Recombinant Bdellovibrio bacteriovorus 50S ribosomal protein L6 (rplF)

What is Bdellovibrio bacteriovorus and why is ribosomal protein L6 significant in this organism?

Bdellovibrio bacteriovorus is a predatory bacterium that invades and consumes Gram-negative bacteria, including various pathogens. Its unique life cycle consists of two primary phases: the non-replicating attack phase (AP), where the predator actively searches for prey, and the intraperiplasmic phase (IP), which begins when the predator invades suitable prey. A third transition phase has also been identified where prey-derived cues trigger specific bdellovibrio transcription profiles .

Ribosomal protein L6 (rplF) is an essential component of the 50S ribosomal subunit involved in the late stage assembly of functional ribosomes. In B. bacteriovorus, L6 likely plays crucial roles in:

• Ribosome assembly and stability during predatory growth

• Translation efficiency during the rapid protein synthesis required for prey invasion and digestion

• Potentially supporting the expression of hydrolytic enzymes necessary for prey consumption

The significance of L6 is heightened in B. bacteriovorus due to the organism's predatory lifestyle, which requires efficient protein synthesis to produce the numerous hydrolytic enzymes needed for prey digestion and utilization .

How is the recombinant B. bacteriovorus 50S ribosomal protein L6 expressed and purified in laboratory settings?

Expression and purification of recombinant B. bacteriovorus 50S ribosomal protein L6 typically follows this methodological workflow:

Expression system selection:

Most commonly, recombinant B. bacteriovorus proteins are expressed in E. coli systems using specialized expression vectors. For ribosomal proteins specifically, BL21(DE3) strains or derivatives are frequently employed due to their reduced protease activity .

Cloning strategy:

• PCR amplification of the rplF gene from B. bacteriovorus genomic DNA

• Addition of appropriate restriction sites or recombination sequences

• Insertion into an expression vector containing a suitable promoter and fusion tag

Expression optimization:

Based on protocols for other B. bacteriovorus proteins, optimal expression conditions typically include:

• Induction at OD600 of 0.6-0.8

• IPTG concentration of 0.5-1.0 mM

• Post-induction growth at 16-25°C to enhance protein solubility

• Expression time of 4-16 hours

Purification approach:

A standard purification protocol would involve:

• Cell lysis via sonication or pressure homogenization in a buffer containing protease inhibitors

• Clarification by centrifugation (20,000 × g for 30 minutes)

• Affinity chromatography using His-tag or other fusion tag

• Size exclusion chromatography to increase purity

• Buffer exchange to a stabilizing buffer

Storage recommendations:

For optimal stability, the purified protein should be stored according to these parameters:

• Liquid form: 6 months at -20°C/-80°C

• Lyophilized form: 12 months at -20°C/-80°C

The protein concentration is typically determined using absorbance at 280 nm with the calculated extinction coefficient or via Bradford/BCA protein assays.

What are the structural and functional characteristics of B. bacteriovorus ribosomal protein L6?

The structural and functional characteristics of B. bacteriovorus ribosomal protein L6 can be inferred from general ribosomal protein properties and specific data from related bacteria:

Structural properties:

• Typically consists of approximately 177 amino acids, similar to E. coli L6

• Contains domains that interact with 23S rRNA, particularly helix 97

• Likely possesses RNA-binding motifs essential for ribosome assembly

• Features a tertiary structure that facilitates interactions with other ribosomal proteins

Functional roles:

• Ribosome assembly: Critical for the late stage assembly of the 50S ribosomal subunit. L6-depleted cells accumulate 45S precursor particles that lack L6, indicating its essential role in ribosome maturation .

• Translation activity: L6 contributes to factor-dependent GTPase activity of the ribosome. Ribosomes lacking L6 show reduced GTPase activity, suggesting L6's involvement in interactions with translation factors .

• Structural stability: Serves as a scaffold protein that helps maintain the integrity of the 50S subunit through interactions with rRNA and other ribosomal proteins.

