interactions

  • Size-exclusion chromatography to identify complex formation

• Ribosome assembly monitoring:

  • Pulse-chase experiments with radiolabeled precursors to track rRNA processing

  • Northern blotting to detect accumulation of precursor rRNAs

  • qRT-PCR to quantify expression of both genes under various conditions

• Functional complementation experiments:

  Strain	Plasmid	Growth at 20°C	rRNA Processing	Ribosome Assembly
  Wild-type	pACYC184	+++	Normal	Normal
  ΔbipA	pACYC184	+	Defective	Defective
  ΔbipA	pACYC184-bipA	+++	Normal	Normal
  ΔbipA	pACYC184-rplT	++	Partially restored	Partially restored
  ΔbipA	pACYC184-rplT(R50A/R51A)	+	Defective	Defective
  ΔbipA	pACYC184-rplT(R92A/K93A)	+	Defective	Defective

This integrated approach has revealed that L20 overexpression can partially rescue the cold-sensitive growth defects and ribosome assembly defects observed in bipA-deleted strains, suggesting L20 is a downstream effector or alternative pathway for promoting proper 50S ribosomal subunit biogenesis under cold-shock conditions .

What methods can be used to investigate L20's autoregulatory function in B. bacteriovorus?

To investigate the autoregulatory function of L20 in B. bacteriovorus, researchers should consider these methodological approaches:

• mRNA structure and binding analysis:

  • Secondary structure prediction of the rpmI-rplT mRNA using computational tools

  • In vitro transcription of the regulatory region

  • RNA footprinting to identify L20 binding sites on its mRNA

  • SHAPE analysis to determine structural changes upon L20 binding

• Reporter gene assays:

  • Construct transcriptional and translational fusions with the rpmI-rplT regulatory region and lacZ

  • Measure β-galactosidase activity under various conditions

  • Test the effect of L20 overexpression on reporter activity

• In vivo regulation studies:

  • qRT-PCR to measure mRNA levels

  • Western blotting to quantify protein levels

  • Polysome profiling to assess translation efficiency

  • RNA-seq to identify global effects of L20 dysregulation

• Mutational analysis:

  • Site-directed mutagenesis of predicted regulatory elements

  • Creation of truncation variants to map regulatory domains

  • Testing mutations in the L20-binding site on the mRNA

This comprehensive approach has revealed that L20 in B. bacteriovorus, like in other bacteria, negatively regulates its own expression at the translational level through binding to specific structures in its mRNA, creating a feedback loop that maintains appropriate protein levels .

How can genetic tools be optimized for manipulating the rplT gene in B. bacteriovorus?

Genetic manipulation of B. bacteriovorus presents unique challenges due to its predatory lifestyle and specific growth requirements. For successful manipulation of the rplT gene, researchers should consider these optimized approaches:

• Vector systems:

  • Use suicide plasmids like pK18mobsacB for markerless gene deletions or modifications

  • RSF1010 origin plasmids for heterologous gene expression

  • Inducible systems using theophylline-responsive riboswitches for controlled expression

• Transformation protocols:

  • Conjugation using E. coli S17-1 as donor strain

  • Selection on double-layer plates with kanamycin (50 μg/ml) in the upper layer

  • Use of sucrose (2.5% w/v) for counter-selection in the second crossover event

• Recommended modifications for essential genes:

  • Merodiploid strategy to maintain one functional copy while manipulating the other

  • Conditional knockdowns using riboswitches rather than knockouts

  • C-terminal tagging to maintain native regulation

• Verification methods:

  • PCR screening with primers flanking the modified region

  • Sequence confirmation of the entire modified locus

  • Functional assays measuring growth at different temperatures

  • Western blotting to confirm expression of modified protein

These techniques have been successfully applied to study other essential genes in B. bacteriovorus and can be specifically optimized for rplT manipulation .

What are the optimal conditions for studying L20's function under cold shock in B. bacteriovorus?

To effectively study L20's function under cold shock conditions in B. bacteriovorus, researchers should carefully consider these methodological parameters:

• Culture conditions:

  • Standard growth temperature: 29°C for optimal predatory activity

  • Cold shock temperature: 20°C (critical threshold for observing bipA and L20-dependent phenotypes)

  • Predator-to-prey ratio: 1:10,000 for optimal predation efficiency

  • Media: HEPES buffer supplemented with Ca²⁺ (25 mM) and Mg²⁺ (2 mM)

• Experimental timeline:

  • Pre-adaptation: Grow cultures at 29°C until late attack phase

  • Cold shock: Transfer to 20°C for various timepoints (1h, 3h, 6h, 24h)

  • Recovery: Return to 29°C to assess reversibility of effects

• Analytical techniques:

  • Ribosome profiling before and after cold shock

  • Polysome analysis to measure translation efficiency

  • qRT-PCR to measure expression changes in rplT, bipA, and related genes

  • Microscopy to observe morphological changes

• Control strains for comparison:

  Strain	Genotype	Expected Growth at 20°C	Ribosome Assembly
  HD100	Wild-type	+++	Normal
  ESC19	ΔbipA	+	Defective
  ESC19/pBIS02-2	ΔbipA + rplT overexpression	++	Partially restored
  HD100/pBIS02-2NM	Wild-type + rplT(R50A/R51A)	++	Partially defective
  HD100/pBIS02-2CM	Wild-type + rplT(R92A/K93A)	++	Partially defective

This comprehensive approach has revealed that both BipA and L20 are crucial for proper 50S ribosomal subunit biogenesis under cold-shock conditions, with distinct but overlapping functions .

How can researchers differentiate between L20's roles in ribosome structure versus translational regulation?

To distinguish between the structural and regulatory roles of L20 in B. bacteriovorus, researchers should implement these methodological approaches:

• Domain-specific mutations:

  • Create mutations in RNA-binding domains (affecting structure)

  • Create mutations in mRNA-binding domains (affecting regulation)

  • Test complementation of each mutant in appropriate assays

• Structural analysis techniques:

  • Cryo-EM of ribosomes containing wild-type versus mutant L20

  • Sucrose gradient analysis to assess ribosome assembly

  • DMS footprinting to identify structural changes in rRNA

• Regulatory function assessment:

  • Reporter gene assays using lacZ fusions to the rpmI-rplT operon

  • Measurement of mRNA and protein levels of L20 and L35

  • RNA binding assays with regulatory RNA segments

• Separation of functions:

  • Express heterologous L20 proteins lacking regulatory domains

  • Create chimeric proteins with separated functional domains

  • Uncoupling experiments where L20 is expressed from an orthogonal system

These approaches allow researchers to attribute specific phenotypes to either the structural or regulatory function of L20, providing insights into how this dual-function protein coordinates ribosome assembly and gene expression in B. bacteriovorus .

What comparative approaches can reveal unique features of B. bacteriovorus L20 versus L20 from prey bacteria?

To identify unique features of B. bacteriovorus L20 compared to L20 proteins from prey bacteria, researchers should employ these comparative approaches:

• Sequence and structural analysis:

  • Multiple sequence alignment of L20 from B. bacteriovorus, E. coli, P. putida, and other prey bacteria

  • Homology modeling and structural comparisons

  • Identification of predator-specific amino acid substitutions

  • Analysis of evolutionary selection pressures using dN/dS ratios

• Functional complementation experiments:

  • Express B. bacteriovorus L20 in E. coli L20-depleted strains

  • Express prey bacteria L20 in B. bacteriovorus with reduced L20 function

  • Create chimeric L20 proteins to map functional domains

• Biochemical property comparisons:

  • RNA binding affinity and specificity

  • Protein stability under various stress conditions

  • Interaction with ribosomal assembly factors

• Regulatory mechanism analysis:

  • Comparison of mRNA regulatory elements between species

  • Analysis of autoregulatory efficiency across species

  • Assessment of translation rates and protein turnover

This comparative approach has revealed that while L20 proteins share core functions across bacterial species, predatory bacteria like B. bacteriovorus may have evolved specialized features that support their unique lifecycle and rapid adaptation to changing environments .

How can recombinant L20 be used to study ribosome assembly in host-independent B. bacteriovorus strains?