• Metabolic adaptation: Given B. bacteriovorus' predatory lifestyle and potential inability to synthesize some amino acids required for protein synthesis, L6 may have adapted to function efficiently under conditions where amino acids are derived from prey digestion .

The positioning of L6 near functional centers of the ribosome suggests it may influence translation efficiency and fidelity, which would be particularly important during the rapid growth phase within prey cells.

How does B. bacteriovorus L6 compare to ribosomal protein L6 in other bacterial species?

Ribosomal protein L6 shows varying degrees of conservation across bacterial species, with both preserved functional domains and species-specific adaptations:

Sequence conservation:

While the search results don't provide direct sequence comparisons for B. bacteriovorus L6, database analysis tools like OrthoInspector can identify evolutionary relationships between L6 proteins from different species. In general, ribosomal proteins maintain conserved core domains involved in rRNA binding and ribosome assembly.

Comparative analysis table:

Functional divergence:

The predatory lifestyle of B. bacteriovorus may have driven specific adaptations in its translational machinery, including L6. These adaptations might include:

• Altered affinity for rRNA or translation factors

• Modified interactions with other ribosomal proteins

• Optimized activity under the nutrient flux conditions experienced during prey consumption

Evolutionary implications:

The conservation of L6 across diverse bacterial phyla underscores its essential role in ribosome function. Comparative genomic analysis could reveal how sequence variations correlate with different bacterial lifestyles, providing insights into the evolutionary pressures shaping ribosomal proteins.

Understanding these similarities and differences is crucial for designing experiments that target L6 function specifically in B. bacteriovorus without affecting non-target organisms.

What are the experimental challenges in expressing and purifying recombinant B. bacteriovorus L6 protein?

Researchers face several technical challenges when expressing and purifying recombinant B. bacteriovorus L6 protein:

Expression challenges:

• Codon usage bias: B. bacteriovorus has a different codon usage pattern than common expression hosts like E. coli, potentially leading to:

  • Translational pausing

  • Premature termination

  • Low yield of full-length protein

  Solution: Codon optimization or use of strains supplemented with rare tRNAs (e.g., Rosetta™)

• Protein toxicity: Ribosomal proteins often interact with RNA and other cellular components, potentially causing toxicity when overexpressed in heterologous hosts.

  Solution: Use of tightly regulated inducible promoters and lower induction temperatures (16-20°C)

• Solubility issues: In the absence of their natural rRNA binding partners, ribosomal proteins often form insoluble aggregates.

  Solution: Expression as fusion proteins with solubility-enhancing tags (MBP, SUMO, etc.) or inclusion of molecular chaperones

Purification challenges:

• Nucleic acid contamination: L6's natural RNA-binding properties can result in co-purification with host RNA.

  Solution: High-salt washes (500 mM-1M NaCl) and nuclease treatment during purification

• Protein instability: Isolated ribosomal proteins may have reduced stability outside their natural complex.

  Solution: Inclusion of stabilizing agents (glycerol 5-50%), optimal buffer conditions, and storage at -80°C

• Protein authenticity: Ensuring the recombinant protein retains native folding and activity.

  Solution: Circular dichroism spectroscopy to verify secondary structure elements and functional binding assays with 23S rRNA

Validation challenges:

• Functional assessment: Verifying that purified L6 maintains its physiological activity is complex since its natural function occurs within the assembled ribosome.

  Solution: In vitro reconstitution assays with 50S ribosomal components or complementation of L6-depleted strains

• Structural integrity: Confirming proper folding is essential for functional studies.

  Solution: Limited proteolysis to assess domain structure and thermal shift assays to evaluate protein stability

These challenges necessitate careful optimization of expression conditions, purification protocols, and validation methods to obtain functionally relevant recombinant B. bacteriovorus L6 protein.

How can genetic tools be used to manipulate L6 expression in B. bacteriovorus?

Several genetic tools and approaches have been developed for B. bacteriovorus that can be adapted to manipulate L6 expression:

Promoter-based expression systems:

Four native B. bacteriovorus promoters have been identified as highly active during the attack phase: P1753, P3184, PAPsRNA5, and PmerRNA . These can be utilized to:

• Drive constitutive expression of modified L6 proteins

• Express additional copies of L6 with reporter tags

• Create expression cassettes with varying strengths

The standard E. coli lac promoter shows weak expression in B. bacteriovorus, making native promoters preferable for robust expression .

Riboswitch-based regulation:

Theophylline-activated riboswitches have been successfully demonstrated in B. bacteriovorus . This approach allows:

• Chemical control of L6 expression levels

• Creation of conditional L6 mutants

• Temporal control of L6 function during predation cycles

The system can be designed by inserting the theophylline-responsive riboswitch (Theo-F) immediately downstream of the transcription start site of the rplF gene .

Chromosomal integration strategies:

The suicide plasmid pK18mobsacB has been used for targeted gene modifications in B. bacteriovorus . For L6 manipulation, this approach enables:

• Precise replacement of the native rplF gene with modified versions

• Introduction of point mutations to study structure-function relationships

• Creation of knock-in mutants with regulated expression

The protocol involves:

• Cloning homologous regions flanking the integration site into pK18mobsacB

• Conjugal transfer from E. coli S17-1 to B. bacteriovorus

• Selection of merodiploids with kanamycin

• Counter-selection with sucrose to identify double crossover events

Plasmid-based expression systems:

Plasmids like pSUP404.2 have been successfully used in B. bacteriovorus . For L6 studies, these vectors allow:

• Expression of tagged L6 variants

• Co-expression of L6 with interacting partners

• Introduction of multiple copies of L6 variants

RBS optimization:

The RBS Calculator tool has been employed to optimize ribosome binding sites for efficient translation in B. bacteriovorus . This approach enables:

• Fine-tuning of L6 expression levels

• Balancing expression of L6 with other experimental components

• Creating an expression gradient for dose-dependent studies

Combined genetic approaches:

For advanced functional studies, combinations of these tools can be implemented:

• Riboswitch-controlled expression of the native L6 combined with constitutive expression of a modified L6

• Dual reporter systems to monitor both L6 expression and predatory activity

• Creation of L6 variant libraries with varying expression levels for high-throughput functional screening

These genetic tools provide researchers with multiple options for manipulating L6 expression to study its role in ribosome assembly, protein synthesis, and predatory behavior in B. bacteriovorus.

What role might L6 play in the unique predatory life cycle of B. bacteriovorus?

Ribosomal protein L6 potentially serves specialized functions throughout the distinct phases of B. bacteriovorus' predatory lifecycle:

Attack Phase Role:

During this non-replicative, high-motility phase, L6 likely contributes to:

• Translation of proteins required for flagellar movement and chemotaxis

• Synthesis of invasion-related proteins maintained in a predation-ready state

• Efficient use of limited internal resources while searching for prey

The high activity of certain promoters during this phase suggests selective protein expression, in which L6-containing ribosomes would participate .

Transition Phase Role:

When B. bacteriovorus attaches to prey and prepares for invasion, L6 may be involved in:

• Rapid translation reprogramming in response to prey-derived cues

• Synthesis of proteins required for pore formation and entry into prey

• Initial production of early-stage hydrolytic enzymes

The transition from free-living to invasion state represents a major metabolic shift that would require adaptable translation machinery .

Growth Phase Function:

Inside the bdelloplast (modified prey cell), L6 becomes especially critical during:

• Extensive synthesis of hydrolytic enzymes that digest prey contents

• Translation of proteins needed for DNA replication and filamentous growth

• Adaptation to changing nutrient availability as prey components are metabolized

The ability of B. bacteriovorus to grow using exclusively prey-derived resources suggests its ribosomes, including L6, may have specialized adaptations for function under fluctuating amino acid and nucleotide pools .