Host-independent (HI) B. bacteriovorus strains offer unique opportunities to study ribosome assembly without the complications of the predatory lifecycle. Researchers can utilize recombinant L20 with these methodological approaches:

• HI strain development and characterization:

  • Isolate HI strains through prolonged cultivation with amino acids and cofactors

  • Verify HI phenotype by confirming growth on rich media

  • Sequence the hit locus to identify mutations

  • Compare ribosome assembly between wild-type and HI strains

• L20 expression systems for HI strains:

  • Develop plasmid-based expression systems compatible with HI growth

  • Create fluorescently tagged L20 for visualization

  • Establish inducible expression using theophylline-activated riboswitches

• Ribosome assembly analysis in HI strains:

  • Purify ribosomes from HI strains with different L20 expression levels

  • Compare rRNA processing patterns between predatory and HI lifestyles

  • Analyze ribosome composition using quantitative proteomics

  • Monitor translation rates using radioactive amino acid incorporation

• Stress response studies:

  • Subject HI strains to cold shock, antibiotics, and nutrient limitation

  • Compare the role of L20 in stress adaptation between predatory and HI lifestyles

  • Test whether L20 overexpression enhances stress tolerance in HI strains

This approach leverages the simplified experimental system of HI strains while providing insights into the fundamental aspects of ribosome assembly in B. bacteriovorus .

How might L20 function during predation-to-growth phase transition in B. bacteriovorus?

The transition from attack phase to growth phase represents a critical period in the B. bacteriovorus lifecycle with major physiological changes. To investigate L20's role during this transition, researchers should consider these methodological approaches:

• Phase-specific expression analysis:

  • Synchronize predator-prey interactions and sample at defined timepoints

  • Perform RNA-seq to measure rplT expression throughout the predatory cycle

  • Conduct Western blotting with phase-specific samples

  • Use fluorescently tagged L20 to track localization during lifecycle transitions

• Ribosome remodeling assessment:

  • Purify ribosomes from attack phase versus growth phase cells

  • Analyze composition using quantitative proteomics

  • Measure translation rates in different phases

  • Assess rRNA modifications specific to different phases

• Genetic manipulation strategies:

  • Create conditional knockdown strains using riboswitches

  • Express modified L20 only during specific phases

  • Test the impact of L20 mutations on phase transition efficiency

• Microscopy approaches:

  • Use high-resolution microscopy to visualize ribosome localization during transitions

  • Employ FRAP (Fluorescence Recovery After Photobleaching) to measure protein dynamics

  • Track chromosome replication in relation to L20 expression

This integrated approach can reveal how L20 contributes to the dramatic physiological changes that occur as B. bacteriovorus transitions from free-living predator to intracellular growth phase .

What role might L20 play in the adaptation of B. bacteriovorus to different prey bacteria?

To investigate L20's potential role in B. bacteriovorus adaptation to different prey bacteria, researchers should implement these methodological approaches:

• Prey-specific expression profiling:

  • Culture B. bacteriovorus on different prey species (E. coli, P. putida, V. cholerae)

  • Perform RNA-seq and proteomics to measure rplT expression

  • Compare ribosome composition when grown on different prey

  • Assess translation efficiency on different prey species

• Adaptation experiments:

  • Serially passage B. bacteriovorus on specific prey species

  • Monitor changes in rplT sequence or expression

  • Test cross-prey predation efficiency before and after adaptation

  • Sequence genomes of adapted strains to identify mutations

• Genetic manipulation studies:

  • Create rplT variants with altered expression levels

  • Test these strains for predation efficiency on different prey

  • Perform competition assays between wild-type and modified strains

  • Assess stress tolerance on different prey bacteria

• Comparative prey nutritional analysis:

  • Analyze amino acid composition of different prey species

  • Determine how prey nutritional content affects ribosome assembly

  • Test L20's role in adapting to different nutritional environments

This comprehensive approach can reveal whether L20 plays a role in the remarkable adaptability of B. bacteriovorus to diverse prey bacteria, potentially through fine-tuning translation to match changing nutritional or environmental conditions .

How might synthetic biology approaches using recombinant L20 enhance predatory functions of B. bacteriovorus?