Septation and Release Phase Contribution:

As the predator divides into progeny cells, L6 likely participates in:

• Translation of cell division proteins

• Production of new ribosomes for progeny cells

• Synthesis of proteins needed for flagella formation and progeny release

The synchronous septation pattern of B. bacteriovorus that allows both odd and even numbers of progeny suggests specialized control of division, possibly including tailored protein synthesis .

Molecular evidence linking L6 to predation:

While direct experimental evidence specifically connecting L6 to predatory functions is limited, parallels can be drawn from related research:

• Studies of the flagellar sigma factor fliA (controlled by riboswitches in experimental settings) showed that its regulation affects predation kinetics

• L6-deficient E. coli shows impaired ribosome assembly and growth defects , suggesting that L6 perturbation in B. bacteriovorus would significantly impact its predatory capability

The essential nature of efficient protein synthesis throughout the predatory lifecycle positions L6 as a critical, if understudied, component of B. bacteriovorus' unique predatory mechanisms.

How can the function of ribosomal protein L6 be studied in the context of B. bacteriovorus predation?

Investigating L6 function in the context of B. bacteriovorus predation requires integrating molecular genetics, biochemistry, and predation assays:

Genetic manipulation approaches:

• Conditional expression systems:

  • Implement theophylline-responsive riboswitches to control L6 expression

  • Design a system where L6 depletion can be triggered at different stages of the predatory cycle

  • Create dose-dependent expression to correlate L6 levels with predatory efficiency

• Domain mapping:

  • Generate targeted mutations in different functional domains of L6

  • Create chimeric L6 proteins combining domains from different bacterial species

  • Introduce fluorescent protein fusions to track L6 localization during predation

Biochemical and structural analyses:

• Ribosome profiling:

  • Isolate ribosomes from different predatory phases

  • Analyze ribosome composition and L6 incorporation

  • Compare translation efficiency between wild-type and L6-modified strains

• Protein-RNA interaction studies:

  • Perform RNA immunoprecipitation to identify L6-interacting RNAs during predation

  • Use crosslinking approaches to capture transient interactions

  • Map binding sites of L6 on 23S rRNA in the context of predatory growth

Predation phenotype assessments:

• Predation kinetics assays:

  • Quantify prey cell lysis rates using optical density measurements

  • Monitor predator population growth within prey using fluorescent markers

  • Compare wild-type with L6-modified strains across multiple prey species

• Microscopy-based analyses:

  • Track predator-prey interactions using time-lapse microscopy

  • Visualize bdelloplast formation and development

  • Quantify septation patterns and progeny numbers in L6-modified strains

Methodological workflow for L6 function during predation:

Specialized experimental considerations:

• Use of synchronized predator populations to isolate specific predatory phases

• Implementation of multicolor fluorescent labeling to distinguish predator-specific translation from prey-specific translation

• Development of in vitro translation systems using isolated B. bacteriovorus ribosomes with modified L6 components

This multifaceted approach would provide comprehensive insights into how L6 contributes to the unique predatory lifestyle of B. bacteriovorus and potentially inform strategies for enhancing its use as a biocontrol agent .

What roles do bacterial ribosomes and their components play in antibiotic resistance mechanisms?

The bacterial ribosome, including ribosomal proteins like L6, is central to antibiotic resistance through multiple mechanisms:

Direct resistance mechanisms involving ribosomal components:

• Target site modifications:

  • Mutations in ribosomal proteins can alter antibiotic binding sites

  • Modifications to rRNA (e.g., methylation) can prevent antibiotic interactions

  • Structural changes in the ribosome can reduce antibiotic affinity

• Protection mechanisms:

  • Specialized proteins can bind to ribosomes, preventing antibiotic access

  • Alterations in ribosomal assembly can create drug-resistant ribosome populations

  • Heterogeneity in ribosome composition can contribute to population-level resistance

Evidence from B. bacteriovorus and other bacteria:

Research on trimethoprim (TMP) resistance in B. bacteriovorus provides insights into ribosome-related resistance mechanisms. One study identified that the gene bd3231 (encoding dihydrofolate reductase) confers high resistance to TMP when expressed in E. coli . While this doesn't directly involve L6, it demonstrates how bacterial species can possess intrinsic resistance mechanisms relevant to protein synthesis.