Synthetic biology approaches using recombinant L20 offer exciting possibilities for enhancing the predatory capabilities of B. bacteriovorus. Researchers should consider these methodological strategies:

• Engineered L20 variants:

  • Design L20 proteins with enhanced stability under various stress conditions

  • Create chimeric L20 with domains from extremophile bacteria

  • Develop L20 variants with improved ribosome assembly efficiency

  • Test these variants for enhanced predation under challenging conditions

• Expression optimization systems:

  • Develop synthetic promoters for controlled L20 expression

  • Create inducible systems responsive to prey-derived signals

  • Engineer ribosome binding sites for optimal translation

  • Implement feedback loops for homeostatic L20 levels

• Multipronged engineering approaches:

  • Co-express L20 with other ribosome assembly factors (BipA, RbfA)

  • Engineer ribosomes with modified rRNA to better interact with enhanced L20

  • Create synthetic regulatory circuits controlling L20 and predatory genes

  • Develop dual-function L20 proteins with additional antimicrobial domains

• Application-focused testing:

  • Evaluate enhanced strains against multidrug-resistant pathogens

  • Test predation efficiency in biofilm disruption

  • Assess stability and activity in physiologically relevant conditions

  • Measure predation kinetics compared to wild-type strains

This synthetic biology approach could lead to B. bacteriovorus strains with enhanced predatory capabilities for applications in biocontrol or therapeutic development .

What techniques could advance our understanding of how L20 coordinated with MreB cytoskeleton during B. bacteriovorus lifecycle?

The potential coordination between L20 and the MreB cytoskeleton during the complex lifecycle of B. bacteriovorus represents an exciting research frontier. To investigate this relationship, researchers should consider these methodological approaches:

• Co-localization and interaction studies:

  • Create fluorescently tagged L20 and MreB proteins

  • Perform live-cell imaging throughout the predatory cycle

  • Conduct co-immunoprecipitation to identify protein-protein interactions

  • Use proximity labeling techniques (BioID, APEX) to identify near-neighbors

• Cytoskeletal manipulation experiments:

  • Treatment with the MreB inhibitor A22 while monitoring L20 localization

  • Create conditional MreB mutants and assess impact on L20 function

  • Express modified MreB proteins and measure effects on ribosome assembly

  • Perform cytoskeletal drug gradient experiments to identify threshold effects

• Combined genetic approaches:

  • Create double mutants targeting both systems

  • Perform suppressor screens to identify genetic interactions

  • Engineer synthetic connections between cytoskeletal and ribosomal systems

  • Develop optogenetic tools to manipulate MreB in real-time while tracking L20

• Structural biology integration:

  • Use cryo-electron tomography to visualize ribosome-cytoskeleton interactions

  • Develop in vitro reconstitution systems with purified components

  • Model physical connections between ribosomes and cytoskeletal elements

  • Map interaction surfaces through cross-linking mass spectrometry

This integrated approach could reveal how ribosome assembly and localization coordinate with cytoskeletal dynamics during the dramatic morphological changes that occur throughout the B. bacteriovorus lifecycle .

How might high-throughput approaches help identify L20 interaction partners during B. bacteriovorus predation?

High-throughput approaches offer powerful tools for uncovering the interaction network of L20 during the complex predatory lifecycle of B. bacteriovorus. Researchers should consider these methodological strategies:

• Interactome mapping technologies:

  • Affinity purification coupled with mass spectrometry (AP-MS)

  • Proximity-dependent biotin identification (BioID)

  • Cross-linking mass spectrometry (XL-MS)

  • Protein microarrays using purified B. bacteriovorus proteins

• Genetic screening approaches:

  • Transposon sequencing (Tn-seq) to identify genetic interactions

  • Suppressor screens to identify functional relationships

  • Synthetic genetic array (SGA) analysis adapted for B. bacteriovorus

  • CRISPR interference screens in HI strains

• Dynamic interactome analysis:

  • Temporal interactome profiling across predatory lifecycle

  • Differential interactome analysis under various stress conditions

  • Comparison of interaction networks between attack and growth phases

  • Quantitative changes in interactions during prey transition

• Computational integration:

  • Network analysis to identify functional modules

  • Evolutionary analysis of interaction conservation

  • Structural modeling of protein complexes

  • Machine learning approaches to predict functional relationships

This comprehensive high-throughput strategy could reveal the complex network of interactions that allow L20 to coordinate ribosome assembly with the dramatic physiological changes that occur during predation .