L6-specific resistance considerations:

• Structural adaptations:

  • Variations in L6 sequence between species may contribute to different antibiotic susceptibility profiles

  • L6 interactions with 23S rRNA could influence binding of macrolide antibiotics that target this region

• Functional adaptations:

  • Changes in L6 that affect ribosome assembly may influence susceptibility to antibiotics targeting the assembly process

  • L6 involvement in GTPase activity suggests potential effects on antibiotics that interfere with translation factors

Implications for antimicrobial development:

• Ribosomal proteins as targets:

  • Understanding species-specific variations in L6 and other ribosomal proteins could enable development of narrow-spectrum antibiotics

  • Targeting ribosomal protein-rRNA interactions represents a potentially underexplored antibiotic strategy

• B. bacteriovorus as a model:

  • Studying intrinsic antibiotic resistance in B. bacteriovorus could reveal novel resistance mechanisms

  • The predatory lifestyle may have driven unique adaptations in ribosomal components to function efficiently within prey cells

• Resistance prediction:

  • Structural analysis of L6 and its interactions could help predict potential resistance mutations

  • Comparative genomics across bacterial species could identify natural variations in L6 that correlate with antibiotic susceptibility

Understanding the complex roles of ribosomal proteins like L6 in antibiotic resistance mechanisms is crucial for developing new antimicrobial strategies and predicting resistance evolution.

How might modifications to L6 affect ribosome assembly and function in B. bacteriovorus?

Modifications to ribosomal protein L6 could profoundly impact B. bacteriovorus ribosome assembly and function through several mechanisms:

Effects on ribosome assembly:

• Assembly defects:
  Modifications to L6 domains that interact with 23S rRNA could disrupt the assembly pathway, potentially leading to accumulation of 45S precursor particles similar to observations in E. coli .

• Altered assembly kinetics:
  Changes affecting L6 incorporation could create a bottleneck in ribosome biogenesis, resulting in:

  • Slower formation of functional ribosomes

  • Accumulation of assembly intermediates

  • Disrupted stoichiometry of ribosomal components

• Structural integrity:
  L6 contributes to the structural stability of the 50S subunit through interactions with rRNA and neighboring proteins. Modifications could lead to:

  • Reduced subunit stability

  • Altered subunit association/dissociation dynamics

  • Formation of ribosomes with suboptimal conformations

Impact on translation processes:

• Translation efficiency:
  L6 modifications that affect GTPase activity could influence:

  • Initiation rates

  • Elongation speed

  • Termination efficiency

  • Ribosome recycling

• Translation fidelity:
  Alterations to L6 might affect:

  • tRNA selection accuracy

  • Reading frame maintenance

  • Stop codon recognition

  • Susceptibility to miscoding events

• Response to regulatory factors:
  Modified L6 could alter interactions with:

  • Translation factors (IF1/2/3, EF-Tu, EF-G)

  • Ribosome-associated quality control systems

  • Specialized regulatory factors

Predation-specific consequences:

• Predation kinetics:
  Similar to the effects observed with fliA regulation , L6 modifications could result in:

  • Altered predation rates

  • Changes in prey preference or range

  • Modified bdelloplast development timeframes

• Prey utilization efficiency:
  B. bacteriovorus relies on prey-derived nutrients, including amino acids . L6 modifications affecting translation could impact:

  • Efficiency of prey resource utilization

  • Ability to adapt to different prey species

  • Yield of progeny cells per prey

• Life cycle regulation:
  Translation reprogramming is likely critical during transitions between predatory phases. L6 modifications might affect:

  • Attack phase to growth phase transition

  • Synchronization of progeny formation

  • Coordination of prey exit and new prey seeking

Experimental consequences table:

These predictions provide a framework for designing experimental studies on L6 function in B. bacteriovorus and understanding how ribosomal protein modifications might be leveraged to modulate predatory behavior for biotechnological applications.